Determination of cell fate by c-Abl activation in the response to DNA damage

The cellular response to DNA damage includes growth arrest and activation of DNA repair. Certain insights into how DNA damage is converted into intracellular signals that control the genotoxic stress response have been derived from the finding that the c-Abl protein tyrosine kinase is activated by ionizing radiation and other DNA-damaging agents. c-Abl associates with the DNA-dependent protein kinase (DNA-PK) and is activated by DNA-PK-dependent phosphorylation. The ataxia telangiectasia mutated (ATM) gene product also contributes to c-Abl activation. The demonstration that c-Abl binds to p53, induces the transactivation function of p53 and activates p21 expression has supported involvement of c-Abl in regulation of the p53-dependent G1 arrest response. Interaction between c-Abl and the Rad51 protein has also provided support for involvement of c-Abl in recombinational repair of DNA strand breaks. Defects in G1 arrest and repair predispose to replication of damaged templates and, in the event of irreparable DNA lesions, induction of apoptosis. The available evidence indicates that c-Abl effects a proapoptotic function by a mechanism largely independent of p53. c-Abl also functions as an upstream effector of the proapoptotic JNK/SAPK and p38 MAPK pathways. In addition, c-Abl-dependent inhibition of PI 3-kinase contributes to the induction of apoptosis. The findings thus suggest that, in response to genotoxic stress, c-Abl functions in determining cell fate, that is growth arrest and repair or induction of apoptosis. The physiologic function of c-Abl may reside in control of the cellular response to DNA strand breaks that occur during DNA replication, genetic recombination and gene rearrangements.     

Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage

Through a differential screening technique, we have identified a cDNA clone with differential expression in normal versus tumor cells. This clone, designated rit42 (reduced in tumor, 42 kDa), was previously isolated as a homocysteine-inducible gene in human endothelial cells (RTP), and the same or a highly related androgen-responsive gene in mouse has also been identified. Both Northern blot analysis and in situ hybridization demonstrated a significantly diminished expression in tumor cells, including those derived from breast and prostate when compared with normal cells. It was shown that RTP/rit42 mRNA cycles with cell division, peaking at G1 and G2-M, with lower expression in S phase. The biphasic expression of RTP/rit42 mRNA was absent in tumor cells. Introduction of rit42 cDNA into human cancer cells reduced cell growth both in vitro and in nude mice. Moreover, analysis of a tetracycline-regulated p53-inducible system in null-p53 cell lines showed that RTP/rit42 mRNA expression increased concomitantly with p53 expression and followed a similar time course. In addition, DNA-damaging agents induced RTP/rit42 expression in a p53-dependent manner but independent of a p53-mediated G1 arrest. Immunofluorescence analysis of a FLAG epitope-tagged RTP/rit42 protein revealed a cytoplasmic localization pattern with redistribution to the nucleus upon DNA damage. We have localized RTP/rit42 to human chromosome 8q24.3. Taken together, these results are consistent with a growth inhibitory role for RTP/rit42, and its down-regulation may contribute to the tumor malignant phenotype.     

Cell cycle re-entry following chemically-induced cell cycle synchronization leads to elevated p53 and p21 protein levels

Mimosine (MIM) and aphidicolin (APH) are two agents frequently used in tissue culture-based experiments to achieve cell synchronization at late G1 and S phases. Following MIM or APH treatment of human cancer cell lines, a reversible growth arrest in late G1 and S phases of the cell cycle was correlated with moderate increases in p53 and p21 protein levels. Both p53-dependent and -independent increases in p21 were observed following treatment with either agent. However, a striking increase in p21 protein levels and a continuous elevation in both p53 and p21 protein levels were observed over 48 h after cells re-entered the cell cycle following the chemically-induced synchronization. In addition, the increase in p21 protein levels typically seen following treatment of cells with DNA damaging agents, was enhanced when cells were treated with genotoxic agents following MIM or APH synchronization. These findings suggest that caution should be exercised when interpreting results from experiments using cell synchronization agents, in particular, studies designed to investigate p53- and p21-regulatory pathways.     

