p53 disruption profoundly alters the response of human glioblastoma cells to DNA topoisomerase I inhibition

A critical challenge in cancer research is to identify genetic lesions that sensitize patients to chemotherapy. p53, which is mutated in nearly one-third to half of glioblastomas, may be such a lesion. In this paper, we demonstrate that p53 disruption dramatically sensitizes glioblastoma cells to DNA topoisomerase I inhibitor-mediated apoptosis. Using 19 glioblastoma cell lines, including 15 low-passage ex vivo cell lines derived from patients, as well as isogenic glioblastoma cells varying in p53 status, we show that clinically relevant levels of SN-38 potently induce cell cycle arrest and temporary senescence in glioblastoma cells with wild-type p53 while causing massive apoptosis in p53-deficient cells (P<0.0002). We demonstrate that glioblastoma cells with wild-type p53 proliferate when recultured in drug-free medium, whereas p53-deficient cells do not. We also show that p16 protein expression is neither necessary nor sufficient for initiation and/or maintenance of SN-38-induced arrest/senescence. These results indicate that p53 disruption has a dramatic effect on how glioblastoma cells process topoisomerase I inhibitor-mediated DNA damage.     

p53-independent WAF1 induction by ACNU in human glioblastoma cells

The induction of WAF1 gene expression after the treatment with the anticancer agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU; nimustine hydrochloride) was studied in two human glioblastoma cell lines: U-87MG, which bears the wild-type p53 gene, and T98G, which bears the mutant p53 gene. A marked accumulation of WAF1 was observed 3 h after ACNU treatment in both cell lines. The induction of WAF1 mRNA by ACNU was detected by northern blot analysis in these cells. Binding activity of p53 to a p53 consensus sequence increased after treatment in U-87MG cells but not in T98G cells. The existence of a p53-independent WAF1 induction pathway was supported by the apparent accumulation of WAF1 after ACNU treatment in the p53-null human osteosarcoma cell line Saos-2. These findings suggest that there are two possible pathways for WAF1 induction: the p53-dependent pathway through the p53-responsive element and the p53-independent pathway through other elements. Mol. Carcinog. 21:171–176, 1998. © 1998 Wiley-Liss, Inc.     

Nitric oxide is an initiator of intercellular signal transduction for stress response after hyperthermia in mutant p53 cells of human glioblastoma.

Nitric oxide is known to be a multifunctional physiological substance. Recently, it was suggested that nitric oxide is involved in p53-dependent response to many kinds of stress, such as heat shock and changes in cellular metabolism. To verify this hypothesis, we examined the effect of nitric oxide produced endogenously by heat-shocked cells on nonstressed cells using a human glioblastoma cell line, A-172, and its mutant p53 (mp53) transfectant (A-172/mp53). The accumulation of inducible nitric oxide synthase was caused by heat treatment of the mtp53 cells but not of the wild-type p53 (wtp53) cells. The accumulation of heat shock protein 72 (hsp72) and p53 was observed in nontreated mtp53 cells cocultivated with heated mp53 cells, and the accumulation of these proteins was suppressed by the addition of a specific inducible nitric oxide synthase inhibitor, aminoguanidine, to the medium. Furthermore, the accumulation of these proteins was observed in the wtp53 cells after exposure to the conditioned medium by preculture of the heated mp53 cells, and the accumulation was completely blocked by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. In addition, the accumulation of hsp72 and p53 in the wtp53 cells was induced by the administration of an nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, to the medium. Finally, the thermosensitivity of the wtp53 cells was reduced in the conditioned medium by preculture of the heated mp53 cells as compared with conventional fresh growth medium. Our finding of the accumulation of hsp72 and p53 in nitric oxide-recipient cells cocultivated with heated nitric oxide-donor cells provides the first evidence for an intercellular signal transduction pathway via nitric oxide as intermediate without cell-to-cell interactions such as gap junctions.     

