Mutation of p53 in Recurrent Hepatocellular Carcinoma and Its Association with the Expression of ZBP-89
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64317-9
ISSN1525-2191
AutoresGeorge G. Chen, Juanita L. Merchant, Paul B.S. Lai, Rocky L.K. Ho, Xu Hu, Morihiro Okada, Sheng F. Huang, A.K.K Chui, David Law, Yong G. Li, Wan Yee Lau, Arthur K.C. Li,
Tópico(s)Ubiquitin and proteasome pathways
Resumop53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment. p53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment. The p53 gene functions as a tumor suppressor and plays a central role in regulating transcriptional activation of crucial growth regulatory genes that control cell cycle progression and cell division. The p53 protein is normally present at low levels in the cell, but it can be up-regulated by stimuli such as DNA damage, hypoxia, viral proteins, or the deregulated cell cycle progression caused by ectopic oncogene expression.1Vousden KH p53: death star.Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar The biological consequence of p53 up-regulation is the induction of pathways that lead to either cell cycle arrest or apoptosis. Mutations in the p53 gene abrogate its normal functions, leading to genomic instability and loss of growth control. Many studies have shown that p53 is frequently mutated in human hepatocellular carcinoma (HCC).2Jeng KS Sheen IS Chen BF Wu JY Is the p53 gene mutation of prognostic value in hepatocellular carcinoma after resection?.Arch Surg. 2000; 135: 1329-1333Crossref PubMed Scopus (54) Google Scholar, 3Katiyar S Dash BC Thakur V Guptan RC Sarin SK Das BC p53 tumor suppressor gene mutations in hepatocellular carcinoma patients in India.Cancer. 2000; 88: 1565-1573Crossref PubMed Scopus (67) Google Scholar, 4Honda K Sbisa E Tullo A Papeo PA Saccone C Poole S Pignatelli M Mitry RR Ding S Isla A Davies A Habib NA p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation.Br J Cancer. 1998; 77: 776-782Crossref PubMed Scopus (102) Google Scholar, 5Heinze T Jonas S Karsten A Neuhaus P Determination of the oncogenes p53 and C-erb B2 in the tumour cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time.Anticancer Res. 1999; 19: 2501-2503PubMed Google Scholar Although altered p53 expression has been reported to correlate with advanced tumor grade, progression, therapy, and survival,4Honda K Sbisa E Tullo A Papeo PA Saccone C Poole S Pignatelli M Mitry RR Ding S Isla A Davies A Habib NA p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation.Br J Cancer. 1998; 77: 776-782Crossref PubMed Scopus (102) Google Scholar, 5Heinze T Jonas S Karsten A Neuhaus P Determination of the oncogenes p53 and C-erb B2 in the tumour cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time.Anticancer Res. 1999; 19: 2501-2503PubMed Google Scholar, 6Malkin D The role of p53 in human cancer.J Neurooncol. 2001; 51: 231-243Crossref PubMed Scopus (49) Google Scholar, 7Ryan KM Phillips AC Vousden KH Regulation and function of the p53 tumor suppressor protein.Curr Opin Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (594) Google Scholar information on how the mutations in the p53 gene is linked to recurrence of HCC is limited and the expression of ZBP-89, a four-zinc finger transcription factor that regulates the expression of several genes related to cell growth through binding to GC-rich DNA elements,11Law GL Itoh H Law DJ Mize GJ Merchant JL Morris DR Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.J Biol Chem. 1998; 273: 19955-19964Crossref PubMed Scopus (76) Google Scholar has not been reported in HCC. HCC is an extremely aggressive disease and the mortality of this disease is very high. Surgical resection of the tumor is currently the most effective treatment for HCC. With the advance of early diagnosis, the short-term prognosis has been improved. However, recurrent tumor after surgical resection remains a serious obstacle to improve the prognosis of patients with HCC. Furthermore, recurrent tumors usually grow faster and have lower chemo- or radiosensitivity, which is believed to be affected by the status of the p53 gene.9McIlwrath AJ Vasey PA Ross GM Brown R Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity.Cancer Res. 1994; 54: 3718-3722PubMed Google Scholar, 10Lowe SW Bodis S McClatchey A Remington L Ruley HE Fisher DE Housman DE Jacks T p53 status and the efficacy of cancer therapy in vivo.Science. 