Detection Of P53 Protein Research Articles

P53 transactivation and protein accumulation are independently regulated by UV light in different phases of the cell cycle.

DNA damage-induced activation of the p53 tumor suppressor gene is suggested to be central in the cellular damage response pathway. In this study, we analyzed the responses of p53 to UVC radiation in synchronized mouse fibroblasts in terms of p53 accumulation, transcriptional activation, and sequence-specific DNA-binding activity. UVC was found to induce accumulation of p53 cell cycle dependently in G1/S- and S-phase cells but not in G0 or G1 cells. In contrast, p53 transcriptional activity and its target genes, p21 and GADD45, were stimulated by UVC in G0 and G1 cells in the absence of detectable p53 protein. The accumulation of p53 and increased p21 and GADD45 expression were replication dependent in S-phase cells. Interestingly, sequence-specific p53 DNA-binding activity was stimulated also replication independently in S phase, though the effect was not conveyed to stimulation of p53 target genes, suggesting that additional events are required for p53-stimulated gene expression. The results show that opposed to the cell cycle dependence of p53 accumulation, the UVC-mediated transactivation by p53 is independent of the cell cycle phase and protein stabilization.

P53 gene mutations in soft-tissue sarcomas--correlations with p53 immunohistochemistry and DNA ploidy.

The significance of p53 mutations in a group of 67 soft-tissue tumors was examined using single-strand conformation polymorphism and direct sequencing analysis. Molecular findings were correlated with immunohistochemical detection of the p53 protein and DNA ploidy status. Mutations of the p53 gene were detected in 13 (19.5%) out of 67 cases of soft-tissue tumors. Only three were localized outside the conservative regions of the p53 gene. Six mutations were described for the first time in these tumors. Most of the mutations were point mutations in exons 5-8 and, in one case, a deletion at the 3'-splice site of exon 5 could be demonstrated. There was no significant correlation between the occurrence of p53 mutations and the histological grade, although a high number of mutations were defined in poorly differentiated tumors (grade 3). Molecular finding of a p53 gene mutation and immunohistochemical detection of p53 expression did not correlate, which may be due to the high percentage of nonsense mutations in our study (50%). We confirm that only DNA sequencing allows a unique identification and differentiation of mutations in the p53 gene. Other factors may be responsible for the detection of p53 protein in many cases. Histological grade correlated with aneuploidy. The frequency of mutations observed was in accordance with values quoted in the literature. Generally, p53 mutations and p53 overexpression are more likely to represent a late event in the oncogenesis of soft-tissue tumors.

Differential induction of growth arrest inducible genes by selenium compounds

The effects of two types of selenium compounds on the expression levels of growth arrest and DNA damage-inducible ( gadd) genes and on selected cell death genes were examined in mouse mammary MOD cells to test the hypothesis that the diversity of selenium-induced cellular responses to these compounds could be distinguished by unique gene expression patterns. Whereas the expression patterns of known cell deathrelated genes ( bcl-2 and bax) were not informative with respect to the cellular response patterns upon exposure to selenium compounds, time-dependent and selenium species-specific induction patterns were observed for gadd34, gadd45 and gadd153 genes. It was also observed that the MOD cells expressed a truncated p53 transcript but no detectable immunoreactive P53 protein, indicating a null p53 phenotype. The fact that selenium compounds induced growth arrest and death of these cells and that these compounds induced specific patterns of expression of gadd genes indicates that these genes may mediate some selenium-induced cellular responses. The findings further imply that selenium compounds may be effective chemopreventive agents for human breast carcinogenesis, in which p53 mutations are frequent.

