bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1931: Impact of bortezomib-induced proteasome inhibition in vivo on antitumor T cell responses
Authors: Shanker Anil , Kluyver Rachel De , Sayers Thomas J.
Published: February 2011

Inhibition of cellular proteasome function crucial for protein turn-over is a recent approach to overcome the resistance of tumors to cell death, a major clinical problem. Bortezomib (also known as Velcade/PS-341), a specific and reversible proteasome inhibitor, has been shown to sensitize some mouse and human tumors to apoptosis induced by the TNF-related apoptosis-inducing ligand (TRAIL) or its receptor (DR5) agonist monoclonal antibody (mAb). In established mouse renal cell (Renca) and breast (4T1) tumor models, the administration of DR5 agonist mAb to tumor-bearing mice pre-treated with bortezomib reduced experimental lung metastases. The combination of bortezomib and DR5 agonist mAb also significantly increased long-term survival of mice bearing Renca tumors compared with either agent alone. The therapeutic effects of bortezomib on tumor metastases and survival of tumor-bearing mice occurred in the absence of overt toxicity. However, little is known concerning the effects of bortezomib on specific immune responses. We thus analyzed the effects of proteasome inhibition on anti-tumor T cell responses in vivo. In mice with established Renca-HA tumor expressing influenza virus hemagglutinin (HA) as a surrogate tumor antigen, we evaluated the antigen-specific proliferation of adoptively transferred monoclonal HA-specific MHC class I or II restricted T cells following various schedules of bortezomib administration. No inhibition of T cell proliferation or cytolytic function in vivo was observed following bortezomib treatments. Nor was there any evidence of defective dendritic cell maturation in bortezomib-treated mice. However, a significant decrease in total T cell yield was noted in the tumor-draining LNs of mice treated with bortezomib. Thus, bortezomib-inhibitory effects on T cell responses seem quantitative rather than qualitative. The basis for this decrease in T cell number is under further investigation. The findings imply that, with an appropriate scheduling, it may be possible to combine bortezomib-induced sensitization of tumors to apoptosis with tumor-specific T cell immunotherapy regimens for increased therapeutic benefit in cancer.

Funded in part by NCI Contract # N01-CO-12400 and HHSN261200800001E.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1931.
Abstract 1933: Adoptive T cell therapy shifts the secretion ability of cytokines to Th1 and decreases the number of Tregs in patients with cancer
Authors: Ishikawa Takeshi , Kokura Satoshi , Matsumoto Tsuguhiro , Funaki Jun , Adachi Satoko , Okayama Tetsuya , Yoshida Naohisa , Uchiyama Kazuhiko , Takagi Tomohisa , Handa Osamu , Konishi Hideyuki , Yagi Nobuaki , Wakabayashi Naoki , Naito Yuji , Yoshikawa Toshikazu , Sakamoto Naoyuki , Tanigawa Mari , Uno Kazuko
Published: February 2011

[Aim] We analyzed the secretion ability of cytokines from peripheral blood mononuclear cells (PBMC) and the number of peripheral blood Tregs before and after adoptive T cell therapy (CD3-LAK) in order to assess the relation of the immunological responses and the efficacy of the treatment.

[Method] Patients who received CD3-LAK treatment more than four times were done an entry of this study. We conducted this study after having obtained the informed consent of the study for these patients. We measured the secretion ability of cytokines from PBMC using the peripheral blood collected from the patients before treatment and 2 weeks later after the 4th treatment. The methods we measured the secretion ability of cytokines are shown below. IFN-alpha: We stimulated the whole blood by Sendai virus for 20 hours, and IFN-alpha of supernatant was measured by bioassay. The other cytokines (IL-2, IL-4, IL-10, TNF-alpha, IFN-gamma etc): We stimulated the whole blood by PHA for 48 hours, and the cytokines of supernatant were measured by BioPlex array. In terms of the change of the number of peripheral blood Tregs, we analyzed Foxp3 and CD4 positive T cells by flow cytometry.

[Result] The values of IFN-gamma and TNF-alpha were significantly high after the treatment. Ratio of Treg to CD4 was significantly low after the treatment. The survival time of the group of patients in whom the number of Treg decreased after the treatment was longer than that of the other group of patients.