1994; 266: 807-810Crossref PubMed Scopus (1564) Google Scholar Our recent studies have further revealed that the inhibitory mechanism of ZBP-89 involves activation of p21wafl stabilization of p53.12Bai L Merchant JL Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21wafl transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (100) Google Scholar, 13Bai L Merchant JL ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (92) Google Scholar ZBP-89 stabilizes p53 through direct protein contact, which leads to retention of p53 in the nucleus. Moreover, tumor cells with a mutation in the p53 gene are resistant to ZBP-89-mediated stabilization.13Bai L Merchant JL ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (92) Google Scholar In the present study we examine the pattern of p53 mutations and ZBP-89 expression to test the possible correlation between ZBP-89 and p53 in recurrent HCC. Thirty-three Hong Kong Chinese patients with HCC were selected for the study. These patients were found to have recurrent intrahepatic or extrahepatic tumors after surgical resection of primary tumor at Prince of Wales Hospital, Hong Kong. All patients studied were found to be positive for hepatitis B virus. The tumors were confirmed by compatible histopathology. The interval between resection surgery and the appearance of recurrence ranged from 2 to 31 months (mean 6.8 months). Tissue samples were collected at the time of surgical resection of primary tumors. After collection, samples were immediately stored in liquid nitrogen (−160°C) until experiments were performed. Tissues underwent immunohistochemical processing, were embedded in paraffin, and were formalin-fixed for 30 hours to preserve the antigen for immunohistochemistry. Tissues were sectioned at 4 mm thick. Immunostaining was performed on paraffin sections according to our previous publications.14Taniuchi T Mortensen ER Ferguson A Greenson J Merchant JL Overexpression of ZBP-89, a zinc finger DNA binding protein, in gastric cancer.Biochem Biophys Res Commun. 1997; 233: 154-160Crossref PubMed Scopus (39) Google Scholar, 15Chen GG Lai PB Chan PK Chak EC Yip JH Ho RL Leung BC Lau WY Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein.Eur J Cancer. 2001; 37: 1695-1702Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar The ZBP-89 antibody was raised against amino acids 1–521 of rat ZBP-89 (dilution 1:1000).14Taniuchi T Mortensen ER Ferguson A Greenson J Merchant JL Overexpression of ZBP-89, a zinc finger DNA binding protein, in gastric cancer.Biochem Biophys Res Commun. 1997; 233: 154-160Crossref PubMed Scopus (39) Google Scholar The p53 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a concentration of 1:200. Negative controls were prepared by using PBS instead of the primary antibody. Genomic DNA was isolated from frozen tissues by proteinase K digestion and spin columns using QIAamp DNA mini kit (QIAGEN, Hilden, Germany). The quality of DNA was checked by running DNA on 1% agarose gel and the concentration of DNA was determined spectrophotometrically at 260 nm. p53 mutations in exons 2 to 8 were analyzed by direct sequencing. The primers listed in Table 1 were designed for sequencing relevant exons. The sequencing was done by ABI PRISM 310 Genetic Analyzer using BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA). Experiment was performed according to the manufacturer's instructions.Table 1Sequences of Primers Used in p53 Mutation AnalysisNameSequenceExonP53-15′-cac gct tcc ctg gat tgg-3′2, 3, 4P53-25′-tca acc cac agc tgc aca-3′5P53-35′-ccc tgc cct caa caa gat-3′6, 7P53-45′-acg cac ctc aaa gct gtt-3′7, 8P53-55′-gcc cat cct cac cat cat-3′8, 9P53-65′-gga ggg agg ctg tca gtg-3′9, 10 Open table in a new tab Hep 3B cells, transfected with wild-type or mutant p53, were infected with ZBP-89-containing recombinant adenovirus.12Bai L Merchant JL Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21wafl transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (100) Google Scholar, 13Bai L Merchant JL ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (92) Google Scholar Co-precipitation of p53 and ZBP-89 was carried out using whole-cell lysate and specific antibodies as described above. The first antibody (p53)-associated proteins were precipitated with protein A-Sepharose. After washing, immunoprecipitates were separated and immunoblotted with the second antibody (ZBP-89). Hep 3B cells is a hepatocarcinoma cell line, whose p53 gene has been deleted.8Friedman SL Shaulian E Littlewood T Resnitzky D Oren M Resistance to p53-mediated growth arrest and apoptosis in Hep 3B hepatoma cells.Oncogene. 