P53-proficient Cells Research Articles

CDK2-Dependent Phosphorylation of FOXO1 as an Apoptotic Response to DNA Damage

The function of cyclin-dependent kinase 2 (CDK2) is often abolished after DNA damage. The inhibition of CDK2 plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences the survival of cells under genotoxic stress is unknown. Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival. CDK2 specifically phosphorylated FOXO1 at serine-249 (Ser249) in vitro and in vivo. Phosphorylation of Ser249 resulted in cytoplasmic localization and inhibition of FOXO1. This phosphorylation was abrogated upon DNA damage through the cell cycle checkpoint pathway that is dependent on the protein kinases Chk1 and Chk2. Moreover, silencing of FOXO1 by small interfering RNA diminished DNA damage-induced death in both p53-deficient and p53-proficient cells. This effect was reversed by restored expression of FOXO1 in a manner depending on phosphorylation of Ser249. Functional interaction between CDK2 and FOXO1 provides a mechanism that regulates apoptotic cell death after DNA strand breakage.

Ribosomal Stress Couples the Unfolded Protein Response to p53-dependent Cell Cycle Arrest

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

E1B55K-Deleted Adenovirus (ONYX-015) Overrides G1/S and G2/M Checkpoints and Causes Mitotic Catastrophe and Endoreduplication in p53-Proficient Normal Cells

In order to take advantage of cell replication machinery, viruses have evolved complex strategies to override cell cycle checkpoints and force host cells into S phase. To do so, virus products must interfere not only with the basal cell cycle regulators, such as pRb or Mad2, but also with the main surveillance pathways such as those controlled by p53 and ATM. Recently, a number of defective viruses has been produced which, lacking the latter ability, are incapable of replicating in normal cells but should be able to grow and finally lyse those cells that, such as the tumor cells, have lost their surveillance mechanisms. A prototype of these oncolytic viruses is the E1B55K-defective Adenovirus ONYX-015, which was predicted to selectively replicate and kill p53-deficient cancer cells. We found that, despite wt p53 and notwithstanding the activation of the checkpoint regulators p53, ATM, and Mad2, ONYX-015 actively replicated in HUVEC cells. Furthermore, ONYX-015 replication induced a specific phenotype, which is distinct from that of the E4-deleted adenovirus dlE4 Ad5, although both viruses express the main regulatory region E1A. This phenotype includes overriding of the G1/S and G2/M checkpoints, over-expression of MAD2 and retardation of mitosis and accumulation of polyploid cells, suggesting the occurrence of alterations at the mitotic-spindle checkpoint and impairment of the post-mitotic checkpoint. Our data suggest that viral E1A and E4 region products can override all host cell-checkpoint response even at the presence of a full activation of the ATM/p53 pathway. Furthermore, the E4 region alone seems to act independently of the E1B55K virus product in impairing the ATM-dependent, p53-independent G2/M checkpoint since dlE4 Ad5-infected cells arrested in G2 while ONYX-015-infected cells did enter mitosis.

UV Radiation Induces Delayed Hyperrecombination Associated with Hypermutation in Human Cells

Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an approximately 5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m2), revealed as mixed GFP+/- colonies. UV-B did not induce DHR at an equitoxic (75 J/m2) dose or a higher dose (150 J/m2). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were approximately 5-fold higher in strains derived from GFP+/- (DHR) colonies than in strains in which recombination was directly induced by UV (GFP+ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP+ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP+/- strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.

Tumor-specific hyperactive low-molecular-weight cyclin E isoform detection and characterization in non-metastatic colorectal tumors

Purpose: Several molecules involved in cancer biology have been studied as potential prognostic markers. Recently, overexpression of cyclin E and its low-molecular-weight (LMW) isoforms has been reported to be the most prominent prognostic marker in breast cancer, surpassing proliferation index, ploidy, and axillary nodal involvement. Furthermore, cyclin E and p53 are considered the main factors controlling the euploid equilibrium in human cells. We investigated the status of cyclin E and p53 in cell lines and tissue samples of colorectal cancer, one of the leading causes of death from a tumor in the Western world.Experimental Design: We analyzed colorectal cancer cells, from established cell lines and patient specimens, to determine the protein levels of cyclin E and p53, and to detect p53 and APC mutations, microsatellite and chromosome instability. In addition, we assessed the presence of cyclin E LMW isoforms and their enzymatic activity.Results: Colorectal cancer cells expressed hyperactive LMW forms both in vitro and in vivo. These tumor-specific isoforms are correlated to genomic instability even in p53-proficient cells, and represented a constant feature in the tumors analyzed.Conclusions: In colorectal cancer, the formation of cyclin E LMW forms is an early event leading to DNA-damage checkpoint-independent proliferation. Collectively, our results provide evidence that evaluation of LMW forms could represent a novel tool in the molecular characterization of colorectal tumors aimed at identifying sensitive prognostic factors and uncovering subsets of high-risk patients within the traditional categories.

