- Original Articles
- Open Access
Transcription Abnormalities Potentiate Apoptosis of Normal Human Fibroblasts
© Picower Institute Press 1997
- Accepted: 15 October 1997
- Published: 1 December 1997
Apoptosis is a natural process by which damaged and potentially tumorigenic cells are removed. Induction of apoptosis is important in chemotherapy aimed at eliminating cancer cells. We address the mechanisms by which this process can be triggered in cells that are recalcitrant to cell death induced by DNA-damaging agents.
Materials and Methods
Normal human fibroblasts and lymphoblasts, and fibroblasts with defined genetic changes, were treated with DNA-damaging agents and inhibitors of transcription. Western blotting was used to study the expression of some of the key factors involved in the response to DNA damage and the induction of apoptosis, namely, p53, p21WAF1,Cip1, Mdm2, Bax, and CD95 (Fas/APO1). Apoptosis was followed by various criteria, including DNA fragmentation, specific proteolysis, cell morphology, viability, and FACS scan for sub-G1 cells.
Normal human fibroblasts were more resistant than lymphoblasts to DNA damage-induced apoptosis. The DNA-damaging agents mitomycin C and cisplatin induced rapid apoptosis of fibroblasts with defects in the repair of transcribed DNA, compared with wild-type cells or those with defects in overall genome repair. Short-term treatment with inhibitors of RNA polymerase II transcription, actinomycin D, and α-amanitin induced rapid cell death of normal fibroblasts. These results show that there is a link between defective transcription and apoptosis. Treatments and genetic backgrounds that favored apoptosis were associated with efficient and prolonged induction of p53 and often altered or unbalanced expression of its downstream effectors p21WAF1,Cip1 and Mdm2, whereas there were no changes in Bax or CD95 (Fas/APO1).
Transcription inhibitors increase p53 levels and are better inducers of apoptosis than DNA-damaging agents in some cell types. Apoptosis might be triggered by blocked polymerases and/or faulty expression of downstream effectors.
Premalignant and aberrant cells are eliminated by mechanisms involving the tumor suppressor p53 (1–5). A variety of cellular stresses, such as damaged DNA, viral infection, hypoxia, and depletion of ribonucleotides pool, can trigger post-translational stabilization and activation of p53 (6–11). Activated p53 suppresses cellular growth through induction of either growth arrest (12,13) and/or apoptosis (1,14).
The mechanisms of p53-mediated growth arrest are fairly well understood and are essentially mediated by transactivation of the gene for the cyclin-dependent kinase inhibitor (CKI) p21WAF1,Cip1 (15). wild-type p53 is also required for Gasl- and c-Abl-mediated growth suppression (16–18). Less well understood are the mechanisms of p53-induced apoptosis, its major tumor-suppressing function (19–23). p53 induces apoptosis through both transcription-dependent (24,25) and -independent mechanisms (26–29). A transcription-independent apoptotic pathways could involve interactions between p53 and transcription/repair factor TFIIH (28,30,31). Transcription-dependent pathways include regulation of the genes for Bax, Bcl-2, the death receptor CD95 (Fas/APO-1), and the mdm2 proto-oncogene (32–36).
Up-regulation of pro-apoptotic Bax and down-regulation of anti-apoptotic Bcl-2 sensitizes certain cell types to apoptotic stimuli (32,37,38). γ-irradiation of mice leads to p53-dependent up-regulation of Bax and apoptosis only in T lymphocytes and epithelial cells of the small intestine (39). In contrast to Bax, Mdm2 inhibits p53-mediated apoptosis through binding to the N-terminal transactivation domain (40,41). p53 is involved in apoptosis of cell types other than thymocytes and epithelia. Fibroblasts from p53 knock-out mice are resistant to various DNA-damaging agents compared with wild-type (42). Rapid stabilization of p53 is associated with the high sensitivity of DNA repair-deficient xeroderma pigmentosum (XP) and Cockayne’s syndrome (CS) fibroblasts to certain DNA-damaging agents (43,44).
In this study, we investigated the factors that increase the sensitivity of fibroblasts to apoptosis. We have found that factors that affect transcription sensitize fibroblasts to apoptosis, including transcription inhibitors and genetic changes that affect transcription-coupled DNA repair. We discuss the possibilities that apoptosis might be triggered by either direct effects of blocked polymerase on p53 and/or faulty expression of downstream effectors. Understanding the mechanisms by which apoptosis can be triggered in recalcitrant cells could greatly help in pathological situations in which therapeutic approaches are aimed at triggering apoptosis.