Spontaneous apoptosis in human colon tumor cell lines and the relation of wt p53 to apoptosis

OBJECTIVE: To examine spontaneous apoptosis of cultured human colon tumor cell lines in vitro and to investigate the role of wild type (wt) p53 in regulation of apoptosis induced by DNA-damaging treatment. METHODS: A model system of human tumor progression involving three cell lines was used in this study for examination of apoptosis. They were originally established from human colon villous adenoma, including an early passage of non-tumorigenic cell line, V235E; a late passage of weakly tumorigenic cell line, V235L; and a spontaneous progressing highly tumorigenic cell line. V411. All of them maintain wt p53 expression. For identification of apoptosis, two tests were performed: 1. morphology study using acridine orange (AO)/ethidium bromide (EB) stainning by fluorescence microscopy; 2. DNA electrophoresis on agarose gel. P53 and WAF-1 (a downstream gene of p53) expressions were analysed at mRNA level using Northern blot technique. Apoptotic index of cell lines examined was measured by DNA fluorescence assay. RESULTS: Spontaneous apoptosis was demonstrated in cell lines of all stages of progression by both morphology and DNA agarose gel electrophoresis. Apoptosis was further induced in V411 after treatment of cells with 137Cs gamma-irradiation and accompanied by increases in p53 and WAF-1 expression. In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen. CONCLUSIONS: Apoptosis can be maintained in some human tumor cell lines despite transformation and carcinogenesis. Wt p53 and WAF-1 products are two of the potential mediators which effect apoptosis. Additionally, since apoptosis was enhanced by irradiation in V411, but not in sw480, it suggests that wt p53 cancer cells are more sensitive to DNA-damaging treatment than mutant p53 cancer cells. These finding may have implications for cancer therapy.     

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.     

Induction of BCL2 family member MCL1 as an early response to DNA damage

When ML-1 human myeloid leukemia cells are exposed to DNA damaging agents, they exhibit dramatic changes in the expression of a variety of gene products. This includes an increase in p53 (wild-type), a decrease in BCL2, a p53-dependent increase in the BCL2 family member BAX, and increases in Growth Arrest and DNA Damage-inducible (GADD) genes such as GADD45; these changes occur as early events in a sequence that culminates in DNA damage-induced apoptosis. DNA damaging agents have now been tested for effects on expression of another BCL2 family member, MCL1, a gene expressed during ML-1 cell differentiation. Expression of MCL1 was found to increase upon exposure of ML-1 cells to various types of DNA damaging agents, including ionizing radiation, ultraviolet radiation, and alkylating drugs. The increase in MCL1 occurred rapidly and was transient, levels of the MCL1 mRNA being elevated within 4 h and having returned to near baseline within 24 h. An increase in the Mcl1 protein was also seen, with the maximal increase occurring at an intermediate dose of IR (5 Gray) and lesser increases occurring at either lower or higher doses. The increase in expression of MCL1 was further studied using a panel of human cell lines that includes cells containing or not containing alterations in p53 as well as cells sensitive or insensitive to the apoptosis-inducing effects of DNA damage. The DNA damage-induced increase in MCL1 mRNA did not depend upon p53 as it was seen in cells lacking functional p53. However, the increase did depend upon susceptibility to apoptosis as it was not seen in cells insensitive to apoptosis-induction by DNA damaging agents. These findings demonstrate that cytotoxic DNA damage causes an increase in the expression of MCL1 along with increases in GADD45 and BAX and a decrease in BCL2. Furthermore, while the increase in GADD45 is seen both in cells that undergo growth arrest and in cells that undergo apoptosis in response to DNA damage, alterations in the profile of expression of BCL2 family members occur exclusively in cells that undergo the apoptotic response, with some family members increasing through p53-dependent (BAX) and others through p53-independent (MCL1) pathways. Overall, expression MCL1 can increase during the induction of cell death as well as during the induction of differentiation.     