The Interaction between Two Radiosensitizers: 5-Iododeoxyuridine and Caffeine

5-Iododeoxyuridine (IUdR) and caffeine are recognized as potential radiosensitizers with different mechanisms of interaction with ionizing radiation (IR). To assess the interaction of these two types of radiosensitizers, we compared treatment responses to these drugs alone and in combination with IR in two p53-proficient and p53-deficient pairs of human colon cancer cell lines (HCT116 versus HCT116 p53-/- and RKO versus RKO E6). Based on clonogenic survival, the three single agents (IR, IUdR, and caffeine) as well as IUdR or caffeine combined with IR are less or equally effective in p53-deficient human tumor cells compared with p53-proficient tumor cells. However, using both radiosensitizers, a significantly greater radiosensitization was found in p53-deficient human tumor cells. To better understand the interaction of these two radiosensitizers, additional studies on DNA repair and cell cycle regulation were done. We found that caffeine enhanced IUdR-DNA incorporation and IUdR-mediated radiosensitization by partially inhibiting repair (removal) of IUdR in DNA. The repair of IR-induced DNA double-strand breaks was also inhibited by caffeine. However, these effects of caffeine on IUdR-mediated radiosensitization were not found in p53-proficient cells. Cell cycle analyses also showed a greater abrogation of IR-induced S- and G2-phase arrests by caffeine in p53-deficient cells, particularly when combined with IUdR. Collectively, these data provide the mechanistic bases for combining these two radiosensitizers to enhance tumor cytotoxicity. This differential dual mode of radiosensitization by combining IUdR and caffeine-like drugs (e.g., UCN-01) in p53-deficient human tumors may lead to a greater therapeutic gain.

Signaling through IFN Regulatory Factor-5 Sensitizes p53-Deficient Tumors to DNA Damage–Induced Apoptosis and Cell Death

Human IFN regulatory factor-5 (IRF-5) is a candidate tumor suppressor gene that mediates cell arrest, apoptosis, and immune activation. Here we show that ectopic IRF-5 sensitizes p53-proficient and p53-deficient colon cancer cells to DNA damage-induced apoptosis. The combination IFN-beta and irinotecan (CPT-11) cooperatively inhibits cell growth and IRF-5 synergizes with it to further promote apoptosis. The synergism is due to IRF-5 signaling since a striking defect in apoptosis and cell death was observed in IRF-5-deficient cells, which correlated well with a reduction in DNA damage-induced cellular events. Components of this IRF-5 signaling pathway are investigated including a mechanism for DNA damage-induced IRF-5 activation. Thus, IRF-5-regulated pathways may serve as a target for cancer therapeutics.

P53 deficiency exacerbates pleiotropic mitotic defects, changes in nuclearity and polyploidy in transdifferentiating pancreatic acinar cells

In a primary culture model for pancreatic acinar-ductal transdifferentiation, cells exhibited increased proliferation, changes in nuclearity and polyploidy. We identify the 'nucleus to centrosome' ratio of the progenitor cell, the dissemination of centrosomes at spindle poles and cytokinesis failure as critical determinants of mitosis outcome and centrosome inheritance. Abortive cytokinesis of mononuclear cells contributes to the binuclear cell pool, whereas enclosure of entire mitotic formations, within a single nuclear envelope, perpetuates polyploidization. Binuclear cell nuclei combine their genomes on a single metaphase plate, doubling descendant ploidy. Moreover, approximately 42% of binuclear and tetraploid cells assemble aberrant spindles with up to 8 centrosomes/poles. These phenotypes were exacerbated in p53-deficient cultures exhibiting increased S-phase entry, giant nuclei, multinucleation, multipolar mitoses and centrosome hyperamplification. The tendency of p53-proficient cells to spontaneously evade the tetraploidy checkpoint degenerates to uncontrolled polyploid progression in p53-deficient cultures, explaining why p53 abrogation alone rapidly descends to aneuploidy in this system. We detected constitutively nuclear mdm2, which may circumvent endogenous cell-cycle checkpoints, and pronounced accumulation of p21 and p27 in multinuclear cells and giant nuclei, consistent with roles in polyploidization. This in vitro model may recapitulate the processes underlying genomic instability in pancreatic tumours in vivo, and attests to the existence of a p53-dependent polyploidy checkpoint acting to limit the degree of polyploidization.