Cells, Chemicals, and Treatments
Human primary cells
Antibodies, Gel Electrophoresis, and Immunodetection
Harvested cells were lysed by heating to 95°C for 5 min in SDS electrophoresis loading buffer containing 1 mM CaCl2. Equal quantities of protein, determined by the Biorad protein assay, were electrophoresed on SDS gels and transferred to nitrocellulose membranes which were probed with appropriate antibodies followed by enhanced chemiluminescence (ECL) reagents (Amersham). Western blots were reprobed with anti-TBP monoclonal antibodies (3G3, Y. Lutz, IGBMC) to verify equal loading. The different antibodies used were DO-1 (p53; 45), 826PAb (Mdm2, rabbit serum containing antibodies against N-terminal 220 amino acids of human Mdm2), 590PAb and 592PAb (p21WAF1,Cip1, purified rabbit antibodies recognizing p21WAF1,Cip1 synthetic peptides amino acids 81–98 and 131–148, respectively), N20 (Bax, Santa Cruz Biotechnology) and C-20 (CD 95 [Fas/APO-1], Santa Cruz Biotechnology). Apoptosis-connected proteolytic cleavage of poly-(ADP ribosylase) polymerase (PARP; 46) was detected with aF2 polyclonal antibodies that recognize full-length PARP and its 25-kDa N-terminal proteolytic fragment (kindly provided by G. de Murcia). A 23-kDa proteolytic fragment of an unknown 42-kDa protein, which is abundant in fibroblasts but undetectable in lymphoblasts, was recognized by the 592PAb antibodies. Proteolytic cleavage of the 42-kDa protein was found to be a marker of apoptosis from studies in CHP234 neuroblastoma cells, where it occurred at the same time as PARP cleavage (data not shown), and from comparisons with other criteria of apoptosis in fibroblasts (morphology, fluorescence-activated cell sorting [FACS] scanning and cleavage of chromosomal DNA, data not shown).
DNA Extraction and Cell Viability Assays
Cells used for extraction of low- to medium-molecular-weight DNA that appears during apoptosis were washed with phosphate-buffered saline (PBS) and permeabilized with DNA extraction buffer (47). Extracted extrachromosomal DNA was treated with phenol/chloroform, ethanol precipitated, separated in 1% agarose gels, and stained with ethidium bromide.
Drug-induced changes in cell viability were determined by the Triazolyl blue assay (MTT assay; 48). Relative cell viability that reflected the number of living cells was calculated as the ratio of (A570:A630) at different times to time zero. Average values from two independent experiments were plotted against time.
Primary Lymphoblasts and Fibroblasts Differ in Their Sensitivity to DNA Damage
Apoptosis Induced by DNA Damage Is Faster in Cells with Deficiencies in Transcription-Coupled DNA Repair
Transcription Inhibitors Induce Apoptosis of Normal Fibroblasts
Altered Expression of Downstream Effectors of p53 Is Associated with Induction of Apoptosis in Normal Fibroblasts
Transcription inhibitors may affect the expression of genes that are normally stimulated by p53 in response to DNA damage. Western blots were used to analyze the expression of p21WAF1,Cip1, Mdm2 (Fig. 3A), Bax, and Fas/APO-1 (not shown). Cisplatin induced rapid accumulation of p53, closely followed by p21WAF1,Cip1, and later Mdm2 (Fig. 3A, see untreated samples for the zero time points for all samples). In contrast, actinomycin D, which also rapidly induced p53, had only a small and barely detectable effect on p21WAF1,Cip1 levds and stimulated Mdm2 with delayed kinetics. α-Amanitin induced p53 and p21WAF1,Cip1 with delayed kinetics and Mdm2 levels hardly varied. DRB produced a transient burst of p53 followed by a slight increase in p21WAF1,Cip1 and linle change in Mdm2. Bax and Fas/APO-1 levels were not altered by any of the treatments (data not shown). These results correlate induction of apoptosis by transcription inhibitors with altered expression of the effectors p21WAF1,Cip1 and Mdm2.
