Effects of XPD Mutations on Ultraviolet-Induced Apoptosis in Relation to Skin Cancer-Proneness in Repair-Deficient Syndromes
2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês
10.1046/j.0022-202x.2001.01533.x
ISSN1523-1747
AutoresSophie Queillé, Christiane Drougard, Alain Sarasin, Leela Daya–Grosjean,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoTo understand the relationship between DNA repair, apoptosis, transcription, and cancer-proneness, we have studied the apoptotic response and the recovery of RNA synthesis following ultraviolet C and ultraviolet B irradiation in nucleotide excision repair deficient diploid fibroblasts from the cancer-prone xeroderma pigmentosum (XP) syndrome patients and the non-cancer-prone trichothiodystrophy (TTD) patients. Analysis of four XPD and four TTD/XPD fibroblast strains presenting different mutations on the XPD gene has shown that XPD cells are more sensitive to ultraviolet-induced apoptosis than TTD/XPD cells, and this response seems to be modulated by the type and the location of the mutation on the XPD gene. Moreover, the other xeroderma pigmentosum fibroblast strains analyzed (groups A and C) are more sensitive to undergo apoptosis after ultraviolet irradiation than normal human fibroblasts, showing that the cancer-proneness of xeroderma pigmentosum patients is not due to a deficiency in the ultraviolet-induced apoptotic response. We have also found that cells from transcription-coupled repair deficient XPA, XPD, TTD/XPD, and Cockayne's syndrome patients undergo apoptosis at lower ultraviolet doses than transcription-coupled repair proficient cells (normal human fibroblasts and XPC), indicating that blockage of RNA polymerase II at unrepaired lesions on the transcribed strand is the trigger. Moreover, XPD and XPA cells are more sensitive to ultraviolet-induced apoptosis than trichothiodystrophy and Cockayne's syndrome fibroblasts, suggesting that both cyclobutane pyrimidine dimers and pyrimidine 6–4 pyrimidone on the transcribed strand trigger apoptosis. Finally, we show that apoptosis is directly proportional to the level of inhibition of transcription, which depends on the density of ultraviolet-induced lesions occurring on transcribed sequences. To understand the relationship between DNA repair, apoptosis, transcription, and cancer-proneness, we have studied the apoptotic response and the recovery of RNA synthesis following ultraviolet C and ultraviolet B irradiation in nucleotide excision repair deficient diploid fibroblasts from the cancer-prone xeroderma pigmentosum (XP) syndrome patients and the non-cancer-prone trichothiodystrophy (TTD) patients. Analysis of four XPD and four TTD/XPD fibroblast strains presenting different mutations on the XPD gene has shown that XPD cells are more sensitive to ultraviolet-induced apoptosis than TTD/XPD cells, and this response seems to be modulated by the type and the location of the mutation on the XPD gene. Moreover, the other xeroderma pigmentosum fibroblast strains analyzed (groups A and C) are more sensitive to undergo apoptosis after ultraviolet irradiation than normal human fibroblasts, showing that the cancer-proneness of xeroderma pigmentosum patients is not due to a deficiency in the ultraviolet-induced apoptotic response. We have also found that cells from transcription-coupled repair deficient XPA, XPD, TTD/XPD, and Cockayne's syndrome patients undergo apoptosis at lower ultraviolet doses than transcription-coupled repair proficient cells (normal human fibroblasts and XPC), indicating that blockage of RNA polymerase II at unrepaired lesions on the transcribed strand is the trigger. Moreover, XPD and XPA cells are more sensitive to ultraviolet-induced apoptosis than trichothiodystrophy and Cockayne's syndrome fibroblasts, suggesting that both cyclobutane pyrimidine dimers and pyrimidine 6–4 pyrimidone on the transcribed strand trigger apoptosis. Finally, we show that apoptosis is directly proportional to the level of inhibition of transcription, which depends on the density of ultraviolet-induced lesions occurring on transcribed sequences. cyclobutane pyrimidine dimers Cockayne syndrome global genome repair, NHF, normal human fibroblasts nucleotide excision repair pyrimidine 6–4 pyrimidone recovery of RNA synthesis transcription-coupled repair trichothiodystrophy xeroderma pigmentosum Xeroderma pigmentosum (XP), trichothiodystrophy (TTD), and Cockayne's syndrome (CS) are genetically inheritable diseases characterized by a defect in nucleotide excision repair (NER) and a hypersensitivity to ultraviolet (UV) radiation (van Steeg and Kraemer, 1999van Steeg H. Kraemer K.H. Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer.Mol Med Today. 1999; 5: 86-94Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar;de Boer and Hoeijmakers, 2000de Boer J. Hoeijmakers J.H. Nucleotide excision repair and human syndromes.Carcinogenesis. 2000; 21: 453-460Crossref PubMed Scopus (534) Google Scholar). In our laboratory, we are particularly intrigued by the wide heterogeneity in clinical symptoms (abnormal growth, neurologic abnormalities, skin cancer susceptibility) manifested by patients with these three diseases. The most striking difference among them is that only the XP patients (classic complementation groups A-G) present a high predisposition to skin cancer development on sun-exposed areas appearing at very early ages (Kraemer et al., 1994Kraemer K.H. Lee M.-M. Andrews A.D. Lambert C. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer.Arch Dermatol. 1994; 130: 1018-1021Crossref PubMed Scopus (447) Google Scholar). The young XP patients typically exhibit exaggerated sunburning, telangiectasia, and atrophy after minimal sun exposure, and some individuals also present progressive neuronal degeneration. There exists a variant form of XP called XP variant (XPV) that is not deficient in NER and these patients are characterized by moderate sensitivity to UV with less numerous skin tumors that appear later in life, between 15 and 40 y of age. XPV cells are proficient in NER but deficient in some kind of "error-free" bypass of lesion because of the defect in the DNA polymerase η (Masutani et al., 1999aMasutani C. Araki M. Yamada A. et al.Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity.EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (374) Google Scholar;Masutani et al., 1999bMasutani C. Kusumoto R. Yamada A. et al.The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.Nature. 1999; 399: 700-704Crossref PubMed Scopus (1096) Google Scholar). The enigma surrounding the three UV-sensitive syndromes is that three of the XP genes (XPB, XPD, and XPG) are also found mutated in TTD and XP/CS patients (exhibiting both XP and Cockayne's symptoms) giving rise to the very specific phenotypes associated with each syndrome. Thus the XP/CS patients mutated in either one of the XPB, XPD, or XPG genes present a combination of the cutaneous abnormalities of XP with the severe neurologic and developmental anomalies typical of CS patients (Lehmann et al., 1993Lehmann A.R. Thompson A.F. Harcourt S.A. Stefanini M. Norris P.G. Cockayne's syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation.J Med Genet. 1993; 30: 679-682Crossref PubMed Scopus (64) Google Scholar). The majority of the TTD patients carrying mutations in the XPD gene, however, only present the TTD phenotype including brittle hair, ichthyosis, as well as physical and mental retardation (Stefanini et al., 1993aStefanini M. Lagomarsini P. Giliani S. et al.Genetic heterogeneity of the excision repair defect associated with trichothiodystrophy.Carcinogenesis. 1993; 14: 1101-1105Crossref PubMed Scopus (86) Google Scholar;Stefanini et al., 1993bStefanini M. Vermeulen W. Weeda G. et al.A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy.Am J Hum Genet. 1993; 53: 817-821PubMed Google Scholar). XPD and TTD/XPD patients who present different mutations mapped on the XPD gene (Taylor et al., 1997Taylor E.M. Broughton B.C. Botta E. et al.Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene.Proc Natl Acad Sci USA. 1997; 94: 8658-8663Crossref PubMed Scopus (224) Google Scholar) have only one common clinical feature, extreme sensitivity to sunlight, yet paradoxically only the XPD patients are predisposed to skin cancer. Recent studies elucidating the structure and function of the TFIIH transcription factor have clearly shown that the XPD protein, besides being involved in NER functions, is also essential for transcription and cell viability (Frit et al., 1999Frit P. Bergmann E. Egly J.M. Transcription factor IIH: a key player in the cellular response to DNA damage.Biochimie. 1999; 81: 27-38Crossref PubMed Scopus (54) Google Scholar). The NER pathway removes UV-induced DNA damage as well as other bulky lesions produced by a variety of genotoxic agents. Thus, UV-induced dimers in genomic DNA, the cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone (6–4PP), are normally efficiently removed by NER. The NER process involves the coordinated action of at least 30 proteins during the four major steps of the mechanism (Petit and Sancar, 1999Petit C. Sancar A. Nucleotide excision repair: from E. coli to man.Biochimie. 1999; 81: 15-25Crossref PubMed Scopus (188) Google Scholar;Wood, 1999Wood R.D. DNA damage recognition during nucleotide excision repair in mammalian cells.Biochimie. 1999; 81: 39-44Crossref PubMed Scopus (204) Google Scholar;Balajee and Bohr, 2000Balajee A.S. Bohr V.A. Genomic heterogeneity of nucleotide excision repair.Gene. 2000; 250: 15-30Crossref PubMed Scopus (117) Google Scholar). In particular, XPA, XPC, and XPE proteins are involved in lesion recognition and the helicase activities of XPB and XPD allow damage demarcation (Batty and Wood, 2000Batty D.P. Wood R.D. Damage recognition in nucleotide excision repair of DNA.Gene. 2000; 241: 193-204Crossref PubMed Scopus (249) Google Scholar). The XPG and XPF endonucleases incise the lesion and repair synthesis by the cell's replicative machinery fills in the gap (Araujo and Wood, 1999Araujo S.J. Wood R.D. Protein complexes in nucleotide excision repair.Mutat Res. 1999; 435 ([published erratum appears in Mutat Res 459: 171–172, 2000]): 23-33Crossref PubMed Scopus (92) Google Scholar). It is important to note that lesions on the transcribed strand of active genes, which block transcription, are quickly removed by transcription-coupled repair (TCR) so that transcription, which is essential for cell survival, can proceed (Mellon et al., 1986Mellon I. Bohr V.A. Smith C.A. Hanawalt P.C. Preferential DNA repair of an active gene in human cells.Proc Natl Acad Sci USA. 1986; 83: 8878-8882Crossref PubMed Scopus (367) Google Scholar;Mellon et al., 1987Mellon I. Spivak G. Hanawalt P.C. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene.Cell. 1987; 51: 241-249Abstract Full Text PDF PubMed Scopus (999) Google Scholar). A defect in any one of the NER proteins results in one of the three rare, recessive syndromes XP, TTD, and CS (de Boer and Hoeijmakers, 2000de Boer J. Hoeijmakers J.H. Nucleotide excision repair and human syndromes.Carcinogenesis. 2000; 21: 453-460Crossref PubMed Scopus (534) Google Scholar). CS cells are deficient in TCR of CPD but are able to repair the remainder of the genome (global genome repair or GGR) (Venema et al., 1990Venema J. Mullenders L.H. Natarajan A.T. van Zeeland A.A. Mayne L.V. The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA.Proc Natl Acad Sci USA. 1990; 87: 4707-4711Crossref PubMed Scopus (479) Google Scholar;Barrett et al., 1991Barrett S.F. Robbins J.H. Tarone R.E. Kraemer K.H. Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells.Mutat Res. 1991; 255: 281-291Crossref PubMed Scopus (39) Google Scholar;van Hoffen et al., 1993van Hoffen A. Natarajan A.T. Mayne L.V. van Zeeland A.A. Mullenders L.H. Venema J. Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells.Nucl Acid Res. 1993; 21: 5890-5895Crossref PubMed Scopus (249) Google Scholar) whereas XP group C cells are deficient in GGR of both CPD and 6–4PP (Venema et al., 1991Venema J. van Hoffen A. Karcagi V. Natarajan A.T. van Zeeland A.A. Mullenders L.H. Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes.Mol Cell Biol. 1991; 11: 4128-4134Crossref PubMed Scopus (285) Google Scholar;van Hoffen et al., 1995van Hoffen A. Venema J. Meschini R. van Zeeland A.A. Mullenders L.H. Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6–4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts.EMBO J. 1995; 14: 360-367Crossref PubMed Scopus (201) Google Scholar;de Laat et al., 1999de Laat W.L. Jaspers N.G. Hoeijmakers J.H. Molecular mechanism of nucleotide exision repair.Genes Dev. 1999; 13: 768-785Crossref PubMed Scopus (887) Google Scholar). The XPD cells are deficient in TCR and GGR of both CPD and 6–4PP (Evans et al., 1993Evans M.K. Robbins J.H. Ganges M.B. Tarone R.E. Nairn R.S. Bohr V.A. Gene-specific DNA repair in xeroderma pigmentosum complementation groups A, C, D, and F. Relation to cellular survival and clinical features.J Biol Chem. 1993; 268: 4839-4847Abstract Full Text PDF PubMed Google Scholar) but some XPD cells have been found to have residual levels of removal of 6–4PP, which may explain the variations in clinical symptoms observed among the patients (Galloway et al., 1994Galloway A. Liuzzi M. Paterson M. Metabolic processing of cyclobutyl pyrimidine dimers and (6–4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.J Biol Chem. 1994; 269: 974-980Abstract Full Text PDF PubMed Google Scholar;van Hoffen et al., 1999van Hoffen A. Kalle W.H. de Jong-Versteeg A. Lehmann A.R. van Zeeland A.A. Mullenders L.H.F. Cells from XP-D and XP-D-CS patients exhibit equally inefficient repair of UV-induced damage in transcribed genes but different capacity to recover UV-inhibited transcription.Nucl Acid Res. 1999; 27: 2898-2904Crossref PubMed Scopus (38) Google Scholar). The TTD cells mutated in the XPD gene (TTD/XPD) are deficient in both TCR and GGR of the CPD lesions with a variable efficiency in 6–4PP repair (Eveno et al., 1995Eveno E. Bourre F. Quilliet X. et al.Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases.Cancer Res. 1995; 55: 4325-4332PubMed Google Scholar). It has already been shown in previous studies that cells deficient in TCR, notably CS as well as XPD and XPA (which are highly deficient in both TCR and GGR), are unable to restore normal transcription following UVC irradiation, whereas TCR-proficient fibroblasts [normal human fibroblasts (NHF), XPC and XPV] recover RNA synthesis after UVC irradiation (Mayne and Lehmann, 1982Mayne L.V. Lehmann A.R. Failure of RNA synthesis to recover after UV irradiation: an early defect in cells from individuals with Cockayne's syndrome and xeroderma pigmentosum.Cancer Res. 1982; 42: 1473-1478PubMed Google Scholar;Yamaizumi and Sugano, 1994Yamaizumi M. Sugano T. UV-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle.Oncogene. 1994; 9: 2775-2784PubMed Google Scholar;Ljungman and Zhang, 1996Ljungman M. Zhang F. Blockage of RNA polymerase as a possible trigger for UV light-induced apoptosis.Oncogene. 1996; 13: 823-831PubMed Google Scholar). Correlation between recovery of RNA synthesis (RRS) and cell survival after UV irradiation suggests that TCR allows expression of genes essential for UV survival. We and colleagues have shown that the unrepaired UV lesions persistent on the transcribed strand, which block transcription, are responsible for triggering the accumulation of the p53 protein following UVC irradiation in human skin fibroblasts (Yamaizumi and Sugano, 1994Yamaizumi M. Sugano T. UV-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle.Oncogene. 1994; 9: 2775-2784PubMed Google Scholar;Ljungman and Zhang, 1996Ljungman M. Zhang F. Blockage of RNA polymerase as a possible trigger for UV light-induced apoptosis.Oncogene. 1996; 13: 823-831PubMed Google Scholar;Dumaz et al., 1997Dumaz N. Duthu A. Ehrhart J.C. et al.Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.Mol Carcinog. 1997; 20: 340-347Crossref PubMed Scopus (56) Google Scholar;McKay et al., 1998McKay B.C. Ljungman M. Rainbow A.J. Persistent DNA damage induced by ultraviolet light inhibits p21waf1 and bax expression: implications for DNA repair, UV sensitivity and the induction of apoptosis.Oncogene. 1998; 17: 545-555Crossref PubMed Scopus (88) Google Scholar;Ljungman et al., 1999Ljungman M. Zhang F. Chen F. Rainbow A.J. McKay B.C. Inhibition of RNA polymerase II as a trigger for the p53 response.Oncogene. 