Analysis Of P53 Mutations Research Articles

Analysis of p53 mutations and Helicobacter pylori infection in human and animal models.

p53 gene mutations are believed to play a critical role in the development of gastric carcinoma. We examined the relation between Helicobacter pylori infection and p53 gene mutations of the gastric mucosa in human and animal models. To detect the original p53 DNA sequences of the Japanese monkey and Mongolian gerbil, the p53 genes of these animals were amplified using the nested polymerase chain reaction method with primers for the human p53 gene. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 genes was performed by the dideoxy terminator method for gastric mucosa of humans, the Japanese monkey, and the Mongolian gerbil. The expression of p53 was examined immunohistochemically in a Japanese monkey model. Mutations of the p53 gene were identified in 52.4% of human H. pylori-positive mucosa and in 100% of monkey H. pylori-positive mucosa. However, no mutations of the p53 gene were found in the H. pylori-positive gastric mucosa of Mongolian gerbils. There were no mutations in H. pylori-negative gastritis mucosa of humans, monkeys, or Mongolian gerbils. Nuclear staining of p53 was seen in the glandular cells of the H. pylori-infected mucosa of Japanese monkeys, especially in the neck region of the glands. These findings demonstrate that the H. pylori infection can induce p53 point mutations in humans and the Japanese monkey and appear to be involved in the pathway leading to dysplasia or carcinoma. However, our direct DNA sequencing method showed no p53 mutations in the Mongolian gerbil model at present. Further studies with this model are needed.

All you need to know about p53

For researchers interested in tumor suppressors, and in particular the p53 protein, this is a useful website; it includes a p53 database, an APC database and a p53 mutation-analysis database

P53 mutation analysis for definite diagnosis of multiple primary lung carcinoma

The detection of a second tumor in patients with lung carcinoma raises the question whether this lesion is a metastasis or a second primary lung carcinoma. Patients cannot always be categorized satisfactorily according the criteria of multiple lung carcinoma proposed by Martini and Melamed. This may result in an inadequate treatment schedule in individual patients. Because p53 mutations can be used as clonal marker, the authors investigated whether p53 mutation analysis can differentiate between primary lung carcinomas and metastatic disease. Sixty-four tumors in 31 patients with synchronous and metachronous lung tumors were investigated by p53 mutation analysis. In 21 patients, the tumors showed different p53 mutations, and therefore a definite diagnosis of multiple primary lung carcinoma was made. One of these patients did not meet the criteria of Martini and Melamed. In two other patients not matching these criteria, identical mutations were demonstrated in both tumors, indicating the presence of metastatic disease. In eight patients, analysis was not conclusive or possible. p53 mutation analysis can be a useful tool to confirm or rule out multiple primary lung carcinoma, and the results confirm the criteria of Martini and Melamed. However, in patients not meeting these criteria, the diagnosis of multiple lung carcinoma still has to be considered, and metastatic disease has to be ruled out. P53 mutation analysis can be helpful for this purpose.

Denaturing High Pressure Liquid Chromatography (DHPLC) for the Analysis of Somatic p53 Mutations

Denaturing High Pressure Liquid Chromatography (DHPLC) for the Analysis of Somatic p53 Mutations

Abnormalities of molecular regulators of proliferation and apoptosis in carcinoma of the oral cavity and oropharynx

Objective: Abnormalities in genes regulating cell proliferation and death may affect disease outcome in squamous cell carcinoma (SCC) of the head and neck. Methods: Proliferative activity (Histone H3 in-situ-hybridization (HISH) labeling index (LI)) and the genes and/or gene products of Cyclin D-1, c-erbB-2, Bcl-2, p21, and p53, were investigated in 35 patients with SCC of the oral cavity and oropharynx, previously studied for p27 expression. Results: Overexpression or very low expression of Cyclin D-1 was associated with unfavorable disease outcome and shorter time-to-recurrence. High c-erbB-2 expression was significantly associated with shorter overall survival and was synergistic with low p27 expression. Bcl-2, HISH LI, p21 expression, and p53 mutation and protein analysis were not significantly predictive, but there were trends suggesting shorter disease-free/overall survival for patients with undetectable Bcl-2, high HISH, and mutant p53. Conclusions: Several cell proliferation and death regulators appeared to predict disease outcome. Limited evidence of cooperativeness among regulators was also seen.

