FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Prost, S.
Right arrow Articles by Harrison, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Prost, S.
Right arrow Articles by Harrison, D. J.
(The FASEB Journal. 1998;12:181-188.)
© 1998 FASEB

Altered DNA repair and dysregulation of p53 in IRF-1 null hepatocytes

S. Prosta,1, C. O. C. Bellamya, D. S. Cunninghama and D. J. Harrisona

a CRC Laboratories, Department of Pathology, University Medical School, Edinburgh EH8 9AG, Scotland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor proteins IRF-1 and p53 are involved in response pathways after DNA damage. In different cell types, IRF-1 and p53 can cooperate to produce cell cycle arrest (embryo fibroblasts) or can independently trigger apoptosis (lymphoid cells). p53 may also regulate DNA repair, but there is no information on IRF-1 and repair. The cell lineage dependency of these effects precludes extrapolation of findings to other tissues of relevance to human cancer. Here, we report the consequences of IRF-1 deficiency for apoptosis, cell cycle arrest, and DNA repair in primary hepatocytes after DNA damage and extend previous work on the role of p53 in hepatocytes. IRF-1-deficient hepatocytes showed reduced DNA repair activity compared with wild-type, as assessed by unscheduled DNA synthesis after UV irradiation (10J/m2) and by host reactivation of a UV-damaged reporter construct. p53-deficient hepatocytes also showed reduced unscheduled DNA synthesis after UV, but there was no impairment of specific repair in host reactivation assays. IRF-1 deficiency did not affect the p53-dependent G1/S arrest after UV irradiation. Hepatocyte apoptosis after UV treatment, previously reported to be independent of p53, was also independent of IRF-1. However, IRF-1 deficiency produced dysregulation of p53, manifested as increased transactivation of a p53-reporter plasmid in undamaged hepatocytes, and accelerated p53 stabilization after DNA damage. Hence, in hepatocytes, IRF-1 is not required for growth arrest or apoptosis after DNA damage, but the results suggest for the first time a role in DNA repair regulation.—Prost, S., Bellamy, C. O. C., Cunningham, D. S., Harrison, D. J. Altered DNA repair and dysregulation of p53 in IRF-1 null hepatocytes. FASEB J. 12, 181–188 (1998)


Key Words: liver • cell cycle • apoptosis • UV irradiation • cell proliferation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE RESPONSES OF CELLS sustaining DNA damage can include DNA repair, cell cycle arrest, or cell death by apoptosis. Together, these activities are believed to prevent the development of mutations that could foster carcinogenesis. The prevalent response to DNA damage differs among cell types, but the molecular controls underlying these differences are still poorly understood. Recent observations suggest that this variability may be determined in part through two tumor suppressor proteins, IRF-1 and p53, for which critical roles have been identified in the regulation of both apoptosis and growth arrest after DNA damage.

The IRF-1 tumor suppressor protein is a transcription factor that mediates many of the growth suppressive effects of type 1 interferon through transactivation of genes including p21WAF1, cyclin D1, and double-stranded RNA-dependent protein kinase (1–3). The involvement of IRF-1 in regulating responses to DNA damage has only recently been recognized (4, 5): IRF-1 regulates a p53-independent pathway triggering apoptosis in mitogen-activated T lymphocytes and Ha-ras-transformed fibroblasts (4). Furthermore, effective cell cycle arrest by embryo fibroblasts after DNA damage required both IRF-1 and p53, cooperating to activate expression of p21WAF1 (6). Thus, IRF-1 has both overlapping and independent activities with p53 in regulating responses to DNA damage, and there is cell type specificity in the particular requirement for IRF-1 or p53 in these responses. Whether IRF-1 also regulates DNA repair activity, as suggested for p53, has not been reported.

Cellular p53 protein concentration commonly increases after exposure to agents that damage DNA, such as ultraviolet (UV) irradiation; however, the precise downstream responses regulated through p53 in cells sustaining DNA injury may depend on the damage stimulus (7, 8), cell type (9), and differentiation state. p53 activates transcription of a specific set of target genes, including the cyclin-dependent kinase inhibitor p21WAF1, which produces growth arrest (10). The downstream pathways to apoptosis that depend on p53 remain less certain, but both transactivation-dependent and independent mechanisms are described (8). In addition to regulation by p53 of apoptosis and cell cycle arrest in certain cell types, recent reports have suggested that p53 can directly regulate proteins involved in nucleotide excision repair, at least in vitro (11–13). However, the issue is not clearly resolved, and there are conflicting data from different systems about the consequences of p53 deficiency for DNA repair (11–19).

p53 is commonly defective in human liver carcinoma, a major cause of cancer death; however, the roles of p53 in normal hepatocytes and the regulation of hepatocyte responses to DNA damage are still incompletely understood. We have reported that proliferating hepatocytes show p53-dependent growth arrest after DNA damage in vivo and in primary culture (20). However, hepatocyte apoptosis after exposure to UV irradiation was p53 independent (20), suggesting that another gene such as IRF-1 may regulate DNA damage-triggered apoptosis in this cell type.

