Modification of the HER2/NEU-derived tumor antigen GP2 improves induction of GP2-reactive cytotoxic T lymphocytes.

GP2 (IISAVVGIL), the p654-662 HER2/neu-derived tumor antigen, induces HLA-A2-restricted cytotoxic T lymphocytes (CTL) reactive to various epithelial cancers. The binding affinity of GP2 for HLA-A2, however, is very low. To improve the immunogenicity of GP2, we tested 10 different amino acid substitutions into GP2 at the C- and N- terminus. Five out of 10 modified peptides, especially those containing phenylalanine at position 1 (1F), showed a significantly improved binding affinity to HLA-A2. 1F-based modified peptides were well recognized by GP2-specific CTL. These peptides were used to stimulate peripheral blood lymphocytes from HLA-A2 healthy donors using peptide-pulsed autologous dendritic cells (DC). After 3 or more weekly stimulations, CTL activity against GP2 pulsed T2 (T2-GP2) and HER2/neu-overexpressing tumor cells was measured in (51)Cr release and IFN-gamma secretion assays. The modified peptides significantly enhanced GP2-specific CTL activity in some donors. In particular, the peptide with phenylalanine at position 1, leucine at position 2 and valine at position 10 (1F2L10V) maximized the CTL activity against both T2-GP2 and HER2/neu-positive tumor cells. Peptide 1F2L10V increased not only the binding affinity to HLA-A2 but also improved recognition of GP2. These data suggest that DC + modified GP2 may improve immune therapies for the treatment of HER2/neu overexpressing tumors.

Retinoic acid receptor-independent mechanism of apoptosis of melanoma cells by the retinoid CD437 (AHPN).

Retinoic acid (RA) induces differentiation of S91 melanoma cells through activation of RA receptor (RAR)gamma without affecting cell viability. The novel RARgamma-agonist CD437 (AHPN), however, also induces concomitant apoptosis through an unknown mechanism which was investigated here. By utilizing DNA microarray analysis, five apoptosis-associated, CD437-induced transcripts (CITs) were identified. Interestingly, all CITs are also regulated by p53 in a DNA damage response, and consistent with this interpretation, CD437 was found to cause DNA adduct-formation. However, p53 is not required for CD437-dependent regulation of CITs. Among this set of genes, induction of p21(WAF1/CIP1) is likely to be responsible for early S-phase growth-arrest of CD437-treated cells, whereas ei24 is a critical mediator of CD437-induced apoptosis in S91 cells. These data suggest an RAR-independent mechanism in which CD437 causes DNA adduct-formation, resulting in induction of a p53-independent DNA damage response, and subsequent growth-arrest and apoptosis. CD437-mediated DNA adduct-formation may also explain its apoptotic effects in other cell types.

Loss of T-cell receptor-CD3zeta and T-cell function in tumor-infiltrating lymphocytes but not in tumor-associated lymphocytes in ovarian carcinoma.

BACKGROUND: Impaired T-cell function has been noted in tumor-infiltrating lymphocytes (TIL). Recently, loss of function was found to be associated with modifications in T-cell receptor complex (TCR)-mediated signaling. A common feature is loss or reduced expression levels of the signaling chain, TCRzeta. We evaluated whether loss of function in TIL and tumor-associated lymphocytes (TAL) from patients with ovarian cancer is associated with changes in TCRzeta expression, and which factors can cause these defects. METHODS: TIL and TAL were isolated from multiple patients and evaluated for their proliferative capacity by stimulation with a polyclonal stimulus. In addition, expression of TCRzeta and CD3epsilon was evaluated in fresh TIL and TAL by the Western blot technique. Finally, various conditions within a tumor environment were tested for their effect on TCRzeta and CD3epsilon. RESULTS: TIL, but not TAL, were significantly impaired in their proliferative response, even when both populations were derived from the same patient (P <.05). Reduced proliferation levels were associated with loss of expression of TCRzeta but not of CD3epsilon. Exposure of normal T cells to relative ischemia or heat shock, or culture in medium without IL-2, did not significantly reduce expression of TCRzeta compared with CD3epsilon. However, coculture of T cells with tumor-derived macrophages or tumor-derived factors led to a selective loss of TCRzeta compared with CD3epsilon (P <.05). Further analysis suggested that oxides such as hydrogen peroxide secreted by macrophages may be responsible for loss of TCRzeta and high molecular weight factors secreted by certain tumors. CONCLUSIONS: TIL but not TAL show impaired T-cell function, which is associated with loss of TCRzeta. In addition to macrophages secreting oxides, loss of TCRzeta may be caused by tumor-derived soluble factors.