[Discussion] We found that even in patients with refractory advanced cancer, the therapeutic intervention of CD3-LAK makes positive change - the secretion ability of cytokines from PBMC shifts to Th1, the number of Treg in peripheral blood decreases. And it suggests that adoptive T cell therapy influences immunoescape mechanism in patients with cancer. It will be necessary to clarify the mechanism of the effect and to develop an adoptive immunotherapy which has more beneficial clinical effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1933.
Abstract 1934: Hyperthermia enhances the anti-tumor activity of adoptive transfer of naïve T cells
Authors: Matsumoto Tsuguhiro , Kokura Satoshi , Ishikawa Takeshi , Funaki Jun , Adachi Satoko , Okayama Tetsuya , Sakamoto Naoyuki , Yoshida Naohisa , Uchiyama Kazuhiko , Takagi Tomohisa , Handa Osamu , Konishi Hideyuki , Wakabayashi Naoki , Yagi Nobuaki , Naito Yuji , Kato Akiko , Tomita Keisuke , Enoki Tatsuji , Yoshikawa Toshikazu
Published: February 2011

Objective: Recently, in the case of the adoptive immunotherapy, less-differentiated T cell (naïve T cells) has been postulated to be more effective for tumor regression than late effector T cells. On the other hand, hyperthermia augments MHC class I antigen presentation via HSPs expression and attenuates the production of immunosuppressive cytokines such as IL-10 and TGF-beta from cancer tissue. There is increasing evidence that the use of hyperthermia is effective as an adjunct to cancer immunotherapy. Therefore, the purpose of this study was to evaluate the effects of hyperthermia on adoptive transfer of naïve T cells in a subcutaneous B16 melanoma model. Experimental design: Male C57BL/6 mice were injected s.c. with B16 murine melanoma cells. Splenocytes for adoptive transfer were isolated from tumor-bearing mice and primary stimulation was accomplished using plate-bound anti-CD3 monoclonal antibody. Cells were expanded in culture media containing IL-2, IL-7 and recombinant human fibronectin fragment (FN-CH296). Prior to adoptive transfer, CD62Lhigh and CD62Llow populations were sorted using a MACS CD62L-positive selection column. Subcutaneous B16 bearing mice were treated with hyperthermia on day 3 and day 4, and then mice were treated with two doses of CD62Lhigh or CD62Llow cells via a tail vein on day 6 and day 10. Tumor volume, body weight and the number of regulatory T cell (Treg) within spleen were examined on day 17. Results: The tumor volume significantly decreased in CD62Lhigh adoptive transfer therapy combined with hyperthermia compared to CD62Llow adoptive transfer therapy combined with hyperthermia or hyperthermia alone. The body weight and the number of Treg within spleen did not have the significant difference among these groups. Conclusion: These data suggest that hyperthermia enhances the anti-tumor effect of adoptive transfer therapy using naïve T cells, but not late effector T cells. Hyperthermia would have potential to activate and expand naïve T cell in vivo after adoptive transfer. These results may benefit further efforts on developing novel adoptive immunotherapies based on hyperthermia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1934.
Abstract 1935: Myeloid derived suppressor cells generated in the absence of Muc1 can inhibit allo expansion of T cells while preserving graft versus tumor activity
Authors: Poh Tze Wei , Gendler Sandra
Published: February 2011

Allogeneic hematopoietic stem cell (HSC) transplantation is a promising area of therapy for various hematological malignancies like chronic/acute myeloid leukemia, myelodysplasia and lymphomas. Its effectiveness at inducing the graft versus tumor (GVT) effect is however, compromised by the graft versus host disease (GVHD) frequently observed in recipients post transplant. One of the strategies utilized in minimizing the effect of GVHD while still preserving GVT activity has been to target immunosuppressive cell types like Treg cells or myeloid derived suppressor cells (MDSCs), in an attempt to correct the balance between GVHD and GVT. MDSCs have been shown to be able to inhibit GVHD but not much is known about the ability of MDSCs to preserve GVT. We have previously shown that myeloid progenitors lacking Muc1 from Muc1 knockout (KO) C57BL/6 (H-2b) mice generate more effective MDSCs in vitro and in vivo. In this current study, we study the potential of using MDSCs generated in the absence of Muc1 to alleviate T cell expansion while preserving CTL activity. Our preliminary data shows that MDSCs generated from Muc1 KO C57BL/6 (H-2b) bone marrow are able to inhibit C57BL/6 (H-2b) T cell proliferation in an allogeneic mixed lymphocyte reaction, using Balb/c (H-2d) stimulators, more effectively than wildtype (WT) MDSCs. Interestingly, despite the reduced T cell proliferation in the presence of KO MDSCs, the CTL activity of these C57BL/6 T cells was still preserved, as demonstrated by their ability to lyse Balb/c A-20 leukemia cells. These results inidicate that co transplant of MDSCs generated in the absence of Muc1 can alleviate GVHD across fully mismatched major histocompatibility complex (MHC) barriers while still preserving the GVT ability of the allograft. This would also validate the clinical desirability of down regulating MUC1 in HSCs for more effective generation of MDSCs in a clinical allogeneic HSC transplantation setting.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1935.
Abstract 1936: Anti CD30 chimeric immune receptor mRNA for therapy of CD30+ lymphomas
Authors: Karkanitsa Pavel , Weissman Sherman , Rabinovich Peter , Abken Hinrich , Hombach Andreas , Komarovskaya Marina , Foss Francine M.
Published: February 2011