Metaplastic Carcinoma of the Breast: p53 Analysis Identified the Same Point Mutation in the Three Histologic Components

A rare case of metaplastic carcinoma of the breast with both squamous metaplasia and cartilaginous metaplasia was reported. Histologically, the neoplasm revealed complex features, which were consisting of invasive ductal carcinoma, squamous carcinomatous component and chondrosarcomatoid component. Gradual transition of each component was recognized microscopically. p53 mutation analysis disclosed the same point mutation in three histologically different components, but not in the normal epithelium. Based on the morphologic findings, immunohistochemical findings and the p53 mutation analysis, we concluded that these three components in the tumor originated from the same duct progenitor cells.

Glioma Classification: A Molecular Reappraisal

The modern clinical practice of neuro-oncology is dependent on accurate tumor classification. No variable predicts prognosis more precisely, and classification is also the basis on which clinicians make critical therapeutic recommendations to their individual patients: neuro-oncologists apply therapies in a relatively uniform way for all patients with a given tumor type. Hence, in a profound way, treatment of brain tumors is dictated by histological diagnosis. Furthermore, classification guides our scientific study of brain tumors, with biological understanding often based on a priori assumptions about specific tumor types. In the future, as specific therapies become based on individual biological alterations within tumors, precise classification will assume even greater importance to guide these distinct treatments. The primacy of accurate classification in neuro-oncology demands that critical attention be directed toward the problem, and encourages periodic re-evaluations of this essential issue. The present reappraisal is directed toward the diffuse gliomas. These are the most common of primary human brain tumors and comprise the bulk of adult neuro-oncology work. Diffuse gliomas are therapeutically vexing: their infiltrative (diffuse) growth pattern essentially prevents surgical cure, and the majority of these gliomas are resistant to standard chemotherapeutic and radiotherapeutic approaches. Nonetheless, some tumors are therapeutically sensitive and rare cures are effected. Paradoxically, these rare successes draw attention to the essential limitation of current glioma classification schemes: responding tumors may be histologically indistinguishable from nonresponding ones. Consequently, existing methods of glioma classification fall short of their ultimate goal of precisely guiding therapy. However, molecular biological studies of gliomas are making inroads toward an improved classification system for gliomas, one in which response to a specific therapy can be predicted for each individual patient. Furthermore, remarkable insights into the origins and behavior of gliomas are beginning to emerge from animal modeling of glial tumors and from basic research in developmental neurobiology. Together, such scientific advances and therapeutic successes provide an opportunity to question and perhaps refine the paradigm for classifying diffuse gliomas. The present reappraisal first defines the problems inherent in current glioma classification systems. Next, by reviewing advances in our molecular understanding of gliomas, we suggest that a more biological approach to glioma classification will provide improved means to type these tumors. Any new classification, however, must be based on clinical significance, and we thus point out the pressing need for better clinical endpoints and outcome measures in the field. Finally, by looking at basic advances in developmental neurobiology and animal modeling, we raise the possibility that we should begin to think of gliomas in a different conceptual framework. In combination, these data suggest that the present is an opportune time to begin to reconsider how glioma classification should advance during the next few years.

P21/waf1/cip1 in gastric cancer: associations with histopathological subtypes, lymphonodal metastasis, prognosis and p53 status.

