High-Frequency Microsatellite Instability is Associated with Defective DNA Mismatch Repair in Human Melanoma

Abstract

Hereditary nonpolyposis colorectal cancers and a steadily increasing number of sporadic tumors display microsatellite instability. In colorectal tumors, high-frequency microsatellite instability is strictly associated with inactivation of the DNA mismatch repair genes hMSH2, hMLH1, or hPMS2, whereas mutations in the mismatch repair gene hMSH6 have been identified in a subset of tumors with low-frequency microsatellite instability. In addition to epithelial tumors of the colon, endometrium, and ovary, microsatellite instability has been reported to occur also in sporadic melanoma. The relationship between microsatellite instability and mismatch repair in melanoma cells, however, has not been investigated so far. In this study, we analyzed microsatellite instability, mismatch repair activity, and expression of the hMSH2, hMSH6, hMLH1, and hPMS2 proteins in five melanoma cell lines and in tumor specimens from which the cells were derived. Four cell lines displayed normal levels of mismatch repair activity and expressed all the mismatch repair proteins. The extracts of the fifth cell line lacked the hMLH1 and hPMS2 proteins, and were correspondingly deficient in the repair of DNA mismatches. This line displayed high-frequency microsatellite instability, whereas the four mismatch-repair-proficient cell lines displayed either no or low-frequency microsatellite instability. These findings could be confirmed in the tumor specimens, in that only the tumor that did not express hMLH1 and hPMS2 displayed high-frequency microsatellite instability. Our data are consistent with the hypothesis that in melanoma, similarly to epithelial tumors, only the high-frequency microsatellite instability phenotype is strictly dependent on a defective mismatch repair system. Further studies on a large series of tumor specimens are required to establish the frequency of mismatch repair loss in human melanoma.

The Effect ofO6-Alkylguanine-DNA Alkyltransferase and Mismatch Repair Activities on the Sensitivity of Human Melanoma Cells to Temozolomide, 1,3-bis(2-Chloroethyl)1-nitrosourea, and Cisplatin

“…We have previously shown that the PR-Mel cells are MMR-deficient, whereas CR-Mel and CN-Mel cells are MMR-proficient (Alvino et al, 2002). All the other cell lines were characterized for MMR activity in the present study.…”

“…Nine human melanoma cell lines were used in this study. M14 and M10 (Golub et al, 1976) were kindly provided by Dr. G. Zupi (Istituto Regina Elena, Rome, Italy); LCP-MEL, GL-MEL, and PD-Mel (Lacal et al, 2000) were kindly provided by Dr. Guadagni (Istituto Regina Elena); CR-Mel, PR-Mel, and CNMel (Alvino et al, 2002) were established in our laboratory; and SK-Mel-28 was obtained from the American Type Culture Collection (Manassas, VA). LCP-Mel was obtained from a primary melanoma; PR-Mel, M14, and M10 were established from cutaneous metastases; and the remaining cell lines were originated from noncutaneous metastases, including lymph node and organ metastases.…”

“…Western blot analysis of MMR protein expression was performed as described previously (Alvino et al, 2002). The mAb against actin was used as an internal standard for protein loading.…”

“…We set out to readdress this situation by evaluating the influence of OGAT and MMR activities on melanoma cell sensitivity to TMZ, BCNU, and CDDP and on the ability of BG to potentiate the cytotoxic effects of the former two drugs. To this end, we used several MMR-proficient melanoma cell lines and clones, as well as the recently established MMR-deficient human melanoma cell lines PR-Mel, which does not express the hMLH1 and hPMS2 proteins (Alvino et al, 2002), and PD-Mel, which lacks hMSH6. We show that in melanoma cells resistance to TMZ is controlled principally by the OGAT and MMR systems, whereas resistance to BCNU and CDDP involves other factors as well.…”

Role of PLZF in melanoma progression

“…The early passage melanoma cells were kindly provided by Dr S D'Atri (IDI IRCCS, Rome). Two cell lines were obtained from primary tumors (GR-mel, ST-mel) and three from lymph node metastases (CRmel, CN-mel, MR/C-mel) (Lacal PM et al, 2000;Alvino et al, 2002). Normal human epidermal melanocytes from foreskin were obtained from Promocell (Heidelberg, Germany).…”