Breast cancer suppressor candidate-1 (BCSC-1) is a melanoma tumor suppressor that down regulates MITF.

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate-1 (BCSC-1) as a novel tumor suppressor in melanoma. BCSC-1 expression is decreased in human MM, and its ectopic expression in MM-derived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC-1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC-1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

Tumor necrosis-like weak inducer of apoptosis as a proinflammatory cytokine in human adipocyte cells: up-regulation in severe obesity is mediated by inflammation but not hypoxia.

CONTEXT Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects. OBJECTIVES The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress. DESIGN In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines. RESULTS Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways. CONCLUSIONS Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation.

Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.

Targeting renal cell carcinoma with NVP-BEZ235, a dual PI3K/mTOR inhibitor, in combination with sorafenib.

Background: Targeted therapies for metastatic renal cell carcinoma (RCC), including mammalian target of rapamycin (mTOR) inhibitors and small-molecule multikinase inhibitors, have produced clinical effects. However, most patients acquire resistance over time. Thus, new therapeutic strategies need to be developed. Here, we evaluated the effect of the dual PI3K/mTOR inhibitor NVP-BEZ235, in combination with the multikinase inhibitor sorafenib on renal cancer cell proliferation and survival in vitro as well as on tumor growth in vivo.Methods: The renal carcinoma cell lines 786-0 and Caki-1 were treated with NVP-BEZ235 or sorafenib, either alone or in combination. Tumor cell proliferation and apoptosis were investigated in vitro. The anticancer efficacy of NVP-BEZ235 alone, or in combination with sorafenib, was also evaluated on RCC xenografts in nude mice.Results: Treatment of 786-0 and Caki-1 cells with NVP-BEZ235 or sorafenib resulted in reduced tumor cell proliferation and increased tumor cell apoptosis in vitro. The combination of NVP-BEZ235 and sorafenib was more effective than each compound alone. Similarly, in vivo, NVP-BEZ235 or sorafenib reduced the growth of xenografts generated from 786-0 or Caki-1 cells. The antitumor efficacy of NVP-BEZ235 in combination with sorafenib was superior to NVP-BEZ235 or sorafenib alone.Conclusions: Our findings indicate that the simultaneous use of NVP-BEZ235 and sorafenib has greater antitumor benefit compared to either drug alone and thus provides a treatment strategy in RCC.

The roles of PKCδ and Src kinases in the glucocorticoid induction and the TGF-β superinduction of the mouse mammary tumor virus transcription in B lymphocytes