Elimination of Hepatic Metastases of Colon Cancer Cells via p53-Independent Cross-Talk between Irinotecan and Apo2 Ligand/TRAIL

The majority of colorectal cancers have lost/inactivated the p53 tumor suppressor gene. Using isogenic human colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders tumor cells relatively resistant to the topoisomerase I inhibitor, irinotecan. Whereas irinotecan-induced up-regulation of the proapoptotic proteins PUMA and Noxa requires p53, we find that irinotecan inhibits Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 and 5 (STAT3/5) signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan inhibits JAK2-STAT3/5-dependent expression of survival proteins (Bcl-x(L) and XIAP) and cooperates with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. Although the synergy between chemotherapy and Apo2L/TRAIL has been ascribed to p53, our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of tumor cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 signaling. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against both p53-proficient and p53-deficient colorectal cancers, including those that have metastasized to the liver.

P53 Prevents the Accumulation of Double-Strand DNA Breaks at Stalled-Replication Forks Induced by UV in Human Cells

To investigate the mechanism by which UV irradiation causes S-phase-dependent chromosome aberrations and thereby genomic instability, we have developed an assay to study the DNA structure of replication forks (RFs) in UV-irradiated mammalian cells, using pulse-field gel electrophoresis for the DNA analysis. We demonstrate that replication stalling at UV-induced pyrimidine dimers results in the formation of single-strand DNA (ssDNA) regions and incomplete RF structures. In normal and in excision-repair-defective xeroderma pimentosum (XP) cells, stalling at dimers is rapid and prolonged and recovery depends on dimer repair or bypass. By contrast, XP variant (XPV) cells, defective in replication of a UV-damaged template due to mutation of bypass-polymerase ?, fail to arrest at dimers, resulting in a much higher frequency of ssDNA regions in the stalled RFs. We show that the stability of UV-arrested RFs depends directly on functional p53, and indirectly on NER and pol ?. In p53-deficient cells, the stalled sites give rise to double-strand DNA breaks (DSBs), at a frequency inversely correlated with repair capacity of the cell. In normal cells only a fraction of the stalled sites give rise to DSBs, while in XPASV, XPDSV and also XPVSV all the sites do. XPVSV cells, although repair proficient, accumulate almost double the number of DSBs, suggesting that a high frequency of ssDNA regions in UV-arrested forks cause RF instability. These replication-associated DSBs do not accumulate in p53-proficient human cells. We propose that a major mechanism by which p53 maintains genome stability is the prevention of DSB accumulation at long-lived ssDNA regions in stalled-replication forks.Supplemental material for this paper can be found at the following link: http://www.landesbioscience.com/journals/cc/squiresCC3-12-sup.pdf

Induction of p21 and nuclear accumulation of TAp73α and c-abl during apoptosis of cisplatin-treated primary pancreatic acinar cells

To understand the role of endogenous p53 and related proteins in pancreatic injury responses, we established primary pancreatic acinar cultures from wild-type and p53-deficient mice and investigated the relationship between apoptosis, proliferation and underlying molecular events in cells exposed to the DNA cross-linking agent cisplatin. This treatment led to a time-dependent elevation in p53 levels, accompanied by phosphorylation at key serine residues. Despite this apparent activation of p53, acinar cells entered growth arrest unaffected by p53 deficiency. Moreover, p53-null cells exhibited only a temporal delay in engaging apoptosis, compared to wild-type counterparts. Whilst p53-proficient cells rapidly accumulated nuclear p21, the kinetics of p21 accumulation in p53-null cells were delayed, correlating with the execution of p53-independent apoptosis. During the course of treatment, c-abl and TAp73alpha, a p53 homologue, accumulated in acinar cell nuclei, irrespective of genotype, indicating that they are induced upon DNA damage and that they may act in parallel or in concert with p53 for the eradication of damaged acinar cells. We also report the nuclear accumulation of c-abl and TAp73alpha in cells, treated with the nuclear export inhibitor leptomycin B, suggesting that these proteins undergo constant nucleocytoplasmic shuttling in normal culture conditions, possibly reflecting a role for TAp73alpha-mediated transactivation or repression in the regulation of in vitro acinar cell growth.

Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition.

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G(1) arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.

Relationship between the radiosensitizing effect of wortmannin, DNA double-strand break rejoining, and p21WAF1 induction in human normal and tumor-derived cells.