1999; 18: 583-592Crossref PubMed Scopus (239) Google Scholar). This was deduced from the finding that TCR-deficient fibroblasts (XPA, XPD, and CS) accumulate p53 protein for prolonged times at a lower UVC dose than TCR-proficient fibroblasts (NHF, XPC, and XPV). The study in our laboratory (Dumaz et al., 1997Dumaz N. Duthu A. Ehrhart J.C. et al.Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.Mol Carcinog. 1997; 20: 340-347Crossref PubMed Scopus (56) Google Scholar) also analyzed TTD/XPD fibroblasts, which are deficient in the repair of CPD and accumulate p53 at low UVC doses, allowing us to postulate that the unrepaired CPD on the transcribed strand were responsible for triggering the accumulation of the p53 protein. Indeed, accumulation of p53 after DNA damage activates transcription from several responsive genes (WAF1, GADD45, cyclin G), which can lead to arrest of cell cycle progression to allow DNA repair. Under certain circumstances, an increase in p53 levels can modulate the transcription of genes implicated in apoptosis (like Bax and Bcl-2), which allows the elimination of damaged premutagenic cells, thus avoiding tumorigenesis. Indeed,Ljungman and Zhang, 1996Ljungman M. Zhang F. Blockage of RNA polymerase as a possible trigger for UV light-induced apoptosis.Oncogene. 1996; 13: 823-831PubMed Google Scholar have shown that accumulation of p53 after UVC irradiation of XPA, XPC, and CS human skin fibroblasts is correlated with apoptosis. In order to better understand the relationship between DNA repair deficiency, apoptosis, transcription, and cancer-proneness, we have analyzed UV-induced apoptosis and the modulation of transcription following UV irradiation in repair-deficient skin fibroblasts focusing on fibroblast strains carrying different mutations in the XPD gene (four XPD and four TTD/XPD cell lines), which up to now have never been studied. We show here for the first time that the XPD cell strains are more sensitive to undergo UVC- and UVB-induced apoptosis than the TTD strains and the type and location of the mutation in the XPD gene seem to influence the UV apoptotic response. Moreover, this is the first study evaluating the role of UVB, the major carcinogenic component of sunlight, in the apoptotic response of skin fibroblasts from patients with cancer-prone and cancer-free DNA repair syndromes. Interestingly, our data show that all the NER-deficient XP skin fibroblasts, notably XPA and XPD, are more sensitive to undergo UV-induced apoptosis than NHF, indicating that predisposition to skin cancer in XP patients is not due to a deficiency in apoptosis. We have also observed that TCR-deficient fibroblasts (XPA, XPD, TTD, and CS) undergo apoptosis at lower doses of UVC and UVB than TCR-proficient fibroblasts (NHF and XPC), confirming that the blockage of the RNA polymerase II at the unrepaired UV lesions on the transcribed strand triggers apoptosis. Surprisingly, at high doses of UVC and UVB, apoptosis levels decline in the majority of TCR-deficient cells in correlation with the inhibition of transcription, confirming that apoptosis requires an active transcription process. We propose that the level of apoptosis is directly proportional to the density of UV lesions on transcribed sequences, but at high UV doses when RNA polymerase is fully blocked the persistence of unrepaired UV lesions on the transcribed strand also impedes apoptosis. The cell lines used in this study are described in Table I. The human primary fibroblast cultures were established from unexposed skin biopsies of normal individuals or TTD, XP, and CS patients. The cells were grown in minimum Eagle's medium (Life Technology, Gaithersburg, MD) supplemented with 15% fetal bovine serum (Dominique Dutcher, Mulhouse, France), 1% antibiotics and fungicides, L-glutamine, and nonessential amino acids.Table ICell lines used in this studyNamePhenotypeLaboratory of origin190VIwildtypeA. Sarasin191VIwildtypeA. Sarasin194VIwildtypeA. SarasinXP17PVXPDM. StefaniniXP1DUXPDC. ArlettXP107LOXPDS. PawseyXP26VIXPDA. SarasinTTD1VITTD/XPDA. SarasinTTD2VITTD/XPDA. SarasinTTD3VITTD/XPDA. SarasinTTD9VITTD/XPDA. SarasinCS1VICSA. SarasinCS2VICSA. SarasinCS360VICSA. SarasinXP111VIXPCA. SarasinXP148VIXPCA. SarasinXP192VIXPAA. Sarasin Open table in a new tab UVC and UVB irradiation was performed as described previously (Robert et al., 1996Robert C. Muel B. Benoit A. Dubertret L. Sarasin A. Stary A. Cell survival and shuttle vector mutagenesis induced by ultraviolet A and ultraviolet B radiation in a human cell line.J Invest Dermatol. 