CD30 is a transmembrane receptor which is overexpressed on Hodgkin lymphoma and large cell anaplastic lymphoma and represents an attractive target for therapy. Current methods of immunotherapy of CD30+ lymphomas include antibody based therapy (immunotoxins, bispecific molecules, radioimmunotherapy), and cellular immunotherapy. Here we describe an approach to cellular immunotherapy of CD30+ lymphomas using a synthetic mRNA of an anti CD30 chimeric immune receptor (anti CD30-CIR) introduced into activated T cells by electroporation. Anti-CD30 CIR consists of a single chain antibody directed against CD30, a transmembrane anchor to localize the protein in the cellular membrane, and the cytoplasmic domain of the TcR receptor chain. In these studies, the cDNA coding sequence was used as a template to produce anti CD30-CIR mRNA. Normal T cells expanded in vitro were electroporated with CIR mRNA and expression of anti CD30-CIR was detected by biotin-conjugated F(ab’)2 followed with Streptavidin-APC. To measure cytotoxicity of the CIR, target cells (HH, K562, Jurkat) were pulsed with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then were cultured with T cells transfected with anti-CD30 CIR mRNA or mock transfected T cells. Cytotoxicity was evaluated at different target::effector (T: E) ratios and normalized by standard algorithm. Twenty-four hours after electroporation almost all viable T cell (>90%) expressed anti-CD30 CIR and were able to specifically kill CD30+ targets in a 4h in vitro assay. These results demonstrate that anti-CD30-CIR mRNA modified T cells can be generated and are capable of killing CD30-expressing cells in vitro and therefore may be an effective strategy for the therapy of refractory CD30-expressing lymphomas.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1936.
Abstract 1939: Augmenting effector function and abrogating Treg function by OX40 costimulation enhances adoptive transfer tumor-specific CTL response
Authors: Murata Satoshi , Ueki Tomoyuki , Kitamura Naomi , Mekata Eiji , Shimizu Tomoharu , Shiomi Hisanori , Abe Hajime , Naka Shigeyuki , Tatsuta Takeshi , Yamaguchi Tsuyoshi , Mori Tsuyoshi , Umeda Tomoko , Kubota Yoshihiro , Cho Hirotomi , Yamamoto Hiroshi , Endo Yoshihiro , Kurumi Yoshimasa , Tani Tohru
Published: February 2011

We have recently shown that OX40 (CD134) costimulation provided with GM-CSF-secreting vaccine can bypass CD8+ T cell tolerance. OX40 costimulation can also abrogate Foxp3+ regulatory T cell (Treg)-mediated suppression of antitumor immunity by reducing Foxp3 expression on Tregs. In addition, OX40 costimulation can directly augment tumor-specific CTL function without CD4+ T cell help in vivo. Here we show that pre-treatment with agonistic anti-OX40 mAb augments the antitumor immune effects of adoptive CD8+ T cell therapy in the clinically relevant wild-type model, not TCR-transgenic, mouse model. CD8+ T cells stimulated with tumor antigen (HER2/neu immunodominant peptide and T2 cells), including a small number of tumor antigen-specific effector CD8+ T cells, were transferred into tumor-bearing FVB/N mice. We could detect the response of tumor antigen-specific CD8+ T cells that were adoptively transferred into the recipient mice, however, the response was not sufficient to eradicate an established tumor. In addition, we showed that pre-treatment with OX40 costimulation in tumor-bearing recipient in order to inhibit the Treg-mediated suppressive function enhanced tumor specific immune response of adoptively transferred CD8+ T cells in the recipients, thus resulting in the eradication of an established tumor. Moreover, adoptively transferred CTLs, with OX40 costimulation in vitro before transfer, significantly enhanced a tumor-specific immune response in the tumor-bearing recipient. Furthermore, when tumor antigen-specific CTLs costimulated through OX40 in vitro were adoptively transferred into the tumor-bearing recipients that had been pre-treated with OX40 costimulation to inhibit Treg function, transferred tumor-specific CTLs were effectively maintained in this recipient 7 times more than in the recipient with CTL transfer alone without OX40 treatment, and about 3 times more than in the recipient with a single treatment of OX40 costimulation. These findings therefore suggest that both direct enhancement of adoptive transfer CTL function and pre-inhibition of Treg-mediated suppressive function in recipients with OX40 costimulation are useful strategy for antitumor adoptive CTL therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1939.
Abstract 194: Loss of imprinting and abnormal expression of insulin-like growth factor II in gastric cancer
Authors: Chen Teng , Zuo Qin-Song , Zhao Ronghua , Feng Dian-Xu , Chen Chao , Jiang Yi-Min , Cruz-Correa Marcia , Casson Alan G. , Han Feng
Published: February 2011

Introduction: Gastric carcinoma (GC) is a leading cause of cancer mortality worldwide, and is highly prevalent in China. Loss of imprinting (LOI) of insulin-like growth factor II (IGF-2) gene is an important epigenetic phenomenon related to carcinogenesis and tumor progression in many human cancers. To date, however, few studies have evaluated the IGF-2 LOI in GC.