Comparison of Immunofluorometry and Immunohistochemistry for the Detection of p53 Protein in Lung Cancer Specimens

Although immunohistochemical techniques are widely used to demonstrate the presence of mutant p53 protein in a wide variety of malignant tissues, quantitative enzyme-linked immunosorbent assay (ELISA)-type immunoassays offer some advantages. In this study we compared immunohistochemistry, performed on formalin-fixed, paraffin-embedded sections of 91 primary lung tumor tissues, with a highly sensitive quantitative two-site immunofluorometric assay, on extracts of fresh-frozen specimens from adjacent regions of the same tissues. Monoclonal DO-7 antibody, and the related monoclonal DO-1 with polyclonal CM-1 antibodies, were used for immunostaining and ELISA, respectively. Concentrations of p53 were expressed relative to total protein, while an immunostaining score reflected the proportion of stained malignant cells, intensity of staining, and tumor cellularity. Strong concordance was shown between the two methods by Spearman correlation (P < .001), Wilcoxon rank sum (P < .001), and contingency table (P < .001) analyses. The use of ELISA-type assays for p53 quantification in lung tumor tissues may be an alternative to the more labor-intensive histologic techniques.

An immunohistochemical analysis of p53 protein expression in pre-malignant and malignant tissues of the oral cavity.

This study looks at p53 protein expression in dysplastic and malignant lesions of the oral cavity using an immunohistochemical staining technique. Archival biopsy specimens of oral dysplasia of squamous cell carcinoma from 64 patients were analysed immunohistochemically. Sections from 90 oral biopsy specimens were examined in all. Positive immunohistochemical detection of the p53 protein, demonstrated by brown nuclear staining, was detectable in over 80% of mild, moderate and severe dysplastic tissues as well as carcinoma-in-situ and squamous carcinoma specimens. We concluded that p53 protein expression occurs frequently in both malignant and dysplastic lesions of the oral cavity, suggesting that abnormally detectable p53 protein is present at the very early stages of development of oral squamous carcinoma. Oral cancer may provide a good model for the study of multistage tumorigenesis in head and neck cancer as the lesions are frequently detected at the pre-invasive stage and are accessible to biopsy.

Immunohistochemical Detection of p53 Protein in Rhabdomyosarcoma

Immunohistochemical Detection of p53 Protein in Rhabdomyosarcoma

Prognostic value of p53 in non-small cell lung cancer: Relationship with proliferating cell nuclear antigen and cigarette smoking

p53 mutations are among the most frequent genetic alterations reported in human lung cancer. Although the prognostic value of altered p53 expression is still debated, it is accepted widely that estimation of the proliferation rate has an important prognostic role. Moreover, an association between certain types of human lung cancers and tobacco use is well known. Drawing from this background, we investigated the immunohistochemical expression of mutant oncogenic p53 protein, and related it to the smoking history of 61 patients with non-small cell lung carcinoma (NSCLC) and to the expression pattern of proliferating cell nuclear antigen (PCNA), which is considered to be an important negative prognostic factor in several neoplasms. We found p53 overexpression in 22 (36.1%) specimens, including 16 squamous carcinomas (41%) and six (27.2%) adenocarcinomas. PCNA nuclear staining was detected in 98.4% of the specimens, and a significantly higher PCNA expression score was found in all of the p53-positive samples. When the patient survival time was compared, p53 accumulation had a statistically significant negative prognostic value ( P < .001). This was supported by a KaplanMeier survival percentage plot of immunohistochemically p53-undetectable specimens and p53-detectable specimens. These latter patients had a greatly reduced survival time. A relationship was established between p53 immunohistochemical detection and the smoking history of the patients. None of the specimens from the nonsmoking patients expressed immunohistochemically detectable p53 protein. Altered p53 expression was detected in 40.7% of smoking patients. Our findings support the hypothesis of involvement of p53 mutations in tobacco-induced carcinogensis and indicate that altered p53 expression plays an important prognostic role in NSCLC in smokers.

Human Papillomavirus Infection and Expression of p53 and c-erbB-2 Protein in Laryngeal Papillomas