Wortmannin (WM) is a potent inhibitor of the catalytic sub-unit of DNA-PK, which is involved in one pathway of DNA double-strand break (DSB) rejoining, and of ATM, which functions upstream in the p53 signaling pathway. WM is known to be an efficient radiosensitizer in a variety of mammalian cell types, to inhibit DSB rejoining following exposure to supralethal doses (> or =30 Gy) of ionizing radiation, and to abrogate the induction of p53 at early times after radiation exposure. We report here that WM is a more effective radiosensitizer in A549 human lung carcinoma cells than in normal human fibroblasts (NHFs). In addition, WM strongly inhibits DSB rejoining in A549 cells exposed to relatively low doses (e.g., 10 Gy) of ionizing radiation, without having any detectable effect in NHFs. We further demonstrate that WM significantly potentiates the induction of p21WAF1, a p53-regulated gene that encodes for a key mediator of cell-cycle/growth arrest, when determined at late times (e.g., 24 h) after irradiation. This late WM-dependent potentiation of p21WAF1 induction following radiation exposure is observed in NHFs and in the p53 wild-type tumor cell lines A549, A172, and SKNSH, but not in the p53-deficient tumor cell lines DLD-1, HeLa, and SKNSH-E6. We conclude that: (i) inhibition of DSB rejoining by WM may be an important contributor to its radiosensitizing effect in A549 cells but not in NHFs; and (ii) radiosensitization of p53-proficient human cells by WM may in part be associated with the delayed induction of p21WAF1, which can lead to a sustained growth-arrested phenotype resembling senescence.

P53 protein regulates the effects of amifostine on apoptosis, cell cycle progression, and cytoprotection

To determine the role of p53 protein on the cellular effects of amifostine, we used molecularly engineered HCT116 colon cancer cells in which the p53 gene was inactivated by targeted homologous recombination or p53 protein was degraded by high-level expression of papillomavirus E6 protein. Amifostine induced a G1 arrest and protected against paclitaxel toxicity in p53-proficient but not in p53-deficient cells. In the absence of p53 protein, amifostine enhanced the cytotoxicity of paclitaxel. In addition, treatment of HCT116 cells with amifostine alone resulted in apoptotic cell death. Compared with p53-deficient cells, p53-proficient cells exhibited low-level resistance to amifostine-induced apoptosis. Amifostine induced the expression of p53 protein in p53-proficient cells and the expression of p21 protein in both p53-proficient and -deficient cells. These findings indicate that amifostine-induced G1 arrest and cytoprotection are mediated via a pathway that is dependent on p53 protein and that amifostine-induced expression of p21 protein is not sufficient to sustain a G1 arrest or to mediate cytoprotection. In addition, these findings identify p53 protein as a mechanism of resistance to amifostine-induced apoptosis.

Radiation-induced Phosphorylation of Chk1 at S345 is Associated with p53-dependent Cell Cycle Arrest Pathways

Because DNA damage -inducible cell cycle checkpoints are thought to protect cells from the lethal effects of ionizing radiation, a better understanding of the mechanistic functions of cell cycle regulatory proteins may reveal new molecular targets for cancer therapy. The two major regulatory proteins of G2 arrest are Chk1 and p53. Yet, it is unclear how these two proteins interact and coordinate their functional roles during radiationinduced G2 arrest. To determine Chk1's role in p53dependent G2 arrest, we used p53 proficient cells and examined expression of G2 arrest proteins under conditions in which G2 arrest was inhibited by the staurosporine analog, UCN-01. We found that UCN-01 inhibited both G1 and G2 arrest in irradiated p53 proficient cells. The arrest inhibition was associated with suppression of radiation-induced expression of both p21 and 14-3-3σ — two known p53-dependent G2 arrest proteins. The suppression occurred despite normal induction of p53 and normal phosphorylation of p53 at S20 and Cdc25C at S216 — the two known substrates of Chki kinase activity. In contrast, we showed that radiation-induced phosphorylation of Chki at S345 was associated with binding of Chki to p53, p21, and 14-3-3σ, and that UCN-01 inhibited S345 phosphorylation. We suggest that DNA damage-induced phosphorylation of Chki at S345, and subsequent p53 binding, links Chki with p53 downstream responses and may provide a coordinated interaction between DNA damage responses and cell cycle arrest functions.

The relationship between radiation-induced G(1)arrest and chromosome aberrations in Li-Fraumeni fibroblasts with or without germline TP53 mutations.

