# Patent

## Title
ARF-BP1 as mediator of p53-dependent and independent tumor suppression and uses thereof

## Abstract

The present invention relates to the mechanism of ARF-mediated cell growth suppression. ARF-BP1 is identified as a novel ubiquitin ligase, and a major component of ARF-containing nuclear complexes in human cells. The present invention discloses a novel mechanism of ARF-mediated p53 activation and that ARF-BP1 is a critical mediator of both p53-independent and p53-dependent tumor suppression functions of ARF. Inactivation of ARF-BP1 in normal cells stabilizes p53 and induces p53-dependent apoptosis. Inactivation of ARF-BP1, but not Mdm2, in p53-wildtype cells promotes cell growth inhibition in a manner reminiscent of ARF induction. ARF-BP1 directly binds and ubiquitinates p53 and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization in Mdm2-null cells. ARF-BP1 is advantageous over Mdm2 as a target for suppressing tumor cell growth regardless of p53 status.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims the benefit of U.S. Provisional Application Ser. No. 60/610,506, filed on Sep. 15, 2004, which is incorporated herein by reference thereto.


STATEMENT OF GOVERNMENT INTEREST
This invention was made, in part, with government support under NIH grant No. CA097403. As such, the United States government has certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates to the mechanism of ARF-mediated cell growth suppression, and more specifically to the p53/Mdm2-independent function of ARF.
BACKGROUND OF THE INVENTION
Neoplasia is a disease characterized by an abnormal proliferation of cell growth known as a neoplasm. Neoplasms may manifest in the form of a leukemia or a tumor, and may be benign or malignant. Malignant neoplasms, in particular, can result in a serious disease state, which may threaten life. Significant research efforts and resources have been directed toward the elucidation of antineoplastic measures, including chemotherapeutic agents, which are effective in treating patients suffering from neoplasia. Effective antineoplastic agents include those which inhibit or control the rapid proliferation of cells associated with neoplasms, those which effect regression or remission of neoplasms, and those which generally prolong the survival of patients suffering from neoplasia. Successful treatment of malignant neoplasia, or cancer, requires elimination of all malignant cells, whether they are found at the primary site, have extended to local/regional areas, or have metastasized to other regions of the body. The major therapies for treating neoplasia are surgery and radiotherapy (for local and local/regional neoplasms) and chemotherapy (for systemic sites).
Despite the various methods for detecting, diagnosing, and treating cancers, the disease remains prevalent in all segments of society, and is often fatal. Clearly, alternative strategies for detection (including the development of markers that can identify neoplasias at an early stage) and treatment are needed to improve survival in cancer patients. In particular, a better understanding of tumor suppressors, and tumor-suppression pathways, would provide a basis from which novel detection, diagnostic, and treatment regimens may be developed.
The p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress (Levine, A. J., Cell 88:323-31, 1997; Oren, M., J. Biol. Chem. 274: 36031-034, 1999). p53 can be thought of as the central node of a regulatory circuit that monitors signaling pathways from diverse sources, including DNA damage responses (e.g., ATM/ATR activation), abnormal oncogenic events (e.g., Myc or Ras activation) and everyday cellular processes (e.g., growth factor stimulation). While p53 mutations have been well documented in more than half of all human tumors (Hollstein et al., Mutat Res. 431:199-209, 1999), defects in other components of the p53 pathway, such as the ARF tumor suppressor, are observed in tumor cells that retain wildtype p53 (Sherr, C. J., Nat Rev Mol Cell Biol 2:731-737, 2001; Sharpless, N. E., DePinho, R. A., J Clin Invest 113:160-8, 2004). Activation of the p53 pathway appears to be a common, if not universal, feature of human cancer.
The mechanisms of p53 activation are not fully understood, but are generally thought to entail post-translational modifications, such as ubiquitination, phosphorylation and acetylation. Ubiquitination of p53 was first discovered in papilloma-infected cells, where p53 degradation is mediated by the viral E6 protein and a HECT-domain containing ubiquitin ligase called E6-AP (Munger, K., Howley, P. M., Virus Res 89:213-228, 2002). In normal cells, Mdm2 acts as a specific E3 ubiquitin ligase for p53, which, if malignantly activated, has the potential to counteract the tumor suppressor activity of p53. The critical role of Mdm2 in regulating p53 is illustrated by studies carried out in mice where inactivation of p53 was shown to completely rescue the embryonic lethality caused by loss of Mdm2 function (Montes de Oca Luna, R., Wagner, D. S., Lozano, G., Nature 378:203-206, 1995).
Although earlier studies suggested that Mdm2 is the primary factor in controlling p53 stabilities, the degradation of p53 is more complex than originally anticipated. The present inventor found that Mdm2 differentially catalyzes either monoubiquitination and polyubiquitination of p53 in a dosage-dependent manner (Li, M., Brooks, C. L., Wu-Baer, F., Chen. D., Baer, R., Gu, W., Science 302:1972-1975, 2003). Low levels of Mdm2 activity induce monoubiquitination and nuclear export of p53, whereas high levels promote polyubiquitination and nuclear degradation of p53. These mechanisms are exploited in different physiological settings. On one hand, Mdm2-mediated polyubiquitination and nuclear degradation may play a dominant role in suppressing p53 function when Mdm2 is malignantly overexpressed or during the late stages of a DNA damage response. On the other hand, Mdm2-mediated monoubiquitination and subsequent cytoplasmic translocation of p53 may represent an important means of p53 regulation in unstressed cells, where Mdm2 is maintained at low levels (Li et al., 2003, supra). Moreover, additional cellular factors may be necessary to facilitate p53 degradation, particularly when endogenous Mdm2 activities are not sufficient to catalyze p53 polyubiquitination directly. It was recently reported that ubiquitin ligases COP1 and Pirh2 are directly involved in p53 degradation (Dornan, D., Wertz, L, Shimizu, H., Arnott, D., Frantz, G. D., Dowd, P., O'Rourke, K., Koeppen, H., Dixit, V. M., Nature 429:86-92, 2004). Therefore, while Mdm2 is a key regulator of p53 function, p53 degradation acts through both Mdm2-dependent and Mdm2-independent pathways in vivo.
ARF (known as p14ARF in humans and p19ARF in mouse) was identified as an alternative transcript of the Ink4a, ARF tumor suppressor locus, a gene that encodes the p16Ink4a inhibitor of cyclin-dependent kinases. By virtue of its unique first exon, the ARF transcript encodes a protein that is unrelated to p16Ink4a. Nevertheless, ARF, like p16Ink4a, exhibits tumor suppression functions, as demonstrated by the tumor susceptibility phenotype of p14ARF-deficient mice. ARF suppresses abherrant cell growth in response to oncogene activation, at least in part, by inducing the p53 pathway (Sherr, et al., 2001, supra). The ARF induction of p53 appears to be mediated through Mdm2, since overexpressed ARF interacts directly with Mdm2 and inhibits its ability to promote p53 degradation (Zhang, Y., Xiong, Y., Yarbrough, W. G., Cell 92:125-34, 1998). The mechanisms by which ARF modulates the Mdm2/p53 pathway appears to be complex, both stabilizing p53 by binding and sequestering Mdm2 and activating p53 function by directly inhibiting the ubiquitin ligase activity of Mdm2.
Interestingly, ARF also has tumor suppressor functions that do not depend on p53 or Mdm2. For example, although ARF can induce cell growth arrest in tumor cells that lack a functional p53 gene (Normand, G., Hemmati, P. G., Verdoodt, B. et al., J. Biol Chem 280:7118-30, 2005) or a gene encoding the p21 cyclin-dependent kinase inhibitor, a key transcriptional target of p53, ARF can also suppress the proliferation of MEFs lacking both Mdm2 and p53. Consistent with these findings, the tumor susceptibility of triple knockout mice that lack ARF, p53 and Mdm2 is significantly greater than that associated with mice lacking any one of these genes alone. It was recently shown that ARF suppresses the growth, progression, and metastasis of mouse skin carcinomas through both p53-dependent and p-53 independent pathways (Kelly-Sprat, K. S., Gurley, K. E., Yasui, Y., Kemp, C. J., PLoS Biol. 2:E242, 2004). Distinct downstream factors may exist that mediate the p53-independent functions of ARF. The identity of these factors and the mechanisms by which they mediate p53-independent tumor suppression by ARF are unknown. Accordingly, while regulation of the p53 pathway is of intense interest and presents a potential means of diagnosing and treating cancers, a greater understanding of this pathway and the factors and mechanisms that mediate the p53 independent functions of ARF would provide a valuable basis upon which new diagnostic and therapeutic methods may be developed.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery of a novel protein, ARF-BP1, which, when inactivated induces cell growth inhibition in p53 null cells and p53-dependent apoptosis in p53 wild-type cells. This discovery has broad implications in the diagnosis, monitoring, and treatment of neoplasias, particularly cancers associated with p53.
According to the invention, it has surprisingly been found that inactivation of ARF-BP1 induced cell growth arrest in p53 null cells, indicating that ARF-BP1 is a critical mediator of the p53-independent pathway of tumor suppression. Inactivation of endogenous ARF-BP1, but not Mdm2, in p53-null cells induces cell growth repression to a manner reminiscent of ARF induction. Inactivation of ARF-BP1 in p53 positive cells induced p53 stabilization and activated a p53-dependent apoptotic response. Accordingly, one aspect of the invention features a novel regulatory pathway involving ARF-BP1 in mediating both the p53-independent and p53-dependent tumor suppressor functions of ARF.
Accordingly, the present invention provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for ARF-BP1 peptide expression, wherein detection of ARF-BP1 expression is diagnostic of neoplasia in the subject.
The present invention provides a method for screening for preneoplastic and genetic predisposition for carcinomas, by assaying a diagnostic sample of the subject for ARF-BP1 peptide expression, wherein detection of ARF-BP1 expression is diagnostic of preneoplasia and genetic predisposition for carcinomas in the subject.
The present invention also provides a method for assessing the efficacy of therapy to treat neoplasia in a subject who has undergone or is undergoing therapy for neoplasia, by assaying a diagnostic sample of the subject for ARF-BP1 expression, wherein decreased or normal ARF-BP1 expression in the diagnostic sample is indicative of successful therapy, and ARF-BP1 expression elevated above normal in the diagnostic sample is indicative of a need to continue therapy to treat neoplasia.
The present invention further provides a method for assessing the prognosis of a subject who has neoplasia, by assaying a diagnostic sample of the subject for ARF-BP1 expression, wherein the subject's prognosis improves with a decrease in ARF-BP1 expression in the diagnostic sample, the subject's prognosis worsens with an increase in ARF-BP1 expression in the diagnostic sample.
The present invention also provides a kit for use in detecting neoplasia, comprising: (a) an agent reactive with ARF-BP1; and (b) reagents suitable for detecting expression of ARF-BP1.
Additionally, the present invention provides a method for treating neoplasia in a subject in need of treatment, by decreasing activity of ARF-BP1 in the subject. Also provided is a pharmaceutical composition, comprising an inhibitor of ARF-BP1 expression or an ARF-BP1 protein, in an amount effective to treat neoplasia in a subject to whom the composition is administered, and a pharmaceutically acceptable carrier.
The present invention further provides a method for deubiquitinating p53 in a cell, by contacting the cell with ARF-BP1, in an amount effective to deubiquitinate p53. Also provided is a method for treating neoplasia in a subject in need of treatment, by deubiquitinating p53 in a cell of the subject.
Additionally, the present invention is directed to a method for identifying an agent that is reactive with p53, by: (a) contacting a candidate agent with p53, in the presence of ARF-BP1; and (b) assessing the ability of the candidate agent to inhibit ARF-BP1-p53 interaction. Optionally, this method of the present invention may further comprise the steps of: (c) contacting the candidate agent with one or more cells containing p53; and (d) determining if the agent has an effect on a p53-associated biological event in the one or more cells.
The present invention further provides a method for treating a p53-associated condition in a subject in need of treatment, by administering to the subject an amount of an ARF-BP1 inhibitory agent effective to treat the p53-associated condition in the subject.
In one aspect of the invention ARF-BP1 directly binds ARF and its ubiquitin ligase activities are strongly inhibited by ARF. Accordingly, the present invention also provides a complex comprising ARF and ARF-BP1, and a mutant ARF-BP1 comprising the ARF-BP1 amino acid sequence.
Finally, the present invention is directed to a transgenic non-human animal whose genome comprises a disruption in its endogenous ARF-BP1 gene, wherein the transgenic animal exhibits decreased expression of functional ARF-BP1 protein relative to wild-type.
According to the invention, ARF-BP1 has been identified as a major component of ARF-containing protein complexes from p53-null human cells. In particular, the present invention characterizes ARF-BP1, a HECT (homology to E6-AP-C-terminus)-containing ubiquitin ligase.
Another aspect of the invention provides that ARF-BP1 interacts with both ARF and p53, respectively, but not with Mdm2. ARF-BP1 is required for ARF-mediated p53 stabilization in Mdm2 nulls
The present invention provides a practical approach for therapeutic intervention in tumors regardless of p53 status where ARF-BP1 may serve as a universal target.
Yet another aspect of the invention provides that ARF-BP1 is widely expressed and contains signature motifs (HECT and UBA) commonly associated with protein ubiquitination. ARF-BP1 catalyzes in vitro ubiquitination of p53 and RNAi-mediated inactivation of endogenous ARF-BP1 in p53-wild-type cells and stabilizes p53 and activates p53 function.
Additional aspects of the present invention will be apparent in view of the description which follows.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-C illustrate identification of ARF-BP1 as a major component of the ARF-associated nuclear complexes in human cells. FIG. 1A is a schematic representation of the HA-ARF-Flag-protein. FIG. 1B depicts the expression levels of HA-ARF-Flag and endogenous ARF in ARF-stable lines using Western blot analysis of cell extracts from parental H1299 cell line (lane 1), ARF stable cell line clone #1 (lane 2), and ARF stable cell line clone #2 (lane 3) with an anti-ARF antibody. FIG. 1C illustrates the silver staining of affinity-purified ARF-complexes from a nuclear extract of the HA-ARF-Flag/H1299 stable cell line (lane 2) and a control eluate from a parental H1299 nuclear cell extract (lane 1). Specific ARF-interacting protein bands were analyzed by mass spectrometry, and the p500/ARF-BP1 and B23/NPM (Nucleophosmin) peptide sequences (SEQ ID NOS. 3-6 from top to bottom in FIG. 1C).
FIGS. 2A-C demonstrate that ARF-BP1 contains a signature HECT-motif and a UBA domain. FIG. 2A is a schematic representation of the ARF-BP1 polypeptides. FIG. 2B depicts an alignment of the HECT domain of human ARF-BP1 (SEQ ID NO:7) with mouse ARF-BP1 (SEQ ID NO:8) and human E6-AP (SEQ ID NO:9) where the homologous amino acid residues are highlighted in outline and shadow. FIG. 2C depicts the expression of ARF-BP1 in different types of human tissue. A multiple tissue Northern filter was hybridized with ARF-BP1 (upper) or actin (lower) cDNA probes.