T-cell response to p53 tumor-associated antigen in patients with colorectal carcinoma

Despite the radical surgical resection performed in patients with colorectal carcinoma, there is a high rate of tumor recurrence. Over an observation period of 3 years, 18% of the patients in our collective suffered a tumor relapse with local or distinct metastases after initial R0-resection. Some evidence suggests that this may be due to suppression of anti-tumor responses, a phenomenon that might be attributed to regulatory T cells. The aim of our study was to investigate the tumor-specific immune response depending on the UICC stage of patients with colorectal cancer. The cellular immune responses against defined antigens that are overexpressed in most of the patients with colorectal cancer were characterized. For this purpose, the tumor suppressor gene, p53, was chosen as the tumor-associated antigen that exhibits mutations and overexpression in up to 60% of colorectal carcinoma. We observed that p53 induced both IFN-gamma and IL-10 secretion. The predominance of IL-10 production indicated that regulatory T cells directly participate in modulating the anti-tumor immune response. IL-10 levels in the blood as well as the expression of regulatory T-cell specific genes at the tumor site correlate with the UICC stage of the disease. These results may provide an explanation for the poor prognosis and increased recurrence rate in patients with advanced carcinoma.     

Humoral immune response against p53 protein in patients with colorectal carcinoma

p53Aberrations are frequent in colorectal carcinogenesis (40–70%). Because p53 gene mutations typically result in increased p53 protein concentration in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, serum p53 antibodies were quantitatively analyzed in 229 patients with colorectal cancer, using an immunofluorometric procedure. Circulating antibodies against p53 were found in 23% (53/229) of the patients. We quantified antibody concentrations in all positive sera and found that they varied from 300 to 500,000 arbitrary units/l. Sequential analysis of positive sera from 3 patients showed that p53 antibody concentrations change during the course of the disease, reflecting progression or regression. No association was found between the presence of p53 antibodies and age, sex, stage, histological grade and patient relapse-free or overall survival. These data demonstrate that antibody generation against the p53 tumor-suppressor protein is a relatively common event in colorectal cancer and that serological analysis for p53 antibodies may have some value for patient monitoring. The test has no value for prognosis. © 1997 Wiley-Liss, Inc.     

Induction of p53-mediated apoptosis and recovery of chemosensitivity through p53 transduction in human glioblastoma cells by cisplatin

Cisplatin is a DNA-damaging chemotherapeutic drug that may have a role in the adjuvant chemotherapy of several solid tumors, such as malignant glioblastoma, and the status of p53 tumor suppressor protein is a critical determinant of cisplatin chemosensitivity. In the present study, we showed the relationship of p53 status and chemosensitivity of cisplatin between two human malignant glioblastoma cell lines, A172 and T98G, harboring wild-type and mutant-type p53, respectively. Cisplatin was found to be more cytotoxic to A172 than T98G cells in a time- and concentration-dependent manner. Cisplatin-induced cytotoxicity manifested as apoptosis, characterized by genomic DNA fragmentation, nuclear condensation and an increase in sub-G1 population. Cisplatin induced the accumulation of p53 and p21 proteins in A172 cells, but not in T98G cells. The introduction of the adenovirus-mediated wild-type p53 gene into T98G cells resulted in the decrease of viability as well as the increase in sub-G1 population with p53 accumulation, activation of caspase-3 protease and release of cytochrome c from the mitochondria. These data strongly suggest that the expression of p53 is essential for the cytotoxic effect of cisplatin in human malignant glioblastoma cells, A172 and T98G, and the introduction of apoptotic signal molecules, such as p53, will be beneficial to achieve chemosensitivity in malignant glioma.     