1997; 15: 63-70Crossref PubMed Scopus (45) Google Scholar Student's t-test or Mann-Whitney U-test was used for continuous variables. χ2 or Fisher exact test was carried out for categorical variables. Regression and correlation analyses of parametric and nonparametric data were performed by Pearson and Spearman methods respectively. A P value of less than 0.05 was considered statistically significant. Mutations in the p53 gene were frequently detected in recurrent HCC. Of 33 recurrent cases studied, 16 (48.5%) had mutations in the p53 gene. All except one mutation detected was a single nucleotide mismatch that occurred in the core domain between codons 68 and 281 (from exon 4 to exon 8) (Table 2). One mutation in exon 3 resulted from a deletion of 7 nucleotides with a 2-nucleotide insertion. Among all mutations detected, 7 cases (43.8%) were located in exon 7, especially in codon 249, which accounted for 5 cases (31.3%) of all mutations found.Table 2Mutation of the p53 Gene in Recurrent HCCCaseExonCodonNucleotide changesAmino acid substitution16193CAT → CCTHis → Pro25157GTC → TTCVal → Phe38274GTT → TTTVal → Phe47245GGC → GACGly → Asp5468GAG → TAGGlu → STOP67249AGG → AGTArg → Ser75157GTC → TTCVal → Phe85175CGC → CACArg → His9325TGAAAAC → AGFrameshift107242TGC → TACCys → Tyr117249AGG → AGTArg → Ser127249AGG → AGTArg → Ser137249AGG → AGTArg → Ser148281GAC → GAGAsp → Glu157249AGG → AGTArg → Ser165159GCC → CCCAla → Pro Open table in a new tab Patient demographics are shown in Table 3. The patients with recurrent HCC and mutations in the p53 gene were younger than those with the wild-type p53 gene. However, the difference did not reach statistical significance, probably due to the wide variations in age, which ranged from 14 to 75 years. The level of α-fetoprotein (AFP) in patients with mutations in the p53 gene was significantly lower than those without mutations. There was no difference in other clinical values between patients with and without mutations in the p53 genes.Table 3Demographic Data on Patients with Recurrent HCCDemographic datap53 Mutation (n = 16)p53 Wild-type (n = 17)Significance (P)Age (years)>0.05 Range32–7437–75 Mean50.354.5Sex>0.05 Male1314 Female33Histological differentiation Well56>0.05 Moderately910>0.05 Poorly11AFP (ng/ml)0.0063 Range3–13,50010–27,170 Mean1366.42337.5HBsAg>0.05 Positive1115 Negative42HCV>0.05 Positive21 Negative1416Liver cirrhosis>0.05 Positive1210 Negative47Portal involvement>0.05 Positive33 Negative1314 Open table in a new tab The interval between surgical resection and the appearance of recurrence was significantly longer in patients with the wild-type p53 gene than those with mutations in the p53 gene (Figure 1), strongly suggesting a pathological role for the p53 gene in HCC recurrence. Since codon 249 in exon 7 was the most frequent site for mutation, we specifically calculated the interval for recurrent HCC with this particular mutation. The mean interval for recurrent HCC with a mutation in codon 249 of exon 7 was 5.4 months, which was much shorter than that in all other codons (9.6 months, P < 0.05). The detailed distribution of intervals between the two groups of recurrent HCC is shown in Figure 2.Figure 2Distribution of interval in recurrent HCC with or without mutations in p53.