Epigenetic silencing of novel tumor suppressors in malignant melanoma.

Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on survival, emphasizing the need for a better understanding of the genetic, epigenetic, and phenotypic changes in melanoma formation and progression. We identified 17 genes that were not previously known to be silenced by methylation in melanoma using a microarray-based screen following treatment of melanoma cell lines with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Eight of these genes have not been previously shown to undergo DNA methylation in any form of cancer. Three of the genes, QPCT, CYP1B1, and LXN, are densely methylated in >95% of uncultured melanoma tumor samples. Reexpression of either of two of the silenced genes, HOXB13 and SYK, resulted in reduced colony formation in vitro and diminished tumor formation in vivo, indicating that these genes function as tumor suppressors in melanoma.

Penetrating keratoplasty in Nepal.

PURPOSE: To identify indications and outcomes in a large series of penetrating keratoplasty surgeries performed in Nepal. METHODS: A retrospective case series of 472 consecutive penetrating keratoplasty surgeries (408 patients) performed at Tilganga Eye Center, Kathmandu, Nepal from June 1994 to September 1999. RESULTS: Mean recipient age was 39.2 years (+/- 19.7 years). Main indications for PKP were corneal scar (37%), adherent leukoma (35%), perforation or impending perforation (9%), pseudophakic bullous keratopathy (6%), keratoconus (4%), and aphakic bullous keratopathy (3%). Mean duration of follow-up was 27.6 +/- 25.1 months. Sixty-five percent of available grafts were clear at 6 months, and 70% of available grafts were clear at 3 years. Six months postoperatively, 15% of patients had acuity better than 6/18, 37% had acuity between 6/18 and 6/60, and 17.7% had acuity between 6/60 and 3/60. Common causes of graft failure were endothelial failure (43%), increased intraocular pressure (15%), ulcer (14%), and trauma (7%). CONCLUSIONS: The corneal diseases and indications for transplant surgery in Nepal are different from those in the Western world. Despite these differences, penetrating keratoplasty is a successful and reasonable way to reduce corneal blindness in developing nations.

Amplification of CDK4 and MDM2 in malignant melanoma.

Amplification of the 12q13-15 region is a common event in several human tumors including liposarcomas, gliomas, and osteosarcomas. We have demonstrated high-level amplification of 12q14 in a subset of uncultured malignant melanomas (3 of 53). High-resolution mapping of the amplicon using quantitative PCR revealed a bipartite amplicon consisting of a primary 50-kb amplicon centered on CDK4 and a secondary amplicon centered on MDM2, without amplification of the intervening 11 Mb of genomic DNA. Analysis of mRNA and protein levels in melanomas with 12q14 amplification demonstrated overexpression of target genes CDK4 and MDM2 without loss of CDKN2A-P16 (P16INK4A) or CDKN2A-P14ARF (P14ARF) expression, important regulators of the RB1 and TP53 pathways, which are commonly lost or mutated in melanoma. These results suggest that coamplification of CDK4 and MDM2 may substitute for loss of P16INK4A and P14ARF function in a subset of melanomas.

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice.

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.