EGFR inhibition in glioblastoma cells induces G2/M arrest and is independent of p53

Mutations involving the TP53 gene are frequently identified in up to 50% of all human tumors, including glioblastomas. Analysis of expression patterns of TP53 in glioblastomas shows that it is mainly mutated in secondary glioblastomas and is less common in primary GBMs. However, the prognostic significance of TP53 loss of function in astrocytomas has always been controversial. In contrast, EGFR/erbB2 complexes have been implicated in the poor prognosis of several cancers, including glioblastomas. Our previous work showed that transforming phenotypes could be inhibited by interfering with active EGFR/erbB2 complex using mutant erbB2 proteins in wild-type p53 GBM cells. To assess the dependence of EGFR inhibited phenotype on p53, we used three mutant p53 glioblastoma cell lines in the present study and showed that mutant erbB2 can be exploited to inhibit EGFR-mediated oncogenic transformation irrespective of p53 status. Ectopic expression of a mutant erbB2 receptor (T691S) in mutant p53 GBM cells resulted in slower growth rate than empty vector controls. T691S-expressing clones exhibited a more flattened and nontransformed morphology. Consistently, T691S inhibited transformation in soft agar assays and tumor formation in nude mice independent of p53 status. Biochemical analysis showed reduced Akt and GSK-3 alpha/beta, but not p42/44MAPK phosphorylation, in T691S-expressing cells, when compared to parental controls, suggesting the P13-K pathway may be more relevant than MAPK for glial cell transformation. Cell cycle analysis showed reduced cyclin D1 and CDK6 and increased phospho-Cdc-2 (Tyr15) and p15INK4B in erbB2-inhibited cells, suggesting that nonfunctional EGFR/erbB2 complexes exert their inhibitory effects at various stages of the cell cycle to block the progression of cells through G2/M via Akt/GSK-3/Cdc2 pathway. Collectively, these observations provide a basis for receptor-based therapies that disable erbB receptors and inhibit proliferative signals in erbB-expressing human cancers including glioblastomas, regardless of their TP53 status.     

Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

Background  

Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.     

Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo

Alterations in the p53 tumor-suppressor gene occur in 35–60% of human glioblastomas, and re-introduction of p53 can suppress neoplastic growth. To evaluate the potential for p53 gene therapy of glioblastoma, we have analyzed the response of human glioblastoma cell lines in vitro and in vivo to experimental therapy with replication-deficient recombinant adenoviruses encoding wild-type p53 (rAd-p53). Western blot analyses showed high-level expression of p53 protein after treatment with rAd-p53, and transgene expression was dependent on promoter strength. A p53-specific dose-dependent inhibition of in vitro cellular proliferation was observed in 5 of 6 cell lines, and growth inhibition corresponded to adenovirus-mediated gene transfer and expression. p53-specific cell death was quantitated by release of the lactate dehydrogenase enzyme. Fragmentation of DNA into nucleosomal oligomers and the occurrence of a hypodiploid cell population detected by flow cytometry provided evidence for apoptosis. Studies in nude mice demonstrated that ex vivo infection with rAd-p53 suppressed the tumorigenic potential of human glioblastoma cells. Furthermore, direct injection of rAd-p53 into established s.c. xenografts inhibited tumor growth. Our observations suggest that re-introduction of wild-type p53 may have potential clinical utility for gene therapy of glioblastoma. © 1996 Wiley-Liss, Inc.     

Phenylbutyrate sensitizes human glioblastoma cells lacking wild-type p53 function to ionizing radiation

PURPOSE: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. METHODS AND MATERIALS: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. RESULTS: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G(1) arrest, increase in sub-G(1) fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios (+/- SE) of 1.5 (+/- 0.2) and 1.3 (+/- 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. CONCLUSIONS: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53.     

Inhibition of hypoxia inducible factor 1alpha causes oxygen-independent cytotoxicity and induces p53 independent apoptosis in glioblastoma cells