Aims: This study aimed: (1) to examine the prevalence of LOI of IGF-2 in tumor, matched normal gastric mucosa, and peripheral blood lymphocyte (PBL) from a well characterized series of patients with GC, and in PBL from a control group, (2) To examine the association of LOI of IGF-2 with circulating IGF-2 and tissue IGF-2 expression.

Methods: IGF-2 genomic imprinting status was analyzed by PCR and RT-PCR followed by restrictive endonuclease (Apa I) digestion. Prevalence of LOI in PBL from 33 Apa I informative GC patients (21 males, age: 59.91±13.87) was compared with that from 21 informative controls (13 males, age: 56.81±13.28). Findings were also associated with the clinicopathologic parameters in patients with GC. IGF-2 level in peripheral blood (ng/ml) and tumor were determined with ELISA and immunohistochemical staining, respectively.

Results: LOI of IGF-2 was positive in 48.48% (16/33) GC tumor, 21.21% (7/33) adjacent mucosa (AM), 12.12% (4/33) distant mucosa (DM), and 15.15% (5/33) PBL. The prevalence of LOI in PBL was not significantly different between GC patients (5/33, 15.15%) and controls (2/21, 9.52%) (p=0.693). Patients with LOI-positive tumor were more likely to be advanced stage (93.75% vs. 58.82%, p=0.039) or have positive lymph nodes (87.50% vs. 52.94%, p=0.057). Significantly increased blood IGF-2 level was seen in GC patients with LOI-positive tumor (808.07±255.25) when compared with those with LOI-negative tumor (396.21±198.94) (p<0.01). The positive rate of IGF-2 staining was significantly higher in tumor (10 of 16, 62.50%), AM (7 of 7, 100.00%), and DM (3 of 4, 75.00%) with LOI than that in tissues without LOI (tumor: 2 of 17, 11.76%; AM: 2 of 26, 7.69%; DM: 2 of 29, 6.90%) (p<0.01).

Conclusions: The results of this study suggest that IGF-2 is a critical factor in carcinogenesis and progression of GC, with LOI of IGF-2 modulating tissue and circulating expression of IGF-2. IGF-2 LOI may represent a potential epigenetic marker for gastric cancer risk, prognosis and treatment stratification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 194.
Abstract 1941: Cancer-driven exhaustion of tumor-reactive γδ-T cells: Relevance to immunosurveillance and immunotherapy
Authors: Beck Benjamin H. , Kim Hyung-Gyoon , Haines Hilary , Feeley Kyle , Lopez Richard
Published: February 2011

In contrast to antigen-specific αβ-T cells (adaptive immunity), γδ-T cells can recognize and lyse malignantly-transformed cells almost immediately upon encounter in a manner that does not require the recognition of tumor-specific antigens (innate immunity). Given the well-accepted capability of γδ-T cells to innately kill a variety of cancerous cells, efforts are now actively underway to develop and refine the means by which to exploit the antitumor properties of γδ-T cells for clinical purposes. Here, we investigated a critical issue surrounding γδ-T cell-based cancer immunotherapies, which relates to the findings that γδ-T cells are numerically rare in the peripheral blood of some cancer patients, and only in a proportion of patients is it possible to activate and/or expand γδ-T cells either in vivo or ex vivo regardless of the methodology employed. This is in stark contrast to what is observed in healthy individuals, as γδ-T cells reliably respond to proliferative stimuli. Indeed, it is evident that such numerical or functional deficits may greatly hinder attempts to employ γδ-T cells in clinical settings. Therefore, the objective of the studies performed here was to examine the biological underpinnings that may explain the γδ-T cell impairments observed in cancer patients. To this end, we employed murine models of different malignancies (breast cancer, myeloma, and melanoma) and systematically examined γδ-T cell numbers in peripheral circulation, γδ-T cell proliferative capacity, and overall γδ-T viability and apoptosis status in healthy mice and mice bearing different tumors. We found that tumor-bearing mice had fewer γδ-T cells in peripheral blood, and γδ-T cells derived from tumor-bearing mice expanded poorly ex vivo using published expansion regimens. Importantly, we show that tumor cells were responsible for this numerical and functional exhaustion of γδ-T cells. Using both in vitro and in vivo models of different cancers we demonstrate that γδ-T cells undergo apoptosis after encounter with tumor cells and we have identified several putative genes involved in this γδ-T cell exhaustion. We propose that this cancer-associated γδ-T cell impairment could serve as a critical early event leading to impaired γδ-T cell anti-tumor immunosurveillance, which may contribute to the accelerated development or progression of different malignancies. Collectively, these findings will help facilitate the development of the next generation of clinical trials designed to exploit the tumor-reactive properties of γδ-T cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1941.
Abstract 1942: Vδ1+ γδ T cells are cytotoxic against glioblastoma multiforme
Authors: Knight Andrea , Arnouk Hilal , Britt William , Gillespie G Yancey , Lowdell Mark W. , Cloud Gretchen A. , Markert James M. , Langford Catherine P. , Su Yun , Lamb Lawrence S.
Published: February 2011

Prior evidence of cytomegalovirus (CMV) infection has been documented in a large proportion of high-grade brain tumors such as glioblastoma multiforme (GBM), suggesting a potential role for CMV infection in GBM pathogenesis. Vδ2+ γδ T cells, which recognize tumor-derived mevolonate metabolites, have been shown to be highly cytotoxic to GBM cell lines and primary tumor explants. The specific role of Vδ1+ T cells, which respond to CMV infected cells in immunosuppressed patients and recognize NKG2D ligands MIC A/B and UL-16 binding proteins (ULBP), has not previously been investigated.

