cytidylyl-3--5--guanosine and Melanoma

Topics: Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Dacarbazine; Dinucleoside Phosphates; Fatal Outcome; Female; Humans; Interferon-beta; Lymphatic Metastasis; Melanoma; T-Lymphocytes

Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity.. We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16.F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation.. MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B- and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clinic.

Topics: Adjuvants, Immunologic; Aminoquinolines; Animals; CD8-Positive T-Lymphocytes; Cell Proliferation; Dinucleoside Phosphates; Hydroquinones; Imiquimod; Immunoglobulin G; Immunotherapy; Injections, Subcutaneous; Killer Cells, Natural; Leukocyte Count; Lymphocyte Activation; Lymphocyte Depletion; Melanoma; Mice; Mice, Inbred C57BL; Pigmentation; Skin Neoplasms

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Topics: Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 9; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Genes, p16; Humans; Melanoma; Mutation; Polymerase Chain Reaction; Promoter Regions, Genetic; Restriction Mapping; Skin Neoplasms; Tumor Cells, Cultured

Human gene MAGE-1 encodes tumor-specific antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. This gene is expressed in a significant proportion of tumors of various histological types, but not in normal tissues except male germ-line cells. We reported previously that reporter genes driven by the MAGE-1 promoter are active not only in the tumor cell lines that express MAGE-1 but also in those that do not. This suggests that the critical factor causing the activation of MAGE-1 in certain tumors is not the presence of the appropriate transcription factors. The two major MAGE-1 promoter elements have an Ets binding site, which contains a CpG dinucleotide. We report here that these CpG are demethylated in the tumor cell lines that express MAGE-1, and are methylated in those that do not express the gene. Methylation of these CpG inhibits the binding of transcription factors, as seen by mobility shift assay. Treatment with the demethylating agent 5-aza-2'-deoxycytidine activated gene MAGE-1 not only in tumor cell lines but also in primary fibroblasts. Finally, the overall level of CpG methylation was evaluated in 20 different tumor cell lines. It was inversely correlated with the expression of MAGE-1. We conclude that the activation of MAGE-1 in cancer cells is due to the demethylation of the promoter. This appears to be a consequence of a genome-wide demethylation process that occurs in many cancers and is correlated with tumor progression.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Azacitidine; Base Sequence; Decitabine; Dinucleoside Phosphates; DNA; DNA Modification Methylases; DNA Primers; DNA Probes; DNA, Neoplasm; Enzyme Inhibitors; Genome, Human; Humans; Male; Melanoma; Melanoma-Specific Antigens; Methylation; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; T-Lymphocytes, Cytotoxic; Transcription, Genetic; Tumor Cells, Cultured