Expression and function of galectin-3, a beta-galactoside-binding protein in activated T lymphocytes.

A soluble beta-galactoside-binding lectin, galectin-3 has been shown to be involved in cell adhesion and activation of immune cells. Although galectin-3 is known to be expressed in various types of cells, it has not been shown whether galectin-3 is expressed in T lymphocytes. We present evidence here that galectin-3 is expressed in activated murine T lymphocytes including CD4+ and CD8+ T cells but not in resting T cells. Galectin-3 expression was induced by anti-CD3 mAb or mitogen and enhanced by common gamma-chain signaling cytokines, IL-2, IL-4, and IL-7, in activated T lymphocytes, whereas the inflammatory cytokines including TNF-alpha and IFN-gamma did not. Galectin-3 expression and proliferation were down-regulated by withdrawal of IL-2 and gamma irradiation. Antisense but not sense phosphorothioated oligonucleotides for galectin-3 inhibited galectin-3 expression and blocked proliferation of T cells significantly. This study suggests that up-regulation of galectin-3 plays an important role in proliferation of activated T lymphocytes.

Soft-tissue sarcomas.

Sarcomas of the soft tissues are challenging lesions for the surgical oncologist. Careful planning must be done at all stages of diagnosis and treatment, because every sarcoma is unique with respect to histologic type, size, and location. Pretreatment discussions in a multidisciplinary format are useful to ensure appropriate and effective management of these tumors.

B7.1 costimulation increases T-cell proliferation and cytotoxicity via selective expansion of specific variable alpha and beta genes of the T-cell receptor.

BACKGROUND: Optimal T-cell activation requires not only ligation of the T-cell receptor (TcR) but also delivery of costimulatory signals by various accessory molecules. The interaction of the costimulatory molecule B7.1 (CD80) with its receptor CD28 provides a strong positive signal to T cells. METHODS: The B7.1 gene was transduced into cultured human ovarian, breast, and pancreatic tumor cells by using a retroviral vector. Autologous as well as allogeneic naive T-cells were stimulated with either wild-type or B7.1-transduced tumor cells in a mixed lymphocyte tumor cell culture (MLTC). In addition to cytolytic activity, T-cell proliferation, T-cell subset composition, and the frequencies of TcR variable (V) alpha and beta genes were compared in T cells from both types of MLTC. RESULTS: Introduction of the B7.1 gene into tumor cells was successful in all tumors to a varying degree. Those tumors expressing high levels of B7.1 induced significantly higher levels of T-cell proliferation than wild-type tumor cells. T-cell subset composition did not markedly differ between T cells stimulated with wild-type tumor cells or B7.1-expressing tumor cells. However, T cells stimulated with B7.1-expressing tumor cells showed a significantly increased cytolytic potential. The increased cytotoxic T lymphocyte activity was associated with a higher frequency of specific TcR V alpha and V beta genes. In addition, B7.1 costimulation promoted oligoclonality among the responding T cells. CONCLUSIONS: These data suggest that costimulation through B7.1 promotes T-cell proliferation and cytotoxic activity through clonal expansions of T cells bearing antigen-specific TcR V alpha and V beta genes and through promotion of oligoclonality. The data also suggest that promoting B7.1-mediated costimulation is an important aspect of immune therapies.

Pancreatic cancer associated ascites-derived CTL recognize a nine-amino-acid peptide GP2 derived from HER2/neu.

BACKGROUND: The proto-oncogene HER2/neu encodes a 185 kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that CTL derived from breast, ovarian, and non-small cell lung cancer recognized a peptide derived from HER2/neu. The aim of this study was to evaluate whether this HLA-A2-binding peptide is a TAA in pancreatic cancer and if pancreatic cancer associated T-lymphocytes (TAL) are useful to generate tumor- and peptide-specific CTL. MATERIALS AND METHODS: TAL from malignant ascites of a HLA-A2+ pancreatic cancer patient whose tumor overexpressed HER2/neu were stimulated on solid-phase anti-CD3 and cultured in low-dose IL-2. Using repetitive autologous tumor cell stimulation, CTL were generated. RESULTS: CTL recognized autologous and allogeneic HER2/neu+ tumor cells in an HLA-A2 restricted fashion significantly. Furthermore, all CTL recognized p654-662 (GP2) derived from HER2/neu, but not the control peptide. CONCLUSIONS: These results demonstrate that this HER2/neu derived peptide is a TAA in pancreatic carcinoma. The identification of the HER2/neu derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies. The possibility of generating peptide-specific CTL from malignant ascites enables future studies to identify more antigens in this disease.

