Signalling via receptor tyrosine kinase modulates the expression of the DNA repair enzyme XPD in cultured cells.
Oxidative damage to DNA has been documented in cells isolated from subjects with diabetes. Herein, we evaluate the mechanism(s) that regulate the expression of the DNA repair enzyme XPD. CHO cells transfected with the human insulin receptor (CHO/HIRc) showed a threefold increase in the level of XPD mRNA when compared to control CHO/neo cells (P < 0.01). The addition of insulin to serum-starved cells led to an increase in XPD mRNA levels in both CHO/neo and CHO/HIRc cells, in a time and dose dependent fashion. Insulin acted primarily by inducing XPD transcription. Moreover, inhibition of protein synthesis by cyclohexamide induced a marked degradation of XPD mRNA levels in insulin treated cells. Site-directed mutagenesis of the tyrosine-kinase domain of the insulin receptor abolished the increase in XPD mRNA resulting from the transfection with wild type insulin receptors (P < 0.001). Western blot analysis of cell extracts from CHO/neo and CHO/HIRc cells revealed an increase in XPD counterpart protein was also induced by transfecting cells with the human insulin receptor. Evaluation of DNA damage by means of internucleosomal fragmentation showed a dramatic decrease in DNA fragmentation in CHO cells transfected with wild-type insulin receptor compared to control CHO/neo cells. DNA fragmentation was further decreased by the addition of insulin in the culture medium. In summary, our data indicates that activation of the insulin receptor plays an important role in the cellular response leading to repair of damaged DNA.
Perfetti
R
R
Division of Endocrinology and Metabolism, Cedars-Sinai Medical Centre, Burns and Allen Research Institute and University of California, Los Angeles 90048, USA. perfettir@csmc.edu
Aggarwal
S
S
D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain / Q000235:genetics; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D053938:DNA Fragmentation / Q000187:drug effects; D004265:DNA Helicases; D011088:DNA Ligases / Q000096:biosynthesis* / Q000187:drug effects; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D014176:Protein Biosynthesis; D011506:Proteins / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects; D020794:Receptor Protein-Tyrosine Kinases / Q000494:pharmacology* / Q000502:physiology*; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors; D051759:Xeroderma Pigmentosum Group D Protein
D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D007328:Insulin; D011506:Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; D011088:DNA Ligases