Résumé : Le virus tumoral de la glande mammaire de la souris (MMTV) est un rétrovirus provoquant le développement de tumeurs dans les glandes mammaires des souris susceptibles femelles. Au cours de son évolution, le virus s'est adapté et s'exprime dans des cellules spécialisées. Les lymphocytes B sont les premières cellules infectées et elles sont essentielles pour la propagation de l'infection aux glandes mammaires. Dans notre étude, le virus MMTV a été utilisé afin d'examiner les voies de signalisation induites par les glucocorticoïdes (dexaméthasone (dex), une hormone stéroïdienne) et le transforming growth factor-f3 (TGF-P, une cytokine), deux molécules impliquées dans l'activation de la transcription à partir du promoteur du MMTV dans les cellules B. Le TGF-P seul n'influence pas l'activité du promoteur du MMTV. Par contre, en synergie avec dex, le TGF-P provoque une super-induction de l'expression du promoteur par rapport à une stimulation par le glucocorticoïde seul. Cette super-induction est régulée par une famille de protéines, les Smads. Ainsi, dans les lymphocytes B, l'utilisation du MMTV a permis de mettre en évidence une nouvelle synergie entre les glueocortieoïdes et le TGF-p. pans ce travail, l'utilisation d'inhibiteurs pharmacologiques et de mutants « dominant-négatifs » nous a pet mis de démontrer qu'une Protéine Kinase C delta (PKC5) active est impliquée dans la transduction du signal lors de la réponse au dex ainsi que celle au TGF-P. Néanmoins, la PKC5 est régulée différemment dans chaque voie spécifique : la voie du TGF-p nécessitait l'activation du PKC5 par diacylglycerol (DAG) et la phosphorylation de tyrosines spécifiques, alors que la voie impliquant les glucocorticoïdes ne le nécessitait pas. Nous avons aussi démontré qu'une tyrosine kinase de la famille Src est responsable de la phosphorylation des tyrosines sur la PKC5. Les essais de kinase in vitro nous ont permis de découvrir que plusieurs Src kinases peuvent phosphoryler la PKC6 dans les cellules B et qu'elles étaient constitutivement actives. Enfin, nous avons montré qu'il existe une interaction protéine - protéine induite par dex, entre le récepteur aux glucocorticoïdes (GR) et la PKC5 dans les cellules B, une association qui n'a pas été démontrée auparavant. Par ailleurs, nous avons analysé les domaines d'interactions entre PKC5 et GR en utilisant les essais de «GST pull-down». Nos résultats montrent que le domaine régulateur de la PKC5 et celui qui interagit avec l'ADN du GR sont impliqués. En résumé, nous avons trouvé que dans une lignée lymphocytaire B, le virus MMTV utilise des mécanismes pour réguler à la fois la transcription et la voie de signalisation qui sont différents de ceux utilisés dans les cellules mammaires épithéliales et les fibroblastes. Nos découvertes pourraient être utilisées comme modèles pour l'étude de gènes cellulaires impliqués dans des processus tels qu'inflammation, immunité ou cancérogénèse. Summary: Mouse Mammary Tumor Virus (MMTV) is a retrovirus that causes tumors in the mammary glands of susceptible female mice and has adapted evolutionarily to be expressed in specialized cells. The B lymphocytes are the first cells to be infected by the MMTV and are essential for the spread of infection to the mammary glands. Here, we used the MMTV as a model system to investigate the signalling cascade induced by giucocorticoids (dexamethasone, "dex", a steroid hormone), and by Transforming Growth Factor-beta (TGF-P, a cytokine) leading to its transcriptional activation in B lymphocytes. By itself, TGF-I3 does not affect the basal activity of the MMTV promoter. However, TGF-13 significantly increases glucocorticoid-induced expression, through its effectors, the Smad factors. Thus, MMTV in B cells demonstrates a novel synergism between glucocorticoids and TGF-16. In this thesis project, we present evidence, based on the use of pharmacological inhibitors and of dominant-negative mutants, that an active Protein Kinase C delta (PKC6) is required as a signal transducer for the dex response and for the TGF-P superinduction as well. The PKC6 is differentially regulated in each specific pathway: whereas the TGF-13 superinduction required PKC6 to be activated by diacylglycerol (DAG) and to be phosphorylated at specific tyrosine residues, the glueocorticoid-induced pathway did not. We also showed that a protein tyrosine kinase of the Src family is responsible for the phosphorylation of tyrosines on PKC6. By performing in vitro kinase assays, we found that several Src kinases of B cells were able to phosphorylate PKC6 and that they were constitutively active. Finally, we demonstrate a dex-dependent functional protein-protein interaction between the glucocorticoid receptor (GR) and PKC6 in B cells, an association that has not been previously described. We further analysed the interacting domains of PKG6 and GR using in vitro GST pull-down assays, whereby the regulatory domain of PKC6 and the extended DNA-binding domain of the GR were involved. In summary, we found that in B-lymphoid cell lines, MMTV uses novel mechanisms of transcriptional control and signal transduction that are different from those at work in mammary epithelial or fibroblastic cells. These findings will be used as model for cellular genes involved in cellular processes such as immune functions, inflammation, or oncogenic transformation that may have a similar pattern of regulation.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence.

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells.

Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.

The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1).

Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Novel Agents Targeting the IGF-1R/PI3K Pathway Impair Cell Proliferation and Survival in Subsets of Medulloblastoma and Neuroblastoma.

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

A sensitized RNA interference screen identifies a novel role for the PI3K p110γ isoform in medulloblastoma cell proliferation and chemoresistance.

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.

Increase of [(18)F]FLT tumor uptake in vivo mediated by FdUrd: toward improving cell proliferation positron emission tomography.

PURPOSE: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts. METHODS: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation. RESULTS: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis. CONCLUSION: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.

BIRO1, a cell-permeable BH3 peptide, promotes mitochondrial fragmentation and death of retinoblastoma cells.

Retinoblastoma is the most common pediatric intraocular neoplasm. While retinoblastoma development requires the inactivation of both alleles of the retinoblastoma tumor suppressor gene (RB1) in the developing retina, additional genomic changes are involved in tumor progression, which progressively lead to resistance of tumor cells to death. Therapeutics acting at very downstream levels of death signaling pathways should therefore be interesting in killing retinoblastoma cells. The BH3-only proteins promote apoptosis by modulating the interaction between the pro- and antiapoptotic members of the BCL2 protein family, and this effect can be recapitulated by the BH3 domains. This report analyzes the effect of various BH3 peptides, corresponding to different BH3-only proteins, on two retinoblastoma cell lines, Y79 and WERI-Rb, as well as on the photoreceptor cell line 661W. The BH3 peptide BIRO1, derived from the BCL2L11 death domain, was very effective in promoting Y79 and WERI-Rb cell death without affecting the 661W photoreceptor cells. This cell death was efficient even in absence of BAX and was shown to be caspase independent. While ROS production or AIF release was not detected from mitochondria of treated cells, BIRO1 initiated mitochondria fragmentation in a short period of time following treatment. IMPLICATIONS: The BIRO1 peptide is highly effective at killing retinoblastoma cells and has potential as a peptidomimetic.