We previously showed that cultured fibroblasts from patients with the cancer-prone Li-Fraumeni (LF) syndrome, having heterozygous germline TP53 mutations, sustain less ionizing radiation-induced permanent G1 arrest than normal fibroblasts. In contrast, fibroblast strains from LF patients without TP53 mutations showed normal G1 arrest. We have now investigated the relationship between the extent of G1 arrest and the level of structural chromosome damage (mainly dicentrics, rings and acentric fragments) in cells at their first mitosis after G1 irradiation, in 9 LF strains with TP53 mutations, 6 without TP53 mutations and 7 normal strains. Average levels of damage in the mutant strains were 50% higher than in normals, whereas in non-mutant LF strains they were 100% higher. DNA double strand breaks (dsb) are known to act as a signal for p53-dependent G1 arrest and to be the lesions from which chromosome aberrations arise. These results suggest that a minimal level of dsb is required before the signal for arrest is activated and that p53-defective cells have a higher signal threshold than p53-proficient cells. Dsb that do not cause G1 blockage can progress to mitosis and appear as simple deletions or interact to form exchange aberrations. The elevated levels in the non-mutant strains may arise from defects in the extent or accuracy of dsb repair. In LF cells with or without TP53 mutations, the reduced capacity to eliminate or repair chromosomal damage of the type induced by ionising radiation, may contribute to cancer predisposition in this syndrome. © 2001 Cancer Research Campaign http://www.bjcancer.com

Transcriptional Regulation of the TFIIH Transcription Repair Components XPB and XPD by the Hepatitis B Virus x Protein in Liver Cells and Transgenic Liver Tissue

Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.

The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations.

The effect of caffeine was studied on the radioresponses of undifferentiated mouse embryonal carcinoma cells (EC cells) with or without the functional p53. The radioresponses studied included radiosensitivity, the activation of p53, apoptosis with characteristic DNA ladder formation and cell cycle progression. An undifferentiated mouse EC cell line, ECA2, and a newly established p53-deficient EC cell line, p53 delta, were used in the present study. The status of the p53 gene did not significantly affect the colony survivals of undifferentiated EC cells to X-rays and UV. Although a post-irradiation treatment with caffeine sensitized both lines to X-rays marginally, the sensitization was prominent for UV regardless of the p53 status of the cells. The activation of a p53 responsible lacZ reporter construct was observed in stably transfected ECA2 cells after X-ray and UV irradiations. Caffeine suppressed the X-ray induced activation of the lacZ reporter, while it drastically enhanced the activation after UV irradiation. X-rays and UV readily triggered the apoptosis of ECA2 cells with the characteristic DNA ladder. Although UV-induced DNA ladder formation was enhanced by caffeine, that induced by X-rays was unaffected. Therefore, the effects of caffeine on the p53-dependent radioresponses were found to be agent specific: suppression for the X-ray induced and augmentation for the UV induced. In contrast to p53-proficient ECA2 cells, smear-like DNA degradation was observed for irradiated p53 delta cells, suggesting the presence of a mode of cell death without DNA ladder formation. UV induction of the smear-like DNA degradation was enhanced in the presence of caffeine. Regardless of the state of the p53 gene, G1/S arrest was not observed in X-ray and UV irradiated EC cells. X-ray induced G2/M arrest in both lines, which was abrogated by caffeine, while G2/M arrest after UV was unaffected by a caffeine treatment. These results indicate that the radioresponses of undifferentiated EC cells differ considerably from those of somatic cells, and that these radioresponses were modulated by a post-irradiation treatment with caffeine.

HBV transcription repression in response to genotoxic stress is p53-dependent and abrogated by pX

Transcription of hepatitis B Virus (HBV), an important risk factor of hepatocellular carcinoma (HCC), is controlled by cellular transcription activators including some of the cellular signaling targets. Consequently, HBV transcription rate changes in response to the cellular physiological conditions. In this report we investigated HBV gene expression and the role of physiological levels of the viral X protein (pX) under cisplatin induced genotoxic stress. We show that under these conditions the RNA level of an HBV mutant which does not express pX is sharply reduced. Studies revealed that transcription repression is responsible for the observed reduction in HBV RNA level. Repression of HBV transcription was obtained only in the p53 proficient cells. Furthermore, HBV transcription rate is recovered by the cotransfected p53 dominant negative plasmid, indicating that p53 is directly responsible for HBV transcription repression. Unexpectedly, p73, the recent p53 homologue, does not repress but rather activates HBV transcription. Interestingly, pX produced either by the HBV genome or by a cotransfected plasmid, relieves the p53 mediated repression. Collectively, these results attribute a physiological role to p53-inactivation by pX, and explain how pX may support HCC development.

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells.

Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV-HBx reporter plasmid and the pCMV-CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in approximately 50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and approximately 25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.