The work presented here sought to better define the responses of hepatocytes to DNA injury. Specifically, we have used primary hepatocytes from IRF-1-deficient mice to test whether IRF-1 is important in hepatocyte responses to DNA damage and whether IRF-1 deficiency affects p53 responses. We have also extended our previous work on the role of p53 in hepatocytes to evaluate a possible role for p53 in regulation of DNA repair. We report for the first time the effects of IRF-1 deficiency on hepatocyte p53 induction and transactivation, cell cycle arrest, and DNA repair compared with both wild-type and p53-null primary hepatocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primary hepatocytes
The male IRF-1-deficient (21) and p53-deficient (22) mice used were outbred. Primary hepatocytes were isolated from adult male mice 6–10 wk old by a standard two-step EDTA/collagenase retrograde perfusion protocol and cultured as previously described (20).

Cell proliferation
Monolayer primary cultures of hepatocytes on chamber slides (Lab Teck) were cultured with 40 µM BrdU for 6 h before fixing in 70% ethanol at 4°C. The fixed cultures were treated with 5 M HCl for 1 h before immunocytochemistry to identify BrdU incorporation into nuclei, using a standard indirect peroxidase technique. The primary antibody was a purified monoclonal rat anti-BrdU IgG2a (Sera Labs, Sussex, U.K.), and the secondary was a horseradish peroxidase-conjugated rabbit anti-rat IgG (Sigma, St. Louis, Mo.). Negative controls omitted the primary antibody. For evaluation of BrdU positivity, 500 hepatocytes were counted (sufficient to achieve a running mean), and the number of BrdU positive cells expressed as a percentage of cells were counted.

Evaluation of apoptosis
Twenty-four hours after plating, hepatocyte monolayers were irradiated with 0, 15, or 50 J/m2, and at various times after treatment (24, 48, 72, 96, 120 h) fixed in acid methanol/formalin. After Feulgen staining and a light green counterstain, apoptotic cells were counted under light microscopy. Five hundred hepatocytes were evaluated on cultured monolayers for each observation.

p53 immunocytochemistry
Immunocytochemistry for p53 protein was performed on acetone:methanol (1:1 v/v) -fixed primary hepatocyte monolayers on chamber slides (Lab Teck) and stored dry at -80°C. Cells were fixed at various times after 10J/m2 UV irradiation (performed 24 h after plating, using a Spectrolinker XL1500 (Spectronics Corporation, Westbury, N.Y.). Immunoreactive p53 was labeled by a standard avidin-biotin peroxidase technique using a primary monoclonal anti-p53 antibody pAB 421 (Oncogene Science, Cambridge, U.K.; 1/1000 dilution), as previously described (23).

p53 reporter plasmid
Primary hepatocytes were cultured for 48 h on fibronectin-coated 24-well plates, then transfected with either a p53 reporter plasmid, pRGC{Delta}FosLacZ (which contains two copies of an RCG p53-specific binding site placed upstream of a nonfunctional fos promoter and a lacZ gene), or a negative control plasmid, p{Delta}FosLacZ, which lacks the RGC p53 binding site (both plasmids were a kind gift from Prof. S. Friend). Transfections were performed using lipofectin (Gibco-BRL, Paisley, U.K.), as previously described (23). Briefly, plasmid DNA (1 µg) and lipofectin (6 µg), each diluted in culture medium (200 and 100 µl respectively), were incubated for 45 min at room temperature. Plasmid and lipofectin were then mixed and further incubated for 45 min before laying the complex over the cells. After 6 h of incubation at 37°C, the DNA-containing medium was replaced by fresh medium. Twenty-four hours after transfection, cells were UV-C irradiated or not, cultured for another 3, 6, 9, or 14 h, and then lysed in 100 µl of reporter lysis buffer (Promega, Southampton, U.K.). ß-Galactosidase activity was determined by using an ONPG substrate assay (Promega ß-galactosidase enzyme assay) and is expressed relative to the amount of protein (Biorad protein assay) recovered from each well, as previously described (23).