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The frequency of ZBP-89 protein detection was significantly higher in cancerous tissues (28 cases) than in noncancerous ones (18 cases, P = 0.0148). ZBP-89 protein was detected in both the cytoplasm and the nucleus. The intensity was stronger in the nucleus than in the cytoplasm in HCC patients regardless of the presence of p53 mutations (Figure 3A). However, the expression of ZBP-89 was more readily detected in the cytoplasm of recurrent HCC when p53 mutations were present (Figure 3A) whereas it was more likely to be present in the nucleus of recurrent HCC when there were no mutations in the p53 gene (Figure 3B). The expression of p53 protein was detected in 21 of 33 cases (63.6%). The positive rate of p53 protein detection was significantly higher in recurrent HCC patients with mutations in the p53 gene than those without (P < 0.01). All 16 cases of recurrent HCC with p53 mutations showed p53 signals and only 5 cases with p53 expression did not have underlying mutations in the p53 gene. p53 protein was more likely to accumulate in the cytoplasm of recurrent HCC if mutations were present in the p53 gene (Figure 3C). Nuclear localization of p53 was also observed, especially in those with the wild-type p53 gene (Figure 3D). Since ZBP-89 was able to bind to p53 and co-localize to the nucleus,13Bai L Merchant JL ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (92) Google Scholar we performed double-staining of both proteins in tissues positive for p53 protein. Indeed, ZBP-89 was present in the nuclear compartment where p53 protein was found (Figure 3E). In most cases where ZBP-89 and p53 proteins co-localized in the nucleus, there were no mutations in the p53 genes. Co-localization of ZBP-89 and p53 proteins was also observed in the cytoplasm. Cytoplasmic co-localization was detected in recurrent HCC tumors primarily with mutant p53 gene. However, ZBP-89 localization could also be observed with some tumors with cytoplasmic wild-type p53 (Figure 3F). Since ZBP-89 and p53 were co-localized in most recurrent HCC, we examined whether this occurred by direct contact. Co-immunoprecipitation was performed to assess whether both proteins could bind each other. Hep 3B, a p53-negative liver cancer cell line,8Friedman SL Shaulian E Littlewood T Resnitzky D Oren M Resistance to p53-mediated growth arrest and apoptosis in Hep 3B hepatoma cells.Oncogene. 1997; 15: 63-70Crossref PubMed Scopus (45) Google Scholar was infected with ZBP-89-containing recombinant adenovirus. Cell extracts were subjected to immunoprecipitation with the p53 antibody followed by immunoblotting with ZBP-89 antibody. Figure 4 shows that ZBP-89 co-precipitated with wild-type 53 and some p53 mutants whose mutations occurred in the N-terminal domains. Mutations in the p53 transactivation domain (35 frameshift) and PXXP domain (G68A) did not affect the binding between ZBP89 and p53. All mutations in the DNA binding domain, except those located in the most N-terminal region of DNA binding domain (V157P and A159P), abolished the direct interaction between ZBP-89 and p53. It is well known that mutations in the p53 gene are closely associated with HCC.2Jeng KS Sheen IS Chen BF Wu JY Is the p53 gene mutation of prognostic value in hepatocellular carcinoma after resection?.Arch Surg. 2000; 135: 1329-1333Crossref PubMed Scopus (54) Google Scholar, 3Katiyar S Dash BC Thakur V Guptan RC Sarin SK Das BC p53 tumor suppressor gene mutations in hepatocellular carcinoma patients in India.Cancer. 2000; 88: 1565-1573Crossref PubMed Scopus (67) Google Scholar, 4Honda K Sbisa E Tullo A Papeo PA Saccone C Poole S Pignatelli M Mitry RR Ding S Isla A Davies A Habib NA p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation.Br J Cancer. 1998; 77: 776-782Crossref PubMed Scopus (102) Google Scholar, 5Heinze T Jonas S Karsten A Neuhaus P Determination of the oncogenes p53 and C-erb B2 in the tumour cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time.Anticancer Res. 1999; 19: 2501-2503PubMed Google Scholar However, the pattern of mutations varies, depending on the geographic areas and types of patients selected. The present study focuses on recurrent HCC in a Chinese population in Hong Kong. Within the population examined, mutations in the p53 gene were detected in 48.5% of recurrent HCC, which is much higher than the 23.1% reported in a previous study of HCC in Hong Kong Chinese.16Ng IO Srivastava G Chung LP Tsang SW Ng MM Overexpression and point mutations of p53 tumor suppressor gene in hepatocellular carcinomas in Hong Kong Chinese people.Cancer. 1994; 74: 30-37Crossref PubMed Scopus (50) Google Scholar The pattern of p53 mutations detected between these two studies was also different. In the present study, mutations in exon 7 of the p53 gene, which is a hot spot in many studies performed in Asia,17Lee YI Lee S Das GC Park US Park SM Lee YI Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma.Oncogene. 