Vδ1+ T cells were purified (93-95%) from CMV+ and CMV- donors by immunomagnetic selection and cultured on irradiated allogeneic MNC in RPMI + 10% human serum (HS) supplemented with phytohemagglutinin (PHA) and 200U IL-2. Vδ2+ T cells were expanded from mononuclear cells in RPMI/Clicks + 7.5% HS, 1mM Zoledronic acid, and 200U IL-2, all over 14-21 days. Cytotoxicity was assessed by flow cytometry against two primary GBM explants and CMV+/- GBM cell lines U251 and U373. The expression of MIC A/B and UL-16 binding proteins was assessed using flow cytometry.

Vδ1+ T cells from CMV+ donors were significantly more cytotoxic to primary GBM explants than Vδ1+ cells from a CMV- donor (p=0.0021 for GBM 1042 and p= 0.0053 for GBM 1016) but did not significantly differ against CMV+ and CMV- U251 and U373 cells, although CMV infected cells significantly down-regulated ULBP-2 and −3 expression. Blocking of MIC A/B and ULBP-1, −2, and −3 decreased cytotoxicity of γδ T cells by 10-40% based on the expression of the respective antigen. ZOL/IL-2 expanded Vδ2+ T cells were cytotoxic to primary GBM and GBM lines regardless of donor CMV status. These findings suggest that Vδ1+ T cells, particularly Vδ1+ cells expanded from CMV+ donors, recognize GBM independently from Vδ2+ cells and and have a potential role in strategies involving CMV-directed cellular immunotherapy of high-grade brain tumors.

This study was supported by NIH NCI Grant # P50 CA 097247, NINDS Grant # 5R21 NS 57341 and the National Brain Tumor Society Samuel Gerson Leadership Chair.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1942.
Abstract 1943: A comparison of three techniques to induce efficient ex vivo T-cell expansion
Authors: Langsdorf Chris L. , Liu Jixiang , Bradford Jolene , Buller Gayle
Published: February 2011

The current study was undertaken to evaluate three common methods of inducing proliferation in a T lymphocyte population. In the first method cells are treated with purified monoclonal mouse anti-human CD3 antibody followed by addition of interleukin-2. The second approach involves treatment with the mitogen Concanavalin A. The third technique makes use of monoclonal mouse antibodies against human CD3 and CD28 immobilized on the surface of superparamagnetic polystyrene microspheres. Human peripheral blood mononuclear cells were isolated from fresh whole blood using a centrifuge density gradient. Cells were stained with CellTrace™ Violet, a novel cell proliferation tracing dye with a mode of action similar to carboxyfluorescein diacetate succinimidyl ester (CFSE), and resuspended in OpTmizer T-Cell Expansion Buffer (GIBCO) containing L-glutamine, penicillin and streptomycin. Stained cells were treated with one of the three stimulation techniques and incubated at 37°C and 5% CO2 for 7 days. A cell impermeant nucleic acid dye was used to evaluate the effect of each treatment on cell viability. The specific subpopulations induced to proliferate with each treatment were characterized in multicolor flow cytometry using monoclonal antibody conjugates against human CD3, CD4, and CD8. Cells treated with CD3/IL-2 or Concanavalin A showed a moderate, heterogeneous level of stimulation, with some cells undergoing 9 cell divisions, others remaining senescent, and approximately equal amounts remaining in generations 1 through 8. Microsphere-bound antibodies provided a much stronger, more uniform stimulus as evidenced by the very high proliferative index of cells. Concanavalin A treatment was mildly cytotoxic, while viability of cells treated with free or bound antibodies was similar to unstimulated cells. The strong, homogeneous stimulus provided by superparamagnetic polystyrene expansion beads is useful when massive numerical expansion of T lymphocytes is desired for subsequent research or adoptive transfer in vivo, but does not appear to be as useful when a population containing several discrete generations of cells is desired. The more moderate stimuli resulting from treatment with CD3/IL-2 or Concanavalin A result in a less efficient numerical expansion, but provide an excellent tool to evaluate the phenotype and pedigree of cells as they proliferate through several generations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1943.
Abstract 1947: Identification of microRNAs that regulate DNA damage response
Authors: Wang Yemin , Taniguchi Toshiyasu
Published: February 2011