Local secretion of IFN-gamma induces an antitumor response: comparison between T cells plus IL-2 and IFN-gamma transfected tumor cells.

Previously we described that the adoptive transfer of tumor-infiltrating lymphocytes (TIL) + interleukin-2 (IL-2) leads to eradication of established methylcholanthrene (MCA)-105 fibrosarcoma pulmonary metastases in a congenic murine model. The in vivo efficacy of TIL was associated with their ability to secrete interferon-gamma (IFN-gamma), and to a lesser extent granulocyte-macrophage colony-stimulating factor. The local secretion of these cytokines resulted in recruitment of naive host immune cells to the tumor and eventually in a successful host antitumor immune response. In the present study, to further evaluate the role of IFN-gamma in the induction of a host antitumor immune response, we compared the treatment efficacy of adoptively transferred T cells and IFN-gamma gene transfected tumor cells (MCA-105/IFN-gamma) as delivery systems of IFN-gamma. Treatment with TIL-IL-2 or irradiated MCA-105/IFN-gamma induced a similar reduction in pulmonary metastases of MCA-105 tumor. In contrast, irradiated wild-type MCA-105 or TIL from IFN-gamma gene knockout mice did not cause tumor eradication. MCA-105 tumor-bearing mice treated with MCA-205/IFN-gamma showed a partial reduction in the number of pulmonary metastases. Histologically, lungs of successfully treated mice showed that initially activated macrophages expressing inducible nitric oxide synthase (iNOS) and dendritic cells infiltrated the tumor bed. Subsequently, CD4+ and CD8+ T cells infiltrated tumors. The therapeutic efficacy of IFN-gamma transfected tumor cells was eliminated when either CD4+ T cells or CD8+ T cells were depleted. These results suggest that local secretion of IFN-gamma induces a tumor-specific host antitumor immune response mediated through activated macrophages, dendritic cells, and tumor-specific T cells. This may be a common component of successful immunotherapy.

Soluble MUC1 secreted by human epithelial cancer cells mediates immune suppression by blocking T-cell activation.

Solid tumors may secrete factors that mediate immune suppression in patients. We investigated the effect of supernatants from 25 human tumor cell lines on T-lymphocytes from healthy donors. A profound inhibition of proliferation, cytokine secretion and cytotoxic activity was seen when T-cells were cultured in concentrated tumor supernatants from 6 cell lines fractionated into high (>100 kDa) m.w. molecules. Interestingly, the inhibitory effects were reversed when the tumor supernatant was removed. Cell cycle studies of inhibited T-cells showed most of them were growth arrested in the G(0)/G(1) phase similar to naïve T-cells. In addition, these T-cells did not express IL2-receptors and expression of CD54 (ICAM-1) and CD58 (LFA-3) resembled that of resting T-cells. Protein gel electrophoresis of the tumor supernatants and western blot analysis demonstrated the presence of soluble MUC1 in the inhibitory tumor supernatants but not in control supernatant. Most importantly, depletion of soluble MUC1 by immunoprecipitation from the tumor supernatants neutralized the inhibitory effects on T-lymphocytes. Therefore, our results show that MUC1 shed by cultured epithelial tumor cells mediates inhibition of T-cell proliferation and function by inducing cell growth arrest.

Tumor-specific cytokine release by donor T cells induces an effective host anti-tumor response through recruitment of host naive antigen presenting cells.

