Cloning and identification of EDD gene from ultraviolet-irradiated HaCaT cells.
Ultraviolet (UV) radiation is one of the most important external stimuli that affects skin by inducing cancer, inflammation and cell death. To identify the regulation of genes regulated by UV during transformation, normal human keratinocyte cell line, HaCaT, was exposed to multiple doses of UVA+B (UVA - 150-200 mJ/cm2 and UVB - 15-20 mJ/cm2 x 6). Malignant transformation was confirmed by formation of colonies on soft agar and DNA methylation assay. To identify the genes involved in this process, random amplification of polymorphic DNA using RNA from unexposed and multiple exposed cells was performed after each exposure. A few up-regulated genes were identified, cloned and sequenced. One of the genes had homology to EDD (E3 identified by differential display) that was up-regulated at second exposure but was down-regulated in colony-forming cells (cells that received six or more exposures) as determined by RT-PCR. This is a progesterone-induced gene and progesterone treatment reduced the extent of colony formation on soft agar plate. It is possible that hormone therapy may have some effects on skin cancer in vivo.
Gupta
Nishma
N
Madurai Kamraj University, Madurai, India.
Chakrobarty
Amit
A
Raman
Govindarajan
G
Banerjee
Gautam
G
D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004249:DNA Damage; D017931:DNA Primers; D004307:Dose-Response Relationship, Radiation; D005786:Gene Expression Regulation / Q000528:radiation effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000502:physiology / Q000528:radiation effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D014466:Ultraviolet Rays
D017931:DNA Primers; C117617:UBR5 protein, human; D044767:Ubiquitin-Protein Ligases