Mammalian cells have developed an elaborate DNA damage response system and DNA repair machinery, which play a critical role in the prevention of cancer and development of resistance to anti-cancer DNA damaging agents. Many proteins involved in DNA damage response, such as phosphorylated histone H2AX (γH2AX) and 53BP1, form nuclear foci at sites of DNA damage and facilitate DNA damage response and repair. The precise regulation of DNA damage response is crucial for cellular survival after DNA damage and its abrogation often results in genomic instability. MicroRNAs (miRNAs) are short, non-coding RNA molecules, which post-transcriptionally regulate gene expression by repressing translation and/or degrading mRNA. Some miRNAs function as oncogenes or tumor suppressors. However, the role of miRNAs in DNA damage response remains largely unknown. Here, we identified miRNAs that regulate DNA damage response, especially γH2AX/53BP1 foci formation, and modulate chemo-/radio-sensitivity of tumor cells. We developed a cell-based screening assay utilizing ionizing radiation (IR)-induced γH2AX/53BP1 foci formation in a human osteosarcoma cell line, U2OS, as a readout. The human miRNA Mimics and Inhibitors Libraries (Dharmacon), consisting of 810 miRNA mimics or inhibitors, were transfected in U2OS cells and screened with an automated high-throughput fluorescence microscope and an automatic foci counter. We found that two and five miRNA mimics reproducibly inhibited IR-induced γH2AX and 53BP1 foci formation, respectively, while none of the miRNA inhibitors inhibited their foci formation. Among these hits, we focused on miR-138, because miR-138 is reported to be deregulated in a subset of cancer and may be involved in tumorigenesis and chemosensitivity of cancer. We found that miR-138 decreased the expression of H2AX and reduced H2AX phosphorylation upon DNA damage. Consistent with a defect in DNA damage response, miR-138 sensitized U2OS cells to DNA damaging agents, such as IR and cisplatin. Importantly, the 3′UTR of H2AX contains a putative miR-138 binding site, suggesting that H2AX may be a direct target of miR-138. Taken together, we have identified some miRNAs which inhibit IR-induced foci formation of γH2AX/53BP1. In particular, miR-138 strongly reduced H2AX-mediated DNA damage response and enhanced the chemo-/radio-sensitivity of cancer cells, suggesting that miR-138 might serve as an important regulator of genome instability and a possible therapeutic target for cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1947.
Abstract 1957: Loss of TGFβ1 alters host-microbial interactions, predisposing the colonic epithelium to inflammation
Authors: Ball Corbie , Azhar Mohamad , Gard Constance , Chen Dora , Mast Thomas , Aronow Bruce , Besselsen David , Doetschman Thomas
Published: February 2011

Introduction: The TGFB pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Tgfb1−/−Rag2−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer.

Methods: To determine the function of TGFβ1 on colonic bacterial composition we used GEM models, Tgfb1−/−Rag2−/− and Tgfb1+/+Rag2−/−. The cecal microflora of 10 Tgfb1−/−Rag2−/− and 10 Tgfb1+/+Rag2−/− mice were isolated and serially diluted onto brucella (BRU), bacteroides bile esculin (BBE), and laked kanomycin-vancomycin (LKV) media under anaerobic conditions. Colony forming units (CFUs) were enumerated. Individual colony types were then streaked onto trypitcase soy agar with 5% sheep blood and grown anaerobically and aerobically. The bacteria were then gram-stained and biotyped utilizing a Dade Berhing MicroScan instrument. To further examine the bacterial composition we designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on fecal DNA.

Results: The Dade Berhing Instrument showed that Tgfb1−/−Rag2−/− mice had a 4 fold increase in bacterial load and a 28 fold increase in Bacteroides species when compared with Tgfb1+/+Rag2−/− mice. The qRT-PCR results showed an increase in Bacteroides fragilis and Bacteroides distasonis and a significant decrease in Bacteroides thetaiotaomicron in the Tgfb1−/−Rag2−/− mice. These data suggest that loss of TGFβ1 alters the colonic microflora. Previous studies illustrated the importance of bacterial nutrient sources on bacterial composition. To examine if TGFβ1 is altering nutrient availability in the colon a previous micro-array was analyzed for candidate genes associated with glycoprotein metabolism. This showed changes in fucose metabolizing enzymes with the loss of TGFβ1.

Conclusion: These findings suggest that TGFβ1 plays a role in bacterial load maintenance, possibly by altering available nutrient sources and when disrupted, can cause abnormalities in pathobionts (commensal bacterial with pathogenic potential) which could then lead to increased inflammation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1957.
Abstract 1958: Signals activating mutant p53 in vivo
Authors: Suh Young-Ah , Post Sean , Elizondo Cathalina , El-Naggar Adel K. , Xiong Shunbin , Lozano Guillermina
Published: February 2011

The p53 protein is activated in response to stress signals and leads to cell cycle arrest and apoptosis. As thus, missense mutations in the p53 tumor suppressor are common alterations in tumors. p53 activity is tightly regulated by murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets wild type p53 for degradation. The Arf tumor suppressor also regulates p53 activity by binding Mdm2. Arf is activated by oncogenes, and the retinoblastoma (Rb) signaling pathway, connecting the p53 and Rb tumor suppressor pathways.

