Latexin: A Potential Tumor Suppressor Figure 3. Overexpression Lxn suppresses growth of A20 lymphoma cell lines. (A) In vitro growth inhibitory effects of Lxn overexpression on A20 cells. A20 cells uninfected (A20 control) or infected with empty (vector control) or Lxn-containing vector (Lxn vector) were cultured for 20 days and counted by the trypan blue exclusion method. The absolute cell numbers at indicated time-points (X axis) were represented as log scale (Y axis) and were shown in left panel. The relative tumor growth in Lxn-overexpression A20 cells to controls was shown in the right panel. One representative experiment out of 3 independent ones is shown. (B) Proportion of GFP+ A20 cells infected with empty (light grey column) and Lxn-containing vector (black column) throughout 20 days of culture. Flow cytometric analysis was performed to detect GFP signal. (C) Western blot analysis with LXN antibody in total protein lysate from A20 cells at day 20 culture showing durable LXN overexpression. (D) In vivo tumor formation and growth with injection of 100,000 A20 cells. A20 cells were subcutaneously injected and tumor growth was monitored and measured by all three dimensions of tumors (Y axis) on days indicated on X axis. Shown is the compilation of 2 independent experiments with 6 SD (n = 12). The statistical significance is represented by * (p,0.005) and ** (p,0.05). (E) In vivo tumor formation and growth with injection of graded doses of A20 cells. Graded doses of A20 cells (X axis) were subcutaneously injected and tumor growth was monitored and measured in all three tumor dimensions (Y axis) on days indicated on Z axis. Unfilled columns represent A20 cells infected with empty vector and filled columns represent cells with Lxn-containing vector. A representative experiment is shown (n = 4/group). (F) The fraction of GFP-expressing A20 cells in tumors biopsied at 21 days. Single tumor cell suspensions were made on day 21 post injection and subject to flow cytometric analysis for GFP-expressing cells. doi:10.1371/journal.pone.0044979.g003 inducing cytochrome C release and caspase activation, and apoptosis. Dysregulation of Bcl-2 is implicated in a variety of hematopoietic malignancies. [32] Three Pim-2 protein isoforms are active kinases which phosphorylate pro-apoptotic protein BAD and dissociate its binding from Bcl-xL, resulting in the inhibition of Bad-induced cell death. [33,34] Enhanced expression of Pim-2 was detected in multiple B-cell lymphoma types [35], T-cell lymphoblastic leukemia/lymphoma [36], and acute myeloid leukemia [37]. Thus our studies identify Lxn as a novel regulator of the apoptotic pathway, in which it suppresses tumor survival through the down-regulation of Bcl-2 and Pim-2 anti-apoptotic factors. The well- known function of Lxn is the sole carboxypeptidase A inhibitor in mammals. [12] Therefore, it was plausible that other carboxypeptidase A inhibitors might mimic Lxn’s anti-tumor effects. A CPA inhibitor from potato tubers has been shown to be an effective inhibitor of mammalian CPA and was therefore chosen to test its anti-tumor properties. We found that it freely entered A20 lymphoma cells but did not have suppressive growth effects via apoptosis, irrespective of dose. Since CPAs are not expressed in A20 cells, we conclude that non-canonical functions of Lxn were responsible, yet unknown intracellular functions of Lxn account for its anti-tumor effects. In addition, it is unlikely that tumor suppressive effect of Lxn is through the regulation of ABGL2-mediated microtubule tyrosination cycle, like TIG1, because we did not detect the expression of ABGL2 in hematopoietic cells (data not shown). These results are in line with findings of other investigations in which gene expression or protein abundance regulated by Lxn were not related to carboxypeptidase inhibition [18,19]. Thus, the action of mode of Lxn in regulating normal and malignant hematopoiesis is not through its canonical CPA inhibitor activity. Future studies aimed at identifying direct targets, or downstream signaling pathways, of Lxn will be critical for our understanding of its function in normal and malignant hematopoiesis. Addressing the issue of why and how Lxn expression is down- regulated in tumor cells, we found that CpG dinucleotides in regulatory regions of the Lxn gene were methylated. Most human leukemia and lymphoma cell lines tested showed strong patterns of methylation involving most of the 15 CpG dinucleotides in Lxn promoter. This, in turn, led to a loss of gene expression that was at least partially reversible. When cell lines were treated with de- methylating reagent, 5-aza-29-deoxycytidine, Lxn expression was re-initiated or up-regulated. Thus, hypermethylation of the CpG island in the Lxn promoter may contribute to silencing or down- regulation of Lxn expression in leukemia and lymphoma cells. Ongoing studies will focus on the genetic and epigenetic regulation of Lxn transcription in cancer stem cells, with promise for improved targeted approaches for cancer prevention, diagnosis, and therapy. Materials and Methods Animals Young 8-to 12- week old female C57BL/6 (B6) and 7-week old female BALB/c mice were purchased from the Jackson Labora- tories (Bar Harbor, ME). Mice were kept in the animal facilities of the University of Kentucky under pathogen-free conditions according to NIH-mandated guidelines for animal welfare. They were fed with acidified water and food ad libitum. All animal work in this study was approved by Institutional Animal Care and Use committee (IACUC) at the University of Kentucky (Identification number: 2010-0753). Leukemia Cell Lines Nine human leukemic cell lines (K562, Molt4, CCRF-CEM, J45.01, Jurkat, U937, HL-60, KG-1 and SupB15) and two mouse lymphoma cell lines (WEHI-231 and A20) were included in our study. Human leukemic cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Mouse lymphoma cell lines are gifts from Dr. Bondada [38]. The leukemia cell lines were maintained in IMDM supplemented with either 10% (K562) or 20% (KG-1, HL-60 and Sup-B15) fetal bovine serum (FBS), or RPMI medium with 10% FBS, 10 mM Hepes (Molt4, CCRF-CEM, J45.01, Jurkat and U937), 0.05 mM 2-mecaptoethanol, 80 U/mL penicillin, and 80 mg/mL streptomycin. The cells were incubated in a humidified atmo- sphere of 5% CO2 in air at 37uC. Isolation of CD34+ Cells Primary AML cells were obtained from the peripheral blood and bone marrow of patients at the Markey Cancer Center and Northwestern University. Normal bone marrow was obtained as discarded material following pathologic analysis, surgical mar- row harvest, or from the National Disease Research Interchange (NDRI). CB was obtained from patients at the University of Kentucky Obstetrics Department or from the NDRI. All tissues were obtained with the approval of the respective institutional review boards and appropriate informed consent (confirmation number is 11-0315-F3R from University of Kentucky). Some samples were collected form Northwestern University and transferred to University of Illinois at Urbana-Champaign with approval of Material Transfer Agreement (MTA). We processed 9 normal and 9 leukemia and lymphoma samples, the age and disease type of each sample was described in the figure 5 legends. Marrow and blood cells were depleted of erythrocytes by suspending in 150 mM NH4Cl plus 10 mM NaHCO3 for 5 minutes, followed by 2 washes with phosphate-buffered saline (PBS). Blood cells were subjected to Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) density gradient separation to isolate PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44979