Postreplicative mismatch repair plays a major role in mediating the cytotoxicity of agents generatingO⁶-methylguanine in DNA. We previously showed that a methylating antitumor triazene compound, temozolomide, induces apoptosis and that the persistence ofO⁶-methylguanine in DNA is required to trigger the process. We wanted to test whether the latter apoptotic signal is dependent on a functional mismatch repair system. To this end, we used two human lymphoblastoid cell lines (i.e., the mismatch repair-proficient TK6 line and its mismatch repair-deficient subline MT1) that are both deficient inO⁶-methylguanine repair. Temozolomide treatment of TK6 cells brought about efficient cell growth inhibition, G₂/M arrest, and apoptosis, as indicated by the results of cytofluorimetric analysis of 5-bromo-2′-deoxyuridine incorporation and DNA content and evaluation of DNA fragmentation. The drug treatment resulted also in the induction of p53 and p21/waf-1 protein expression. In contrast, MT1 cells were highly resistant to the drug and no p53 and p21/waf-1 induction was observed. Importantly, we could show that MT1 cells are not deficient in the p53-dependent apoptosis pathway; treatment with etoposide, a topoisomerase II inhibitor, resulted in p53 and p21/waf-1 protein expression and apoptosis in both cell lines. In conclusion, we demonstrate the existence of a link between a functional mismatch repair system and the trigger of apoptosis in cells exposed to clinically relevant concentrations of temozolomide. The results also suggest that p53 induction in response toO⁶-guanine methylation involves the mismatch repair system.

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3σ, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3σ–dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16INK4a tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses.

Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. A crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

The use of IFN-α in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-α also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-α, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8⁺ T cells recognizing modified and native MART-1 and gp100 peptides and MART-1⁺gp100⁺ melanoma cells was observed. Moreover, vaccination induced an increase in CD8⁺ T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA⁺CCR7⁻ (terminally differentiated effectors) and CD45RA⁻CCR7⁻ (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14⁺ monocytes and particularly of the CD14⁺CD16⁺ cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8⁺ T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-α, the present data represent an important starting point for considering a new clinical use of IFN-α and new immunologic end points, potentially predictive of clinical response. (Cancer Res 2006; 66(9): 4943-51)

BACKGROUND: Melanoma metastasizes by different mechanisms comprising direct invasion of the surrounding tissue and spreading via the lymphatic or vascular system. Despite their clinical relevance, the molecular mechanisms that guide the route of spreading and localization of the metastases in different tissues are not well known. Recent studies in different tumor types have shown that vascular endothelial growth factor‐C (VEGF‐C), which displays a high specificity for lymphatic endothelium, is involved in tumor‐induced lymphangiogenesis and lymphatic metastatic spread. The authors studied the expression of VEGF‐C in cultured human melanoma cells derived from cutaneous and lymph node metastases as well as in metastatic melanoma tissue specimens to assess a possible involvement of this growth factor in lymph node localization of melanoma metastases.METHODS: VEGF‐C expression was evaluated in vitro on human melanoma cell lines established from cutaneous and lymph node metastasis specimens by reverse transcriptase‐polymerase chain reaction, Northern blot analysis, and immunofluorescence analysis. Immunohistochemical analysis of 42 tissue specimens of melanoma metastases and 10 tissue specimens of primary skin melanomas was also performed.RESULTS: Preferential expression of VEGF‐C was detected in lymph node‐derived tumor cell lines at both the mRNA and protein levels. The association between VEGF‐C production and lymph node localization of metastases was confirmed by the in vivo analysis. In addition, analysis of 10 patients, from whom specimens of both the primary skin melanoma and melanoma metastases were available, indicated a correlation between VEGF‐C expression in the primary tumor and lymph node localization of metastases.CONCLUSIONS: The findings of the current study demonstrate that VEGF‐C expression is correlated with localization of melanoma metastases in the lymph nodes and suggest that VEGF‐C expression in primary skin melanoma may be predictive of lymph node metastatic dissemination. Cancer 2003;98:789–97. © 2003 American Cancer Society.DOI 10.1002/cncr.11583