Unscheduled DNA synthesis
Primary hepatocytes were cultured on fibronectin-treated chamber slides (Lab Teck) for 24 h, then irradiated with the indicated doses of UV-C and incubated for a further 3 h in medium supplemented with 10 µCi/ml of tritiated thymidine. The radioactive medium was removed and cells were incubated in fresh medium containing 1 mM of nonradioactive thymidine for another hour. The cultures were then fixed in Boum's fixative, Feulgen-counterstained, then dipped in LM-1 autoradiographic emulsion (Amersham, Slough, U.K.) as recommended by the manufacturer. The slides were placed in a sealed box in a refrigerator for 7 days. Processing was performed according to the manufacturer's protocol, using solutions at 13°C and a developing time of 3 min.

Nuclear grain counting was performed on autoradiographed cultures using an image analysis system (Kontron V2.0), and two to four replicate cultures were evaluated for each data point given. For each culture, 10 high-power (x400) fields were assessed, each field containing 20 to 40 nuclei. The num~ber of grains within nuclei was corrected for background (nonnuclear) grain density by subtraction; the results represent the mean (±95% confidence limit) of nuclear grain density for all replicate cultures.

Reactivation of a UV-C irradiated reporter plasmid
The pOP13 CAT reporter plasmid (Stratagen, Cambridge, U.K.) was treated with UV-C at various doses and then cotransfected with unirradiated pßCMV (1:1 ratio) into primary hepatocytes as described above. Twenty-four hours after transfection, the cells were lysed in reporter lysis buffer (Promega).

The CAT activity in transfected cells was measured by liquid scintillation counting assay. Briefly, 30 µl of extract was incubated for 1 h in the presence of [3H]chloramphenicol and n-butyryl coenzyme A in 0.25 M Tris-HCl, pH 8.0. Extraction was performed using 300 µl of mixed xylenes, and for maximum sensitivity back-extracted once after addition of 100 µl of Tris-HCl, pH 8.0. The xylene phase was then transferred into a scintillation vial and counted.

The results for each lysate were corrected for the transfection efficiency, given by the ß-galactosidase activity resulting from the undamaged pCMVß expression (Promega assay) per µg of protein (Biorad protein assay). Reactivation of CAT activity from the irradiated plasmid was calculated by using the average of the activity obtained for ‘n’ independent transfections relative to the average of activity for an undamaged transfected plasmid.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IRF-1-deficient hepatocytes retain G1/S cell cycle arrest after UV irradiation
The proportion of hepatocytes entering S phase after UV irradiation was assessed by BrdU immunocytochemistry on cultures exposed to a pulse of BrdU at various times after UV treatment (10J/m2). Comparison was made between IRF-1 null, wild-type, and p53-null primary hepatocytes. Six to 12 h after irradiation, there was a significant decrease in BrdU uptake in both wild-type and IRF-1 null cultures, indicating a cell cycle arrest at G1/S. This response was not observed in p53-deficient cells, as previously reported (20). These results therefore indicate that p53-dependent G1/S growth arrest by hepatocytes is IRF-1 independent ( Fig. 1).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. The effect of IRF-1 genotype on hepatocyte growth arrest after UV irradiation. BrdU incorporation into hepatocytes was measured at the indicated times after isolation and plating. 60 h after plating, hepatocytes cultures were UV-irradiated (10J/m2). The results are the mean with standard error for duplicate cultures of one typical experiment where all three genotypes were studied simultaneously. The difference in proliferation observed between IRF-1 null and wild-type untreated hepatocytes was not observed in other experiments and is not significant. IRF-1 null ({circ}); wild-type ({square}); p53 null ({triangleup}); open symbols are untreated controls; closed symbols are UV treated.

Apoptosis after UV damage is IRF-1 independent in primary hepatocytes
We have shown that primary hepatocytes undergo apoptosis after UV injury in a dose-dependent manner. No significant increase in apoptosis was detected in either wild-type or IRF-1 null primary hepatocytes treated with 15 J/m2 UV, the level remaining around 5%, as observed in untreated cells. UV irradiation at a higher dose (50 J/m2) produced in both wild-type and IRF-1 null hepatocytes a wave of apoptosis starting 48 to 72 h (20%) and peaking around 96 h after irradiation (50–60%). There was no significant difference between genotypes in the apoptotic responses to UV-C.