We recently reported that tumor eradication induced by immunotherapy (IT) in a congenic mouse model using tumor infiltrating lymphocytes (TIL) + recombinant interleukin-2 (rIL-2) is dependent on recruitment of naive host immune cells at the tumor sites. The recruitment of host immune cells was induced mainly through a local secretion of interferon-gamma (IFN-gamma) produced by donor T cells. We now further investigated how a non-specific inflammatory response progresses to a host T-cell-mediated tumor-specific response. In cross-over experiments using MCA-105 and MCA-205 sarcoma tumors, pulmonary metastatic disease was eradicated only in mice treated with tumor-matched TIL + rIL-2. In vitro, TIL stimulated with the tumor of origin secreted relatively high levels of IFN-gamma and granulocyte-macrophage colony stimulating factor (GM-CSF) compared to TIL stimulated with mismatched tumor cells. In lungs of tumor-bearing mice treated with matched TIL + rIL-2, significant increases in the percentages of IFN-gamma, GM-CSF and tumor necrosis factor-alpha (TNF-alpha) positive cells were detected, as well as of macrophages, natural killer (NK) cells and dendritic cells. Depletion of macrophages or NK cells did not inhibit the efficacy. In contrast, depletion of dendritic cells partially inhibited the efficacy of the treatment. Combined depletion of dendritic cells and macrophages abrogated more than 80% of the efficacy. Our data suggest that successful IT may require 3 steps: (1) release of inflammatory cytokines by donor TIL after restimulation by tumor cells; (2) infiltration of host immune cells in response to local cytokine production; and (3) activation of tumor-specific host immune cells by dendritic cells and to a lesser extent by macrophages.

Pancreatic cancer cells can evade immune surveillance via nonfunctional Fas (APO-1/CD95) receptors and aberrant expression of functional Fas ligand.

BACKGROUND: The Fas (APO-1/CD95) receptor/Fas ligand (FasR/FasL) system plays a key role in immune surveillance. We investigated the possibility of a tumor escape mechanism involving the FasR/FasL system in pancreatic cancer cells. METHODS: Fourteen pancreatic cancer cell lines and 3 pancreatic cancer surgical specimens were studied for their expression of FasR and FasL by flow cytometry, immunoblotting, and immunohistochemistry, FasR function was tested with an anti-FasR antibody. FasL function was assessed by coculture assays using pancreatic cancer cells and FasR-sensitive Jurkat T-cells. RESULTS: FasR was expressed in normal pancreas, in 14 of 14 pancreatic cancer cell lines, and in 3 of 3 surgical specimens. However, only 1 of 14 cancer cell lines expressed functional FasR when grown in monolayer, although 3 additional cell lines displayed functional FasR when cultured in suspension. Normal pancreas did not express FasL, whereas 14 of 14 cancer cell lines and 3 of 3 surgical specimens expressed FasL. FasL expressed by pancreatic cancer cells mediated killing of Jurkat T-cells in coculture assays (P < .005). CONCLUSIONS: These data suggest that pancreatic cancer cells have 2 potential mechanisms of evading Fas-mediated immune surveillance. A nonfunctional FasR renders them resistant to Fas-mediated apoptosis. The aberrant expression of functional FasL allows them to "counterattack" activated Fas-sensitive T-cells. Alone or in unison, these tumor escape mechanisms may contribute to the malignant and often rapid course of pancreatic cancer disease.

T cells mediate treatment of six-day-old cytokine-gene-transfected mouse mammary tumor.

We have previously shown that immunologically different mouse mammary cancer cell lines induce antitumor responses after IL2 or IL4 gene transfection. We now report the ability of cytokine-transfected tumors to induce eradication of established wild-type tumor. Animals with 6-day-old tumor treated with IL2-transfected cells also had significantly smaller tumors 2.8 and 1.7 cm2 (EMT6 and 410.4). Findings were similar for IL4-transfected cells. Tumor infiltrating lymphocytes or cells from draining lymph nodes demonstrated tumor-specific in vitro cytotoxicity. Immunohistochemical studies revealed T cell infiltrates in transfected tumors.

Treatment of established tumor is associated with ICAM-1 upregulation and reversed by CD8 depletion in a tumor necrosis factor-alpha gene transfected mouse mammary tumor.