Transcription-Coupled DNA Repair Deficiency Alters the Response to Transcription Inhibitors
We have examined an important question relating to both normal cellular functions and the treatment of pathologies such as cancer: what makes a cell more or less sensitive to induction of apoptosis. We have found an interesting link between defects in transcription and induction of apoptosis. Furthermore, we found that inhibitors of transcription induce accumulation of p53.
p53 protein levels are usually low in normal cells and increase in response to various stress signals. The best characterized signal is DNA damage of various forms (4); others are hypoxia (50) and depletion of ribonucleotide triphosphate pools (11). We show that p53 levels rise in response to transcription inhibitors and cell death is associated with prolonged induction of p53, as would be expected for p53 monitoring of correct transcription that is required for normal progression through the cell cycle.
Normal B lymphoblasts are more sensitive than primary fibroblasts to the DNA-damaging agents mitomycin C and cisplatin, which trigger rapid accumulation of p53 and apoptosis. Proliferating EBV-transformed B cells are known to be sensitive to DNA-damaging agents but growth-stimulated primary B lymphoblasts are apparently resistant to cisplatin treatment (51,52). The B lymphoblasts that were used in our experiments were not transformed with EBV but still readily underwent apoptosis after challenge with mitomycin C. Their sensitivity to DNA-damaging drugs could be connected with their inability to induce p21WAF1,Cip1 expression (see below).
Primary fibroblasts with defined genetic alterations in DNA repair pathways helped us to identify factors that participate in induction of apoptosis. The resistance of fibroblasts to apoptosis is not simply related to the extent of DNA damage. XPC fibroblasts, which are deficient in repair of transcriptionally silent DNA but not in transcription-coupled DNA repair, are as resistant to DNA damage induced apoptosis as normal fibroblasts. These results are in apparent contradiction with their poor clonogenic survival after UV treatment (43). A possible explanation is that unrepaired DNA maintains higher levels of p53 and extended growth arrest, which prevents colony formation but not survival. XPD, XPG, and CS fibroblasts, which are deficient in transcription-coupled DNA repair, underwent rapid apoptosis when treated with mitomycin C or cisplatin. Apoptosis may be triggered by the persistence of damage to essential genes or by the failure to restore RNA synthesis (43,44). CS fibroblasts are deficient in transcription (53–55) as well as transcription-coupled DNA repair (56,57), both of which may influence their response to DNA-damaging agents. Defects in transcription may signal cell death in the absence or presence of DNA damage and may also have effects through altered expression of downstream targets of p53.
The kinetics of p53 induction and expression of its target genes p21WAF1,Cip1, mdm2, Bax, and Fas/APO-1 were compared in treated cells. Bax and Fas/APO-1 have been shown to be p53-inducible in ML-1 and BRK cells (Bax; 38,58) and in an adenocarcinoma cell line (Fas; 33). However, their levels did not changed in any of the fibroblasts strains and most probably did not contribute to induction of apoptosis. We observed that sensitivity to apoptosis of fibroblasts and lymphoblasts was associated with prolonged induction of p53 often accompanied by altered expression of either p21WAF1,Cip1, Mdm2, or both. There are a number of examples. Actinomycin D treatment of normal fibroblasts (Fig. 3A) leads to cell death and a rapid increase in p53 that is not accompanied by the increases in p21WAF1,Cip1 or Mdm2 seen in cells resistant to apoptosis, such as normal fibroblasts treated with cisplatin (Fig. 3A) or CSA, or CSB fibroblasts treated with actinomycin D (Fig. 4). Mitomycin C treatment of lymphoblasts induces apoptosis and p53 with no increase in p21WAF1,Cip1, whereas treatment of fibroblasts does not induce cell death but upregulates both p53 and p21WAF1,Cip1. There is a graded response to α−amanitin in that there is extensive cell death when p53 is induced strongly, but p21WAF1,Cip1 and Mdm2 are hardly detectable (CSB, Fig. 4B); there is less cell death when there is a more pronounced increase in p21WAF1,Cip1 and Mdm2 (normal fibroblasts, Fig. 3A), which is nonetheless delayed compared with normal fibroblasts treated with cisplatin which survive (Fig. 3A). Apoptosis has always been associated with an increase in p53. For example, α-amanitin stimulated p53 and apoptosis in CSB, but did not affect p53 levels nor stimulate apoptosis in CSA (Fig. 4). However, high p53 levels per se were not sufficient to induce apoptosis. The rapidity rather than the level of p53 induction was correlated with the sensitivity of repair-deficient cells to apoptosis induced by mitomycin C (see Fig. 2). Equivalent amounts of p53 were induced in cisplatin- and actinomycin D-treated CSA and CSB fibroblasts (Fig. 4), but only cisplatin induced apoptosis. These results suggest that imbalances in the relative levels of induction of p21WAF1,Cip1 and Mdm2 could affect p53’s ability to induce apoptosis. Experiments are in progress to test this hypothesis, using antisense or HPV E6 to modulate the different effectors. Interestingly, rapid down-regulation of p21WAF1,Cip1 in Baf-3 cells after interleukin 3 (IL-3) withdrawal is associated with rapid apoptosis (59). Furthermore, several recent studies have shown that the proapoptotic function of p53 is inhibited by p21WAF1,Cip1-mediated growth arrest (25,60–63) and Mdm2 overexpression (40,41). Mdm2 and p21WAF1,Cip1 regulate the p53 and Rb pathways, respectively, and imbalances in the two pathways are associated with apoptosis (4).