IRF-1 deficiency affects DNA repair in primary hepatocytes
DNA repair by hepatocytes was assessed in two ways. 1) Host reactivation of a transfected, damaged plasmid (HCR); this assay measures the repair capacity of cells that are not themselves exposed to a genotoxic agent. 2) The quantification of unscheduled DNA synthesis (UDS); this assay measures repair synthesis after whole cell irradiation.

We compared the ability of IRF-1-null, wild-type, and p53-deficient hepatocytes to repair a reporter plasmid damaged by various doses of UV-C. As expected, a UV dose-related impairment of the recovery of the reporter function was shown by all genotypes ( Fig. 2). However, recovery of the reporter activity by IRF-1 null hepatocytes was significantly lower than that of wild-type and p53-null cells, which were not significantly different from each other, suggesting a role for IRF-1 in this type of DNA repair.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. Host reactivation of a damaged plasmid. Hepatocytes in culture for 24 h were cotransfected with a CAT reporter plasmid damaged with the indicated doses of UV-C (500, 750, 1000, 1500 J/m2) and a control plasmid, as described in Methods. The CAT activities 24 h after transfection, corrected according to the transfection efficiency, are given relative to the activity of an untreated plasmid transfected under the same conditions. The figure shows the average CAT activity from ‘n’ independent transfections ±SEM from three (IRF-1-/-), six (wt), or three (p53-/-) mice.

Unscheduled DNA synthesis was measured by image analysis of nuclear grain density after autoradiography of cultures that had been treated with different doses of UV, then allowed to incorporate tritiated thymidine. After UV irradiation, grain density was increased in hepatocyte nuclei regardless of genotype, showing that the cells were initiating UDS. However, the level of UDS was significantly lower in IRF-1 null hepatocytes than that in wild-type cells ( Fig. 3a), in keeping with the results obtained for the host reactivation assay. p53 null hepatocytes also showed a reduced level of UDS after UV irradiation compared with wild-type ( Fig. 3b), to a level similar to that shown by IRF-1 null cells ( Fig. 3c).




View larger version (48K):
[in this window]
[in a new window]
  		Figure 3. Unscheduled DNA synthesis. Unscheduled DNA synthesis was quantified as described in Methods in hepatocytes treated with 0, 10, or 20 J/m2 UV. Shown is the mean of two or four independent cultures (±95% confidence limit) from three typical experiments. a) Dose response for wild-type and IRF-1 null hepatocytes, b) dose response for wild-type and p53 null hepatocytes, c) UDS for wild-type, IRF-1, and p53 null hepatocytes treated by 20 J/m2 UV. d, e) Examples of the autoradiography of untreated (d) and UV-irradiated (e) hepatocytes. Hepatocyte nuclei are highlighted by the Feulgen stain, allowing easy discrimination and counting of nuclear grains (black) by image analysis. The experiment was performed three times for each genotype (three mice).

IRF-1 deficiency affects p53 protein level and activity
Nonirradiated cultures of both genotypes showed a low prevalence of weak nuclear p53 immunopositivity (5–8% of cells). The proportion of immunopositive cells did not change with time in culture for either genotype (3 days); however, the intensity of positive nuclear staining was increased noticeably in the IRF-1 null cultures compared with wild-type.

After UV treatment (10J/m2, 254 nm), strong p53 immunopositivity developed in up to 70% of hepatocytes for both genotypes; however, the increase in p53 immunopositivity was more rapid and was sustained longer in IRF-1 null hepatocytes compared with wild-type ( Fig. 4a) : 60% of IRF-1-deficient hepatocytes were p53 immunopositive within 3–5 h after UV irradiation, and 20 h later, 40% of the cells had sustained positivity; by contrast, in wild-type hepatocytes, there was a significant increase in the proportion of p53 immunopositive cells by only 6 h after UV treatment, with a sharp peak 12 h after UV, already declining by 24 h.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. p53 in IRF-1 null hepatocytes. a) Time course of p53 immunopositivity after treatment with UV-C at 10 J/m2. Results are mean ±SEM for duplicate cultures for three independent experiments. IRF-1 null ({circ}); wild-type ({square}); open symbols are untreated controls; closed symbols are UV treated. b) p53 transactivation function. Hepatocytes cultured for 24 h were transfected with the p53 reporter plasmid (RGC{Delta}FosLacZ) or the negative control plasmid ({Delta}FosLacZ), and ß-galactosidase expression was measured at various times after transfection. Results are the mean of three independent transfections ±SEM. This experiment was performed twice with similar findings.