INTRODUCTION: We have performed TNF-alpha gene transfection in a mouse mammary cancer line and found significant antitumor effects. We hypothesize that the antitumor effects observed in this model are mediated by ICAM-1 and by the recruitment of CD4+ and CD8+ T cells. In vivo (Balb/c mice) tumor growth inhibition, treatment of established tumor and the effects of ICAM-1 and CD4+ and CD8+ T cells were evaluated. METHODS AND RESULTS: Gene transfection with highly efficient vectors resulted in secretion of large amounts of TNF-alpha (ELISA). In vivo antitumor effects were tested. The number of cells required to generate palpable tumor 7-10 days after subcutaneous injection was determined (1 x 10(6)). The same number of transfected cells were injected subcutaneously and compared to nontransfected controls. Tumors were measured blindly and size was analyzed on day 30 by the Wilcoxon rank sum test. Mean tumor size after injection of transfected cells is compared to that of controls. Control tumors reached the maximum allowable size by day 30 (4 cm(2)). On day 30 EMT6-TNF-alpha tumors were 0.48 cm(2) (p < 0.05). The effect of repeat injection (challenge was also tested. Animals were injected with transfected cells or wild-type control on day -6 and challenged with the same number of wild-type tumor cells on day 0. Significant immune protection against subsequent challenge was seen after first time injection with EMT6-TNF-alpha but not after first time EMT6 wild-type injection (1.62 vs. 4 cm(2)). Treatment of 6-day-old tumor was also evaluated. On day 30, mean tumor size in animals treated with EMT6-TNF-alpha was 0.9 cm(2) compared to 4 cm(2) for controls. In all experiments, CD8+ T cell depletion and CD4+ T cell depletion caused a reversal of TNF-alpha-induced inhibitory effects. In addition, in vivo antibody blocking of ICAM-1 in tumor growth experiments reversed antitumor effects (control 4 cm(2), TNF-alpha 0.2 cm(2) and ICAM-1 blocking 3.14 cm(2)). Using flow cytometry, MHC class I and II and ICAM-1 adhesion molecule expression of transfected tumor was tested. ICAM-I expression was significantly upregulated. MHC class II antigen expression was also increased. TNF-alpha-transfected human breast cancer was also evaluated. Three cell lines and fresh tumor were transfected to express TNF-alpha. In vitro analysis revealed ICAM-1 upregulation following transfection. Histologic analysis and immunohistochemical staining revealed TNF-alpha and ICAM-1 in transfected tumors and not in wild-type tumors. CONCLUSION: Highly significant in vivo tumor growth inhibition and immune protection after injection with TNF-alpha-transfected tumors appears to be mediated predominantly by CD8+ T cells and ICAM-1 upregulation. These findings suggest that TNF-alpha increases recruitment and adhesion of effector T cells.

Successful adoptive cellular immunotherapy is dependent on induction of a host immune response triggered by cytokine (IFN-gamma and granulocyte/macrophage colony-stimulating factor) producing donor tumor-infiltrating lymphocytes.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and systemic low dose rIL-2 effectively eradicates pulmonary metastases of the murine MCA-105 sarcoma. We described earlier that host CD8+ T cells are critical for tumor eradication and that successful treatment is associated with production of high levels of IFN-gamma and granulocyte/macrophage (GM)-CSF by donor TIL in vitro. Here, we propose the mechanism through which adoptively transferred Thy-1.1+ TIL induce a host antitumor response in congenic Thy-1.2+ tumor-bearing mice. Donor Thy-1.1+ TIL were detected at the tumor site 12 h after transfer. These Thy-1.1+ cells produced IFN-gamma and GM-CSF in situ. The percentage of Thy-1.1+ TIL at the tumor site increased up to 16.4 +/- 4.9% 24 h after transfer but decreased to undetectable levels thereafter. In contrast, the percentages of host cells producing IFN-gamma and GM-CSF continued to increase at the tumor site. These increases were significantly higher in TIL + rIL-2-treated mice compared with untreated mice and rIL-2-treated mice 48 h after TIL transfer. The appearance of IFN-gamma+ and GM-CSF+ cells was followed by a large influx of host CD4+, CD8+, and Thy-1.2+ TIL and eventually by tumor eradication. This response was tumor specific since TIL obtained from MCA-205 did not induce high levels of IFN-gamma and GM-CSF and did not induce tumor eradication of MCA-105 tumor. Coinjection of Thy-1.1+ TIL and anti-IFN-gamma or anti-GM-CSF mAb significantly inhibited antitumor efficacy of the TIL + rIL-2 treatment. We conclude that successful adoptive immunotherapy in this model is mediated through cytokine production by adoptively transferred TIL that induce a host T cell-dependent antitumor response.

[Significance if ICAM-1 on lysis of human pancreatic carcinoma cells by autologous, cytotoxic T-lymphocytes]. / Die Bedeutung von ICAM-1 auf die Lyse von humanen Pankreaskarzinomzellen durch autologe, zytotoxische T-Lymphozyten.