Luzar B, Gale N, KambiČ V, Poljak M, Zidar N, Vodovnik A. Human papillomavirus infection and expression of p53 and c-erbB-2 protein in laryngeal papillomas. Acta Otolaryngol (Stockh) 1997; Suppl 527: 120-124.Laryngeal papilloma (LP) is the most frequent benign laryngeal epithelial tumor caused by human papillomaviruses (HPV) types 6 and 11. In the present study, we were interested in whether we can find any prognostic markers which might reflect the biological behavior of the covering epithelium in LP. We focused our attention on the determination of HPV infection, the detection of p53 protein, and c-erhB-2 protein in 24 biopsy specimens of LP. We confirmed the HPV 6 and 11 etiology in 23 of 24 LP. In these lesions the overexpression of p53 protein increased with the grade of epithelial abnormalities. The distribution of positive cells changed from scattered and focal, in simple and abnormal hyperplasia, to diffuse in atypical hyperplasia. It has been shown that in the presence of HPV types 6 and 11 found in LP, p53 can still preserve its tumor suppressor activity. Infection with HPV types 6 and 11 might therefore account for the significantly lower rate of malignant transformation in LP. Two staining patterns for c-erbB-2 protein were observed in the hyperplastic epithelium covering LP: membranous and cytoplasmic. With the increasing grade of epithelial abnormalities, cytoplasmic staining became predominant, and c-erhB-2 positivity sometimes occupied the whole epithelial thickness. This may represent either an alteration in the processing stability of the c-erbB-2 mRNA, gene amplification, or even an artefact.

Clinical Significance of Immunohistochemically Detectable p53 Protein in Renal Cell Carcinoma

To elucidate the clinical significance of p53 protein in renal cell carcinoma (RCC). The p53 protein in the paraffin-embedded materials taken from 72 patients with RCCs was evaluated immunohistochemically and was compared with the histological findings, expression of proliferating cell nuclear antigen (PCNA), genetic instability as assessed by 2c deviation index (2cDI) and 5c exceeding rate (5cER) as well as clinical outcome. The p53 positivity was demonstrated only in a localized and/or focal area of the cancerous tissue. The positive rate of p53 protein was 40.3% in this study. The p53 protein significantly correlated with nuclear grade as well as PCNA expression (p < 0.001 and p < 0.01, respectively). Although there was a wide scatter of 2cDI and 5cER values between p53 positive and negative RCCs, the RCC with positive p53 exhibited significantly higher values in 2cDI as well as 5cER, as compared to that with negative p53 (p < 0.02 and p < 0.005, respectively). However, some of the RCCs with negative p53 showed relatively higher values in 2cDI and 5cER. Using univariate analysis, the prognostic relevance was noted in T, N, M categories, age and p53 positivity, while it was not in 2cDI, 5cER and PCNA expression. Multivariate analysis demonstrated that N category and p53 positivity were independently significant indicators in predicting survival. The presence of p53 protein might reflect the genetic instability already occurred. The p53 positivity reflecting a high cellular proliferation could afford an additional but useful information when predicting survival in patients with RCC.

From Epithelial Dysplasia to Squamous Carcinoma of the Head and Neck Region: Evolutive and Prognostic Histopathological Markers

Silvestri F, Bussani R, Pavletic N, Mannone T, Bosatra A. From epithelial dysplasia to squamous carcinoma of the head and neck region: evolutive and prognostic histopatliological markers.Ki67 immunoreactivity, p53 expression and apoptotic index were examined in 26 non-malignant lesions of the head and neck region, 22 dysplastic lesions of patients without evidence of head and neck carcinoma during follow-up time, 24 dysplastic lesions of patients who subsequently developed a squamous carcinoma in the same area, and 42 squamous cancer cases. A directly proportional relation between Ki67 immunoreactive pattern, apoptotic index and histological evolution from normal to dysplastic or neoplastic mucosa was evident, As far as p53 protein is concerned, its expression became higher and frequently transmural in neoplastic mucosa. A strict correlation between frequency and density of Ki67/p53 immunoreactivity according to invasive cell grading (ICC) scores and poor prognosis of patients were found. On the contrary, malignant cells highly expressing p53 seemed not to undergo apoptosis. Ki67 antigen and p53 protein detection in premalignant lesions and in carcinomas of the head and neck tract could be a useful marker for the management of patients at risk.