FIG. 3 sets forth the amino acid sequence of ARF-BP1 and is identified as SEQ ID NO: 2.
FIG. 4 illustrates the presence of a UBA domain of ARF-BP1 with alignment of human ARF-BP1 (SEQ ID NO:10), mouse ARF-BP1 (SEQ ID NO:11), yeast rad23 (SEQ ID NO:12), human HHR23A (SEQ ID NO:13), human Cb1 (SEQ ID NO:14) and Cb1-b (SEQ ID NO:15) and homologous amino acid residues highlighted in outline and shadow.
FIGS. 5A-D shows that ARF interacts with ARF-BP1 in vitro and in vivo and ARF-BP1-mediated ubiquitin ligase activity is inhibited by ARF. FIG. 5A depicts the direct interaction of ARF-BP1 with GST-ARF using the wild-type GST-ARF full-length protein (GST-ARF) (lanes 3, 9), the mutant GST-ARF (GST-ARF (GST-ARF Δ1-14) (lane 4), the N terminus of ARF protein (1-64) (lane 5), the C terminus of ARF (65-132) (lane 6), or GST alone (lanes 2, 8) in a pull-down assay either with an in vitro translated 35S-labeled ARF-BP1 (1015-4374) (lanes 1-6), or with in vitro translated 35S-labeled ARF-BP1 (1-1014) (lanes 7-9). FIG. 5B depicts coimmunoprecipitation of ARF with ARF-BP1 from H1299 cells using a Western blot analysis of indicated whole cell extract (lane 1) and immunoprecipitates with an ARF-BP1-specific antibody (lane 3) or a control IgG (lane 2) by anti-ARF monoclonal antibody (lower) or anti-ARF-BP1 antibody (top). FIG. 5C depicts coimmunoprecipitation of ARF-BP1 with ARF from H1299 cells using a Western blot analysis of whole cell extract (lane 1) or immunoprecipitates with anti-ARF polyclonal antibody (lane 3) or a control anti-serum (lane 2) by an ARF-BP1-specific antibody (lower) or anti-ARF monoclonal antibody (top). FIG. 5D shows the ubiquitination activity of ARF-BP1 is inhibited by ARF using Western blot analysis of the ubiquitin conjugates by anti-GST antibody. The in vitro ubiquitination assay was set up by incubating GST-ARF-BP1 (3760-4374) with E1, E 2 (His-UBCH5a), and ubiquitin (lane 2), or in the presence of GST-ARF (lane 3), GST-NARF (lane 4) or GST-CARF (lane 5), respectively.
FIGS. 6A-E illustrate that inactivation of endogenous ARF-BP1, but not Mdm2, induces cell growth arrest in p53-null H1299 cells. FIG. 6A depicts ablation of endogenous ARF-BP1 and Mdm2 proteins by RNAi using Western blot analysis of cell extracts of H1299 cells treated with a control RNAi (GFP-RNAi) (lane 1), Mdm2 RNAi (lane 2), or ARF-BP1 RNAi #1 (lane 3) with the antibodies against ARF-BP1, Mdm2, p21 and actin. FIG. 6B depicts overall cell growth of H1299 cells treated with a control RNAi (GFP-RNAi), Mdm2 RNAi, or ARF-BP1 RNAi # 1 stained with crystal violet three days after siRNA treatment. FIG. 6C depicts BrdU incorporation of H1299 cells treated with a control RNAi (GFP-RNAi), Mdm2 Nai, or ARF-BP1 RNAi #1 with labeling and staining of the cells one day after RNAi treatment. FIG. 6D is a bar graph showing that RNAi-mediated ablation of ARF-BP1 induces cell growth inhibition in p53 null SaoS-2 cells and the percentages of BrdU positive cells, 24 hours after transfection with control RNAi, Mdm2-RNAi or ARF-BP1-RNAi where the cells were counted and averaged in three independent experiments. FIG. 6E is a bar graph showing that RNAi-mediated ablation of ARF-BP1 induces cell growth inhibition in p53 null SaoS-2 cells and the number of Saos2 cells after being treated with control RNAi, Mdm2-RNAi or ARF-BP1-RNAi where the cells were counted and averaged in three independent experiments.
FIGS. 7A-D demonstrate that inactivation of endogenous ARF-BP1, but not Mdm2, induces cell growth arrest in p53-null H1299 cells and stabilizes p53 in U20S cells. FIG. 7A depicts ablation of endogenous ARF-BP1 and Mdm2 proteins by RNAi and shows Western blot analysis of H1299 cell extracts treated with a control RNAi (GFP-RNAi) (lane 1), Mdm2 RNAi (lane 2), or ARF-BP1 RNAi #2 (lane 3) with the antibodies against ARF-BP1, Mdm2, p21 and actin. FIG. 7B is a line graph showing the growth curves of H1299 cells treated with a control RNAi (GFP-RNAi), Mdm2 RNAi, or ARF-BP1 RNAi#2. After treatment with different types of siRNA, the cells were seeded with 2×106 cells per plate in fresh medium, and counted each day. FIG. 7C depicts overall cell growth of the H1299 cells treated with a control RNAi (GFP-RNAi), Mdm2 RNAi, or ARF-BP1 RNAi#2 where the cells were stained with crystal violet three days after siRNA treatment. FIG. 7D depicts endogenous ARF-BP1 ablated by RNAi in human U2OS cells and shows Western blot analysis of cell extracts of native U2OS cells (lane 1), U2OS cells treated with a control RNAi (GFP-RNAi) (lane 2), or ARF-BP1 RNAi#2 (lane 3) with the antibodies against ARF-BP1, P53, p21, bax and actin.
FIGS. 8A-B illustrate that inactivation of ARF-BP1 induces G2M arrest in H1299 cells, similar to overexpression of ARF. FIG. 8A shows the cell cycle profile of control RNAi plus control virus treatment (i), ARF-BP1 RNAi (ii), adenovirus-ARF treatment (iii) and ARF-BP1 RNAi plus adenovirus-ARF treatment. FIG. 8B is a bar graph representation of G2M arrest in H1299 cells.
FIGS. 9A-E demonstrate that inactivation of ARF-BP1 stabilizes p53 and induces p53-dependent apoptosis. FIG. 9A depicts endogenous ARF-BP1 knockdown by RNAi in human U2OS cells and shows Western blot analysis of cell extracts of native U2OS cells (lane 1), the U2OS cells treated with a control RNAi (GFP-RNAi) (lane 2), or ARF-BP1 RNAi # (lane 3) with the antibodies against ARF-BP1, p53, p21, bax and actin. FIG. 9B depicts the inactivation of ARF-BP1 extending the half life of endogenous p53 protein and showing Western blot analysis of cells extracts with an anti-p53 (DO-1) antibody from ARF-BP1-RNAi, or control-RNAi-transfected cells, harvested at indicated time points (min.) after cyclohexamide (CHX) treatment. FIG. 9C shows that inactivation of ARP-BP1 induced apoptosis. U2OS cells transfected with either ARF-BP1-RNAi or control-RNAi were analyzed for apoptotic cells (sub-G1) according to DNA content (PI staining). FIG. 9D depicts the reintroduction of ARF-BP1 (R) as abrogating ARF-BP1 RNAi-mediated p53 upregulation and shows Western blot analysis of cell extracts of U2OS cells treated with control-RNAi (GFP-RNAi) (lane 1), ARF-BP1-RNAi #2 (lane 2), or a combination of ARF-BP1-RNAi #1 and ARF-BP1 (R) (lane 3), ARF-BP1-RNAi #1 and ARF-BP1M (R) (lane 4) with the antibodies against ARF-BP1, p53, p21 and actin. FIG. 9E shows Western blot analysis of cell extracts from parental HCT116 cells (lane 1, 2) or HCT116-p53−/− cells (lane 3, 4) treated with either control RNAi (lanes 1, 3) or ARF-BP1-RNAi (lanes 2, 4), with the antibodies against ARF-BP1, Mdm2, p53, p21, Myc and actin.
FIG. 10 is a line graph showing the quantitation of the p53 half-life in the cells treated with control RNAi or ARF-BP1 RNAi.
FIG. 11 depicts the effects of different ARF-BP1 RNAi oligonucleotides and shows Western blot analysis of cell extracts from U2OS cells treated with control-RNAi (GFP-RNAi) (lane 1), ARF-BP1-RNAi #1 (lane 2), ARF-BP1-RNAi#2 (lane 3), or ARF-BP1-RNAi#1 mutant (lane 4).
FIG. 12 depicts that RNAi-mediated ablation of ARF-BP1 induces p53 activation in A549 and MCF-7 cells showing whole cells extracts from human lung adenocarcinoma A549 (lane 1, 2) or human breast cancer MCF-7 cells (lanes 3, 4) treated with either ARF-BP1-RNAi (lane 2, 4) or control-RNAi (lane 1, 3) immunoblotted with anti-ARF-BP1, anti-p53 (DO-1), anti-Mdm2, anti-p21, and anti-actin (AC-15) antibodies.
FIG. 13 demonstrates that RNAi-mediated ablation of ARF-BP1 induces p53 activation in normal human fibroblast (NHF-1) cells. Western blot analysis of cell extracts from normal human fibroblast cells (NHF-1) treated with ARF-BP1-RNAi (lane 2) or control-RNAi (lane 1) by anti-ARF-BP1, anti-p53 (DO-1), anti-p21, and anti-actin antibodies.
FIGS. 14A-D demonstrate that the HECT domain is critical for the ubiquitin ligase activity of ARF-BP1 and that re-introduction of ARF-BP1(R) abrogates cell growth arrest by ARF-BP1 RNAi in p53-null H1299 cells. FIG. 14A is a schematic of ARF-BP1 mutation (ARF-BP1(M)), ARF-BP1#1RNAi resistant construct (ARF-BP1(R)) and its mutation (ARF-BP1M(R)). The nucleic acid AATTGCTATGTCTCTG (SEQ ID NO:16) has been mutated to AATTGATATCCTCTG (SEQ ID NO:17) for both ARF-BP1(R) and ARF-BP1M(R).
FIG. 15 is a bar graph showing that inactivation of ARF-BP1 induces p53-dependent apoptosis in HCT116 cells. Quantitation of apoptotic cells from HCT116 and HCT116-p53 (−/−) cells treated with either ARF-BP1-RNAi or control-RNAi (GFP-RNAi) where apoptotic cells were counted by FACS analysis (SubG1) and averaged from three independent experiments.
FIGS. 16A-D illustrate that ARF-BP1 binds and ubiquitates p53, and ARF-BP1-mediated ubiquitation of p53 is inhibited by ARF. FIG. 16A shows direct interactions of ARF-BP1 with GST-p53. The GST-p53 protein (lane 3, 7), the GST-Mdm2 (line 4, 8), or GST alone (lanes 2, 6) were used in a GST pull-down assay with in vitro translated 35S-labeled ARF-BP1 (1015-4374) (lane 1-4) or ARF-BP1 (1-1014) (lane 5-8). FIG. 16B depicts coimmunoprecipitation of p53 with ARF-BP1 from U2OS cells. Western blot analysis of whole-cell extract (WCE) (lane 1) or immunoprecipitates with anti-ARF-BP1 specific antibody (lane 3) or a control IgG (lane 2) by a p53 monoclonal antibody DO-1 (lower) or ARP-BP1 specific antibody (top). FIG. 16C depicts coimmunoprecipitation of ARF-BP1 with p53 from U2OS cells. Western blot analysis of indicated whole-cell extract (WCE) (lane 1) and immunoprecipitates with a p53 monoclonal antibody DO-1 (lane 3) or control antibody (lane 2) by anti-ARF-BP1 specific antibody (lower) or anti-p53 DO-1 antibody (top). FIG. 16D depicts that ARF-BP1-mediated ubiquitination of p53 is inhibited by ARF. After incubation of Flag-p53 with GST-ARF-BP1 (3760-4374) in the presence of E1, E2 and ubiquitin (HA-Ub), the generated ubiquitin-conjugates were immunoprecipitated by the Flag/M2 beads and analyzed by Western blot with the anti-p53 DO1-antibody. The recombinant bacteria expressed protein GST-ARF, NARF (1-64), or CARF-(65-132) were added in the reactions in lanes 3, 4 or 5 respectively.
FIGS. 17A-C demonstrate that ARF induces p53 stabilization in an Mdm2-independent manner, and ARF-BP1 is critical for ARF-mediated p53 stabilization in Mdm2-null cells. FIG. 17A shows ARF stabilizes p53 in Mdm2-null cells. Western blot analysis of cell extracts from MEF p53/Mdm2-double null cells transfected with expression vectors of p53 and ARF with a p53 antibody (DO-1). FIG. 17B shows inactivation of ARF-BP1 stabilizes p53 in Mdm2-null cells. Western blot analysis of cell extracts from MEF p53/Mdm2-double null cells transfected with the p53 expression vector together with either ARF-BP1 RNAi or Mdm2 RNAi, by a p53 antibody (DO-1). FIG. 17C demonstrates that ARP-BP1 is required for ARF-mediated p53 stabilization in Mdm2-null cells. Western blot analysis of cell extracts from MEF p53/Mm2-double null cells transfected with expression vectors of p53 and ARF, together with either ARF-BP1 RNAi or Mdm2 RNAi, by a p53 antibody (DO-1), anti-ARF, anti-ARF BP1 and anti-GFP antibodies.
FIGS. 18A-B demonstrate that inactivation of ARF-BP1 extends the half-life of transfected p53 protein. FIG. 18A shows Western blot analysis of cells extracts with an anti-p53 (DO-1) antibody, from ARF-BP1-RNAi, or control RNAi transfected MEF p53/Mdm2-double null cells, harvested at indicated time points (hr) after cyclohexamide (CHX) treatment. The exposure time in the left panel (lane 1-4) is longer than that in the right panel (lane 5-8) so that the base intensity of p53 at time 0 between control RNAi and ARF-BP1 RNAi is comparable. FIG. 18B is a line graph showing the quantitation of the p53 half life in the MEF p53/Mdm2-double null cells treated with control RNAi or ARF-BP RNAi as shown in FIG. 18A.
