Drug-induced apoptosis is delayed and reduced in XPD lymphoblastoid cell lines: possible role of TFIIH in p53-mediated apoptotic cell death.
The tumor suppressor gene product p53 can bind to and inhibit the helicase activity of the multisubunit transcription-repair factor TFIIH. We previously reported that p53-mediated apoptosis is attenuated in primary human fibroblasts from individuals with Xeroderma Pigmentosum (XP) that harbor mutations in the TFIIH DNA helicases XPD or XPB. In this study we show that apoptosis is reduced and delayed in three XPD lymphoblastoid cell lines (LCLs), but not in an XPD heterozygote LCL, after exposure to doxorubicin, a DNA-damaging agent and topoisomerase II inhibitor frequently used in cancer therapy. Apoptosis was assessed by quantitation of Annexin V binding to exposed phosphatidylserine residues and by caspase-mediated cleavage of Poly(ADP)Ribose Polymerase (PARP). Apoptosis induced by doxorubicin was suppressed in LCLs retrovirally transduced with the Human Papillomavirus 16 E6 oncoprotein, consistent with the hypothesis that this is a p53-dependent process. PARP cleavage was not delayed in XPD LCLs in response to anti-Fas (CD95) antibody-mediated apoptosis, thus, the defect in the apoptotic pathway in these cells lies upstream of caspase activation. Similar changes in the expression of apoptosis-effector genes, p53, and p53-responsive genes p21Cip1/WAF-1/Sid1 (p21), gadd45, bcl-2 and bax were observed in normal and XPD LCLs after treatment with doxorubicin, indicating that delayed apoptosis was not a consequence of defective transcription of these genes. Thus, our studies provide further support to the hypothesis that XPD and p53 can functionally interact in a p53-mediated apoptotic pathway.
Robles
A I
AI
Laboratory of Human Carcinogenesis, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, MD 20892-4255, USA.
Wang
X W
XW
Harris
C C
CC
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D002460:Cell Line; D004265:DNA Helicases / Q000235:genetics*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells; D006579:Heterozygote; D006801:Humans; D008214:Lymphocytes; D011506:Proteins / Q000235:genetics*; D059005:Topoisomerase II Inhibitors; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000235:genetics*; D019473:Transcription Factors, TFII; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051759:Xeroderma Pigmentosum Group D Protein
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D011506:Proteins; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D016159:Tumor Suppressor Protein p53; D051758:Transcription Factor TFIIH; D004317:Doxorubicin; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human