1996; 106: 721-728Crossref PubMed Scopus (103) Google Scholar;Dumaz et al., 1997Dumaz N. Duthu A. Ehrhart J.C. et al.Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.Mol Carcinog. 1997; 20: 340-347Crossref PubMed Scopus (56) Google Scholar). All the primary diploid fibroblast cell lines were irradiated in the exponential growth phase and flow cytometry allowed us to evaluate the fraction of cells in G1/S/G2/M. All cell lines were seen to have the majority (70%-80%) of cells in the G1 phase considered to be typical of cultured fibroblasts. At various times after irradiation, attached cells were trypsinized and collected together with detached cells by centrifugation at 800 rpm for 10 min. The pellets were washed with phosphate-buffered saline (PBS), fixed in 70% ethanol, and stored at -20°C. Cells were incubated with RNase (20 µg per ml) in PBS for 30 min at 37°C and stained with propidium iodide (70 µg per ml) for 30 min at 37°C prior to analysis in a Coulter EPICS Profile II cytometer. Cell cycle distributions were calculated with Multicycle AV software (Phoenix Flow Systems, San Diego, CA). Fluorescence values significantly below the normal G1 values (sub-G1) are taken as an indication of apoptosis (Nicoletti et al., 1991Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunol Meth. 1991; 139: 271-279Crossref PubMed Scopus (4340) Google Scholar;Darzynkiewicz et al., 1992Darzynkiewicz Z. Bruno S. Del Bino G. Gorczyca W. Hotz M.A. Lassota P. Traganos F. Features of apoptotic cells measured by flow cytometry.Cytometry. 1992; 13: 795-808Crossref PubMed Scopus (1870) Google Scholar). The necrotic cell populations including cell debris do not take up propidium iodide and are easily identified on the DNA histogram in front of the hypodiploid or sub-G1 peak. All experiments were carried out at least twice for each cell type studied. Coverslip cultures of the different fibroblast cell lines were irradiated with UV, fixed, and stained after 72 h with Hoechst 33342. Nuclear morphologic changes were visualized microscopically under UV illumination. Protein extract samples were prepared as previously described (Dumaz et al., 1997Dumaz N. Duthu A. Ehrhart J.C. et al.Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.Mol Carcinog. 1997; 20: 340-347Crossref PubMed Scopus (56) Google Scholar). Specific proteins were revealed with either a mouse monoclonal p53 (DO-7) antibody (Dako, Trappes, France), a mouse monoclonal Bcl-2 antibody (Dako), or a rabbit polyclonal Bax antibody (N-20, Santa Cruz, Biotech, CA). The reaction was completed with a peroxidase-conjugated secondary antispecies (mouse or rabbit) antibody followed by enhanced chemiluminescence according to the manufacturer's instructions (ECL, Amersham, Courtaboeuf, France). Cells grown on glass coverslips and irradiated with UVC or UVB were incubated for 23 h and then labeled 1 h in a medium containing 3H-uridine at 10 µCi per ml (specific activity of 27 Ci per mmol, Amersham). Coverslips were mounted on glass slides with the cells on the upper surface, dipped in EM-1 emulsion (Amersham), exposed overnight at 4°C, and revealed according to instructions from the manufacturer. The mean number of grains per nucleus was obtained by counting 30 nuclei. Results are expressed in percentage of grains per nucleus compared to the control, where the number of grains per nucleus corresponds to 100%. To enable a quantitative evaluation of apoptosis and in order to analyze a large number of fibroblast strains, we quantified the sub-G1 DNA content of cells by flow cytometry after staining of