FIGS. 19A-C depict that ARF-BP1 is a critical mediator of ARF tumor suppressor function. FIG. 19A shows that reduction of ARF-BP1 has the most significant effect on p53 levels when compared with the known ligases for p53, including Mdm2, COP1, Pirh2. Western blot analysis of cell extracts from U2OS treated with control RNAi (lane 1), Mdm2 RNAi (lane 2), COP1 RNAi (lane 3), Pirh2 RNAi (lane 4) and ARF-BP1 RNAi (lane 5), with the antibodies against ARF-BP1, Mdm2, Pirh2, p53, p21, Bax and actin. FIG. 19B is a model for cooperative controls of the p53-dependent and p53-independent functions of ARF by ARF-BP1 and Mdm2. FIG. 19C shows ARF-BP1 expressions in breast cancer cell lines. Western blot analysis of cell extracts from a number of breast cancer cell lines compared with the normal breast cell line MCF-10A as well as the normal human fibroblast NHF cell line by anti-ARF-BP1 specific antibody and anti-actin antibody.
FIG. 20 depicts overexpression of ARF activates p53 and other transcriptional factors (p21, Mdm2, Bax ) via Western blot analysis of cell extracts from U2OS infected with adenovirus-GFP (lanes 1), or adenovirus-ARF (lane 2) with the antibodies against ARF, Mdm2, p53, p21, Bax and actin.
FIG. 21 demonstrates that ARF-BP1 does not target the NPM/B23 protein for degradation and that the NPM/B23 protein is not the enzymatic target of the ARF-BP1 ubiquitin ligase activity. Proteasome inhibition stabilized p53 beyond what is achieved by ARF or ARF-BP1 RNAi as shown via Western blot analysis of cell extracts from MEF p53/Mdm2-double null cells transfected with expression vectors of p53 and ARF (lane 2, 4), together with either ARF-BP1 RNAi (lane 3, 5) or Mdm2 RNAi (lane 2, 4), by anti-p53 (DO-1) and anti-GFP antibodies.
FIGS. 22A-D illustrate that ARF-BP1 interacts with B23 in vivo and in vitro, but does not ubiquitinate and degrade B23. FIG. 22A shows ARF-BP1 interacts with B23 in vitro. The GST-B23 protein (lane 3), or GST alone (lane 2) were used in a GST pull-down assay with in vitro translated 35S-labeled ARF-BP 1 (1015-4374) (lanes 1-3) or ARF-BP1 (1-1014) (lanes 4-6). FIG. 22B shows ARF-BP1 interacts with B23 in vivo. Western blot analysis of whole cell extracts (lanes 1 and 2) or immunoprecipitates with the anti-FLAG M2 beads (IP/M2) (lanes 3 and 4) from cells stably transfected with FLAG-B23 (lanes 2 and 4) or control without transfection (lane 1 and 3) by anti-ARF-BP1 specific antibody. FIG. 22C shows that ARF-BP1 does not mediate B23 ubiquitination. After incubation of GST-B23 with GST-ARF-BP1 in the presence of E1, E2 and ubiquitin (HA-Ub), the reactions were analyzed by Western blot with the anti-B23-antibody. FIG. 22D shows ablation of ARF-BP1 does not affect B23 levels. Western blot analysis of cell extracts of SJSA (lane 1-3) or U2OS cells (lane 4-6) treated with a control RNAi (GFP-RNAi) (lanes 2, 5), ARF-BP 1 RNAi (lanes 3, 6), or without treatment (lanes 1, 4) with the antibodies against ARF-BP1, p53, B23 and actin.



DETAILED DESCRIPTION OF THE INVENTION
Since ARF can stabilize p53 in an Mdm2-independent manner and inactivation of ARF-BP1 directly leads to p53 activation, this invention modifies the current view about how ARF activates p53 in vivo, whose primary target has been presumed to be Mdm2. Moreover, given that inactivation of ARF-BP1 induces cell growth inhibition in p53 null cells and p53-dependent apoptosis in p53-wild-type cells, ARF-BP1 is a universal target for therapeutic intervention in tumors regardless of p53 status.
Beside use for regulating p53 protein and hence application in the control of cell proliferation, the ARF-BP1 peptide of the invention is also useful for in vitro screening methods for therapeutic agents (e.g., antineoplastic agents), for diagnosis and treatment of neoplastic or preneoplastic pathological conditions and genetic diseases.
The Identification of ARF-BP1 Reveals a Novel Aspect of ARF-Mediated Effect In p53 Activation
It is well accepted that the ARF polypeptide, as a product of an alternative reading frame of the INK4a locus, is a bona fide tumor suppressor (Sherr et al., 2001, supra; Sharpless, N. E., DePinho, R. A., 2004, supra). The first clue for ARF in activating the p53 pathway came from the tissue culture experiments showing that p53 stabilization is crucial for ARF-mediated function (Kamijo, T., et al., Cell 91:649-659, 1997). At the time, the role of Mdm2 in ubiquitination and degradation of p53 was just discovered (Haupt et al., Nature 387:296-299, 1997; Honda, R., Tanaka, H., Yasuda, H., FEBS Lett 420:25-27,1997; Kubbutat, M. H., Jones, S. N., Vousden, K. H., Nature 387:299-303, 1997). Mostly based on the results derived from over-expression settings, this seemingly obvious connection between ARF and Mdm2 was immediately accepted as the primary pathway for ARF-mediated p53 activation (Sherr et al., 2001, supra), which apparently leaves no room for the possibility of other factors involved in this pathway.
However, several lines of evidence indicate that ARF-mediated activation of p53 is much more complicated than a simple ARF-Mdm2 model. For example, the ARF-Mdm2 interaction was discovered in overexpression settings. However, the expression levels of Mdm2 in normal cells are low; whether endogenous ARF interacts with endogenous Mdm2 in normal cells remains an unsolved issue. Furthermore, low levels of Mdm2, which are commonly observed in normal cells, preferentially catalyze monoubiquitination of p53 (Li et al., 2003, supra); interestingly, however, recent studies from Lane's group showed that ARF can block polyubiquitination of p53 but is incapable of inhibiting Mdm2-mediated monoubiquitination of p53 in cells (Xirodimas, D. P., Stephen, C. W., Lane, D. P., Oncogene 20:4972-83, 2001). These studies raise a critical question: how does ARF stabilize p53 in the cells where the levels of Mdm2 are low? Recent studies showing an important role of Pirh2 and COP1 in p53 degradation further support the notion that stabilization of p53 may act through different pathways in vivo (Leng, R. P., Lin, Y., Ma, W., et al., Cell 112:779-91, 2003; Dornan, D. et al., Nature 429: 86-92, 2004).
The present invention has discovered a novel, Mdm2-independent pathway for ARF-mediated activation of p53. The present invention discloses that ARF-BP1 interacts directly with p53 both in vitro and in vivo and catalyzes ubiquitination of p53 in an Mdm2-independent manner. Moreover, ARF-BP1-mediated ubiquitination of p53 is severely inhibited by ARF and inactivation of endogenous ARF-BP1 is critical for ARF-mediated p53 stabilization in Mdm2-null cells. Thus, ARF-mediated p53 stabilization may act through both Mdm2-dependent and Mdm2-independent manners.
The invention identifies the relationship between ARF-BP1- and Mdm2-mediated regulations on p53 by ARF. Since ARF-BP1 was identified as a major target for ARP through biochemical purification and the interaction between the endogenous ARF-BP1 and ARF proteins is easily detected in normal cells, ARF-mediated p53 activation in normal cells acts, at least in part, though inhibiting ARF-BP1 function in vivo. Thus, ARF-mediated regulation on both ARF-BP1 and Mdm2 may cooperatively control the stability of p53 and more effectively activate p53-mediated functions. For example, when the levels of endogenous Mdm2 are high, p53 may be mainly degraded by Mdm2-mediated polyubiquitination. Thus, the ARF-Mdm2 interaction might be critical for up-regulating p53 activities. In contrast, when the levels of endogenous Mdm2 are low, ARF-mediated regulation of ARF-BP1 may become the key factor to activate p53 (FIG. 19). Several recent studies support the notion that p53 degradation is mediated by both Mdm2-dependent and Mdm2-independent pathways in vivo (Leng et al., 2003, supra; Dornan et al., 2004, supra). By using RNAi knocking down approaches, the differential effects on p53 stabilization by each of known E3 ligases of p53 were evaluated. As expected, inactivation of Mdm2 promoted p53 stabilization while inactivation of either COP1 or Pirh2 also modestly stabilized p53. Notably, inactivation of ARF-BP1 strongly induced p53 stabilization and activated p53-mediated transcription (FIG. 8); the levels of p21 and Bax induction induced by ARF-BP1 RNAi were higher than the levels induced by other types of E3 siRNAs for p53 (FIG. 19A), and very close to the effects by ARF over-expression (FIG. 20). These data indicate that ARF-BP1 is one of the major ubiquitin ligases of p53 in human cells and more importantly, is a key target for ARF-mediated tumor suppressor function.
The existence of two distinct pathways for ARF-mediated p53 activation, one based on the ARF-BP1 ubiquitin ligase and another on the Mdm2 ubiquitin ligase, allows for more versatile control of p53 functions (FIG. 19B) but also raises the question regarding their biological significances. For example, the critical role of Mdm2 in tumorigenesis is well established. Gene amplification and protein overexpression of Mdm2 are found in varies types of tumors (Michael, D., and Oren, M., Semin Cancer Biol 13:49-58, 2003). Thus, the ARF-Mdm2 interaction might be particularly important in the cells expressing high levels of Mdm2. Interestingly, the inventor found that ARF-BP1 is highly expressed in 80% (16/20) of breast cancer lines while the expression level of ARF-BP1 in normal breast cells (MCF-10A) is low (FIG. 19C), suggesting a potential role of ARF-BP1 in breast cancer tumorigenesis.
ARF-Mediated Inhibition of ARF-BP1 is Critical for P53-Independent Cell Growth Regulation
Although the role of ARF in stabilizing and activating p53 is well accepted, ARF is also found mutated or down-regulated in the tumors that lack functional p53 (Sherr et al., 2001, supra), suggesting that ARF mediated p53-independent function is also critical for its tumor suppression function. Consistent with this notion, a number of studies indicate that ARF can induce p53-independent cell growth repression (Weber, J. D., et al., Genes Dev. 14:2358-65, 2000; Rocha, S., Campbell, K. J., Perkins, N. D., Mol Cell, 12:15-25, 2003). Based on our observations that ARF-BP1 is the major binding partner of ARF in p53-null cells, the inventor proposes that ARF-BP1 is a critical target for ARF-mediated, p53-independent function. This is supported by the fact that inactivation of ARF-BP1, but not Mdm2, in p53-null cells induces cell growth repression in a manner reminiscent of ARF induction.
The precise mechanism by which ARF-mediated regulation of ARF-BP1 leads to p53-independent cell growth arrest needs future investigation. Since ARF-BP1 is a bona fide ubiquitin ligase, ARP-mediated p53-independent function may act by regulating unidentified substrates of ARF-BP1 (FIG. 19). In the light of recent studies showing that nucleoplasmin/B23 and ribosomal subunits are involved in the regulation of ARF-mediated ribosomal RNA processing (Itahana, K et al., Mol Cell 12:1151-64, 2003; Bertwistle, D, Sugimoto, M. Sherr, C. J., Mol Cell Biol 24:985-96, 2004), whether ARF-BP1 is directly involved in regulating B23 function or ribosomal RNA processing was examined. The present invention determined that ARF-BP1 interacts with B23 in vivo and in vitro, but does not ubiquitinate and degrade B23 (FIG. 22B). ARF-BP1 did not mediate B23 ubiquitination (FIG. 22C) and ablation of ARF-BP1 did not affect B23 levels. These results indicate that B23 is not the target for ARF-BP1 ubiquitin ligase activity. Moreover, since the ARF pathway is intimately linked with oncogene activation in vivo (Sherr, 2001, supra; Nilsson, J. A. Cleveland, J. L. Oncogene 22:9007-21, 2003; Sharpless and Depinho, 2004, supra), the ARF-BP1 and ARF interaction as well as the ubiquitin ligase activity of ARF-BP1 may be regulated upon oncogene activation or other types of stress in the cells.
Potential Implications in Cancer Therapy
Activation of the p53 pathway is a critical and perhaps obligatory step in cancer development. Numerous studies have shown that p53 activation is crucial for the function of many cancer therapeutic agents and that p53-dependent function plays a crucial role in the clinical effectiveness of these agents (Lane and Fisher, Nature 427:789-90, 2004; Lowe et al, 1994; Chresta and Hickman, Nature Medicine 2:745-6, 1996; Lutzker and Levine, Cancer Treat Rep, 87:345-56, 1996). Recent studies on new drug discovery related to p53 also shed light on this matter. For example, the p53 gene therapy was approved for cancer treatment (Surendran, A., Nature Medicine, 10:9, 2004). Nutlin, a small molecule the blocks the p53-Mdm2 interaction, was found to effectively kill the tumor cells in vivo by activating the p53 pathway (Vassilev L. T. et al., Science 303:844-8, 2004). However, the 53 gene is found mutated in more than 50% of human tumors and many tumor derived p53 point-mutants even have dominant negative effects (Vogelstein et al., Nature 408:307-310, 2000). The drugs specifically targeting the p53 pathway may encounter difficulty in killing tumor cells that lack functional p53.
In several aspects, the ARF-BP1 protein is an appropriate candidate target for therapeutic interventions in tumors. Inactivation of ARF-BP1 in the tumor cells expressing wild-type p53, leads to p53 stabilization and activates p53-mediated apoptosis. Importantly, inhibition of ARF-BP1 in the tumor cells that lack functional p53 induces cell growth inhibition. ARF-BP1 is also an enzyme and its ubiquitin ligase activity is critical for its mediated function. Thus, drug screening for inhibitors of its enzymatic activity will be a promising strategy for therapeutic interventions in tumors. In contrast to targeting the proteins specifically inhibiting p53 function such as Mdm2, inhibitors of ARF-BP1 should be effective in preventing tumor cells growth regardless of p53 status.
In view of the foregoing, the present invention provides a method for determining whether a subject has neoplasia. As used herein, the “subject” is a mammal, including, without limitation, a cow, dog, human, monkey, mouse, pig, or rat. Preferably, the subject is a human. The inventor has demonstrated herein (see, e.g., FIGS. 16A-D) the detection of significant enhancement of ARF-BP1 interaction, and enhanced ARF-BP1 expression, in cells subjected to DNA damage, as compared with normal (undamaged) cells. Accordingly, the method of the present invention comprises assaying a diagnostic sample of the subject for expression of ARF-BP1, wherein detection of ARF-BP1 expression elevated above normal is diagnostic of neoplasia in the subject.
As used herein, “ARF-BP1” includes both a ARF-BP1 protein and an ARF-BP1 analogue, including “conservative substitutions”. Unless otherwise indicated, “protein” shall include a protein, protein domain, polypeptide, or peptide. As further used herein, the ARF-BP1 protein has the amino acid sequence set forth in FIG. 3 (SEQ ID NO: 2).