Cyclins and cyclin-dependent kinases are determining factors of the cell cycle. In the present study, we investigated the role of p21 and p53 in the biology of gastric cancer, focusing on its influence on progression and prognosis (n = 195). P21 and p53 immunoreactivity was analysed immunohistochemically, applying monoclonal antibodies. The p53 status was comparatively evaluated by PCR-SSCP analysis of p53 mutations in selected tumours. Fifty-eight percent of the carcinomas were p21+ in more than 5% of the cancer cell nuclei, whereas 19% exhibited a p21 immunoreactivity in more than 20% of the nuclei. On the other hand, p53 was over-expressed (in more than 50% of the nuclei) in about 45% of the specimens. P21 immunoreactivity in more than 5% of the nuclei was inversely related to the pN as well as pTNM cancer stage, whereas only a strong p21 expression (in >20% of the nuclei) was correlated with a better survival probability in a univariate analysis. The p53 status was associated with lymphonodal metastasis, but not with prognostic data. In multivariate survival analyses, neither p21 nor p53 emerged as independent prognostic factors. Compared with the results of p53 mutation analysis by PCR-SSCP. p21 immunoreactivity was reduced in p53-mutated cases. These features show an association of p21 over-expression with certain clinico-pathological parameters of gastric cancer. In this context, our data suggest that p21 immunoreactivity in more than 5% of the tumour cells has a predictive value for the course of adenocarcinoma of the stomach.

Analysis of p53 mutation and expression in pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is a rare, superficially situated tumor that most frequently occurs in the temporal lobe of young adults and is often associated with seizures. It generally has a relatively favorable prognosis. Prior studies have shown that TP53 mutations may occur in up to 25% of PXAs, suggesting that PXA may have an etiology similar to diffuse astrocytoma rather than pilocytic astrocytoma. In the present study, we performed immunostaining for p53 protein and examined the mutation status of exons 5-8 of the p53 gene in 55 PXAs, 8 of which had undergone one or multiple recurrences. Of 55 primary PXAs, 35 (64%) showed staining in <1% of tumor cells, 15 (27%) in 1-10%, 4 (7%) in 11-50%, and only 1 (2%) in >50%. No significant increase in p53 protein expression was noted in recurrences, even when associated with increased histological anaplasia. We found a TP53 heterozygous mutation in exon 7 in 1 of 47 primary tumors that yielded useable DNA, and in its recurrence 3 years later. This tumor, a grade II PXA, did not show signs of anaplastic transformation at recurrence. Eleven additional recurrences from 7 patients, 5 of which showed signs of histological anaplasia, did not show TP53 mutations in exons 5-8. Based on our data, the p53 mutation appears to be an uncommon (2%) genetic event in PXA formation and does not appear to be involved in tumor progression. Consequently, our findings suggest that the genetic events that underlie PXA formation differ from those involved in diffuse astrocytoma.

Immunoexpression of Ultraviolet Photoproducts and p53 Mutation Analysis in Atypical Fibroxanthoma and Superficial Malignant Fibrous Histiocytoma

p53 mutation is one of the major results of ultraviolet (UV) radiation. UV photoproducts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6–4) photoproducts (64PPs) also play an important role in skin cancer development. Atypical fibroxanthoma (AFX), which mimics malignant fibrous histiocytoma (MFH) histologically, occurs in the sun-exposed skin of the elderly, and therefore, an association with UV has long been suspected. Eighteen fibrohistiocytic skin lesions comprising AFX (n = 7), storiform-pleomorphic type MFH centered in the subcutis (superficial MFH; S-MFH; n = 4) and benign fibrous histiocytoma (BFH; n = 7) were used for immunohistochemical and molecular analysis. Eight cases of deep MFH (D-MFH) were also analyzed for UV photoproduct expression for the purposes of comparison. Immunohistochemically, the CPD scores of AFX (3.6 ± 0.4) were significantly higher than those of S-MFH (1.3 ± 0.8), D-MFH (0.8 ± 0.5), or BHF (1.4 ± 0.7); however, the 64PP scores were extremely low in all these tumors (AFX, 0.1 ± 0.1; S-MFH, 0.0 ± 0.0; D-MFH, 0.0 ± 0.0; and BHF, 0.0 ± 0.0). AFX, S-MFH, and BFH showed immunoexpression for p53 (2/7, 2/4, and 0/7), respectively. p53 mutations were detected in AFX (4/6; 67%) and S-MFH (1/4; 25%), but not in BFH (0/5; 0%) using polymerase chain reaction–single-strand conformation polymorphism, and all of the mutations in AFX were either C-T transitions or at dipyrimidine sites. In conclusion, AFX and S-MFH are both similar fibrohistocytic lesions; however, AFX has high immunoreactivity for CPDs compared with S-MFH, D-MFH, or BFH. These data suggest that CPDs may play an important role in the pathogenesis of AFX.