fixed cells with propidium iodide (see Materials and Methods). In fact, the sub-G1 DNA content arises from the leaking out of DNA fragments from ethanol-fixed cells and characterizes apoptotic cells. All the cells were collected (attached and detached) for fluorescence-activated cell sorter analysis. We analyzed the effect of UVC and UVB irradiation in four XPD, two XPC, and one XPA fibroblast strains from XP patients presenting skin cancer predisposition as well as in four TTD/XPD and three CS fibroblast strains from patients who were not predisposed to skin cancer development. In a time course experiment we first observed that normal and NER-deficient fibroblasts all underwent apoptosis around 72 h after irradiation whatever the UV dose given. Figure 1 and Figure 2 present the percentage of apoptotic cells 72 h after UVB and UVC irradiation using sub-G1 DNA content analysis. It was found that variations existed in the responses of the different strains of XPD analyzed, probably due either to the genetic background of the cells or to the effect of the different XPD mutations. The general tendency to undergo UV-induced apoptosis remained constant, however, with all XPD strains as well as the XPA cell line (the most NER deficient) being very sensitive. In fact, XPD and XPA fibroblasts, which had a greater level of apoptosis at 300 J per m2 of UVB and 5 J per m2 of UVC, were found to start undergoing apoptosis at a very low UV dose (2 J per m2 of UVC and 100 J per m2 of UVB, data not shown). At 1000 J per m2 of UVB and 20 J per m2 of UVC, we observed a decrease of the UVB/C-induced apoptosis level in XPA and XPD fibroblasts except for the XP1DU cell line. It is important to note that at low UV fluences apoptosis is very high in XPA and XPD fibroblasts and the majority of the dead cells are detached. In contrast, we have clearly observed that these cells irradiated at higher doses remained attached and alive (Trypan blue viability test) 72 h after irradiation, and only at later times (up to 5 d after irradiation) did the cells begin to detach and die by necrosis. Indeed, DNA histograms of the cell cycle obtained by flow cytometry of propidium iodide treated cells (see Materials and Methods) show that these cells are never seen as a sub-G1 apoptotic population but are observed as a nonfluorescent necrotic population. Therefore, it can be postulated that fibroblasts irradiated at high UV doses were still alive when apoptosis was evaluated (72 h after irradiation) but were so badly damaged in all vital cell compartments, blocking replication, transcription, etc., that they remained in a numbed state for a long time but finally died by necrosis resulting in low survival levels. Both TTD/XPD and CS fibroblast strains started to undergo apoptosis at 5 J per m2 of UVC and 300 J per m2 of UVB, which increased at 10 J per m2 of UVC and 500 J per m2 of UVB (data not shown), and a maximum apoptosis level was reached at 1000 J per m2 of UVB and 20 J per m2 of UVC irradiation (Figure 1, Figure 2). It was only at the higher UVC/B doses (40 J per m2 of UVC and 1500 J per m2 of UVB) that NHF were seen to undergo apoptosis, whereas the level of apoptosis was found to greatly decrease in the majority of XPD and TTD/XPD cells. Taken together, our results showed clearly that there was no deficiency in the apoptotic response of skin fibroblasts from patients with cancer-prone DNA repair syndromes. Moreover, apoptosis was found to be directly dependent on the DNA repair level of cells because NER-deficient fibroblasts (XPA, XPD, and XPC) were more sensitive to undergo apoptosis than NER-proficient NHF where only a high dose of UV induced apoptosis. Finally, we confirmed that apoptosis was seen in unfixed cells (data not shown) by using Annexin labeling to detect the externalization of the phosphatidylserine, which confirmed the data from the sub-G1 analysis. Tunnel assay on coverslip cultures did not prove to be useful as we did not detect strand breaks and internucleosomal breaks typical of apoptosis by electrop
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