To test whether differences between genotypes in p53 immunopositivity were correlated with differences in p53 function, p53 transcriptional activity was studied in the primary hepatocytes, using a transiently transfected p53 reporter plasmid, pRGC{Delta}FosLacZ. Unexpectedly, unirradiated IRF-1 null transfectants showed a high level of p53 reporter activity, indicating the presence of transcriptionally active p53; wild-type transfectants showed no evidence of significant p53 reporter activity (i.e., comparable with transfectants carrying the negative control plasmid). p53 reporter activity in the IRF-1 null hepatocytes was three- to sixfold greater than comparable wild-type transfectants between 27 and 39 h after transfection ( Fig. 4b). This observation is in keeping with the increased intensity of p53 immunostaining observed in the non~transfected cultures, and along with other evidence suggests an increased basal level of physiological p53 activity in IRF-1-deficient hepatocytes compared with wild-type, rather than a response of the IRF-1 null cells to the transfection procedure itself.

When IRF-1 null transfectants carrying the p53 reporter plasmid were UV-irradiated (10 J/m2), there was only a slight relative increase in the reporter activity (data not shown). This is in contrast to findings for UV-irradiated wild-type transfectants, which show a fourfold increase in p53 reporter activity (23). However, as the basal level of reporter activity in IRF-1 null cells was already similar to the maximum seen in the irradiated wild-type cells, the failure to see a further increase after irradiation might simply reflect limitations of the reporter system (i.e., already maximally active).

In summary, IRF-1 deficiency in hepatocytes produces a state of increased basal p53 activity and accelerated, prolonged p53 protein responses to DNA damage.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present results show a new role for IRF-1 in regulating responses to DNA injury through DNA repair, and that IRF-1 deficiency has consequences for regulation of p53. Moreover, taken together with reports on fibroblasts and lymphoid cells (4, 5), the data highlight fundamental and lineage-specific differences in the roles of IRF-1 and p53 in the control of responses to DNA damage. The observations have implications for carcinogenesis, suggesting that suppression of normal lineage differentiation in preneoplastic clones could change the pattern of response to injury from that typical of the tissue, even without mutation in these critical genes.

The altered p53 regulation observed in IRF-1-deficient hepatocytes might reflect changes in the p53 phosphorylation state (24–27). Such a change could explain the increased basal p53 reporter activity observed without a commensurately increased propor~tion of p53 immunoreactive hepatocytes. Alternatively, the elevated p53 reporter activity in IRF-1-deficient cells could be due to an increased basal level of p53 protein, just below the threshold for immunodetection yet detectable by the reporter assay. After DNA damage, the stabilization of more preexisting molecules would manifest as a quicker conversion to immunopositivity (28), as was indeed observed here after UV treatment. Activation of p53 by DNA damage is a very efficient process: one strand break is sufficient to trigger p53 accumulation (29, 30). Hence, the increased p53 activity in IRF-1 null hepatocytes may be caused by unrepaired DNA lesions induced by either endogenous genotoxins or DNA-damaging treatments (UV).

We have reported that when apoptosis is triggered in hepatocytes after UV injury, it is p53 independent, although p53 is stabilized and activated by the injury (8, 20). IRF-1 seemed a reasonable candidate gene to regulate such apoptosis, given its role in p53-independent apoptosis of activated T lymphocytes after {gamma}-irradiation (4). We are now able to exclude such a role in UV-injured hepatocytes. Conversely, the failure of UV-injured IRF-1 null hepatocytes to show a significant increase in apoptosis compared with wild-type, despite accelerated, prolonged stabilization of p53, lends further support to the hypothesis that a p53-dependent pathway to apoptosis after DNA damage is not enabled in hepatocytes, regardless of the concentration or timing of p53 protein changes that have been suggested to determine whether a cell undergoes p53-dependent growth arrest or apoptosis (31).

The relationships between growth arrest, apoptosis, and DNA repair in cells sustaining DNA damage are poorly defined, yet of fundamental importance to understanding the mechanisms underlying the suppression of carcinogenesis. The repair data presented here suggest a novel and intriguing link between cytokines and DNA repair regulation through IRF-1, and also support reports that p53 can regulate repair. The discrepancies between the HCR and UDS assays are suggestive: HCR predominantly reflects transcription-coupled repair, whereas UDS is a measure of global repair. Thus, our results in hepatocytes suggest that p53 is required for a normal level of global repair but not for transcription-coupled repair. This observation agrees with findings in Li-Fraumeni skin fibroblasts (15), but differs from reports on other cell types (16, 18). Indeed, there is no clear consensus as to whether p53 regulates DNA repair, perhaps reflecting variability between cell types (11–19). By contrast, the IRF-1 null cells are deficient in both global and transcription-coupled repair, as measured here, despite remaining competent to produce a relatively high level of functional p53 as indicated by the reporter assay. It seems probable therefore that any effects of IRF-1 on repair are not acting through p53.