A “ARF-BP1 analogue”, as used herein, is a functional variant of the ARF-BP1 protein, having ARF-BP1 biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the ARF-BP1 protein. An ARF-BP1 “analogue” includes a variant of the ARF-BP1 protein that has a homologous three-dimensional conformation. ARF-BP1 and ARF-BP1 analogues may be produced synthetically or recombinantly, or may be isolated from native cells. ARF-BP1 is preferably produced recombinantly, using conventional techniques and cDNA encoding ARF-BP1 (SEQ ID NO:1).
As used herein, “conservative substitutions” are those amino acid substitutions which are functionally equivalent to the substituted amino acid residue, either because they have similar polarity or steric arrangement, or because they belong to the same class as the substituted residue (e.g., hydrophobic, acidic, or basic). The term “conservative substitutions”, as defined herein, includes substitutions having an inconsequential effect on the ability of ARF-BP1 to interact with p53, particularly in respect of the use of said interaction for the identification and design of p53 inhibitors, for molecular replacement analyses, and/or for homology modeling.
The method of the present invention may be used to determine whether a subject has neoplasia, thereby permitting the diagnosis of such neoplasia in the subject. As used herein, “neoplasia” refers to the uncontrolled and progressive multiplication of cells of a neoplasm (i.e., neoplastic cells, such as tumor cells), under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasia results in a “neoplasm”, which is defined herein to mean any new and abnormal growth, particularly a new growth of tissue, in which the growth of cells is uncontrolled and progressive. Thus, neoplasia includes “cancer”, which herein refers to a proliferation of neoplastic cells having the unique trait of loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis.
As used herein, neoplasms include, without limitation, morphological irregularities in cells in tissue of a subject, as well as pathologic proliferation of cells in tissue of a subject, as compared with normal proliferation in the same type of tissue. Additionally, neoplasms include benign tumors and malignant tumors (e.g., breast tumors) that are either invasive or noninvasive. Malignant neoplasms are distinguished from benign in that the former show a greater degree of anaplasia, or loss of differentiation and orientation of cells, and have the properties of invasion and metastasis. Examples of neoplasms or neoplasias which may be assessed, detected, diagnosed, monitored, or treated in accordance with inventions described herein include, without limitation, carcinomas, particularly those of the bladder, breast, cervix, colon, head, kidney, lung, neck, ovary, prostate, and stomach; lymphocytic leukemias, particularly acute lymphoblastic leukemia and chronic lymphocytic leukemia; myeloid leukemias, particularly acute monocytic leukemia, acute promyelocytic leukemia, and chronic myelocytic leukemia; malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, peripheral neuroepithelioma, and synovial sarcoma; and mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease (Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy, 17th ed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991).
As indicated above, over 50% of all cancer cases are associated with p53 mutations. Accordingly, in one embodiment of the present invention, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of p53-associated neoplasias, including neoplasias associated with a defect in the p53 pathway. In another embodiment of the present invention, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of breast cancer, colon cancer, leukemia, lung cancer, malignant melanoma, ovarian cancer, or prostate cancer.
In another embodiment of the present invention, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of p53-independent neoplasias, including neoplasias not associated with a defect in the p53 pathway. In another embodiment of the present invention, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of breast cancer, colon cancer, leukemia, lung cancer, malignant melanoma, ovarian cancer, or prostate cancer.
According to the method of the present invention, the diagnostic sample of a subject may be assayed in vitro or in vivo. Where the assay is performed in vitro, a diagnostic sample from the subject may be removed using standard procedures. The diagnostic sample may be tissue, including any bone, brain tissue, breast tissue, colon tissue, muscle tissue, nervous tissue, ovarian tissue, prostate tissue, retinal tissue, skin tissue, or soft tissue, which may be removed by standard biopsy. In addition, the diagnostic sample may be a bodily fluid, including cerebrospinal fluid, pericardial fluid, peritoneal fluid, saliva, serum, and urine. Furthermore, the diagnostic sample taken from the subject or patient may be, for example, any tissue known to have a neoplasm, any tissue suspected of having a neoplasm, or any tissue believed not to have a neoplasm.
Protein may be isolated and purified from the diagnostic sample of the present invention using standard methods known in the art, including, without limitation, extraction from a tissue (e.g., with a detergent that solubilizes the protein) where necessary, followed by affinity purification on a column, chromatography (e.g., FTLC and HPLC), immunoprecipitation (e.g., with an antibody to ARF-BP1), and precipitation (e.g., with isopropanol and a reagent such as Trizol). Isolation and purification of the protein may be followed by electrophoresis (e.g., on an SDS-polyacrylamide gel). Nucleic acid may be isolated from a diagnostic sample using standard techniques known to one of skill in the art.
In accordance with the method of the present invention, neoplasia in a subject may be diagnosed by assaying a diagnostic sample of the subject for expression of ARF-BP1, wherein expression of ARF-BP1 elevated above normal is diagnostic of neoplasia. As used herein, “expression” means the transcription of a gene into at least one mRNA transcript, or the translation of at least one mRNA into a protein. For example, “expression of ARF-BP1” means the transcription of the ARF-BP1 gene into at least one mRNA transcript, or the translation of at least one mRNA into a ARF-BP1 protein, as defined above. Accordingly, a diagnostic sample may be assayed for ARF-BP1 expression by assaying for ARF-BP1 protein, ARF-BP1 cDNA, or ARF-BP1 mRNA. The appropriate form of ARF-BP1 will be apparent based on the particular techniques discussed herein.
Furthermore, it is contemplated that the diagnostic sample may be assayed for expression of any or all forms of ARF-BP1 protein (including precursor, endoproteolytically-processed forms, and other forms resulting from post-translational modification) in order to determine whether a subject or patient has neoplasia. It is also contemplated that the diagnostic sample may be assayed for expression of ARF-BP1 elevated above normal by detecting an increase in p53-ARF-BP1 interaction, as disclosed herein. Accordingly, in one embodiment of the present invention, ARF-BP1 expression elevated above normal is detected by detecting p53-ARF-BP1 interaction elevated above normal.
As used herein, the term “elevated above normal” refers to detection (e.g., of expression of ARF-BP1, of p53-ARF-BP1 interaction, of ARF-BP1-ARF interaction, etc.) at a level that is significantly greater than the level expected for the same type of diagnostic sample taken from a nondiseased subject or patient (i.e., one who does not have neoplasia) of the same gender and of similar age. As further used herein, “significantly greater” means that the difference between the level (e.g., of expression of ARF-BP1, of p53-ARF-BP1 interaction, of ARF-BP1-ARF interaction, etc.) that is elevated above normal, and the expected (normal) level (e.g., of expression of ARF-BP1, of p53-ARF-BP1 interaction, etc.), is of statistical significance.
Preferably, ARF-BP1 expression (or p53-ARF-BP1 interaction) elevated above normal is expression of ARF-BP1 (or p53-ARF-BP1 interaction) at a level that is at least 10% greater than the level of ARF-BP1 expression (or p53-ARF-BP1 interaction) otherwise expected. Where ARF-BP1 expression (or p53-ARF-BP1 interaction) is expected to be absent from a particular diagnostic sample taken from a particular subject or patient, the normal level of ARF-BP1 expression (or p53-ARF-BP1 interaction) for that subject or patient is nil. Where a particular diagnostic sample taken from a particular subject or patient is expected to have a low level of constitutive ARF-BP1 expression (or p53-ARF-BP1 interaction), that low level is the normal level of ARF-BP1 expression (or p53-ARF-BP1 interaction) for that subject or patient.
Expected or normal levels of ARF-BP1 expression for a particular diagnostic sample taken from a subject or patient may be easily determined by assaying nondiseased subjects of a similar age and of the same gender. For example, diagnostic samples may be obtained from at least 30 normal, healthy men between the ages of 25 and 80, to determine the normal quantity of ARF-BP1 expression in males. A similar procedure may be followed to determine the normal quantity of ARF-BP1 expression in females. Once the necessary or desired samples have been obtained, the normal quantities of ARF-BP1 expression in men and women may be determined using a standard assay for quantification, such as flow cytometry, Western-blot analysis, or an ELISA for measuring protein quantities, as described below. For example, an ELISA may be run on each sample in duplicate, and the means and standard deviations of the quantity of the ARF-BP1 protein may be determined. If necessary, additional subjects may be recruited before the normal quantities of ARF-BP1 expression are quantified. A similar type of procedure may be used to determine expected or normal levels of p53-ARF-BP1 interaction for a particular diagnostic sample taken from a subject or patient.
In accordance with the method of the present invention, a diagnostic sample of a subject may be assayed for ARF-BP1 expression (or p53-ARF-BP1 interaction), and ARF-BP1 expression (or p53-ARF-BP1 interaction) may be detected in a diagnostic sample, using assays and detection methods readily determined from the known art (e.g., immunological techniques, hybridization analysis, fluorescence imaging techniques, and/or radiation detection, etc.), as well as any assays and detection methods disclosed herein (e.g., immunoprecipitation, Western-blot analysis, etc.). For example, a diagnostic sample of a subject may be assayed for ARF-BP1 expression using an inhibitor of ARF-BP1. As used herein, “inhibitor” means the agent has affinity for, binds to, inhibits or is directed against a target of interest (e.g., ARF-BP1). As further used herein, an “agent” shall include a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab′)2 fragment, molecule, compound, antibiotic, drug, and any combinations thereof. A Fab fragment is a univalent antigen-binding fragment of an antibody, which is produced by papain digestion. A F(ab′)2 fragment is a divalent antigen-binding fragment of an antibody, which is produced by pepsin digestion. Preferably, the agent of the present invention is labeled with a detectable marker or label.
In one embodiment of the present invention, the inhibitor of ARF-BP1 is an antibody. As used herein, the antibody of the present invention may be polyclonal or monoclonal. In addition, the antibody of the present invention may be produced by techniques well known to those skilled in the art. Polyclonal antibody, for example, may be produced by immunizing a mouse, rabbit, or rat with purified protein (e.g., ARF-BP1). Monoclonal antibody then may be produced by removing the spleen from the immunized mouse, and fusing the spleen cells with myeloma cells to form a hybridoma which, when grown in culture, will produce a monoclonal antibody.
The antibodies used herein may be labeled with a detectable marker or label. Labeling of an antibody may be accomplished using one of a variety of labeling techniques, including peroxidase, chemiluminescent labels known in the art, and radioactive labels known in the art. The detectable marker or label of the present invention may be, for example, a nonradioactive or fluorescent marker, such as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine, which can be detected using fluorescence and other imaging techniques readily known in the art. Alternatively, the detectable marker or label may be a radioactive marker, including, for example, a radioisotope. The radioisotope may be any isotope that emits detectable radiation, such as 35S, 32P, 125I, 3H, or 14C. Radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from the radioisotope may be detected using gamma imaging techniques, particularly scintigraphic imaging. Preferably, the agent of the present invention is a high-affinity antibody (e.g., α-ARF-BP1) labeled with a detectable marker or label.
Where the agent of the present invention is an antibody reactive with ARF-BP1, a diagnostic sample taken from the subject may be purified by passage through an affinity column which contains ARF-BP1 antibody (e.g., α-ARF-BP1) as a ligand attached to a solid support, such as an insoluble organic polymer in the form of a bead, gel, or plate. The antibody attached to the solid support may be used in the form of a column. Examples of suitable solid supports include, without limitation, agarose, cellulose, dextran, polyacrylamide, polystyrene, sepharose, or other insoluble organic polymers. The ARF-BP1 antibody (e.g., α-ARF-BP1) may be further attached to the solid support through a spacer molecule, if desired. Appropriate binding conditions (e.g., temperature, pH, and salt concentration) for ensuring binding of the agent and the antibody may be readily determined by the skilled artisan. In a preferred embodiment, the ARF-BP1 antibody (e.g., α-ARF-BP1) is attached to a sepharose column, such as Sepharose 4B.
Where the agent is an antibody, a diagnostic sample of the subject may be assayed for ARF-BP1 expression using binding studies that utilize one or more antibodies immunoreactive with ARF-BP1, along with standard immunological detection techniques. For example, the ARF-BP1 protein eluted from the affinity column may be subjected to an ELISA assay, Western-blot analysis, flow cytometry, or any other immunostaining method employing an antigen-antibody interaction. Preferably, the diagnostic sample is assayed for ARF-BP1 expression using Western blotting.
Alternatively, a diagnostic sample of a subject may be assayed for ARF-BP1 expression using hybridization analysis of nucleic acid extracted from the diagnostic sample taken from the subject. According to this method of the present invention, the hybridization analysis may be conducted using Northern-blot analysis of mRNA. This method also may be conducted by performing a Southern-blot analysis of DNA using one or more nucleic acid probes, which hybridize to nucleic acid encoding ARF-BP1. The nucleic acid probes may be prepared by a variety of techniques known to those skilled in the art, including, without limitation, the following: restriction enzyme digestion of ARF-BP1 nucleic acid; and automated synthesis of oligonucleotides having sequences which correspond to selected portions of the nucleotide sequence of the ARF-BP1 nucleic acid, using commercially-available oligonucleotide synthesizers, such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
The detection of ARF-BP1 expression (or p53-ARF-BP1 or ARF-BP1-ARF interactions) in the method of the present invention may be followed by an assay to measure or quantify the extent of ARF-BP1 expression in a diagnostic sample of a subject. Such assays are well known to one of skill in the art, and may include immunohistochemistry/immunocytochemistry, flow cytometry, mass spectroscopy, Western-blot analysis, or an ELISA for measuring amounts of ARF-BP1 protein. For example, to use an immunohistochemistry assay, histological (paraffin-embedded) sections of tissue may be placed on slides, and then incubated with an antibody against ARF-BP1. The slides then may be incubated with a second antibody (against the primary antibody), which is tagged to a dye or other calorimetric system (e.g., a fluorochrome, a radioactive agent, or an agent having high electron-scanning capacity), to permit visualization of ARF-BP1 that is present in the sections.
It is contemplated that the diagnostic sample in the present invention frequently will be assayed for ARF-BP1 expression (or p53-ARF-BP1 interaction) not by the subject or patient, nor by his/her consulting physician, but by a laboratory technician or other clinician. Accordingly, the method of the present invention further comprises providing to a subject's or patient's consulting physician a report of the results obtained upon assaying a diagnostic sample of the subject or patient for ARF-BP1 expression.
Similarly, the present invention provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for ARF-BP1 expression, wherein detection of ARF-BP1 expression elevated above normal in the diagnostic sample is diagnostic of neoplasia in the subject. As discussed above, cancer has been associated with defects in the p53 pathway, including defects in ARF-BP1, Mdm2, and/or p53. Accordingly, in one embodiment, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of p53-associated neoplasias. In another embodiment, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of p-53 independent neoplasias.