Mice inheriting p53 missense mutations that mimic common hot spots in human cancers present with a gain-of-function metastatic phenotype. An analysis of tumors from these mice indicated that some but not all had stable mutant p53 implying that other alterations besides mutation in p53 are responsible for stabilization and the gain-of-function nature of the mutant protein. Additionally, mutant p53 in normal tissues and in some tumors of homozygous mutant mice is unstable. However mutant p53 was detected in normal tissues in mutant mice lacking Mdm2 showing an increased tumor incidence and decreased survival as compared to p53 mutant mice. Importantly, stabilization of p53 correlated with a metastatic phenotype. Disruption of the Rb pathway via p16INK4a deletion, a common event in human tumors, also stabilized mutant p53 in vivo. These observations indicate that mutant protein is also regulated in the same way as wild-type p53.

Here, we examined the effects of DNA damage and oncogene activation on mutant p53 stability. Mice with Ras or c-Myc activation have stable mutant p53 and show a decreased survival in vivo. Surprisingly, stable mutant protein is also detected in mice treated with sub-dose of chemotherapeutic agent or low-dose of radiation implying that the factors affecting wild-type p53 are also responsible for mutant protein stability. Treated mice also showed decreased survival as compared to p53 mutant mice. Characterization of the mechanism by which the mutant p53 protein is stabilized in tumors is important to our understanding of the gain-of-function phenotype in these mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1958.
Abstract 196: UHRF1 is upregulated in prostate cancer and induces epigenetic silencing of tumor suppressor genes
Authors: Kunderfranco Paolo , Curti Laura , Babbio Federica , Pistore Christian , Magistri Marco , Chiorino Giovanna , Bonapace Ian Marc , Catapano Carlo V. , Carbone Giuseppina M.
Published: February 2011

Cancer of the prostate is the most common cancer and a leading cause of cancer death in Europe and North America. At the present there is a need to understand the mechanisms involved in the pathogenesis of this disease and discover alternative therapeutic targets. UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, has been previously reported to acts as a dominant negative effectors of cell growth. UHRF1 is involved in epigenetic mechanisms by virtue of its interaction with DNMTs and HMTs. In this study, analysis of gene expression profiles of prostate tumors and normal prostate showed that UHRF1 is frequently over-expressed in tumors compared to normal tissues. UHRF1 expression was very low in immortalized prostate epithelial cells (LH) while it was higher in the Ras transformed counterpart LHSR cells. Furthermore, we observed low level of UHRF1 in the androgen-dependent prostate cancer cell lines LNCaP and 22Rv1, while higher levels were present in the androgen-independent cell lines PC3 and DU145. These data suggested that over-expression of UHRF1 is associated with malignant transformation and prostate cancer progression. To understand the role of UHRF1 we performed knockdown experiments with UHRF1 specific siRNA. Transient transfection in PC3 cells reduced UHRF1 mRNA and protein level. UHRF1 knock-down resulted in reversion of the transformed phenotype with significant inhibition of clonogenic growth in anchorage dependent and independent condition. Reversion of the transformed phenotype occurred concomitantly with restoration of the expression of several tumor suppressor genes relevant for prostate differentiation, proliferation and epithelial-mesenchymal transition such as CDH1 and RARB2. Moreover, chromatin immunoprecipitation showed binding of UHRF1 to these gene promoter and UHRF1 knockdown resulted in significant reduction of repressive histone marks on gene promoters. These studies suggest that deregulated UHRF1 expression results in epigenetic silencing of relevant tumor suppressor genes, contributing to prostate cancer progression. Targeting UHRF1 may result in re-expression of tumor suppressor genes and thus may be a valid strategy for therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 196.
Abstract 1964: Genetic instability and mammary tumor formation in mice carrying mammary-specific disruption of Chk1
Authors: Li Yuing , Fischler Thomas , Xu Xiaoling , Wang Rui-Hong , Kim Hyun-Seok , Sengupta Kundan , Vassilopoulos Athanassios , Lahusen Tyler , Liu Qinghua , Elledge Steve , Ried Thomas , Deng Chuxia
Published: February 2011