2000; 19: 3717-3726Crossref PubMed Scopus (82) Google Scholar, 18Yu MW Yang SY Chiu YH Chiang YC Liaw YF Chen CJ A p53 genetic polymorphism as a modulator of hepatocellular carcinoma risk in relation to chronic liver disease, familial tendency, and cigarette smoking in hepatitis B carriers.Hepatology. 1999; 29: 697-702Crossref PubMed Scopus (136) Google Scholar accounted for 43.8% of all alterations found, and therefore the present study suggests that the exon 7 should be considered a prevalent site of p53 mutation. In the previous study, mutations in exon 7 of the p53 gene were detected in 33.3% of cases; however, these two studies may not be comparable, as one was performed on newly diagnosed and recurrent HCC patients in general while the other was conducted only on recurrent HCC. The mean interval between surgical resection and the appearance of recurrence in patients with mutations in the p53 gene was 7.8 months, whereas those with the wild-type p53 gene was about two-fold as long (16.4 months). This finding establishes a potential link between mutations in the p53 gene and recurrent tumor. Such a link may be of clinical significance if it can be further confirmed in a large number of patients. The behaviors of the recurrent tumor usually differ from that of the primary tumor. Of particular importance, recurrent tumors grow faster and are less sensitive to chemotherapy and radiotherapy.10Lowe SW Bodis S McClatchey A Remington L Ruley HE Fisher DE Housman DE Jacks T p53 status and the efficacy of cancer therapy in vivo.Science. 1994; 266: 807-810Crossref PubMed Scopus (1564) Google Scholar, 11Law GL Itoh H Law DJ Mize GJ Merchant JL Morris DR Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.J Biol Chem. 1998; 273: 19955-19964Crossref PubMed Scopus (76) Google Scholar, 19Chao C Goldberg M Hoffman JP Surgical salvage therapy: abdominoperineal resection for recurrent anal carcinoma, metastasectomy of recurrent colorectal cancer, and esophagectomy after combined chemoradiation.Curr Opin Oncol. 2000; 12: 353-356Crossref PubMed Scopus (11) Google Scholar, 20Easson EC: General principle of radiotherapy. The Radiotherapy of Malignant Disease Pointon RCS eds. ed 2: pp 111-130 Springer-Verlag, London, New YorkGoogle Scholar These features may be strongly linked to the function of p53 protein. Mutant p53 provides tumor cells with a selective survival advantage during non-surgical therapy and thus decrease the sensitivity to chemo- and radiodrugs.21Matsuzoe D Hideshima T Kimura A Inada K Watanabe K Akita Y Kawahara K Shirakusa T p53 mutations predict non-small cell lung carcinoma response to radiotherapy.Cancer Lett. 1999; 135: 189-194Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 22Blandino G Levine AJ Oren M Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy.Oncogene. 1999; 18: 477-485Crossref PubMed Scopus (386) Google Scholar Functioning as a transcription factor, p53 binds to DNA in a sequence-specific manner and regulates the transcription of the gene responsible for the promotion of apoptosis and the inhibition of angiogenesis,23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar both of which are believed to be critical for the prevention or the inhibition of the development of tumors. This concept is supported by both animal and human data. Although p53-null mice are able to develop normally, they are susceptible to an array of spontaneous tumors in early adult life.24Harvey M McArthur MJ Montgomery CA Butel JS Bradley A Donehower LA Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice.Nat Genet. 1993; 5: 225-229Crossref PubMed Scopus (501) Google Scholar When the wild-type p53 allele is lost, a variety of mesenchymal and epithelial tumors develop in patients with Li-Fraumeni syndrome, which carries heterozygous p53 mutations in the germline.25Srivastava S Wang S Tong YA Pirollo K Chang EH Several mutant p53 proteins detected in cancer-prone families with Li-Fraumeni syndrome exhibit transdominant effects on the biochemical properties of the wild-type p53.Oncogene. 