In accordance with the method of the present invention, a diagnostic sample may be assayed for ARF-BP1 expression by assaying for ARF-BP1 protein, ARF-BP1 cDNA, or ARF-BP1 mRNA, as described above. Expected or normal levels of ARF-BP1 expression for a particular diagnostic sample taken from a subject or patient may be easily determined by assaying nondiseased subjects of a similar age and of the same gender, as described above in connection with ARF-BP1.
It is contemplated that the diagnostic sample may be assayed for expression of any or all forms of ARF-BP1 proteins (including precursor, endoproteolytically-processed forms, and other forms resulting from post-translational modification) in order to determine whether a subject or patient has neoplasia. It is also contemplated that the diagnostic sample may be assayed for expression of p53 elevated above normal and ARF-BP1 elevated above normal by detecting an increase in p53-ARF-BP1 interaction. Accordingly, in one embodiment of the present invention, expression of p53 elevated above normal and expression of ARF-BP1 elevated above normal are detected in the diagnostic sample by detecting p53-ARF-BP1 interaction elevated above normal in the diagnostic sample. It is also contemplated that the diagnostic sample may be assayed for expression of ARF decreased below normal and ARF-BP1 elevated above normal by detecting an increase in ARF-ARF-BP1 interaction. Accordingly, in one embodiment of the present invention, expression of p53 elevated above normal and expression of ARF-BP1 elevated above normal are detected in the diagnostic sample by detecting p53-ARF-BP1 interaction elevated above normal in the diagnostic sample. In another embodiment of the present invention, expression of ARF below normal and expression of ARF-BP1 elevated above normal are detected in the diagnostic sample by detecting ARF-ARF-BP1 interaction elevated above normal in the diagnostic sample.
A diagnostic sample of a subject may be assayed for ARF, ARF-BP1 expression, and/or p53-ARF-BP1 interaction in accordance with methods described herein. p53 expression, ARF-BP1 expression, and/or ARF-ARF-BP1 interaction may also be detected in a diagnostic sample using assays and detection methods readily determined from the known art, as well as any assays and detection methods disclosed herein.
For example, a diagnostic sample of a subject may be assayed for ARF-BP1 expression using hybridization analysis of nucleic acid extracted from the diagnostic sample taken from the subject. This method preferably utilizes a nucleic acid probe which hybridizes to nucleic acid encoding ARF-BP1. In one embodiment, the nucleic acid probe is labeled with a detectable marker or label. In the alternative, a diagnostic sample of a subject may be assayed for ARF-BP1 expression using an agent reactive with ARF-BP1. Preferably, the agent of the present invention is labeled with a detectable marker or label. In one embodiment of the present invention, the agent reactive with ARF-BP1 is an antibody (e.g., anti-ARF-BP1 monoclonal antibody).
When the agent of the present invention is an antibody reactive with ARF-BP1, a diagnostic sample taken from the subject may be purified by passage through an affinity column which contains anti-ARF-BP1 antibody as a ligand attached to a solid support, such as an insoluble organic polymer in the form of a bead, gel, or plate, in accordance with techniques described above for detecting ARF-BP1. Additionally, where the agent is an anti-ARF-BP1 antibody, a diagnostic sample of the subject may be assayed for ARF-BP1 expression using binding studies that utilize one or more antibodies immunoreactive with ARF-BP1, along with standard immunological detection techniques, as described herein in connection with ARF-BP1.
The detection of ARF-BP1 expression, and/or ARF-ARF-BP1 interaction, and/or p53-ARF-BP1 interaction in the method of the present invention may be followed by an assay to measure or quantify the extent of ARF-BP1 expression, and/or ARF-ARF-BP1 or p53-ARF-BP1 interaction in the diagnostic sample of a subject. Additionally, the method of the present invention may further comprise the step of providing to a subject's or patient's consulting physician a report of the results obtained upon assaying a diagnostic sample of the subject or patient for ARF-BP1 expression, ARF-ARF-BP1 interaction, and/or p53-ARF-BP1 interaction.
The present invention further provides a method for assessing the efficacy of therapy to treat neoplasia in a subject or patient who has undergone or is undergoing treatment for neoplasia. The method of the present invention comprises assaying a diagnostic sample of the subject or patient for ARF-BP1 expression, wherein detection of a normal level of ARF-BP1 expression is indicative of successful therapy to treat neoplasia, and detection of ARF-BP1 expression elevated above normal is indicative of a need to continue therapy to treat neoplasia. In one embodiment of the present invention, ARF-BP1 expression elevated above normal is detected by detecting p53-ARF-BP1 or ARF-ARF-BP1 interactions elevated above normal. The neoplasia may be any of those described above, including p53-dependent and p53-independent neoplasias. The diagnostic sample may be a tissue or a bodily fluid, as described above, and may be assayed for expression of ARF-BP1 (or p53-ARF-BP1 and/or ARF-ARF-BP1 interaction) in vitro or in vivo. In addition, the diagnostic sample may be assayed for expression of ARF-BP1 (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) using all of the various assays and methods of detection and quantification described above. This method of the present invention provides a means for monitoring the effectiveness of therapy to treat neoplasia by permitting the periodic assessment of levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in a diagnostic sample taken from a subject or patient.
According to the method of the present invention, a diagnostic sample of a subject or patient may be assayed, and levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) may be assessed, at any time following the initiation of therapy to treat neoplasia. For example, levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) may be assessed while the subject or patient is still undergoing treatment for neoplasia. Where levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) detected in an assayed diagnostic sample of the subject or patient continue to remain elevated above normal, a physician may choose to continue with the subject's or patient's treatment for the neoplasia. Where levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample of the subject or patient decrease through successive assessments, it may be an indication that the treatment for neoplasia is working, and that treatment doses could be decreased or even ceased. Where levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample of the subject or patient do not rapidly decrease through successive assessments, it may be an indication that the treatment for neoplasia is not working, and that treatment doses could be increased. Where ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) is no longer detected in an assayed diagnostic sample of a subject or patient at levels elevated above normal, a physician may conclude that the treatment for neoplasia has been successful, and that such treatment may cease.
It is within the confines of the present invention to assess levels of ARF-BP1 expression following completion of a subject's or patient's treatment for neoplasia, in order to determine whether the neoplasia has recurred in the subject or patient. Accordingly, an assessment of levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample may provide a convenient way to conduct follow-ups of patients who have been diagnosed with neoplasias. Furthermore, it is within the confines of the present invention to use assessed levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample as a clinical or pathologic staging tool, as a means of determining the extent of neoplasia in the subject or patient, and as a means of ascertaining appropriate treatment options.
The present invention also provides a method for assessing the efficacy of therapy to treat neoplasia in a subject who has undergone or is undergoing treatment for neoplasia, by assaying a diagnostic sample of the subject for ARF-BP1 expression and ARF-BP1 expression, wherein detection of normal ARF-BP1 expression in the diagnostic sample is indicative of successful therapy to treat neoplasia, and detection of ARF-BP1 expression elevated above normal in the diagnostic sample is indicative of a need to continue therapy to treat neoplasia. In one embodiment of the present invention, ARF-BP1 expression elevated above normal are detected in the diagnostic sample by detecting p53-ARF-BP1 interaction elevated above normal in the diagnostic sample. The neoplasia may be any of those described above, including p53-dependent and p-53 independent neoplasias. Suitable diagnostic samples, assays, and detection and quantification methods for use in the method of the present invention have already been described.
A correlation exists, in general, between tumor burden and the survival of a patient who has cancer. Therefore, it is also contemplated in the present invention that assaying a diagnostic sample of a subject for ARF-BP1 expression may be a useful means of providing information concerning the prognosis of a subject or patient who has neoplasia. Accordingly, the present invention further provides a method for assessing the prognosis of a subject who has neoplasia, comprising assaying a diagnostic sample of the subject for ARF-BP1 expression, wherein the subject's prognosis improves with a decrease in ARF-BP1 expression in the diagnostic sample of the subject, and the subject's prognosis worsens with an increase in ARF-BP1 expression in the diagnostic sample of the subject. In one embodiment of the present invention, ARF-BP1 expression elevated above normal is detected by detecting p53-ARF-BP1 interaction elevated above normal. Suitable diagnostic samples, assays, and detection and quantification methods for use in the method of the present invention have already been described. This method of the present invention provides a means for determining the prognosis of a subject or patient diagnosed with neoplasia based upon the level of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample of the subject or patient.
According to the method of the present invention, a diagnostic sample of a subject or patient may be assayed, and levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) may be assessed, at any time during or following the diagnosis of neoplasia in the subject or patient. For example, levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample may be assessed before the subject or patient undergoes treatment for neoplasia, in order to determine the subject's or patient's initial prognosis. Additionally, levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample may be assessed while the subject or patient is undergoing treatment for neoplasia, in order to determine whether the subject's or patient's prognosis has become more or less favorable through the course of treatment.
By way of example, where levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) detected in an assayed diagnostic sample of the subject or patient are, or continue to remain, significantly high, a physician may conclude that the subject's or patient's prognosis is unfavorable. Where ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample of the subject or patient decreases through successive assessments, it may be an indication that the subject's or patient's prognosis is improving. Where levels of ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) in an assayed diagnostic sample of the subject or patient do not decrease significantly through successive assessments, it may be an indication that the subject's or patient's prognosis is not improving. Finally, where ARF-BP1 expression (or p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) is low, or is normal, in a diagnostic sample of the subject or patient, a physician may conclude that the subject's or patient's prognosis is favorable.
The discovery that ARF-BP1 can be detected in cells displaying neoplasias provides a means of identifying patients with neoplasias, and presents the potential for commercial application in the form of a test for the diagnosis of neoplasias. The development of such a test could provide general screening procedures. Such procedures can assist in the early detection and diagnosis of neoplasia, or preneoplasia or genetic predisposition to neoplasia and can provide a method for the follow-up of patients in whom ARF-BP1 expression (including p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction) elevated above normal have been detected.
Accordingly, the present invention further provides a kit for use as an assay of neoplasia, comprising an agent reactive with ARF-BP1 and reagents suitable for detecting expression of ARF-BP1 (and p53-ARF-BP1 interaction and/or ARF-ARF-BP1 interaction). The present invention also provides a kit for use in detecting neoplasia, comprising: (a) at least one agent reactive with ARF-BP1; and (b) reagents suitable for detecting expression of ARF-BP1. The agents may be any of those described above, and may be used in any of the above-described assays or methods for detecting or quantifying ARF-BP1 expression, p53-ARF-BP1 interaction, and ARF-ARF-BP1 interaction. Preferably, at least one agent of the present invention is labeled with a detectable marker or label.
As indicated above, over 50% of all cancer cases are associated with p53 mutations. Therefore, p53 is the key for treating many cancers, and the p53 pathway is a particular focus of interest. p53 is generally not a stable protein; it has a half-life of approximately 20 min, and is degraded very rapidly by proteosomes in the protein-degradation pathway following ubiquitination (the binding of ubiquitin). It is believed that the stabilization of p53 is important for the protein's efficiency as a tumor suppressor.
It is expected that some cancers associated with defects in the p53 pathway result not from a defect in p53, but from a mutated ARF-BP1 (e.g., a mutation resulting from a genetic alteration at the coding region) and/or a defect in ARF-BP1 regulation at the expression level (e.g., a defect resulting from a genetic alteration at the promoter region of the ARF-BP1 gene). In view of the foregoing, it is clear that modulation of the levels of ARF-BP1 in cells provides a means for enhancing p53's tumor-suppressor function, and for supplementing this function with ARF-BP1's own tumor-suppressor activity. Accordingly, the present invention further provides a method for treating neoplasia in a subject in need of treatment therefore, comprising decreasing activity of ARF-BP1 in the subject. The neoplasia may be any of those described above, including p53 independent and p53-dependent neoplasia.
In accordance with the method of the present invention, activity of ARF-BP1 in a subject may be decreased by targeting ARF-BP1 directly. Additionally, activity of ARF-BP1 in a subject may be decreased indirectly, by targeting an enzyme or other endogenous molecule that regulates or modulates the functions or levels of ARF-BP1 in the subject. Preferably, ARF-BP1 activity in the subject is decreased by at least 10% in the method of the present invention. More preferably, ARF-BP1 activity is decreased by at least 20%.
For example, activity of ARF-BP1 in a subject may be decreased by directly or indirectly deactivating, inhibiting, binding or neutralizing one or more functions of ARF-BP1 in the subject (e.g., by the modulation or regulation of proteins that interact with ARF-BP1). The term “inhibiting”, as used herein, means decreasing or negating the functions of ARF-BP1 in the subject, particularly the ubiquitination, and resulting destabilization, of p53. In the method of the present invention, ARF-BP1 in a subject may be inhibited, for example, by administering to the subject a small molecule or protein mimetic that inhibits ARF-BP1 or that is reactive with ARF-BP1, as defined above.
Activity of ARF-BP1 in a subject also may be decreased by directly or indirectly prohibiting, suppressing, or inhibiting the upregulation of ARF-BP1 expression within a subject. Accordingly, in one embodiment of the present invention, activity of ARF-BP1 is decreased in a subject by administering to the subject an inhibitor of ARF-BP1 expression in an amount effective to treat the neoplasia in the subject. As used herein, a “inhibitor of expression” may be any agent or combination of agents that that has an antagonistic (inhibitory) or agonistic (facilitatory) effect on expression of a specified protein. Thus, a modulator of expression may be an agonist or an antagonist. The modulators of the present invention include any protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab′)2 fragment, molecule, compound, antibiotic, and drug, and an agent reactive with a protein of interest (e.g., ARF-BP1) that inhibits or downregulates expression of that protein.
Inhibitors of ARF-BP1 may be identified using a simple screening assay. For example, to screen for candidate inhibitors of ARF-BP1, human lung carcinoma cells (H1299) may be plated onto microtiter plates, then contacted with a library of drugs. Any resulting decrease in, or down regulation of, ARF-BP1 expression then may be detected using nucleic acid hybridization and/or immunological techniques known in the art, including an ELISA. Additional inhibitors of ARF-BP1 expression may be identified using screening procedures well known in the art or disclosed herein. Inhibitors of ARF-BP1 will be those drugs which prevent or downregulate expression of ARF-BP1. In this manner, candidate inhibitors also may be screened for their ability to inhibit proliferation of neoplasms using ARF-BP1 expression as an indicator that cell division or growth of cells in a neoplasm is decreasing in rate, or has stopped.
It is within the confines of the present invention that the inhibitor of ARF-BP1 expression may be linked to another agent, or administered in combination with another agent, such as an antineoplastic drug or a ribozyme, in order to increase the effectiveness of the treatment of neoplasia, increase the efficacy of targeting, and/or increase the efficacy of p53 deubiquitination. Examples of antineoplastic drugs to which the inhibitor of ARF-BP1 expression may be linked include, without limitation, carboplatin, cyclophosphamide, doxorubicin, etoposide, and vincristine.