Inhibition of transcription might trigger apoptotic functions of p53 that do not require transcription. It is possible that transcription complexes, stalled by DNA damage or inhibitors of transcription elongation (actinomycin D and α−amanitin), recruit p53 and convert it to a proapoptotic form. p53 may be modified by components of the transcription and DNA repair machinery, such as the kinase associated with TFIIH. This working model is a framework for further experiments that explain our observations and the known properties of p53. Ljungman and Zhang (44) independently suggested that persistent transcription-blocking lesions, due to defective transcription-coupled repair, may trigger apoptosis. We show in addition that inhibitors of transcription can induce apoptosis. p53 has apoptotic functions that are distinct from transcription regulation. For example, p53 has an N-terminal proline-rich region that is required for growth suppression but not transcription (64,65). We found that actinomycin D treatment leads to apoptosis of normal but not CSA or CSB fibroblasts. Both CSA and CSB proteins interact with TFIIH (66,67), and CSB and TFIIH interact with p53 (30,31); they could affect recruitment and modification of p53.
Stabilized p53 might not always be competent to induce apoptosis. Treatment of fibroblasts with DRB resulted in a strong increase in p53 protein levels but did not lead to apoptosis. DRB inhibits TFIIH-mediated phosphorylation of the C-terminal repeats of the largest subunit of RNA polymerase II and consequently, elongation of transcription (68). p53 interacts with TFIIH, and it is conceivable that the TFIIH kinase could directly (through phosphorylation of p53) (69) or indirectly (phosphorylation of an auxiliary factor) affect the proapoptotic function of p53. SAP kinases may also influence p53-mediated apoptosis, since they can phosphorylate the N terminus of p53 (70) and are connected with the induction of apoptosis (71,72). Finally, downstream effectors of p53 could modulate its apoptotic functions: p21WAF1,Cip1, by its ability to inhibit cyclin-dependent kinases that phosphorylate p53 (73,74), and Mdm2, by complex formation (40,41). Our results, in accordance with these studies, suggest that deregulation of the expression of the p53 target genes p21WAF1,Cip1 and Mdm2, and possibly also phosphorylation of p53, could affect the ability of p53 to induce apoptosis in damaged or stressed cells.
In summary, a stressed transcriptional state in the cell may be a trigger for p53 and apoptosis in a manner similar to that in DNA damage or metabolism. Inhibitors of transcription may be useful in treatments aimed at inducing apoptosis of cells that are resistant to DNA-damaging agents.
We are grateful to Jan H. Hoeijmakers, Nicholas J. Jaspers, David P. Lane, and Gilbert De Murcia for generously providing us with cells and antibodies and to Sagar Sengupta for kind help. We thank Thierry Léveillard and Sagar Sengupta for critical reading of the manuscript, members of the Gene Medecine Department of Rhone-Poulenc Rohrer (especially Bruno Tocque, Laurent Bracco, Laurent Debussche, and Emmanuel Conseiller) for continual help and encouragement, the staff of the IGBMC facilities for their invaluable help, and various funding agencies, including: the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Centre Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Ligue Nationale Française contre le Cancer, the Ligue Régionale (Haut-Rhin) contre le Cancer, the Ligue Régionale (Bas-Rhin) contre le Cancer (the Legs Meyer), and the Bioavenir Program (Ministère de la Recherche et Ministère de l’Industrie).
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