Nevertheless, cooperation between these genes to affect a common target gene such as p21, itself a possible repair regulator (32–36), remains a possibility. The decreased ability of IRF-1 null hepatocytes to repair DNA damage might explain the persistently increased p53 immunopositivity after UV treatment compared with wild-type. Similar observations have been made in DNA repair-deficient XP, CS, and TTD fibroblasts, in which the p53 protein level remains high for days (28, 37). Indeed, in normal cells, p53 stabilization shows dose-dependent kinetics (28) and hepatocytes of mice lacking the DNA repair gene ERCC1 have a high level of cellular p53 (38), suggesting that the concentration of p53 is influenced by the level and persistence of genomic DNA damage.

In conclusion, the present results show intriguing links between IRF-1 and p53, but also reveal independent functions in different responses to DNA damage. Dysregulation of IRF-1 in hepatocytes, either directly or through cytokine signaling in diseased liver, may reduce cellular capacity to repair DNA damage and alter the sensitivity of cells to DNA damage thus favoring selection pressure toward the outgrowth of potentially neoplastic clones of cells. In the presence of wild-type p53, such hepatocytes are likely to remain growth arrested; however, in hepatocytes lacking fully functional p53 (such as in chronic hepatitis B infection or with aflatoxin-induced mutation), the consequences of defective or suppressed IRF-1 for reduced DNA repair and hence accumulation of significant mutations may become significant.


   ACKNOWLEDGMENTS
 
This work was supported by the Scottish Hospital Endowments Research Trust.


   FOOTNOTES
 
1 Correspondence: Department of Pathology, University Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland. E-mail: s.prost{at}ed.ac.uk

2 Abbreviations: UV, ultraviolet; UDS, unscheduled DNA synthesis; HCR, host cell reactivation; CAT, chloramphenicol acethyl transferase; BrdU, 5-bromo-2'-deoxyuridine.