Activity of ARF-BP1 in a subject also may be decreased in a subject by directly or indirectly decreasing levels of ARF-BP1 in vivo within the subject. By way of example, the level of ARF-BP1 in a subject may be decreased by administering a ARF-BP1 binding-protein to the subject, in an amount effective to treat neoplasia in the subject.
In accordance with the method of the present invention, ARF-BP1 inhibitors may be administered to a subject who has neoplasia, either alone or in combination with one or more antineoplastic drugs used to treat neoplasias. Examples of antineoplastic drugs with which the ARF-BP1 binding protein may be combined include, without limitation, carboplatin, cyclophosphamide, doxorubicin, etoposide, and vincristine.
In the method of the present invention, an inhibitor of ARF-BP1 expression, a ARF-BP1 protein, or a nucleic acid sequence encoding ARF-BP1 is administered to a subject who has neoplasia in an amount effective to treat the neoplasia in the subject. As used herein, the phrase “effective to treat the neoplasia” means effective to ameliorate or minimize the clinical impairment or symptoms resulting from the neoplasia. For example, the clinical impairment or symptoms of the neoplasia may be ameliorated or minimized by diminishing any pain or discomfort suffered by the subject; by extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment; by inhibiting or preventing the development or spread of the neoplasia; or by limiting, suspending, terminating, or otherwise controlling the maturation and proliferation of cells in the neoplasm. The amount of inhibitor of ARF-BP1 expression, ARF-BP1 protein, or nucleic acid encoding ARF-BP1 that is effective to treat neoplasia in a subject will vary depending on the particular factors of each case, including the type of neoplasia, the stage of neoplasia, the subject's weight, the severity of the subject's condition, and the method of administration. These amounts can be readily determined by the skilled artisan.
In the method of the present invention, the inhibitor of ARF-BP1 expression, the ARF-BP1 protein, or the nucleic acid sequence encoding ARF-BP1 may be administered to a human or animal subject by known procedures, including, without limitation, oral administration, parenteral administration (e.g., epifascial, intracapsular, intracutaneous, intradermal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, or subcutaneous administration), transdermal administration, and administration by osmotic pump. One preferred method of administration is parenteral administration, by intravenous or subcutaneous injection.
For oral administration, the formulation of the ARF-BP1 inhibitor, protein, or nucleic acid may be presented as capsules, tablets, powders, granules, or as a suspension. The formulation may have conventional additives, such as lactose, mannitol, corn starch, or potato starch. The formulation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins. Additionally, the formulation may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose. The formulation also may be presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the formulation may be presented with lubricants, such as talc or magnesium stearate.
For parenteral administration, the ARF-BP1 inhibitor, protein, or nucleic acid may be combined with a sterile aqueous solution, which is preferably isotonic with the blood of the subject. Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation may be presented in unit or multi-dose containers, such as sealed ampules or vials. The formulation also may be delivered by any mode of injection, including any of those described above.
For transdermal administration, the ARF-BP1 inhibitor, protein, or nucleic acid may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increase the permeability of the skin to the modulator, protein, or nucleic acid, and permit the modulator, protein or nucleic acid to penetrate through the skin and into the bloodstream. The composition of enhancer and modulator, protein, or nucleic acid also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which may be dissolved in solvent, such as methylene chloride, evaporated to the desired viscosity, and then applied to backing material to provide a patch. The inhibitor, protein, or nucleic acid may be administered transdermally, at or near the site on the subject where the neoplasm is localized. Alternatively, the inhibitor, protein, or nucleic acid may be administered transdermally at a site other than the affected area, in order to achieve systemic administration.
The ARF-BP1 inhibitor, protein, or nucleic acid of the present invention also may be released or delivered from an osmotic mini-pump or other time-release device. The release rate from an elementary osmotic mini-pump may be modulated with a microporous, fast-response gel disposed in the release orifice. An osmotic mini-pump would be useful for controlling release, or targeting delivery, of the inhibitor, protein, or nucleic acid.
In the method of the present invention, where the inhibitor of ARF-BP1 expression is a protein, or where ARF-BP1 protein is the therapeutic of choice, the protein also may be administered or introduced to the subject by introducing into a sufficient number of cells of the subject a nucleic acid encoding the protein, in a manner permitting expression of the protein in the subject. The amount of nucleic acid encoding the therapeutic protein is an amount that will produce the protein in an amount effective to treat neoplasia, as defined above, in the subject. This amount may be readily determined by the skilled artisan.
Nucleic acid encoding the inhibitor of ARF-BP1 expression, or the ARF-BP1 protein, as well as any nucleotide modulators of ARF-BP1 expression, all may be introduced to the subject using conventional procedures known in the art, including, without limitation, electroporation, DEAE Dextran transfection, calcium phosphate transfection, lipofection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors, and naked DNA transfer, or any combination thereof. Recombinant viral vectors suitable for gene therapy include, but are not limited to, vectors derived from the genomes of such viruses as retrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus, and vaccinia virus.
It is within the confines of the present invention that a nucleic acid encoding an inhibitor of ARF-BP1 expression, or encoding the ARF-BP1 protein itself, may be introduced into suitable cells in vitro, using conventional procedures, to achieve expression of the therapeutic protein in the cells. Cells expressing the inhibitor of ARF-BP1 expression, or the ARF-BP1 protein, then may be introduced into a subject to treat neoplasia in vivo. In such an ex vivo gene therapy approach, the cells are preferably removed from the subject, subjected to DNA techniques to incorporate nucleic acid encoding the therapeutic protein, and then reintroduced into the subject.
It is also within the confines of the present invention that a formulation containing a ARF-BP1 inhibitor, protein, or nucleic acid may be further associated with a pharmaceutically-acceptable carrier, thereby comprising a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition, comprising an inhibitor of ARF-BP1 expression, or a ARF-BP1 protein or a nucleic acid sequence encoding ARF-BP1, and a pharmaceutically-acceptable carrier. The pharmaceutically-acceptable carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc, and water, among others. Formulations of the pharmaceutical composition may be conveniently presented in unit dosage.
The formulations of the present invention may be prepared by methods well-known in the pharmaceutical arts. For example, the ARF-BP1 inhibitor, protein, or nucleic acid may be brought into association with a carrier or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g., buffers, flavoring agents, surface active agents, and the like) also may be added. The choice of carrier will depend upon the route of administration. The pharmaceutical composition would be useful for administering the ARF-BP1 inhibitor, protein, or nucleic acid of the present invention to a subject to treat neoplasia. The ARF-BP1 inhibitor, protein, or nucleic acid is provided in an amount that is effective to treat neoplasia in a subject to whom the pharmaceutical composition is administered. That amount may be readily determined by the skilled artisan, as described above.
The present invention further provides a method for treating neoplasia in a subject, by increasing or enhancing activity of p53 in the subject, wherein activity of p53 is increased or enhanced in the subject by inhibiting ARF-ARF-BP1 or p53-ARF-BP1 interaction in the subject. Preferably, p53 activity in the subject is increased or enhanced by at least 10% in the method of the present invention. More preferably, p53 activity is increased or enhanced by at least 20%. The neoplasia may be any of those described above, without regard to p53 status.
As disclosed herein, the inventor has used mass-spectrometry analysis of affinity-purified p53-associated factors to determine that ARF-BP1 bind and ubiquitates p53. ARF-BP1 inhibition strongly activates p53, even in the presence of excess Mdm2, and induces p53-dependent and p-53 independent cell-growth repression and apoptosis. Significantly, ARF-BP1 has an intrinsic enzymatic activity that specifically ubiquitinates p53, both in vitro and in vivo. In contrast, expression of a catalytically-inactive ARF-BP1 point mutant in cells increases the decreased levels of p53 ubiquitination, and stabilizes p53. These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination. In view of the foregoing, the present invention further provides a method for deubiquitinating and/or stabilizing p53 in a cell containing p53. The method comprises contacting the cell with an ARF-BP1 inhibitor, in an amount effective to deubiquitinate and/or stabilize p53.
The method of the present invention may be used to deubiquitinate p53, or remove ubiquitin from p53, in vitro, or in vivo in a subject. Deubiquitination of p53 may be detected by known procedures, including any of the methods, molecular procedures, and assays disclosed herein. The ability of ARF-BP1 inhibition to modulate deubiquitination of p53 renders ARF-BP1 particularly useful for treating neoplasias, particularly p53-associated neoplasias, as described above. Accordingly, in one embodiment of the present invention, the subject is a human with neoplasia, and the ARF-BP1 inhibition treats the neoplasia.
The method of the present invention may be used to modulate deubiquitination of p53 (i.e., by removing ubiquitin from p53, or adding ubiquitin to p53) in vitro, or in vivo in a subject. As disclosed herein, where deubiquitination of p53 is increased, stability of p53 will also be increased. The ubiquitination and deubiquitination of p53 may be detected by known procedures, including any of the methods, molecular procedures, and assays disclosed herein.
The present invention also provides a method for identifying an agent that is reactive with p53, by assessing the ability of a candidate agent to inhibit ARF-BP1-p53 interaction. Unless otherwise indicated, “p53” includes both a p53 protein (GenBank Accession No. CAA38095), including conservative substitutions thereof, and a p53 analogue. A “p53 analogue” is a functional variant of the p53 protein, having p53 biological activity, that has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the p53 protein. As further used herein, the term “p53 biological activity” refers to the activity of a protein or peptide that demonstrates detectable binding with ARF-BP1 (i.e., binding of approximately two fold, or, more preferably, approximately five fold, above the background binding of a negative control), under the conditions of the assays described herein, although affinity may be different from that of p53.
In one embodiment, in a competitive binding assay, standard methodologies may be used in order to assess the ability of a candidate agent to displace or replace ARF-BP1 in its binding to p53, thereby inhibiting the interaction of ARF-BP1 and p53. In such a competitive binding assay, the candidate agent competes with ARF-BP1 for binding to p53, and, once bound to p53, may sterically hinder binding of ARF-BP1 to p53, thereby preventing ubiquitination of p53 by ARF-BP1, otherwise stabilizing p53. A competitive binding assay represents a convenient way to assess inhibition of ARF-BP1-p53 interaction, since it allows the use of crude extracts containing p53 and ARF-BP1.
Accordingly, the present invention further comprises the steps of: (c) contacting the candidate agent with one or more cells comprising ARF, ARF-BP1, or p53; and (d) determining if the agent has an effect on one or more ARF, ARF-BP1-, or p53-associated biological events in the one or more cells. As used herein, a “ARF-BP1-associated biological event” includes a biochemical or physiological process in which ARF-BP1 activity has been implicated (e.g., neoplasia). In one embodiment of the present invention, for example, the method may further comprise the steps of: (c) contacting the candidate agent with one or more cells of a neoplasm (neoplastic cells); and (d) determining if the agent has an effect on proliferation of the neoplastic cells. As further used herein, a cell “comprising ARF-BP1” is a cell in which ARF-BP1, or a derivative or homologue thereof, is naturally expressed or naturally occurs.
The following materials and methods were used to generate the data described herein.
Plasmids and Antibodies
To clone the cDNA of ARF-BPI, five overlapped cDNA sequences that cover the full-length ARF-BPI were amplified by PCR from Marathon-Ready HeLa cDNA (Clontech, BD) and subcloned into pcDNA3.1/V5-His-Topo vector (Invitrogen). After sequence verification, the cDNA sequences were assembled and further cloned into expression vectors. To prepare mutant constructs (ARF-BPI (M), ARF-BP1(R), ARF-BP1M (R), cDNA sequences corresponding to different regions were amplified by PCR from above constructs using QuikChange Site-Directed Mutagenesis Kit (Stratagene), and subcloned into full length ARF-BP1 using specific restriction enzymes. For the HA-ARF-Flag construct, the HA and Flag sequence were introduced to the N terminus and C terminus ARF respectively by PCR and subcloned into the pCIN4 vector. To construct the Flag-p53, GST-ARF and GST-Mdm2 vectors, cDNA sequences corresponding to the full-length proteins were amplified by PCR from other expression vectors, and subcloned into either a pET-Flag or pGEX (GST) vector for expression in bacteria (Li et al., 2003, supra). To prepare GST-ARF mutant constructs, cDNA sequences corresponding to different regions were amplified by PCR from the ARF (wt) constructs. To construct adenovirus-ARF, the cDNA ARF was first cloned into pShuttle-IRES-hrGFP-1 vector (Stratagene). The resulting plasmid was then transformed for recombination into E. coli strain BJ5183 containing the adenoviral backbone plasmid pAdEasy-1. AD-293 cells were used for amplification of recombinant adenoviral ARF.
To prepare the ARF-BP1 antiserum, DNA sequences corresponding to 191 amino acids of ARF-BPI (residues 3435-3626) were amplified by PCR and subcloned into pGEX-2T (Luo, J. et al., Cell 107:137-48, 2001). α-ARF-BP1 antiserum was raised in rabbits against the purified GST-ARF-BP1 (3435-3626) fusion protein (Covance) and further affinity-purified on the antigen column. p53-specific monoclonal (DO-1) and polyclonal (FL-393) antibodies, anti-p21 polyclonal, anti-Mdm2 (SMP40) monoclonal antibody, Myc polyclonal antibody were purchased from Santa Cruz. Anti-GFP monoclonal and anti-GST monoclonal antibody were purchased from Clontech. Anti-V5 monoclonal antibody was purchased from Invitrogen. Mouse monoclonal (4C 6/4) p14ARF and rabbit polyclonal p14ARF (ab470) antibodies were purchased from Abcam.
Purification of ARF-Complexes From Human Cells
The epitope-tagging strategy to isolate ARF-containing protein complexes from human cells was performed essentially as previously described with some modifications (Luo, J. et al., Nature 408:377-81, 2000; Nikolaev, A. Y. et al., Cell 112:29-40, 2003). In brief, to obtain an HA-ARF-Flag expressing cell line, p53 null H1299 cells were transfected with pCIN4-HA-ARF-Flag and selected for 2 weeks in 1 mg/ml G418 (GIBCO). The tagged ARF protein levels were detected by Western blot analysis. The stable cell lines were chosen to expand for complex purification based on the fact that the expression levels of the ectopic ARF protein in H1299 cells were very close to the levels of endogenous protein. Thus, the cells were grown in DMEM with 10% fetal bovine serum and harvested near confluence. The cell pellet was resuspended in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF and protein inhibitor mixture [Sigma]). The cells were allowed to swell on ice for 15 min, after which 10% NP 40 (Fluka) was added until a final concentration of 0.5%. The tube was vigorously vortex for 1 min. The homogenate was centrifuged for 10 min at 4,000 rpm. The nuclear pellet was resuspended in ice-cold buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and protein inhibitor mixture) and the tube was vigorously rocked at 4° C. for 45 min. The nuclear extract was diluted with buffer D (20 mM HEPES [pH 7.9], 1 mM EDTA) to the 100 mM final NaCl concentration, ultra-centrifuged 25,000 rpm for 2 hr at 4° C. After filtered with 0.45/μm syringe filters (NALGENE), the supernatants were used as nuclear extracts for M2 immunoprecipitations by anti-FLAG antibody-conjugated agarose (Sigma). The bound polypeptides were eluted with the FLAG peptide and were further affinity purified by anti-HA antibody-conjugated agarose (Sigma). The final elutes from the HA-beads with HA peptides were resolved by SDS-PAGE on a 4%-20% gradient gel (Novex) for silver staining or colloidal-blue staining analysis. Specific bands were cut out from the gel and subjected to mass-spectrometry peptide sequencing.
