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Expression of 25‐hydroxyvitamin D 3 ‐1α‐hydroxylase in malignant melanoma: implications for growth control via local synthesis of 1,25(OH) 2 D 3 and detection of multiple splice variants
Author(s) -
Seifert M.,
Diesel B.,
Meese E.,
Tilgen W.,
Reichrath J.
Publication year - 2005
Publication title -
experimental dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.108
H-Index - 96
eISSN - 1600-0625
pISSN - 0906-6705
DOI - 10.1111/j.0906-6705.2005.0266c.x
Subject(s) - autocrine signalling , biology , paracrine signalling , alternative splicing , melanoma , vitamin d and neurology , microbiology and biotechnology , du145 , splice , prostate cancer , gene , cell culture , messenger rna , cancer research , cancer , genetics , endocrinology , lncap , receptor
1,25‐Dihydroxyvitamin D 3 , 1,25(OH) 2 D 3 ) and analogs have been shown to inhibit proliferation and to induce differentiation in various cell types, including human melanocytes. There are two principal enzymes involved in the formation of circulating 1,25(OH) 2 D 3 from vitamin D, the hepatic microsomal or mitochondrial vitamin D 25‐hydroxylase (25‐OHase) and the renal mitochondrial enzyme 25‐hydroxyvitamin D 3 ‐1α‐hydroxylase (1α‐OHase) for vitamin D and 25(OH)D 3 , respectively. Recently, extra‐renal activity of 1α‐OHase has been reported in various cell types including macrophages, keratinocytes, prostate and colon cancer cells. Increasing evidence indicates that extra‐renal 1α‐OHase may act in many tissues via the local production of 1,25(OH) 2 D 3 as a major regulator of cell growth in an autocrine or paracrine fashion. In human malignant glioma we have demostrated a correlation between amplification of the gene and its overexpression. Using Reverse transcription (RT)‐PCR with primers amplifying a part of the 1α–OHase sequence, we have shown now for the first time several splice variants, including transcripts encoding truncated proteins in melanoma cell lines. To arrive at a more complete description of 1α–OHase expression we developed and employed a highly specific approach that combinded nested and touchdown PCR to clone full length 1α‐OHase. In addition, we identified several new splice variants in human melanoma. The new splice variants that were termed Hyd‐V5, ‐V6, ‐V7 and ‐V8 were cloned and sequenced. All but one of the new variants showed an insertion of intron 1 leading to a premature termination signal and to truncated proteins without ferredoxin and haem‐binding site of the P450 protein. In conclusion our findings indicate that (1) local synthesis of 1,25(OH) 2 VitD 3 mediated via 1α‐OHase may be of high importance for growth characteristics of melanoma cells and (2) 25(OH)VitD 3 or other percusors of biologically active vitamin D metabolites may be effective in the palliative treatment of metastasizing melanoma.