P53 Gene Mutation and Protein Accumulation during Neoplastic Progression in Barrett's Esophagus

The aim of the present study was to characterize expression and mutation of p53 during the neoplastic progression from Barrett's esophagus to adenocarcinoma and to test the reliability of immunohistochemistry for p53 overexpression as an indicator of p53 mutation in this context. The association of both gene mutation and protein accumulation with clinicopathological findings and survival was also studied. A total of 77 samples from 30 esophagectomy specimens with Barrett's esophagus and adenocarcinoma of patients in longitudinal clinical follow-up were analyzed. Different lesions (intestinal metaplasia, dysplasia, and adenocarcinoma) as well as normal squamous-cell esophageal epithelia were sampled from formalin-fixed, paraffin-embedded tissues by microdissection. Mutations in p53 Exons 5 to 9 were detected by polymerase chain reaction–single-strand conformation polymorphisms (PCR-SSCP) and confirmed by direct DNA sequencing. Nuclear accumulation of p53 protein was analyzed immunohistochemically from tissue sections adjacent to those used for microdissection. p53 gene mutations were found in 17 and p53 protein accumulation were found in 20 tumor samples. Of the 17 adenocarcinomas with a p53 mutation, 16 stained positive for p53 protein. p53 mutations were detected significantly more frequently in high-grade dysplastic than in low-grade dysplastic lesions (77% versus 29%, P < 0.01). In contrast, nuclear accumulation of p53 was detected in 85% of high-grade and 71% of low-grade dysplastic lesions. In eight cases with p53 mutation, the mutation identified in the tumors was also detected in premalignant lesions, mainly in high-grade dysplasia. In four cases of p53-mutated tumors, clones with different p53 mutations were detected in premalignant lesions. Neither p53 mutations nor p53 protein accumulations were found in metaplastic lesions. In summary, we found that p53 mutations occurred mainly during the transition from low-grade to high-grade dysplasia in the neoplastic progression of Barrett's esophagus but not in the nondysplastic Barrett's mucosa. Mutational analysis of p53 by PCR-SSCP and p53 accumulation by immunohistochemistry were mostly concordant in adenocarcinoma and high-grade dysplastic lesions but frequently discordant in low-grade dysplastic lesions. No correlation between p53 gene mutation or p53 accumulation and clinicopathological findings was observed in this study.

Loss of heterozygosity at 11p15 and p53 alterations in malignant gliomas.

Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with prognostic implications. In the later steps of tumor-progression, deletions on chromosome 11p15 and mutations of the tumor suppressor gene p53 were determined for different malignancies. To elucidate the involvement of 11p15 deletions in the tumorigenesis of malignant gliomas, we analyzed a series of 50 glioblastomas for loss of heterozygosity (LOH). Paired tissue and blood samples from 50 patients with glioblastoma multiforme were included. Microsatellite markers located on 11p15.1-11p15.5 were used for LOH analysis. Additionally, mutation analysis of the tumor suppressor gene p53 was performed, which might correlate with favorable survival in glioblastomas. The region 11p15.4-5 was deleted heterozygously in 28% of cases representing 15 cM. Twenty-six glioblastomas did not show allelic loss for any locus. Our data revealed close association of LOH 11p15 with p53 mutations, and survival analysis showed a trend indicating better prognosis in glioblastomas characterized by LOH 11p15. In the tumorigenesis of malignant gliomas, p53 mutations and 11p15 deletions seem to indicate a genetic subset of tumors with favorable prognostic value.

Analysis of p53 mutations and Helicobacter pylori infection in human and animal models

Background. p53 gene mutations are believed to play a critical role in the development of gastric carcinoma. We examined the relation betweenHelicobacter pylori infection andp53 gene mutations of the gastric mucosa in human and animal models.Methods. To detect the originalp53 DNA sequences of the Japanese monkey and Mongolian gerbil, thep53 genes of these animals were amplified using the nested polymerase chain reaction method with primers for the humanp53 gene. Direct DNA sequencing of exons 5, 6, 7, and 8 of thep53 genes was performed by the dyedeoxy terminator method for gastric mucosa of humans, the Japanese monkey, and the Mongolian gerbil. The expression ofp53 was examined immunohistochemically in a Japanese monkey model.Results. Mutations of thep53 gene were identified in 52.4% of humanH. pylori-positive mucosa and in 100% of monkeyH. pylori-positive mucosa. However, no mutations of thep53 gene were found in theH. pylori-positive gastric mucosa of Mongolian gerbils. There were no mutations inH. pylori-negative gastritis mucosa of humans, monkeys, or Mongolian gerbils. Nuclear staining of p53 was seen in the glandular cells of theH. pylori-infected mucosa of Japanese monkeys, especially in the neck region of the glands.Conclusions. These findings demonstrate that theH. pylori infection can inducep53 point mutations in humans and the Japanese monkey and appear to be involved in the pathway leading to dysplasia or carcinoma. However, our direct DNA sequencing method showed nop53 mutations in the Mongolian gerbil model at present. Further studies with this model are needed.

Analysis of p53 mutations and Helicobacter pylori infection in human and animal models

Analysis of p53 mutations and Helicobacter pylori infection in human and animal models

Analysis of p53 and BAX mutations, loss of heterozygosity, p53 and BCL2 expression and apoptosis in basal cell carcinoma in Korean patients.

Apart from some Japanese studies, there are few data on the gene mutations involved in the development of basal cell carcinomas (BCC) in Koreans or other Asians. Objective To gain insight into the molecular pathogenesis of BCC in Koreans. A collection of 33 cases of BCC were screened for mutations of p53 and BAX genes, p53 and BCL2 expression, loss of heterozygosity (LOH) and apoptosis. Mutations of p53, found in 9% (three of 33) of the cases, were all mis-sense mutations (G-->C transversions) at codon 246 on exon 7. In 6% (two of 33), BAX gene showed frameshift mutations resulting from deletions in the poly(G) tract. LOH on chromosome 9q was seen in 58% (14 of 24), and p53 mutations developed only among the 9q LOH+ cases; LOH on chromosome 18q, where BCL2 gene is located, was found in 13% (four of 30). Immunohistochemical expression of p53 was seen in 27% (nine of 33), and its expression did not coincide with p53 mutations. BCL2 expression was seen in 39% (13 of 33). Apoptosis was revealed in 21%. In BCC, 9q LOH and p53 mutations seem to be closely related; the immunoreactivity of p53 and its mutations were not directly related; and p53 and BCL2 expression were negatively correlated. Frameshift mutations of the BAX gene in BCC are documented for the first time. Various molecular mechanisms operate with redundant complexity in the pathogenesis of BCC. The LOH on chromosome 9q is the most frequent genetic alteration, as in other races; however, p53 mutations are much less frequent in Koreans than in Caucasians and suggest aetiologies other than ultraviolet radiation.

Novel method for simultaneous analysis of p53 and K-ras mutations and p53 protein expression in single histologic sections.