PURPOSE: Hypoxia, which activates the hypoxia inducible factor-1 alpha (HIF-1alpha) pathway, is a common feature in malignant gliomas and has been linked with tumor cell survival and therapy resistance. In this study, we examined the effect of antisense inhibition of HIF-1alpha on the survival, apoptosis and responses to chemotherapy in U-87 malignant glioma cells. MATERIALS AND METHODS: Hypoxia (1% oxygen) was achieved in a tri-gas incubator with intermittent N(2) gas flushing or in a gas tight-module sealed with 94% N(2), 1% O(2) and balance CO(2). HIF-1alpha inhibition was achieved with antisense phosphorothioate oligodeoxynucleotide (AS-HIF ODN), delivered using cytofectin GSV3815. HIF-1alpha expression level was monitored by a hypoxia-responsive luciferase reporter assay and verified by northern blot and immunoblot analyses. Cell viability was quantified by a colorimetric microtiter plate MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. Apoptotic cell death was detected with a colorimetric caspase-3 assay, as well as using terminal transferase-catalyzed in situ end labeling (TUNEL) staining. RESULTS: Antisense HIF-1alpha phosphorothioate oligodeoxynucleotide (AS-HIF ODN) treatment suppressed HIF-1alpha expression by up to 80% under both normoxic and hypoxic conditions as measured by a hypoxia-responsive reporter assay and confirmed by northern and western blot analyses. Antisense knockdown of HIF-1alpha resulted in significant reduction in U-87 cells survival and an acceleration of apoptosis, which did not involve p53 transactivation. Pretreatment of cells with Z-Val-Ala-Asp (-OCH(3))-fluoromethylketone (Z-VAD), a broad-spectrum caspase inhibitor largely eliminated this effect of AS-HIF. Caspase-3 specific activity was markedly induced 3 days after AS-HIF treatment when increased cell death was also noted. Transient overexpression of HIF-1alpha in U-87 cells neutralized apoptosis-inducing effect of AS-HIF. AS-HIF treatment did not affect viability of primary astrocytes and was selectively more toxic to U-87 glioma cells than normal human fibroblasts. The HIF-1alpha antisense treatment exerted an oxygen-independent, and additive but not synergistic effect to the cytotoxicity of cisplatin, etoposide, and vincristine. CONCLUSIONS: These results together indicate that suppression of HIF-1alpha-expression may be a promising strategy that is selective for reducing the survival and facilitating chemotherapeutic efficacy of malignant glioma.     

Expression of p53 and 17p allelic loss in colorectal carcinoma.

Mutations in the p53 gene are the most common genetic changes in cancer thus far. Many p53 mutations result in a protein product having a prolonged half-life compared to wild-type p53. The mutant protein is frequently detectable immunohistochemically, whereas the wild-type p53 present in normal cells is not. We examined 90 colorectal carcinomas for increased expression of p53 using 3 p53 specific monoclonal antibodies, PAb1801, PAb421, and PAb240. Overall, 70% of the colorectal carcinomas stained for p53. Each tumor's DNA was also assessed for loss of heterozygosity on chromosome 17p, the location of the p53 gene. Of those tumors that reacted with the anti-p53 antibodies, 76% showed loss on chromosome 17p. Tumors with loss of heterozygosity on 17p generally stained with all 3 antibodies, whereas those without loss tended to stain with just one antibody, typically PAb240. Fifteen tumors were examined for the presence of specific p53 mutations. A total of 10 mutations were found, 6 were missense and 2 were deletions, and all but one of the tumors with missense mutations stained for p53.     

大肠癌组织中p53蛋白和基因的表达及意义

目的:观察以抑癌基因p53编码蛋白在正常肠黏膜、肠上皮化生及结直肠癌组织中的表达,探讨结直肠癌组织中p53和mRNA的表达及意义。方法:用半定量逆转录聚合酶链反应(RT-PCR)检测64例结直肠癌及其癌旁肠上皮化生组织中p53蛋白的表达水平,比较其与结直肠癌组织学分型、临床病理分期的关系。结果:1)结直肠癌组织中p53蛋白的表达明显低于正常组织(66.7%,26/39)。结直肠癌组织p53蛋白mRNA的表达水平与正常黏膜组织相比较,显著偏低(1.12±1.07,2.32±1.05,P<0.05)。2)蛋白和基因表达呈显著正相关(r=0.875,P<0.01)。3)黏液腺癌组为3/4,管状、乳头状腺癌为19/35,p53蛋白和基因表达与结直肠癌临床病理、组织学分型无明显相关性,P>0.05。结论:p53失活或低表达与结直肠癌的发生有密切相关性。提示p53 mRNA表达水平的检测可作为判定结直肠癌发生及侵袭转移能力的一项客观指标。     

Overexpression of p53 protein is an independent prognostic indicator in human endometrial carcinoma.