Received for publication August 20, 1997. Accepted for publication October 27, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kirchhoff, S., Schaper, F., and Hauser, H. (1993) Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes. Nucleic Acids Res. 21, 2881–2889[Abstract/Free Full Text]
  2. Kirchhoff, S., Koromilas, A. E., Schaper, F., Grashoff, M., Sonenberg, N., and Hauser, H. (1995) IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein PKR. Oncogene 11, 439–445[Medline]
  3. Taniguchi, T., Harada, H., and Lamphier, M. S. (1995) Regulation of the interferon system and cell growth by the IRF transcription factors. J. Cancer Res. Clin. Oncol. 121, 416–520
  4. Tamura, T., Ishihara, M., Lamphier, M. S., Tanaka, N., Oishi, I., Aizawa, S., Matsuyama, T., Mak, T. W., Taki, S., and Taniguchi, T. (1995) An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature (London) 376, 596–599[Medline]
  5. Tanaka, N., Ishihara, M., Kitagawa, M., Harada, H., Kimura, T., Matsuyama, T., Lamphier, M. S., Aizawa, S., Mak, T. W., and Taniguchi, T. (1994) Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 77, 829–840[Medline]
  6. Tanaka, N., Ishihara, M., Lamphier, M. S., Nozawa, H., Matsuyama, T., Mak, T. W., Aizawa, S., Tokino, T., Oren, M., and Taniguchi, T. (1996) Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature (London) 382, 816–818[Medline]
  7. Lu, X., and Lane, D. P. (1993) Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell 75, 765–778[Medline]
  8. Bellamy, C. O. C. (1997) p53 and apoptosis. Br. Med. Bull. 53, 522–538[Abstract/Free Full Text]
  9. MacCallum, D. E., Hupp, T. R., Midgley, C. A., Stuart, D., Campbell, S. J., Harper, A., Walsh, F. S., Wright, E. G., Balmain, A., Lane, D. P., Hall, D. J., and Hall, P. A. (1996) The p53 response to ionising radiation in adult and developing murine tissues. Oncogene 13, 2575–2587[Medline]
  10. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816[Medline]
  11. Wang, X. W., Yeh, H., Schaeffer, L., Roy, R., Moncollin, V., Egly, J.-M., Wang, Z., Friedberg, E. C., Evans, M. K., Taffe, B. G., Bohr, V. A., Weeda, G., Hoeijmakers, J. H. J., Forrester, K., Harris, C. C., Egly, J. M., Freidberg, E. C., et al. (1995) p53 modulation of TFIIH-associated nucleotide excision repair activity. Nature Genet. 10, 188–195[Medline]
  12. Wang, X. W., Vermeulen, W., Coursen, J. D., Gibson, M., Lupold, S. E., Forrester, K., Xu, G. W., Elmore, L., Yeh, H., Hoeijmakers, J. H. J., and Harris, C. C. (1996) The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway. Genes & Dev. 10, 1219–1232[Abstract/Free Full Text]
  13. Marx, J. (1994) New link found between p53 and DNA repair. Science 266, 1321–1322[Free Full Text]
  14. Ishizaki, K., Ejima, Y., Matsunaga, T., Hara, R., Sakamoto, A., Ikenaga, M., Ikawa, Y., and Aizawa, S. (1994) Increased UV-induced SCEs but normal repair of DNA damage in p53-deficient mouse cells. Int. J. Cancer 58, 254–257[Medline]
  15. Ford, J. M., and Hanawalt, P. C. (1995) Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance. Proc. Natl. Acad. Sci. USA 92, 8876–8880[Abstract/Free Full Text]
  16. Smith, M. L., Chen, I. T., Zhan, Q., O'Connor, P. M., and Fornace, A. J., Jr. (1995) Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene 10, 1053–1059[Medline]
  17. Li, G., Mitchell, D. L., Ho, V. C., Reed, J. C., and Tron, V. A. (1996) Decreased DNA repair but normal apoptosis in ultraviolet-irradiated skin of p53-transgenic mice. Am. J. Pathol. 148, 1113–1123[Abstract]
  18. Li, G., Ho, V. C., Mitchell, D. L., Trotter, M. J., and Tron, V. A. (1997) Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes. Am. J. Pathol. 150, 1457–1464[Abstract]
  19. Mirzayans, R., Enns, L., Dietrich, K., Barley, R. D. C., Paterson, M. C., and Barley, R. D. (1996) Faulty DNA polymerase {delta}/{epsilon}-mediated excision repair in response to gamma radiation or ultraviolet light in p53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome. Carcinogenesis 17, 691–698[Abstract/Free Full Text]
  20. Bellamy, C. O. C., Clarke, A. R., Wyllie, A. H., and Harrison, D. J. (1997) p53 deficiency in liver reduces local control of survival and proliferation, but does not affect apoptosis after DNA damage. FASEB J. 11, 591–599[Abstract]
  21. Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham, A., et al. (1993) Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75, 83–97[Medline]
  22. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., and Wyllie, A. H. (1993) Thymocyte apoptosis induced by p53-dependent and independent pathways [see comments]. Nature (London) 362, 849–852[Medline]
  23. Bellamy, C. O. C., Prost, S., Wyllie, A. H., and Harrison, D. J. (1997) UV but not gamma irradiation induces specific transcriptional activity of p53 in primary hepatocytes. J. Pathol. 183, 177–181
  24. Scheidtmann, K. H., and Landsberg, G. (1996) UV irradiation leads to transient changes in phosphorylation and stability of tumor suppressor protein p53. Int. J. Oncol. 9, 1277–1285