Abnormal protein expression and gene mutation should be examined on exactly identified lesions. To perform simultaneous analyses of oncogene or tumor suppressor gene mutations and related protein expression in single histologic sections, we have developed a novel method using an antigen-retrieval solution for a polymerase chain reaction template before immunohistochemical staining. Using 20 cases of sporadic colorectal carcinoma, several kinds of antigen-retrieval solutions were tested after heating rehydrated, 4-microm-thick, formalin-fixed, paraffin-embedded histologic sections at 96 degrees C for 20 minutes. Polymerase chain reaction-single-strand conformation polymorphism analysis was conducted for p53 (exons 5 through 9) and K-ras (exons 1 and 2), and the histologic sections were then immunostained with monoclonal antibody against p53. DNA analysis of antigen-retrieval solutions was possible in all 20 cases and revealed completely consistent results (100%) with fresh cancer tissue and microdissected cancer tissue of paraffin-embedded histologic sections. With this method, K-ras mutations were positive in 10 of 20 cases (exon 1 in 9 cases and exon 2 in 1 case) and p53 mutations were positive in 9 of 20 cases (exon 5 in 4 cases, exon 6 in 1, exon 7 in 3, and exon 8 in 1 case), with 8 of the 9 p53 mutation cases showing diffuse p53 protein expression on immunostaining. Base alterations of all abnormal conformers were confirmed with direct sequencing. For polymerase chain reaction-single-strand conformation polymorphism analysis, sodium citrate buffer (pH 6.0) was found to be the optimal antigen-retrieval solution. This newly developed method can be used for routine immunostaining and genetic analysis with single histologic sections.

Mutation analysis of p53 in ovarian tumors by DHPLC

Up to now, ovarian carcinomas represent a major health problem among female cancers because they are the leading cause of death from gynecological malignancy. A high proportion of these tumors selects for mutations in the p53 gene. There is evidence that inactivation of the p53 protein could indicate poor prognosis and chemoresistance of patients. To set up a fast and sensitive test for p53 defects in tumor tissues, we analyzed ovarian cancer cells by denaturing high-performance liquid chromatography (DHPLC). A primer set spanning the whole coding region of p53 with seven fragments was designed and appropriate heteroduplex detection in DHPLC analysis was elaborated. The analysis of 45 ovarian tumor specimens yielded 17 genetic alterations (38%) occurring exclusively in the malignant tissue of the patients. In addition, frequent polymorphisms present in normal compared to tumor tissue could serve as a tool for the rapid identification of loss of heterozygosity (LOH) in the tumor. We observed that LOH in intron 2 or 3 correlated well with a lack of one allele in mutated fragments. In conclusion, DHPLC screening appears to be a sensitive and effective test for genetic alterations in tumors with p53 involvement. Since p53 mutations point to a poor prognosis state in several cancers, a fast screening of tumor material for genetic variations may have implications for further individual treatment of patients.

Bax expression in benign and malignant thyroid tumours: dysregulation of wild-type P53 is associated with a high Bax and P21 expression in thyroid carcinoma.

The purpose of our study was to determine the expression of the pro-apoptotic BAX protein in relation to the mutational status of BAX and p53 (as transcriptional activator of the BAX gene) in benign and malignant thyroid tissue. In 47 patients with thyroid tumours (14 follicular and 3 papillary carcinomas, 14 adenomas and 16 goitres), the DNA was screened for mutations of BAX (exon 1-6) and p53 (exon 5-8) by single-strand conformation polymorphism polymerase chain reaction (SSCP-PCR). Furthermore, the protein expression of BAX, p53 and p21 (which is also increased transcriptionally by p53) was investigated by immunohistochemistry. Surprisingly, we observed elevated BAX levels in patients with thyroid carcinomas compared with patients with adenomas (unpaired t-test: p<0.05) or with goitres (p<0.02). This is in clear contrast to other carcinomas where BAX is frequently inactivated which correlates to a poor prognosis (Sturm et al. J. Clin. Oncol. 1999;17:1364-74.). There were no significant differences of the BAX levels between goitres or the adenomas. In the SSCP-PCR analysis, no BAX mutations were detectable. P53 mutation analysis by SSCP-PCR did not reveal any functional p53 mutations in the patients with carcinomas, adenomas or goitres. Nevertheless, patients with carcinomas showed an overexpression (preferentially cytoplasmic) of p53 protein compared with patients with benign tumours (p<0.05). The absence of p53 mutations suggests that the overexpressed p53 is wild type. This is in line with the expression profile of BAX and p21, which showed a higher protein expression in these p53 positive tumours (p<0.05 in the carcinomas compared with the non-malignant lesions). Consequently, the overexpressed p53 might be a correlate for dysregulation without loss of function. This, in turn, might be a reason for the good outcome of some patients with thyroid cancer.