Ablation of Endogenous ARF-BP1 by RNAI in Both P53-Null Cells and p53 Expressing Cells
p53-null cell lines (H1299 and Saos-2), and p53-expressing cells (U2OS, MCF-7 and A549) were maintained in DMEM medium supplemented with 10% fetal bovine serum. The HCT116 and HCT116-p53(−/−) cell lines were kindly provided by B. Vogelstein's lab. The RNAi-mediated ablation of endogenous ARF-BP1 was performed essentially as previously described (Elbashir, S. M. et al., Nature 411:494-498, 2001). A 21-nucleotide siRNA duplex with 3′dTdT overhangs corresponding to ARF-BP1 mRNA (ARF-BP1 #1) (AAUUGCUAUGUCUCUGGGACA (SEQ ID NO:18)) or (ARF-BP1 #2) (AAGUAUCCCUACCACCUCAUG (SEQ ID NO:19)) was synthesized (Dharmacon). The same sequence (ARF-BP1 #1 mutant) with 2 nucleotides changed (AAUUGCCAUGUAUCUGGGACA (SEQ ID NO:20)) was used as a specific RNAi control. The sequence (AAGAGGACUCCGCUACUGACA (SEQ ID NO:21)) was used as mouse ARF-BP1 RNAi for MEF cells. The sequence AAGGUGGGAGUGAUCAAAAGG (SEQ ID NO:22) was used for Mdm2 RNAi.
BRDU Labeling
The BrdU incorporation assay was performed essentially as previously described (Yarbrough, W. G. et al., Cancer Res 62:1171-7, 2002). In brief, cells were grown in medium containing 20 gM BrdU (Calbiochem) for 2 h and then fixed in 70% ethanol. DNA was denatured, and cells were permeabilized in 2N HCl, 0.5% Triton X-100 (Sigma), neutralized in 0.1 M Na2B4O7 (pH 8.5), and then blocked with 1% BSA in PBS. Anti-BrdU was added following the manufacturer's protocol (Amersham). After washing with 1% BSA/PBS, the cells were incubated with Alexa488 conjugated anti-mouse IgG (Molecular Probes). Finally, cells were counterstained with DAPI to visualize the nuclei.
Protein Purification of the Components for In Vitro Ubiquitination Reactions
To prepare the purified components for the in vitro ubiquitination assay (Li et al., 2003, supra), Flag-p53, E3 (GST-ARF-BP1 3760-4374), and GST-ARF were induced in Rosetta (DE3) pLys (Novagen) cells at room temperature and proteins were extracted with buffer BC500 (20 mM Tris-HCl, pH7.3, 0.2 mM EDTA, 500 mM NaCl, 10% glycerol, 1 mM DTT and 0.5 mM PMSF) containing 1% NP-40, and purified on either glutathione-Sepharose (Pharmacia) or M2 beads (Sigma). Rabbit E1 was obtained from Calbiochem. Rabbit E2 and His-Ub were purchased as a purified protein from Affinity Inc.
In Vitro Ubiquitination Assays
The in vitro ubiquitination assay was performed as described previously (Li et al, 2003, supra) with some modifications. For the self ubiquitination assay, 200 ng of bacteria-produced GST-ARF-BPI (3760-4374) or its ca mutant was mixed with other components, including E1 (10 ng), E2 (His-UbcH5a, 100 ng), and 5 μg of His-ubiquitin (affinity) in 10 μl of reaction buffer (40 mM Tris, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, pH 7.6). 400 ng of bacteria produced GST-ARF or GST-ARF mutant protein was added as inhibitor. The reaction was stopped after 60 min at 37° C. by addition of SDS sample buffer, and subsequently resolved by SDS-PAGE gels for Western blot analysis.
For p53 ubiquitination, 20 ng of the bacteria produced Flag-p53 was mixed with other components, including E1 (100 ng), E2 (His-UbcH5a, 1 μg), E3 (bacteria produced GST-ARF-BP1 (3760-4374) (400 ng), and 20 μg of bacteria produced His-HA-ubiquitin in 100 μl of reaction buffer (40 mM Tris, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, pH 7.6). 1 μg of bacteria produced GST-ARF or GST-ARF mutant protein was added as an inhibitor. After 2 hr incubation at 37° C., 15 μl of anti-FLAG antibody-conjugated agarose was added following addition of 500 μl Flag lysis buffer, and subsequently rotated at 4° C. overnight. The elutes were analyzed by Western blot with anti-p53 (DO-1) antibody.
The present invention is described in greater detail in the examples which follow, which should be considered as illustrative and nonlimiting.
EXAMPLE 1 
This example demonstrates identification of ARF-BP1 as a major component of the ARF-associated nuclear complexes from p-53-null cells
To identify the in vivo targets for ARF-mediated function an epitope tagging procedure was used to isolate ARF-containing protein complexes from human cells. The method was developed by the present inventor to purify protein complexes such as the HDACI and p53 complexes (Gu, W., Malik, S., Ito, M., Yuan, C. X., Fondeil, J. D., Zhang, X., Martinez, E., Qin, J., Roeder, R. G., Mol Cell 3:97-108, 1999; Luo, J., Su, P., Chen, D., Shiloh, A., Gu. W. Nature 408:377-81, 2000; Nikolaev, A. Y., Li, M., Puskas, N., Qin, J., Gu, W., Cell 11:29-40, 2003); nevertheless, some modifications were made to improve the stoichiometry of the protein complexes. In particular, a derivative of the human lung carcinoma p53-null H1299 cell line that stably expresses a double-tagged human ARF protein containing a N-terminal HA- and C-terminal FLAG epitope (HA-ARF-Flag (FIG. 1A) was generated. To avoid non-physiological interactions that might occur in cells that overexpress ARF, H1299 derivatives that express the ectopic ARF protein at levels similar to those of endogenous ARF (FIG. 1B) were used. As such, the tagged protein complexes reflected native conditions of the endogenous ARF complexes.
To isolate protein complexes containing ARF, nuclear extracts from HA-ARF-Flag expressing H1299 cells and from control cells (parental H1299) were first subjected to affinity chromatography on M2 (Flag antibody) agarose beads. The bound proteins were eluted with the FLAG peptide, and the elutes were chromatographed on a HA-affinity column. Finally, the bound proteins were eluted from the column with an HA peptide, fractionated by SDS-PAGE, and visualized by silver staining (FIG. 1C). B23/necleoplasmin (NPM), a known ARF-binding protein, was identified from the complexes (FIG. 1C). Unexpectedly, a major protein band of  ̃500 kDa (p500) also co-purified with ARF from HA-ARF-Flag-expressing H1299 cells (lane 2) but not from parental H1299 cells (lane 1) was found suggesting that this protein is a specific binding partner of ARF. The protein was designated as ARF-BP1 (ARF-binding protein 1). Significant levels of Mdm2 were not detected in these complexes by Western blot analysis, and mass spectrometric analysis of additional minor bands that co-purified with ARF (FIG. 1C), failed to identify Mdm2 sequences. Thus, these data suggest that ARF-BP1 is a major component of the ARF-associated complexes of these cells.
EXAMPLE 2 
This example demonstrates the initial characterization of ARF-BP1, a novel ubiquitin E3 ligase.
Peptide sequencing of the ARF-BP1 band by mass spectrometry revealed two peptide sequences, matched a single partial cDNA clone in the GeneBank database (accession number Gi 22090626). A small fragment of this protein named UREB1 (upstream initiator-like sequence binding protein 1) was originally identified by the present inventor as a binding protein of the preprodynorphin gene promoter, but its biological functions were previously unknown (Gu, J., Ren, K., Dubner, R., and Iadarola, M. J., Brain Res Mol Brain Res. 24:77-88, 1994).
A full-length human ARF-BP1 cDNA was assembled by RACE (Rapid amplification of cDNA ends) and homology alignment with the partial cDNA sequences in the database. The human ARF-BP1) cDNA encodes a 4374 amino acid protein (FIGS. 2A and 3) and the full length protein of ARF-BP1 is more than 3000 amino acids longer than the published UREB1 sequences (Gu et al., 1994, supra). SEQ ID NO: 1 represents the DNA sequence encoding the human ARF-BP1 protein (FIG. 2A)). SEQ ID NO: 2 represents the amino acid sequence of the human ARF-BP1 protein (FIG. 3).
The C-terminal sequences of ARF-BP1 possess a signature motif (the HECT domain) common to a number of ubiquitin E3 ligases (FIGS. 2A and 2B). The HECT domain sharing a conserved about 350-amino acid, harbors the Cys residue that forms a catalytic thiol ester with Ub and is regarded as a bona fide E3 ligase enzymatic motif. ARF-BP1 also contains the ubiquitin associated domain (UBA) (FIGS. 2A and 4), a small sequence motif found in various proteins linked to the ubiquitination pathway, such as the DNA repair protein Rad23 or the Cb1 ubiquitin ligase (Hicke, L., and Dunn. R., Annu Rev Cell Dev Biol 19:141-172, 2003; Buchberger, A., Trends in Cell Biol. 12:216-221, 2002. Northern blot analysis showed that the ARF-BP1 mRNA is ubiquitously expressed in different types of human tissues (FIG. 2C).
EXAMPLE 3 
This example demonstrates that ARF-BP1 interacts with ARF both in vitro and in vivo.
To confirm the physical interaction between ARF and ARP-BP1, the in vitro binding of ARF-BP1 to ARF was evaluated. The ARF polypeptide can be roughly divided into two major functional domains: an N-terminus region encoded by the unique 1β exon (N-ARF: residues1-64), which are critical for ARF-mediated p53 activation as well as p53-independent ARF functions, and a C-terminal region (C-ARF:residues 65-132), not conserved between human and mouse counterparts and of uncertain function. As shown in FIG. 5A, 35S-labeled ARF-BP1(1-1014), a polypeptide comprising the N-terminal 1014 residues of ARB-BP1, did not associate with immobilized GST-ARF (lanes 7-9). In contrast, however, 35S-labeled ARF-BP1 (1015-4574) strongly bound both full-length ARF (GST-ARF, lane 3) and the N-terminal ARF domain (GST-N-ARF, lane 5) but not the C-terminal ARF domain (GST-C-ARF, lane 6) or GST alone (lane 2). Interestingly, ARF-BP1 weakly bound the ARF mutant (GST-ARFΔ1-14; (lane 4), indicating that deletion of the first 14 amino acids significantly compromises but does not completely eliminate the ARF and ARF-BP1 interaction.
To confirm the interaction between ARF and ARF-BP1 in vivo, an affinity-purified polyclonal antiserum was raised against the 191 amino acid segment of ARF-BP1 (residues 3435-3626), a region that shows no apparent homology with any known proteins. Upon Western blot analysis, this antibody specifically detected ARF-BP1 polypepetides in human cells (lane 1, FIG. 5B). To investigate the interaction between endogenous ARF-BP1 and ARF polypeptides, cell extracts from native H1299 cells were immunoprecipitated with α-ARF-BP1 or with the control IgG. Western blot analysis revealed that this antibody immunoprecipitated endogenous ARF-BP1 (lane 3, upper panel, FIG. 5B); more importantly, ARF was clearly detected in the immunoprecipitations obtained with the α-ARF-BP1 antiserum (lane 3, lower panel, FIG. 5B) but not the control IgG (lane 2, lower panel, FIG. 5B). Conversely, endogenous ARF-BP1 was readily immunoprecipitated with the ARF-specific antibody (lane 3, FIG. 5C), but not with a control antibody (lane 2, FIG. 5C). These data indicate that ARF and ARF-BP1 interact both in vitro and in vivo.
EXAMPLE 4
This example demonstrates that the HECT domain of ARF-BP1 has an ubiquitin ligase activity that is strongly inhibited by ARF.
ARF can stabilize p53 by sequestering Mdm2 in the nucleolus and can also stabilize p53 by directly inhibiting the enzymatic activity of Mdm2. To examine whether ARF can also inhibit the ubiquitin ligase activity of ARF-BP1, ARF-BP1 was tested for enzymatic activity in an in vitro assay using purified components. The GST-ARF-BP1 (3760-4374) polypeptide (FIG. 3; SEQ ID NO: 2), which includes the HECT domain of ARF-BP1, was expressed in bacteria and purified to near homogenicity. As shown in FIG. 5D, ubiquitin-conjugated forms of ARF-BP1 were readily formed when GST-ARF-BP1 (3760-4374) was incubated in the presence of ubiquitin, E1, and an E2 (UbcH5c) (lane 2). Notably, this activity was strongly represented by recombinant full-length ARF (lane 3). Moreover, consistent with the binding results (FIG. 5A), the evolutionarily conserved N-terminal region of ARF, but not the C-terminal region, also inhibited ARF-BP1-mediated autoubiquitination (lanes 4, 5). These data suggest that ARF functions as a potent inhibitor of the ARF-BP1 ubiquitin ligase activity.
EXAMPLE 5 
This example demonstrates that inactivation of ARF-BP1 induces cell growth repression in p53-null cells.
Although in normal cells ARF stabilizes and activates p53 by inhibiting Mdm2 function, ARF can also inhibit the growth of p53-null cells. To determine whether ARF induces p53-independent growth suppression by inhibiting ARF-BP1 function, the present invention examined whether inactivation of endogenous ARF-BP1 also represses cell growth in p53-null cells in a manner reminiscent of ARF induction. p53-null H1299 cells were transfected with either an ARF-BP1-specific (ARF-BP1-RNAi# 1) or a control (GFP-RNAi) siRNA. As shown in FIG. 6A, the levels of endogenous ARF-BP1 polypeptides were severely reduced after three consecutive transfections (upper panels, lane 3 vs. lane 2) with ARF-BP1-RNAi#1. The steady state levels of p21 and Mdm2, two transcriptional targets of p53, were unaffected by ARF-BP1 ablation. Unexpectedly, ARF-BP1-RNAi treatment significantly reduced the growth rate of these cells (FIG. 6B), suggesting that ARF-BP1 inactivation induces cell growth repression. These cells grew slightly faster when endogenous Mdm2 expression was diminished with RNAi in these cells (FIG. 6A, 6B). By monitoring BrdU incorporation (FIG. 6C), the present inventor discovered that ARF-BP1 knockout inhibits, while Mdm2 knockdown modestly promotes, the growth of p53-null cells. Similar results were also obtained with another p53-null cell line (SaoS-2, FIG. 6D and 6E) and another siRNA (RNAi/ARF-BP1 #2) that recognizes a different region of the ARF-BP1 mRNA (FIG. 7A-D).
