sybr-green-i and Skin-Neoplasms

Telomerase activity is primarily determined by transcriptional regulation of the catalytic subunit, human telomerase reverse transcriptase (hTERT). Several mRNA splice variants for hTERT have been identified, but it is not clear if telomerase activity is determined by the absolute or relative levels of full-length (functional) and variant hTERT transcripts. We have developed an SYBR green-based reverse transcription-quantitative polymerase chain reaction assay for the enumeration of the four common hTERT mRNA variants and correlated these with telomerase activity and telomere length in 24 human melanoma cell lines. All except five of the lines expressed four hTERT transcripts, with an overall significant level of co-occurrence between absolute mRNA levels of full-length alpha+/beta+ hTERT and the three splice variants alpha-/beta+, alpha+/beta-, and alpha-/beta-. On average, alpha+/beta+ made up the majority (48.1%) of transcripts, followed by alpha+/beta- (44.6%), alpha-/beta- (4.4%), and alpha-/beta+ (2.9%). Telomerase activity ranged from 1 to 247 relative telomerase activity and correlated most strongly with the absolute amount of alpha+/beta+ (R = 0.791, P = .000004) and the relative amount of alpha+/beta- (R = -0.465, P = .022). This study shows that telomerase activity in melanoma cells is best determined by the absolute expression of full-length hTERT mRNA and indicates a role for the hTERT beta deletion variant in the negative regulation of enzyme activity.

Topics: Alternative Splicing; Benzothiazoles; Diamines; Enzyme Activation; Gene Deletion; HL-60 Cells; Humans; Isoenzymes; Melanoma; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Telomerase; Tumor Cells, Cultured

The participation of BRCA1 (breast cancer 1) in DNA repair is well established, especially in mammary and ovarian cells. Our purpose was to develop a new in vivo radio-sensitizing therapy for melanoma. We therefore investigated the effect of downregulation of BRCA1 on irradiated melanoma cells using an anti-BRCA1 ribozyme. Our results show that BRCA1 downregulation increased radio-sensitivity of the A375 cell line, suggesting that BRCA1 could act as a caretaker in melanoma; however, as BRCA1 functions are not limited to maintaining genomic integrity but also regulate transcription and the cell cycle, we confirmed that the proliferative rate of BRCA1 downregulated clones did not change. We also demonstrate that: (1) among the major pro-angiogenic genes, FGF-2 was not increased before or after irradiation and vascular endothelial growth factor strongly inhibited after irradiation; (2) expression of two important metalloproteinases, matrix metalloproteinase 2 and 9, involved in melanoma metastasis were decreased before and after irradiation; (3) expression of their major inhibitor, tissue inhibitor of metalloproteinase, was mainly upregulated; and (4) that invasion of BRCA1 downregulated cells was modified. Together these data suggest that BRCA1 downregulation in melanoma cells did not make them more aggressive and could lead to new therapeutic strategies for this tumor, which is so difficult to control once metastasized.

Topics: Benzothiazoles; Carrier Proteins; Cell Division; Cell Line, Tumor; Cell Survival; Diamines; Down-Regulation; Fibroblast Growth Factor 2; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Neovascularization, Pathologic; Organic Chemicals; Proteins; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Thrombospondin 1; Thrombospondins; Tissue Inhibitor of Metalloproteinase-1; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Vascular Endothelial Growth Factor A