Ultraviolet‐induced transformation of keratinocytes: possible involvement of long interspersed element‐1 reverse transcriptase

Abstract

The normal human keratinocyte cell line, HaCaT, was transformed using multiple doses of ultraviolet (UV)A+B (UVA, 150-200 mJ/cm(2) and UVB, 15-20 mJ/cm(2) x 6). Malignant transformation was confirmed by upregulation of Cyclin D1 (mRNA) and formation of colonies on soft agar. To identify the genes involved in this transformation process, we have done rapid amplification of polymorphic DNA using RNA from unexposed and multiple-exposed cells. Six percent PAGE showed several differentially regulated genes in exposed cells compared with unexposed cells. Total 19 genes were identified, cloned and sequenced. Three of these 19 cloned genes showed 99% homology at both DNA and protein levels to a stretch of 540 bp (180 aa) of long interspersed element (LINE)-1 reverse transcriptase (RT) open reading frame (ORF-2). Colonies from soft agar showed upregulation of this gene compared with non-colonized (lawn on soft agar) cells as detected by RT-PCR. This data implicates LINE-1 RT (ORF-2) in UV-induced malignancy and can possibly be used as a marker for the diagnosis of UV-induced skin cancer.