The important role of the p53 gene in tumour progression and cellular response to DNA damage has prompted investigation of the clinical significance of alterations to this gene. We examined both p53 overexpression and mutation of the gene in endometrial carcinoma in order to evaluate the prognostic significance of these changes. Of 122 endometrial carcinomas, 33 (27%) showed overexpression of p53 in the nucleus and 66 (54%) in the cytoplasm. Mutation in the p53 gene was found in 16 (13%) cases but showed no significant association with patient survival. Nuclear p53 overexpression was associated with poor survival (48% vs 80% alive in negative tumours 5 years post operatively, P < 0.001). In contrast, cytoplasmic p53 overexpression was associated with better survival (85% vs 55%, P < 0.001). When patients were separated into prognostic subgroups according to established clinical markers, these associations remained significant within most subgroups examined. In multivariate analysis adjusted for surgical stage, histological grade and type and vascular invasion, both nuclear p53 overexpression [hazard ratio 4.9 (95% CI 1.3-17.6). P = 0.016] and cytoplasmic overexpression [0.25 (0.06-0.98), P = 0.047] were independent prognostic factors. Immunohistochemical assessment of p53 overexpression in the nucleus and cytoplasm could provide useful prognostic information for the management of patients with endometrial cancer.     

Nuclear thymidylate synthase expression, p53 expression and 5FU response in colorectal carcinoma.

Thymidylate synthase (TS) is a key enzyme in DNA synthesis and is inhibited by metabolites of the chemotherapeutic agent 5-fluorouracil (5FU). Nuclear expression of TS in human tissue in vivo has not been characterised and its clinicopathological correlates in malignancy are unknown. 52 cases of primary colorectal carcinoma (CRC) and 24 cases of matched metastatic carcinoma were studied immunohistochemically using the monoclonal antibody TS106. The degree of nuclear TS immunostaining correlated closely with levels of TS mRNA expression amongst 10 CRCs studied. Strong nuclear immunostaining was seen in normal basal crypt colonocytes and germinal centre cells, and in a varying proportion of adenocarcinoma cells. Amongst the primary carcinomas, higher TS nuclear expression was associated with prominent extracellular mucin production and right-sided location. Higher TS nuclear expression also showed a significant association with poorer response to protracted venous infusional 5FU therapy. There was no clear association between TS nuclear expression and Ki67 or p53 expression assessed immunohistochemically. There was a strong positive correlation between TS nuclear expression in primary and metastatic CRC but the latter generally showed higher expression than matched primary tumour tissue. These findings confirm the nuclear expression of TS protein in human cells in vivo and provide new insight into how such expression may relate to the behaviour of CRCs.     

Loss of heterozygosity of p53 gene and p53 protein expression in human colorectal carcinomas

The p53 gene is a tumor suppressor gene located on chromosome 17p. Deletions of this chromosome and point mutations of p53 have been implicated in the development of colonic neoplasms. We have analyzed the loss of heterozygosity of the human p53 tumor suppressor gene in 40 cases of colorectal carcinoma using two restriction fragment length polymorphisms detected by BglII and AccII restriction enzymes. p53 gene product expression was studied immunohistochemically in 64 colorectal carcinomas, 18 adenomas, and 40 normal colonic mucosae using an anti-human p53 monoclonal antibody (Pab 1801) and the avidin-biotin-peroxidase complex technique. Twelve of the 40 patients (30%) were polymorphic for the p53 gene. In ten of these informative patients (83%), the tumor samples showed the loss of one allele when compared with normal colorectal samples of the same patient. One of the homozygous patients showed a loss of both p53 alleles. p53 immunostaining was observed in 43 of 64 carcinomas (67%) but only in two adenomas (11%). These two positive adenomas showed areas of carcinoma in situ. The normal mucosa was always negative. No relation could be found between p53 immunostaining and the degree of differentiation, the extension of the tumor, or the Ki-67 proliferative index. Mucinous carcinomas and right-side carcinomas were less p53 immunoreactive (25% and 52%, respectively) than the usual adenocarcinomas (73%) and distal tumors (72%). These findings suggest that p53 may be a target of chromosome 17 deletions and that this gene may play a role in the malignant transformation of adenomas. BglII and AccII restriction fragment length polymorphism analysis of the p53 gene may be a useful and direct technique to detect allelic loss of this gene in tumors.     