1993; 8: 2449-2456PubMed Google Scholar In recurrent HCC, p53 dysfunction caused by the mutation may create a similar scenario to the one observed in p53-null mice or p53 allele deletion, in which the growth of a tumor is promoted. If this is true, it would be easier to understand why HCC patients with a mutant p53 gene are more likely to have tumor recurrence within a short interval after surgical resection for the primary tumor. The central domain of p53, which consists of amino acids 100 to 293, is required for sequence-specific DNA binding.23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar All p53 gene mutations except two found in recurrent HCC of this study are located in the DNA-binding domain. Mutations in the central domain alter either the structural integrity of the domain or the amino acids that directly contact the DNA, as demonstrated by the study of the crystal structure of the p53 protein.26Cho Y Gorina S Jeffrey PD Pavletich NP Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations.Science. 1994; 265: 346-355Crossref PubMed Scopus (2207) Google Scholar The N terminus of the p53 protein encoded by exons 2 to 4 has an abundance of acidic amino acids that are involved in the transcriptional function of p53.23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar It is reported that multiple point mutations in this region are usually required to block the transcriptional transactivation of the p53 gene.27Lin J Chen J Elenbaas B Levine AJ Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein.Genes Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (589) Google Scholar In the present study, two patterns of mutant p53 gene in this region were found in two patients with recurrent HCC, one was a single point mutation in exon 4 and the other was a frameshift in exon 3. The interval between surgical resection and the appearance of tumor in these two patients were 12 months and 5 months respectively, which were much shorter than the mean 16.4 months in those patients without mutations in the p53 gene. Therefore, we believe that these two p53 mutants within the N-terminus are pathologically significant. This is consistent with the report which indicated that a point mutation in codon 68 of exon 4 decreased approximately 35% transcriptional activity of the p53 gene.28Lin SR Yang YC Jung JH Tsai JH A significant decrease of the transcriptional activity of p53 mutants deriving from human functional adrenal tumors.DNA Cell Biol. 1996; 15: 793-803Crossref PubMed Scopus (7) Google Scholar The p53 nuclear import or retention is essential for its normal function in growth inhibition or induction of apoptosis.23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar, 29Knippschild U Milne D Campbell L Meek D p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage.Oncogene. 1996; 13: 1387-1393PubMed Google Scholar A defect in the regulation of p53 nuclear import or export may result in tumorigenesis. Dysfunctional transportation of p53 between the nucleus and the cytoplasm is known to occur in a subset of human tumors and in such a situation, p53 is sequestered either in the cytoplasm or in the nucleus.30Bosari S Viale G Roncalli M Graziani D Borsani G Lee AK Coggi G p53 gene mutations, p53 protein accumulation and compartmentalization in colorectal adenocarcinoma.Am J Pathol. 1995; 147: 790-798PubMed Google Scholar, 31Lee KC Crowe AJ Barton MC p53-mediated repression of α-fetoprotein gene expression by specific DNA binding.Mol Cell Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (156) Google Scholar In normal cells, the wild-type p53 protein is kept at a low concentration by rapid degradation. Therefore, the wild-type p53 protein is usually undetected or detected at a very low level. However, when there are mutations within the p53 gene, they result in a dysfunctional protein product with a prolonged half-life that enables them to accumulate in the cell. Accumulation of p53 protein either in the cytoplasm or in the nucleus is considered a pathological index.23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar In the present study, nearly 58% (19 of 33) of recurrent HCC showed a nuclear localization of p53 protein and about 9% (3 of 33) was positive for the p53 protein in the cytoplasm. Detailed analysis revealed that accumulation of p53 protein in the nucleus