A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage

The tumor suppressor gene p53 encodes a nuclear phosphoprotein which is critical for cell cycle control and prevention of uncontrolled cell proliferation that can lead to cancer. Previous studies have shown that cells respond to DNA damage by increasing their levels of p53, which then acts to prevent replication of damaged DNA. This study examined the effects on p53 protein levels of several different categories of chemical carcinogens. N-Methyl-N'-nitro-nitrosoguanidine and N-ethyl-N-nitrosourea, two direct-acting genotoxic (DNA-reactive) carcinogens, caused p53 induction as early as 2 h following treatment, with peak increases within 4-12 h. Aflatoxin B1 and 2-acetylaminofluorene, indirect-acting genotoxic carcinogens, caused a later induction of p53, with the peak increase appearing between 16 and 24 h following treatment. These observations demonstrate a correlation between p53 induction pattern and DNA damaging mechanism of genotoxins. Phenol, diethylstilbestrol and ethylacrylate also induced increases in cellular p53. The half-life of p53 protein was increased in cells treated with genotoxic agents. On the other hand, the epigenetic (non-DNA-reactive) carcinogens azathioprine and saccharin, as well as two substances generally considered to be non-carcinogens, dimethylsulfoxide and benzethonium chloride, had no effect on p53 protein levels of treated cells. Measurement of the cytotoxic effects of each of these chemicals led to the conclusion that p53 protein induction is not a general, non-specific consequence of the cytotoxic effect of these genotoxins. These results suggest that measurement of p53 protein induction may be an effective tool to identify environmental genotoxins.     

A general role for c-Fos in cellular protection against DNA-damaging carcinogens and cytostatic drugs

One of the earliest responses of cells upon exposure to DNA-damaging agents is the induction of c-fos. To elucidate the biological role of Fos expression, we analyzed cells deficient in c-Fos upon treatment with different DNA-damaging agents, including carcinogens and antineoplastic drugs. We show that cells lacking c-Fos are hypersensitive with regard to reproductive cell death, apoptosis, and chromosomal breakage after treatment with agents inducing methylation lesions, bulky adducts, or crosslinks in DNA. They were not significantly hypersensitive to ionizing radiation. The activities of various repair enzymes and glutathione S-transferase and the level of proliferating cell nuclear antigen were not altered in c-fos-/- fibroblasts. Furthermore, the cells were able to remove the main methylation lesions from DNA. c-Fos-deficient cells exhibited a more severe mutagen-induced block to DNA replication and were compromised in the abolition of replication blockage. The data provide compelling evidence that c-Fos/activator protein-1 plays a decisive and general role in cellular defense against genotoxic agents, which require DNA replication to induce chromosomal instability. They are consistent with the hypothesis that impaired recovery from DNA replication inhibition upon mutagen exposure is causally involved in c-fos-/- hypersensitivity.     

Tumor necrosis factor-induced apoptosis stimulates p53 accumulation and p21WAF1 proteolysis in ME-180 cells