Chk1 is a key element in the DNA damage response pathway that is required for maintaining genomic stability. To study the potential role of Chk1 in mammary tumorigenesis, we disrupted it using a Cre/loxP system. We showed that although Chk1 haploinsufficiency caused abnormal development of the mammary gland, it was not sufficient to induce tumorigenesis. Simultaneous deletion of one copy of p53 failed to rescue the developmental defects; however, it synergistically induced mammary tumor formation in Chk1+/-;MMTV-Cre animals with a median time to tumor latency of about 10 months. Chk1 deficiency caused a preponderance of abnormalities, including prolongation, multipolarity, misalignment, mitotic catastrophe and loss of spindle checkpoint that is accompanied by reduced expression of several cell cycle regulators, including Mad2. On the other hand, we also showed that Chk1 deficiency inhibited mammary tumor formation in mice carrying a homozygous deletion of p53, uncovering a complex relationship between Chk1 and p53. Furthermore, inhibition of Chk1 with a specific inhibitor, SB-218078, or acute deletion of Chk1 using shRNA killed mammary tumor cells effectively. These data demonstrate that Chk1 is critical for maintaining genome integrity and serves as a double-edged sword for cancer: while its inhibition kills cancer cells, it also triggers tumorigenesis when avorable mutations are accumulated for cell growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1964.
Abstract 1965: Cross-talk among the components of DNA damage response and chromatin remodeling
Authors: Ray Alo , Wang Qien , Zhu Qianzheng , Wani Gulzar , Zhao Qun , Wani Altaf A.
Published: February 2011

Chromatin remodeling is integral to the functionality of many DNA-templated processes, including DNA damage repair and damage induced checkpoint signaling. The coordinated response to genotoxic damage begins by recruitment of specific protein factors exhibiting a high degree of cross-talk to orchestrate individual pathway events. The common initial step, i.e., accessing DNA damage embedded within chromatin, is a key factor in determining the course of DNA damage response and efficiency of damage processing. Our recent studies have sought to understand the roles for the mammalian SWI-SNF components in nucleotide excision repair (NER) and checkpoint activation. We have knocked down Brg1, the catalytic component and SNF5, the core component, of the SWI-SNF complex, and assessed the impact of these knockdowns on NER and checkpoint activation. Our data suggest that SWI-SNF complex associates with UV damaged chromatin through its interaction with damage recognition factor XPC and helps stabilization of XPC at the damage site. Furthermore, SWI-SNF influences the access of ATM at the damage site, which promotes assembly of ATM-γH2AX-MDC1-53BP1-BRCA1 complex, and eventually influences NER. We propose that SWI-SNF complex positively modulates NER by facilitating stability and accessibility of NER and checkpoint factors at the UV damaged chromatin sites.

(This work was supported by NIH grants CA93413, ES2388 and ES12991)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1965.
Abstract 1969: Inhibition of poly(ADP-ribose) polymerase downregulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130
Authors: Hegan Denise C. , Lu Yuhong , Stachelek Gregory C. , Crosby Meredith E. , Bindra Ranjit S. , Glazer Peter M.
Published: February 2011

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy based on the role of PARP in recruitment of base excision repair (BER) factors to sites of damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a novel mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1969.
Abstract 1976: The discovery of mechanisms of resistance to SMO antagonists and the therapeutic implications
Authors: Dorsch Marion , Buonamici Silvia , Williams Juliet , Wang Anlai , Guo Ribo , Vattay Anthony , Hsiao Kathy , Yuan Jing , Ostrom Lance , Fordjour Paul , Anderson Dustin , Morrissey Michael , Monahan John , Kelleher Joseph , Peukert Stefan , Pan Shifeng , Wu Xu , Maira Sauver-Michel , Garcia-Echeverria Carlos , Briggs Kimberly , Watkins Neil , Yao Yung-mae , Lengauer Christoph , Warmuth Markus , Sellers William
Published: February 2011

Smoothened (Smo) is a G-protein coupled receptor (GPCR)-like molecule that activates the Hedgehog (Hh) signal transduction pathway. In the resting state, the 12-pass transmembrane protein Patched (Ptch) inhibits Smo activity. When Ptch inhibition is attenuated, Smo signals via a cytosolic complex of proteins leading to activation of the Gli family of transcription factors. Genetic activation of the Hh pathway at or upstream of Smo is linked to tumorigenesis in several cancers. In particular, somatic mutations in Ptch and Smo leading to constitutive pathway activation are found in sporadic medulloblastoma (MB) and basal cell carcinoma (BCC). Evidence suggests that antagonists of Smo may abrogate the tumorigenic phenotype engendered by Ptch inactivation.

NVP-LDE225 is a potent and selective orally available Smo antagonist that robustly inhibits Smo-dependent signaling in vitro and in vivo. NVP-LDE225 exerted dose-related anti-tumor activity in vivo in several genetically defined MB models that are driven by mutations in Ptch leading to near complete tumor regression and Hh pathway inhibition. However, following long-term continuous dosing of NVP-LDE225 in medulloblastoma allograft models, evidence of resistance to NVP-LDE225 was observed. Here, we describe our efforts to proactively identify mechanisms of resistance to targeted therapy of Smo. Genome-wide DNA- and RNA-profiling of resistant tumors revealed distinct resistance mechanisms allowing tumors to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, was identified as one mechanism leading to restoration of pathway signaling despite adequate drug exposure. Additional mining of the gene expression data for pathway signatures that are selectively deregulated in resistant tumors identified increased phosphatidylinositol-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTor inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1976.