  25. Zhang, W., McClain, C., Gau, J. P., Guo, X. Y., and Deisseroth, A. B. (1994) Hyperphosphorylation of p53 induced by okadaic acid attenuates its transcriptional activation function. Cancer Res. 54, 4448–4453[Abstract/Free Full Text]
  26. Lohrum, M., and Scheidtmann, K. H. (1996) Differential effects of phosphorylation of rat p53 on transactivation of promoters derived from different p53 responsive genes. Oncogene 13, 2527–2539[Medline]
  27. Steegenga, W. T., Van der Eb, A. J., and Jochemsen, A. G. (1996) How phosphorylation regulates the activity of p53. J. Mol. Biol. 263, 103–113[Medline]
  28. Abrahams, P. J., Schouten, R., Van Laar, T., Houweling, A., Terleth, C., and Van der Eb, A. J. (1995) Different regulation of p53 stability in UV-irradiated normal and DNA repair deficient human cells. Mutat. Res. 336, 169–180[Medline]
  29. Di Leonardo, A., Linke, S. P., Clarkin, K., and Wahl, G. M. (1994) DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes & Dev. 8, 2540–2551[Abstract/Free Full Text]
  30. Huang, L. C., Clarkin, K. C., and Wahl, G. M. (1996) Sensitivity and selectivity of the DNA damage sensor responsible for acti~vating p53-dependent G1 arrest. Proc. Natl. Acad. Sci. USA 93, 4827–4832[Abstract/Free Full Text]
  31. Chen, X., Ko, L. J., Jayaraman, L., and Prives, C. (1996) p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes & Dev. 10, 2438–2451[Abstract/Free Full Text]
  32. Pan, Z. Q., Reardon, J. T., Li, L., Flores-Rozas, H., Legerski, R., Sancar, A., and Hurwitz, J. (1995) Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21. J. Biol. Chem. 270, 22008–22016[Abstract/Free Full Text]
  33. McDonald, E. R., 3rd, Wu, G. S., Waldman, T., and El-Deiry, W. S. (1996) Repair defect in p21 WAF1/CIP1 -/- human cancer cells. Cancer Res. 56, 2250–2255[Abstract/Free Full Text]
  34. Sheikh, M. S., Chen, Y. Q., Smith, M. L., and Fornace AJ Jr (1997) Role of p21(Waf1/Cip1/Sdi1) in cell death and DNA repair as studied using a tetracycline-inducible system in p53-deficient cells. Oncogene 14, 1875–1882[Medline]
  35. Li, R., Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994) Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature (London) 371, 534–537[Medline]
  36. Shivji, M. K., Grey, S. J., Strausfeld, U. P., Wood, R. D., and Blow, J. J. (1994) Cip1 inhibits DNA replication but not PCNA-dependent nucleotide excision-repair. Curr. Biol. 4, 1062–1068[Medline]
  37. Yamaizumi, M., and Sugano, T. (1994) U.V.-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle. Oncogene 9, 2775–2784[Medline]
  38. McWhir, J., Selfridge, J., Harrison, D. J., Squires, S., and Melton, D. W. (1993) Mice with DNA repair gene (ERCC-1) deficiency have elevated levels of p53, liver nuclear abnormalities and die before weaning [see comments]. Nature Genet. 5, 217–224[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
X. He, Z. Zhu, C. Johnson, J. Stoops, A. E. Eaker, W. Bowen, and M. C. DeFrances
PIK3IP1, a Negative Regulator of PI3K, Suppresses the Development of Hepatocellular Carcinoma
Cancer Res., July 15, 2008; 68(14): 5591 - 5598.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. Dornan, M. Eckert, M. Wallace, H. Shimizu, E. Ramsay, T. R. Hupp, and K. L. Ball
Interferon Regulatory Factor 1 Binding to p300 Stimulates DNA-Dependent Acetylation of p53
Mol. Cell. Biol., November 15, 2004; 24(22): 10083 - 10098.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
R. Wessely, L. Hengst, B. Jaschke, F. Wegener, T. Richter, R. Lupetti, M. Paschalidis, A. Schomig, R. Brandl, and F.-J. Neumann
A central role of interferon regulatory factor-1 for the limitation of neointimal hyperplasia
Hum. Mol. Genet., January 15, 2003; 12(2): 177 - 187.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. R. Fagerlie, J. Diaz, T. A. Christianson, K. McCartan, W. Keeble, G. R. Faulkner, and G. C. Bagby
Functional correction of FA-C cells with FANCC suppresses the expression of interferon {gamma}-inducible genes
Blood, May 15, 2001; 97(10): 3017 - 3024.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Bergeron, N. Benlimame, N. Zeng-Rong, D. Xiao, P. J. Scrivens, A. E. Koromilas, and M. A. Alaoui-Jamali
Identification of the Interferon-inducible Double-Stranded RNA-dependent Protein Kinase as a Regulator of Cellular Response to Bulky Adducts
Cancer Res., December 1, 2000; 60(24): 6800 - 6804.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
S. Prost, J. M. Ford, C. Taylor, J. Doig, and D. J. Harrison
Hepatitis B x Protein Inhibits p53-dependent DNA Repair in Primary Mouse Hepatocytes
J. Biol. Chem., December 11, 1998; 273(50): 33327 - 33332.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Prost, S.
Right arrow Articles by Harrison, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Prost, S.
Right arrow Articles by Harrison, D. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS