Combination of chemotherapy with cancer vaccines is currently regarded as a potentially valuable therapeutic approach for the treatment of some metastatic tumors, but optimal modalities remain unknown. We designed a phase I/II pilot study for evaluating the effects of dacarbazine (DTIC) on the immune response in HLA-A2 1 disease-free melanoma patients who received anticancer vaccination 1 day following chemotherapy (800 mg/mq i.v.). The vaccine, consisting of a combination of HLA-A2 restricted melanoma antigen A (Melan-A/MART-1) and gp100 analog peptides (250 lg each, i.d.), was administered in combination or not with DTIC to 2 patient groups. The combined treatment is nontoxic. The comparative immune monitoring demonstrates that patients receiving DTIC 1 day before the vaccination have a significantly improved long-lasting memory CD8 1 T cell response. Of relevance, these CD8 1 T cells recognize and lyse HLA-A2 1 /Melan-A 1 tumor cell lines. Global transcriptional analysis of peripheral blood mononuclear cells (PBMC) revealed a DTIC-induced activation of genes involved in cytokine production, leukocyte activation, immune response and cell motility that can favorably condition tumor antigen-specific CD8 1 T cell responses. This study represents a proof in humans of a chemotherapyinduced enhancement of CD8 1 memory T cell response to cancer vaccines, which opens new opportunities to design novel effective combined therapies improving cancer vaccination effectiveness. '

Methylation of N3-adenine represents a novel pharmacological strategy for the treatment of resistant tumors. However, little is known about the biochemical pathways involved in cell death induced by N3-methyladenine. In the present study, we show that MeOSO 2 (CH 2 ) 2 -lexitropsin (Me-Lex), a compound generating almost exclusively N3-methyladenine (Ͼ99%), provoked a burst of poly(ADP-ribosylation) and loss of mitochondrial membrane potential in leukemia cells. These events were followed by a marked decrease in nuclear poly(ADP-ribose) polymerase-1 (PARP-1) expression and nuclear factor-B (NF-B) activity. Moreover, DNA damage generated by N3-methyladenine induced a marked decrease in telomerase in the cytosol that was accompanied by a transient up-regulation of activity in the nucleus, as a consequence of nuclear translocation of telomerase in response to genotoxic damage. PARP-1 inhibition blocked ADP-ribose polymer formation, preserved mitochondrial membrane integrity, and counteracted the reduction of NF-B activity, thus preventing the appearance of necrosis. On the other hand, because PARP-1 is a component of the base excision repair (BER), the combination of Me-Lex ϩ PARP-1 inhibitor triggered apoptosis as a result of disruption of BER process. In conclusion, the present study provides new insight into the cellular response to N3-adenine-selective methylating agents that can be exploited for the treatment of tumors unresponsive to classical wide-spectrum methylating agents. Moreover, the results underline the central and paradoxical role of PARP-1 in cell death induced by N3-methyladenine: effector of necrosis and coordinator of methylpurine repair.Monofunctional methylating agents include antitumor drugs that interact with DNA, generating a wide spectrum of base adducts endowed with cytotoxic and/or mutagenic activity. The three major modified bases are represented by N7-methylguanine, N3-methyladenine (N3-MeA), and O 6 -methylguanine (O 6

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We describe a novel method for detecting micrometastasis in the blood stream of cancer patients based on RT-PCR amplification of tumor-associated carcinoembryonic antigen (CEA) mRNA. To increase sensitivity and specificity of RT-PCR, CEA transcript was selectively up-regulated in cancer cells by exposure of peripheral blood to non-toxic concentrations of staurosporine (ST). Thereafter, polyA(+) RNA was extracted from tumor cells captured by means of magnetic beads coated with a monoclonal antibody against a common human epithelial antigen. Finally, RNA was subjected to RT-PCR analysis of CEA transcript. Using this approach, we demonstrated an ST-mediated increase in CEA transcript in blood specimens collected from a patient with metastatic colon cancer before receiving treatment with 5-fluorouracil/leucovorin. After a few cycles of chemotherapy, CEA-positive tumor cells were no longer detected. Clinical follow-up of this patient indicated that treatment with chemotherapy induced a dramatic reduction in liver metastasis. Therefore, it can be hypothesized that lack of CEA transcript detection might be consistent with disappearance or at least marked reduction of circulating tumor cells.