ARF-mediated cell growth in p53-null cells is not well characterized and there is evidence that ARF expression induces G2/M arrest in a number of p53-null human cell lines (Normand, G. et al., 2005). To analyze the nature of cell growth arrest mediated by ARF-BP1 inactivation in H1299 cells, the effect of ARF expression in these cells was examined. As shown in FIG. 8, ARF expression induced G2/M accumulation of these cells but no obvious apoptotic cells (Sub-G1) were observed (iii vs i). Unexpectedly, inactivation of ARF-BP1 by ARF-BP1 RNAi in these cells also led to G2/M arrest at similar levels (FIG. 8). These results show that inactivation of ARF-BP1, inhibits the growth of these p53-null cells in a manner reminiscent of ARF induction.
EXAMPLE 6 
This example demonstrates that inactivation of endogenous ARF-BP1 in normal cells stabilizes p53 and induces p53-dependent apoptosis.
To further investigate the role of the ARF and ARF-BP1 interaction in p53-positive cells, the functional consequences of ARF-BP1 inactivation in cells expressing wild-type p53 was tested. Human osteosarcoma U2OS cells were transfected with either an ARF-BP1-specific siRNA (ARF-BP1-RNAi#1) or control siRNA (GFP-RNAi). Unexpectedly, RNAi-mediated knockdown of ARF-BP1 expression elevated the steady-state levels of endogenous p53 (FIG. 9A) and extended the half-life of p53 polypeptides (FIGS. 9B and 10). The expressions of p21 and BAX, the transcriptional targets of p53, were strongly induced by ARF-BP1 inactivation (FIG. 9A). ARF-BP1 ablation also induced programmed cell death; as shown in FIG. 9C, 32.3% of the ARF-BP1-RNAi#1-treated U2OS cells underwent apoptosis (II), while no significant apoptosis was observed in control transfected cells (I). These data indicate that inactivation of ARF-BP1 stabilizes p53 and activates its mediated functions.
Given that Mdm2 is considered the primary target in ARF-mediated p53 activation, these results were unexpected. To verify the specific effects induced by ARF-BP1 ablation, control experiments were conducted.
Using another ARF-BP1-specific siRNA (RNAi/ARF-BP1 #2), which recognizes a different region of ARF-BP1 mRNA (see Methods), but not with a point mutant form of the siRNA (ARF-BP1-RNAi#1mut) (FIGS. 7D and 11), knockdown levels of endogenous ARF-BP1 proteins were performed. The levels of p53 were elevated in those cells. Similar results were obtained using a variety of different cell lines that retain wild-type p53 function, including MCF-7 human breast carcinoma cells (FIG. 12), A549 human lung adenocarcinoma cells (FIG. 12) and normal human fibroblast cells (NHF-1) (FIG. 13).
To further demonstrate the specificity of ARF-BR-RN1-mediated effects, rescue experiments were performed. A new expression vector for ARF-BP1 which contains a point mutation at the RNAi#1 targeting region (ARF-BR1(R) was made (FIG. 14). This mutant was immune to the effect by the ARF-BP1 RNAi#1. To further elucidate the importance of ubiquitin ligase activity of ARF-BP1 another mutant (ARF-BP1 M (R) was made, in which the conserved Cystine residue at the HECT domain was replaced by Alanine (FIG. 14A). Via in vitro ubiquitination assay, it was confirmed that this mutation at the HECT domain abrogates the ubiquitin ligase activity of ARF-BP1 (FIG. 14B).
To perform the “rescue experiments”, the RNAi assay in U2OS cells with ARF-BP1-RNAi#1 was employed. Rescue was attempted by expressing the ARF-BP1 mutant (ARF-BP1 (R)). As indicated in FIG. 9D, after the ARF-BP1 RNAi#1 treatment, endogenous p53 was stabilized and p21 was activated. ARF-BP1 (R) expression reversed the effect on the p53 stabilization and p21 induction induced by the ARF-BP1 RNAi#1 (lane 3 vs. lane 2). The HECT mutant form (ARF-BP1M(R)) which was expressed at similar levels, failed to rescue the effects (lane 4 vs. lane 2) (FIG. 9D). This approach was utilized for p53-independent function in H1299 cells. The cell growth inhibition induced by the ARF-BP1 RNAi#1 treatment was rescued by expression of ARF-BP1(R), but not the HECT mutant form (ARF-BP1M(R)). This demonstrates not only the specificity of the ARF-BP1-RNAi medicated effects but also the importance of the ubiquitin ligase activity in ARF-BP1-mediated functions.
To confirm that these effects of ARF-BP1 are p53-dependent, the siRNA assay was performed in a pair of isogenic human colorectal carcinoma lines that do or do not express wild-type p53. As shown in FIG. 9E, when HCT116 parental cells and HCT116 p53(−/−) cells were subjected to RNAi treatment, ARF-BP1 knockdown stabilized p53 and induced p21 in the parental cells but not in the p53 null cells. In contrast, the levels of control proteins such as c-Mye and actin were unaffected by RNAi treatment. ARF-BP1 ablation induced apoptosis in parental HCT116 cells but not in p53-null HCT116 derivatives (FIG. 15). The steady-state levels of Mdm2 were also induced by ARF-BP1 inactivation in the parental cells, consistent with the fact that Mdm2 is a transcription target of p53. Mdm2 levels in the p53 null cells were unaffected by ARF-BP1 ablation (FIG. 9E), suggesting that inactivation of ARP-BP1 induces p53 stabilization but has no effect on Mdm2 stabilization. By demonstrating that ARF-BP1 inactivation is sufficient to stabilize and activate p53 in normal cells, these data demonstrate that the ARF/ARF-BP1 interaction contributes, at least in part, to p53 activation induced by ARF.
EXAMPLE 7 
This example demonstrates that ARF-BP1 directly binds and ubiquitinates p53, and ARF-BP1-mediated ubiquitination of p53 is inhibited by ARF.
The functional relationship between p53 and ARF-BP1 was evaluated by determining whether ARF-BP1 can bind p53 in the absence of ARF. As shown in FIG. 16, 35S-labeled in vitro-translated ARF-BP1 (1015-4374) strongly bound an immobilized GST-p53 polypeptide but not GST alone (lane 3 vs. lane 2). Conversely, no significant binding was detected between ARF-BP1 and GST-Mdm2 (lane 4).
To test for the interaction between endogenous p53 and ARF-BP1 proteins in human cells, cell extracts from U2OS cells, were immunoprecipitated with α-ARF-BP1 or with control IgG. As seen in FIG. 16B, p53 was clearly detected in the immunoprecipitates obtained with the α-ARF-BP1 antiserum (lane 3) but not the control IgG (lane 2, lower panels). Conversely, endogenous ARF-BP1 was readily immunoprecipitated with the p53-specific monoclonal antibody DO-1 (lane 3, FIG. 17C), but not with a control antibody (lane 2, FIG. 16C). This data indicates that p53 can interact directly with the ARF-BP1 protein both in vitro and in vivo.
To test if ARF-BP1-mediated E3 ubiquitin ligase was involved in p53 degradation, ARF-BP1 direct induction of p53 ubiquitination in the absence of Msm2 by was examined via a standard in vitro ubiquitination assay using all purified components. Flag-p53 was incubated with GST-ARF-BP1 in the presence of HA-tagged ubiquitin (HA-Ub), E1 and an E2 (UbcH5c). The ubiquitin-conjugated p53 products of the reaction were immunoprecipitated with Flag/M2 beads and visualized by Western blot analysis with a p-53-specific antibody. As indicated in FIG. 16D, high levels of ubiquitinated p53 were generated by ARF-BP1 (lane 2). ARF-BP1 mediated p53 ubiquitination was strongly repressed in the presence of ARF (lane 3). Consistent with the binding data shown in FIG. 5A, the N-terminal region of ARF (N-ARF) retained full inhibition of ARF-BP1-mediated p53 ubiquitination whereas the C-terminal region (C-ARF) showed no effect (lanes 4 and5). These data demonstrate that ARF-BP1 is an ubiquitin ligase for p53 and that ARF-BP1 mediated ubiquitination of p53 is repressed by ARF.
EXAMPLE 8
This example demonstrates that ARF-BP1 is critical for ARF-mediated p53 stabilization in Mdm2-null cells.
Since ARF-BP1 binds and unbiquitates p53 in the absence of Mdm2, whether the ARF/BP1 interaction stabilizes p53 in an Mdm2-independent manner was evaluated. To determine whether ARF expression induces p53 stabilization in Mdm2-null cells, p53/Mdm2 double-null MEF cells were transfected with expression vectors encoding p53 alone, or both p53 and ARF. p53 protein levels were significantly elevated in these cells by ARF overexpression (FIG. 17A), indicating that ARF stabilizes p53 in an Mdm2-independent manner.
The role of endogeneous ARF-BP1 in p53 degradation in the absence of Mdm2 was verified by examination of whether inactivation of ARF-BP1 is sufficient to stabilize p53 in Mdm2-null cells. Mdm2/p53 double null cells were co-transfected with a p53 expression vector and siRNA specific for either ARF-BP1 or Mdm2. Ablation of endogeneous ARF-BP1 expression in the cells caused a marked stabilization of p53 (FIGS. 17B and 18A and B). Treatment with Mdm2-specific siRNAs had no effect on p53 levels in Mdm2/p53-null cells (lane 3, FIG. 17B), confirming the specificity of p53 stabilization by ARF-BP1 inactivation.
To provide direct evidence that ARF-BP1 is involved in the Mdm2-independent p53 stabilization induced by ARF, the requirement for ARF-BP1 for ARF-mediated p53 stabilization in Mdm2-null cells was examined. p53/Mdm2 double-null cells were co-transfected with ARF-BP1-specific siRNAs and expression vectors encoding p53 and ARF. As indicated in FIG. 17C, the p53 stabilization induced by ARF was clearly attenuated in ARF-BP1 knockdown cells (lanes 6-9), indicating that ARF-BP1 is critical for ARF-mediated p53 stabilization in these cells. In contrast, ARF-mediated p53 stabilization was intact in cells treated with Mdm2 RNAi (lanes 2-5, FIG. 17C). These results indicate that ARF-BP1 is critical for the ARF-mediated, Mdm2-independent, stabilization of p53.
EXAMPLE 9 
This example demonstrates that NPM/B23 protein is not the enzymatic target of the ARF-BP1 ubiquitin ligase activity.
Several recent studies have indicated that NPM/B23 might be the key target for p53-independent functions mediated by ARF (Bertwistle et al., MCB 23:8097, 2004; Kuo et al., Genes Dev. 18:1862, 2004). Additionally, the data in FIG. 1C shows that NPM/B23 is the major component of the ARF-associated nuclear complexes. To examine how ARF-BP1 loss leads to cell cycle arrest in p53 null cells and whether there are other targets of its E3 activity, as well as whether ARF-BP1 induces degradation of NPM/B23 for p53-independent functional regulation, a series of experiments were conducted. The results are shown in FIGS. 21 and 22. Using the GST-pull-down-assay (FIG. 22A), NPM/B23 was found to interact directly with ARF-BP1. Using the coimmunoprecipitation assay to assess the in vivo interaction between ARF-BP1 and NPM/B23 (FIG. 22B), it was found via Western blot analysis that endogenous ARF-BP1 is coimmunosuppressed with B23. After transfecting the expression vector of Flag-B23 into 293 cells, the B23 protein was immunoprecipitated by M2-beads. To examine whether NPM/B23 is a substrate of ARF-BP1, the enzymatic activity of ARF-BP1 was determined using an in vitro assay. The NPM/B23 polypeptide, which was expressed in bacteria and purified to near homogeneity, was incubated with GST-ARF-BP1 in the presence of ubiquitin, E1, and an E2 (UbcH5C). As shown in FIG. 22C, no significant ubiquitination of NPM/B23 was detected by Western blot analysis with the anti-NPM/B23 antibody. These results indicate that ARF-BP1 fails to induce ubiquitination of NPM/B23. Consistent with the above results, RNAi-mediated knock-down of endogenous ARF-BP1 had no effect on the stability of endogenous NPM/B23 as shown in FIG. 22D. These results indicate that ARF-BP1 is not involved in NPM/B23 degradation. These results further indicate that NPM/B23 is not the enzymatic target for ARF-BP1 ubiquitin ligase activity.
While the foregoing invention has been described in some detail for purposes and understanding, it will be appreciated by one skilled in the art, from a reading of sure, that various changes in form and detail can be made without departing from cope of the invention in the appended claims.

## Claims

1. An isolated ARF-BP1 polypeptide having the amino acid sequence of SEQ ID NO: 2.

2. A method for identifying an inhibitor of p53-ARF-BP1 interaction, comprising the steps of: (a) obtaining or generating an in vitro system comprising p53 and an ARF-BP1 polypeptide having the amino acid sequence of SEQ ID NO:2; (b) contacting the in vitro system with a candidate inhibitor; and (c) determining if the candidate inhibitor inhibits p53-ARF-BP1 interaction in the in vitro system.

3. The method of claim 2, wherein the determination in step (c) is made by comparing p53-ARF-BP1 interaction in the in vitro system of step (b) with p53-ARF-BP1 interaction in a second in vitro system comprising p53 and an ARF-BP1 polypeptide having the amino acid sequence of SEQ ID NO:2 in the absence of the candidate inhibitor.

4. The method of claim 2, wherein the determination in step (c) is made by comparing p53-ARF-BP1 interaction in the in vitro system of step (b) with p53-ARF-BP1 interaction in a second in vitro system comprising p53, an ARF-BPI polypeptide having the amino acid sequence of SEQ ID NO:2, the candidate inhibitor, and an anti-p53 or anti-ARF-BP1 antibody.

5. A method for identifying an agent that inhibits the binding between ARF-BP1 and p53, comprising the steps of: (a) contacting a candidate agent with p53, in the presence of an ARF-BP1 polypeptide having the amino acid sequence of SEQ ID NO:2; and (b) assessing the ability of the candidate agent to inhibit p53-ARF-BP1 interaction.

6. A method for identifying an agent that inhibits the binding between ARF and ARF-BPI, comprising the steps of: (a) contacting a candidate agent with an ARF-BP1 polypeptide having the amino acid sequence of SEQ ID NO:2, in the presence of ARF; and (b) assessing the ability of the candidate agent to inhibit ARF-BP1-ARF interaction.