Expression of p53 and human herpesvirus-8 (HHV-8)-encoded latency-associated nuclear antigen with inhibition of apoptosis in HHV-8-associated malignancies.

Kaposi sarcoma (KS) and primary effusion lymphoma (PEL) cells express human herpesvirus-8 (HHV-8)-encoded latency-associated nuclear antigen (LANA) (open reading frame [ORF] 73 protein), suggesting that LANA plays an important role in the pathogenesis of HHV-8-associated malignancies. Recently, the binding of LANA to p53 was demonstrated in vitro. In the current study, the authors investigated the association between p53 and LANA expression with apoptosis in HHV-8-associated malignancies in vivo. Twenty-six cases of KS, 1 case of HHV-8-associated solid lymphoma, 2 PEL cell lines, and an HHV-8-associated lymphoma engrafted in severe combined immunodeficiency (SCID) mice were examined. Immunohistochemistry using the catalyzed signal amplification system was employed to detect LANA and p53 on paraffin embedded tissues and the immunofluorescence technique was used on cell lines. To detect apoptosis, the TdT-mediated dUTP nick end labeling (TUNEL) method was used. For mutation analysis of p53, exons 5-9 of the p53 gene were amplified by polymerase chain reaction and examined by direct sequencing. Immunohistochemistry revealed that LANA and p53 were expressed in the tumor cells of all these specimens, and apoptotic cells were rarely detected in them using the TUNEL method. Immunofluorescence assay revealed that LANA colocalized with p53 in the nuclei of PEL cells. Sequencing analysis indicated that there was no mutation in the deduced amino acid sequences of p53 in KS tissues. These data suggest colocalization of p53 and LANA and the inhibition of apoptosis in HHV-8-associated malignancies in vivo, supporting the results found in vitro that p53 inhibition by LANA suppresses cell death, as reported previously. These results also suggest that the p53 pathway is crucial in the pathogenesis of HHV-8-associated malignancies.

P21/waf1/cip1 in Gastric Cancer: Associations with Histopathological Subtypes, Lymphonodal Metastasis, Prognosis and p53 Status

Background: Cyclins and cyclin-dependent kinases are determining factors of the cell cycle. In the present study, we investigated the role of p21 and p53 in the biology of gastric cancer, focusing on its influence on progression and prognosis (n = 195). Methods: P21 and p53 immunoreactivity was analysed immunohistochemically, applying monoclonal antibodies. The p53 status was comparatively evaluated by PCR-SSCP analysis of p53 mutations in selected tumours. Results: Fifty-eight percent of the carcinomas were p21+ in more than 5% of the cancer cell nuclei, whereas 19% exhibited a p21 immunoreactivity in more than 20% of the nuclei. On the other hand, p53 was over-expressed (in more than 50% of the nuclei) in about 45% of the specimens. P21 immunoreactivity in more than 5% of the nuclei was inversely related to the pN as well as pTNM cancer stage, whereas only a strong p21 expression (in >20% of the nuclei) was correlated with a better survival probability in a univariate analysis. The p53 status was associated with lymphonodal metastasis, but not with prognostic data. In multivariate survival analyses, neither p21 nor p53 emerged as independent prognostic factors. Compared with the results of p53 mutation analysis by PCR-SSCP, p21 immunoreactivity was reduced in p53- mutated cases. Conclusions: These features show an association of p21 over-expression with certain clinico-pathological parameters of gastric cancer. In this context, our data suggest that p21 immunoreactivity in more than 5% of the tumour cells has a predictive value for the course of adenocarcinoma of the stomach.