Tumor necrosis factor (TNF)-mediated apoptotic signaling has been characterized by activation of specific protease or protein kinase cascades that regulate the onset of apoptosis. TNF has also been shown to induce oxidative or genotoxic stress in some cell types, and apoptotic potential may be determined by the cellular response to this stress. To determine the role of genotoxic stress in TNF-mediated apoptosis, we examined cellular accumulation of p53 in TNF-treated ME-180 cells selected for apoptotic sensitivity (ME-180S) or resistance (ME-180R) to TNF. Although TNF was able to activate receptor-mediated signaling in either cell line, p53 accumulation was measurable only in apoptotically sensitive ME-180S cells. TNF-induced changes in p53 levels were detected 1 h after treatment, and peak levels were measurable 4-8 h after TNF exposure. TNF was unable to induce p21WAF1 in either cell line but affected the stability of this protein in apoptotically responsive ME-180S cells. Evidence of p21WAF1 proteolysis was detected by monitoring the appearance of a 16-kDa immunoblottable p21WAF1 fragment, which became detectable 4 h after TNF addition and increased in content before the onset of DNA fragmentation (16-24 h). The kinetics of p21WAF1 proteolysis closely paralleled those of poly(ADP-ribose) polymerase, suggesting cleavage of p21WAF1 by activation of an apoptotic protease. Pretreatment of ME-180S cells with the apoptotic protease inhibitor YVAD blocked TNF-induced apoptosis and prevented both poly(ADP-ribose) polymerase and p21WAF1 degradation but did not affect p53 induction. These results provide evidence for the early onset of genotoxic stress in cells committed to TNF-mediated apoptosis and for divergence in propagation of this signal in non-responsive cells. In addition, TNF-induced p21WAF1 proteolysis may be mediated by an apoptotic protease and may contribute to the apoptotic process by disrupting p53 signaling, altering cell cycle inhibition, and limiting cellular recovery from genotoxic stress.     

Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage

It is now well established that in response to genotoxic stresses mammalian cells show an increased p53 protein levels and undergo cell cycle arrest at G1/S and G2/M checkpoints. But, the consequences of these cell cycle arrests on cell survival are not yet elucidated. In this study, we have analysed the relationships between p53 protein induction, cell cycle arrest and cell survival following exposure of normal human fibroblasts (NHFs) to various genotoxic agents such as cisplatin, UV radiation and gamma radiation. p53 protein accumulation and G2/M arrest arose at the same time following exposure to DNA damaging agents, suggesting that p53 is responsible for the G2/M block. However, following inhibition of p53 induction by an antisense oligonucleotide, this G2/M arrest is even more important and correlates with an enhanced sensitivity of NHFs to UV radiation. In addition, there appears to be a threshold in the response of NHFs to DNA damaging agents, p53 induction and cell cycle arrest being observed only with lethal UV doses. We show that: 1) there appears to be a threshold in the cellular response to genotoxic agents, below which neither p53 induction, nor cell cycle arrest, nor cell survival alteration occur and beyond which p53 induction is accompanied by cell cycle arrest and decreased cell survival; 2) although there is a tight temporal relationship, the onset of which depends of the DNA damaging agent used, between the start of p53 induction and the occurrence of G2/M arrest, this latter is independent of p53; 3) p53 inhibition enhances NHFs' sensitivity to DNA damaging agents, the extent of the G2/M arrest correlating with decreased cell survival. Finally, the lack of obligatory correlation between p53 inactivation, apoptosis and radio- or chemoresistance is discussed.     

Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells were observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb. G2 arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.     

Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts

The occurrence of DNA double strand breaks induces cell cycle arrest in mortal and immortal human cells. In normal, mortal fibroblasts this block to proliferation is permanent. It depends on the growth regulator p53 and a protein p53 induces, the cyclin dependent kinase inhibitor, p21. We show here that following DNA damage in mortal fibroblasts, the induction of p21 and p53 is to a large degree shortlived. By 8 days after a brief exposure to DNA strand breaking agents, bleomycin or actinomycin D, p53 protein is at baseline levels, while the p53 transactivation level is only slightly above its baseline. By this time the concentration of p21 protein, which goes up as high as 100-fold shortly after treatment, is down to just 2-4-fold over baseline levels. Following the drop in p21 concentration a large increase in the expression level of the tumor suppressor gene p16INK4a is observed. This scenario, where a transient increase in p21 is followed by a delayed induction of p16INK4a, also happens with the permanent arrest that occurs with cellular senescence. In fact, these cells treated with agents that cause DNA double strand breaks share a number of additional markers with senescent cells. Our findings indicate that these cells are very similar to senescent cells and that they have additional factor(s) beside p21 and p53 that maintain cell cycle arrest.