Low dose of wortmannin reduces radiosensitivity of human glioblastoma cells through the p53 pathway

Wortmannin is an inhibitor of PI3-kinase and acts on cultured cells at dosages below 1 microM. Wortmannin also inhibits the gene products of the PI3-kinase family such as ATM or DNA-PK at dosages above 10 microM in cultured cells. There are many reports on the enhancement of radiosensitivity by a high dose of wortmannin inhibiting the proteins of the PI3-kinase family. However, there have been no reports on the effect on radiosensitivity of low doses of wortmannin inhibiting PI3-kinase. We found that low doses of wortmannin reduced the radiosensitivity of human A172 glioblastoma cells. This effect was shown only in wild-type p53 cells, but was not shown in mutant p53 cells such as T98G or A172/248W carrying a dominant point-mutated p53 gene. This result indicates that the PI3-kinase, or another wortmannin-sensitive enzyme, may affect the signal transduction of p53. We examined the response of the p53 pathway by X-ray irradiation. A low dose of wortmannin did not affect the accumulation of p53 and the phosphorylation of p53 at ser-15, but reduced the induction of WAF1 and enhanced the induction of GADD45.     

The effect of p53-function on the sensitivity to paclitaxel with or without hyperthermia in human colorectal carcinoma cells

The importance of p53-function for the sensitivity to paclitaxel with and without hyperthermia (HT) was studied in an isogenic cell line system. The inactivation of p53 decreased sensitivity to paclitaxel (1.1-2.5-fold), which correlated with a lower induction of apoptosis. The magnitude of the G2/M arrest after treatment with paclitaxel was similar in all cell lines. The cytotoxicity of paclitaxel was not enhanced by HT in either wild-type p53 or p53-inactivated cells. In conclusion, cellular sensitivity to paclitaxel depends on p53-function by its ability to induce apoptosis. Irrespective of the p53-function HT was not able to enhance the sensitivity to paclitaxel.     

Prognostic significance of p53 overexpression in gastric and colorectal carcinoma.

p53 expression was examined in 55 gastric and 107 colorectal carcinomas with an immunoperoxidase technique, using the polyclonal antibody CM1 on routinely fixed, paraffin embedded tissue. p53 protein was detected in 47% gastric and in 46% colorectal carcinomas and found to correlate with stage of disease and unfavourable clinical outcome (P less than 0.001). Thus, the proportion of positively reacting neoplasms increased as the stage progressed, tumours which had invaded regional lymph-nodes overexpressed p53 more frequently than localised carcinomas and an elevated level of p53 was associated with early relapse and death. In colorectal carcinoma p53 positivity was also linked with site and macroscopic configuration of the primary tumour and was most frequently expressed in carcinomas from the rectum and in ulcerative tumours. p53 overexpression was irrespective of tumour grade. Uniform negative reactivity with anti-p53 antibody was seen in normal epithelium adjacent to carcinoma, intestinal metaplasia, atrophic gastritis and in colonic adenomas. There was a good correlation between immunohistochemical staining on paraffin and frozen sections. These studies suggest that in gastric and colorectal carcinoma, immunohistochemical detection of p53 protein in routinely fixed tissue can be used along with other established parameters to assess prognostic outcome, especially to identify patients with poor short-term prognosis.