Cytotoxic T-Lymphocytes were generated from tumor-infiltrating lymphocytes of a newly established pancreatic cancer cell line, which lysed autologous tumor cells significantly and HLA-class I restricted. ICAM-1+ tumor clones were significantly higher lysed than an ICAM-1- clone. Stimulation with IFN-gamma resulted in a higher expression of ICAM-1 and significantly higher lysis, which was inhibited by anti-ICAM-1 monoclonal antibodies.

Clone 10d/BM28 (CDCL1), an early S-phase protein, is an important growth regulator of melanoma.

Retinoic acid (RA) induces growth arrest and differentiation of many different tumor cells. RA activates RA receptors, which function as ligand-dependent transcriptional modulators. S91 murine melanoma cells stop proliferating and then reversibly differentiate into a melanocytic cell type after the administration of RA. The genetic changes that take place during this process serve as an excellent model for the etiology of melanoma. The use of subtractive hybridization techniques yielded several differentially expressed cDNAs that are associated with RA-induced growth arrest. One clone, cyclin D1, is repressed and is probably a differentiation marker. Two other cDNAs represent novel, RA-inducible genes. Expression of another cDNA, clone 10d, is strongly down-regulated. It is the homologue of the human gene BM28 (CDCL1) that is indispensable for entry into S phase and cell division. S91 cells that are permanently transfected with a plasmid that constitutively expresses clone 10d become significantly more resistant to RA, suggesting that repression of this gene is a critical event in RA-induced growth arrest. The use of reverse transcription-PCR for the detection of expression in human melanoma in vitro was performed to study the potential role of clone 10d/BM28 in this disease. It is expressed in 80% of melanoma cell lines but is virtually undetectable in primary melanocytes. The expression of BM28 is not regulated by RA in human, RA-resistant melanoma cells. These results suggest that clone 10d/BM28 functions as an important tumor cell growth promoter. The regulation of clone 10d may be directly mediated by RA receptors, and escape from negative regulation may, thus, contribute to the etiology of melanoma.

Generation of peptide-specific cytotoxic T lymphocytes using allogeneic dendritic cells capable of lysing human pancreatic cancer cells.

BACKGROUND: Dendritic cells (DCs) are potent antigen presenting cells (APCs), able to efficiently induce primary T cell-mediated responses to foreign antigens. In earlier studies we were able to identify a histocompatibility antigen (HLA)-A 2-restricted nine amino acid peptide (GP2, peptide 654-662) from the transmembrane portion of the protooncogene HER2/neu as a tumor-associated antigen (TAA) in human pancreatic cancer. METHODS: Peripheral blood mononuclear cells (PBMCs) of HLA-A2+ and HLA-A2 healthy volunteers were isolated. PBMCs were grown with initial anti-CD3, low-dose interleukin-2 (IL-2), and peptide-pulsed DC stimulation. T-cell lines were analyzed in functional studies. RESULTS: After 4 weeks, T-cell cultures were more than 50% CD8+. All peptide-pulsed T cells significantly lysed APC pulsed with the immunizing antigen in an HLA-A2 restricted fashion. Furthermore, HLA-A2+,HER2/neu+ human pancreatic cancer cells were lysed significantly higher than HLA-A2 HER2/neu+ pancreatic cancer cells. Transfection of an HLA-A2 pancreatic cancer cell line with the HLA-A2 gene resulted in a significantly higher lysis of the transfected cell line compared to the wild type. In HLA-A2+ pancreatic cancer targets, specific lysis was HLA-A2 restricted. CONCLUSION: The ability to use DCs for presentation of either tumor or peptide antigen in an HLA-restricted fashion to stimulate T-cell proliferation, as well as cytotoxicity, demonstrates the potential of this technology for future development of a pancreatic cancer vaccine.

Specific activation of retinoic acid receptors (RARs) and retinoid X receptors reveals a unique role for RARgamma in induction of differentiation and apoptosis of S91 melanoma cells.

Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRalpha, RXRbeta, RARalpha, RARgamma, and RARbeta in low levels. Among these, only RARbeta gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARbeta expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARbeta gene expression, and blocks cellular proliferation. However, only the RARgamma-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARbeta, but suggest that RARgamma has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.