occurred in recurrent HCC regardless of whether the p53 gene was mutated, whereas the cytoplasmic p53 protein was found in all recurrent HCC with mutant p53 gene. Mechanisms responsible for the sequestration of a particular mutant p53 protein in the nucleus or the cytoplasm are very complicated and not yet completely known. The nuclear localization signals are located within the carboxyl terminus of p53 protein (amino acids 293 to 393).23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar Mutations in this nuclear signal region can affect the nuclear import or export of p53. However, none of the mutations detected in the current study were within this nuclear signal region. Recently, we have demonstrated that ZBP-89 protein can stabilize p53 through a direct protein contact, leading to its retention in the nucleus.13Bai L Merchant JL ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (92) Google Scholar In the present study it was found that ZBP-89 co-localized with p53 in the nucleus in about 63% (12 of 19) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of p53 protein in a subset of recurrent HCC. Indeed, ZBP-89 is able to bind wild-type p53 and some forms of p53 mutants, as demonstrated by co-immunoprecipitation experiment. Mutation within p53 non-binding N-terminal domains retains its ability to bind ZBP-89, whereas mutation in DNA binding domain (after codon 175) abolishes the direct interaction between ZBP-89 and p53. These p53 mutations may thus eliminate ZBP-89-mediated stabilization of p53.13Bai L Merchant JL ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (92) Google Scholar In agreement with the co-immunoprecipitation experiment reported here, ZBP-89 was found to co-localize not only with wild-type p53 but also with the mutant protein. This further confirms that the ZBP-89-interaction domain in some mutant p53 proteins remains intact. Co-localization of ZBP-89 and p53 in the nucleus may be clinically significant in certain types of patients who possess functional p53. The function of p53 protein depends on the nuclear localization.23Stewart ZA Pietenpol JA p53 signaling and cell cycle checkpoints.Chem Res in Toxicol. 2001; 14: 243-263Crossref PubMed Scopus (171) Google Scholar With accumulation of p53 protein in the nucleus, tumor cells are more likely to undergo apoptosis and are thus more susceptible to radiotherapy and chemotherapy.9McIlwrath AJ Vasey PA Ross GM Brown R Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity.Cancer Res. 1994; 54: 3718-3722PubMed Google Scholar, 10Lowe SW Bodis S McClatchey A Remington L Ruley HE Fisher DE Housman DE Jacks T p53 status and the efficacy of cancer therapy in vivo.Science. 1994; 266: 807-810Crossref PubMed Scopus (1564) Google Scholar Therefore, by co-localizing p53 protein, the expression of ZBP-89 may define a subgroup of recurrent HCC that is more suitable to receive radiotherapy and chemotherapy. The present study also revealed that the level of serum α-fetoprotein (AFP) was much lower in recurrent HCC with mutant p53 than those without. This finding is not consistent with an in vitro study that demonstrated that wild-type p53 inhibits the expression of AFP and that mutant one loses its ability to repress AFP production.31Lee KC Crowe AJ Barton MC p53-mediated repression of α-fetoprotein gene expression by specific DNA binding.Mol Cell Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (156) Google Scholar We are unable to explain the difference at the present, but it may be due to the different model of experiments, one is in vivo and the other in vitro. Although AFP is a well-established conventional tumor marker for HCC, there is a report indicating that recurrent HCC may not have an increase in serum α-fetoprotein level.32Cristani A Cioni G De Santis M Chianese L Gozzetti G Ventura E Increase in serum α-fetoprotein without recurrent disease after hepatectomy for hepatocellular carcinoma.Hepatogastroenterology. 1994; 41: 137-139PubMed Google Scholar Also, serum AFP is in general not disease-stage-dependent.33Johnson PJ The role of serum α-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma.Clin Liv Dis. 2001; 5: 145-159Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar
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