A Comparison of Different Modes for the Detection of p53 Protein Accumulation a Study of Bladder Cancer

The aim of the present study was to evaluate different techniques for the analysis of p53 protein accumulation in human bladder cancer. The accumulation was evaluated in 23 carcinomas by immunoblotting (IB), immunohistochemistry (IHC) and flow cytometry (FCM). The results revealed that six (26%), eight (35%) and ten (43%) of the tumours were p53 protein positive by IB, IHC and FCM, respectively. Mutation analysis of the TP53 gene confirmed mutations in 8 of 9 tumours which showed increased levels of p53 protein by FCM. Our results indicate that IHC could be applied for studies of p53 protein accumulation in archival formalin fixed, paraffin-embedded bladder tumours. However, FCM is a more sensitive and objective method for the detection of p53 protein than IHC and this should be taken into account when routinely evaluating the p53 protein accumulation by IHC.

Immunohistochemical detection of p53 protein in rhabdomyosarcoma: association with clinicopathological features and outcome.

Alteration in the p53 tumor suppressor gene is the most common tumor specific genetic change identified in most major cancer types including rhabdomyosarcomas. To investigate the overexpression of p53 and its relation to clinical features and outcome in patients with rhabdomyosarcoma (RMS), an immunocytochemical study was performed. Formalin-fixed paraffin embedded tissue sections obtained from 42 cases of RMS were immunostained with a mouse monoclonal antibody p53-D07. Staining was assessed by evaluating the percentage of p53 immunopositive cancer cell nuclei. Nuclear accumulation of p53 protein was detected in 8 of 42 (19%) samples. Clinical analyses of patients demonstrated no correlation between positive staining and age, sex, histological subtype, stage and overall survival. This analysis, however, was limited by the small number of patients who demonstrated p53 immunostaining. Nonetheless, a statistically significant association was observed between p53 expression and adverse outcome. Nuclear p53 expression was associated with disease progression or recurrence (p <0.001) and with a worse event free survival (p = 0.0015). The nuclear p53 immunoreaction rate is low in RMS, but p53 expression appears to correlate with poor prognosis.

P53 expression in Reed-Sternberg cells does not correlate with gene mutations in Hodgkin's disease.

Immunohistochemically detectable p53 protein expression is common in the Reed-Sternberg and Hodgkin's (RS-H) cells of Hodgkin's disease, but p53 gene mutations have only rarely been identified. The authors found p53 expression in RS-H cells in 16 of 30 cases of Hodgkin's disease (53%), with the percentage of RS-H positive cells ranging from 4% to 85%. In 12 of 30 cases (40%), at least 10% of the RS-H cells were positive for p53. p53 gene mutations were detected in only two cases (7%) using a single-stranded conformational polymorphism assay with a detection sensitivity of between 1% and 5%. The cellular protein, mdm-2, which can stabilize and functionally inactivate wild-type p53 protein, was expressed in RS-H cells in most of these cases (86%). However, neither case with a p53 gene mutation expressed mdm-2 (P < .005). The two cases with p53 gene mutations had a higher mean proliferative index than cases without detectable mutations (90% versus 72%; P < .02). p53 expression in RS-H cells may be related to concurrent mdm-2 protein expression and a p53-positive, mdm-2-negative immunophenotype may be predictive of gene mutations in RS-H cells.

Immunohistochemical and molecular evaluation of the mdm-2 gene product in bronchogenic carcinoma: Gorgoulis VG, Rassidakis GZ, Karameris AM et al. 53 Antaiou Str., Lamprini Ano Patisia, Athens 11146. Mod Pathol 1996;9:544–554

In this study, we investigated immunohistochemically the expression of mdm-2 protein in 93 surgically resected bronchogenic carcinomas. The findings were correlated with p53 protein detection status and clinicopathologic data (histologic type, differentiation grade of the lesions, lymph node metastases, and smoking history of the patients). Thirty of the 93 immunohistochemically examined specimens were subjected to Northern blot and differential polymerase chain reaction analysis to look into the mechanism of mdm-2 overexpression. Finally, we studied the concordance between p53 immunohistochemical positivity and p53 gene alterations as assessed by the single-strand conformation polymorphism technique. Seventy-three (78%) and 67 (72%) of 93 carcinomas showed nuclear immunoreactivity for mdm-2 and p53 proteins, respectively. We observed a high degree of concordance (75%) between p53 mutations and p53 immunolabelling, which was even higher in the specimens with p53 positively in more than 50% of the cells (90%). Despite the high percentage of mdm-2 and p53 expression, the two molecules were simultaneously detected in 50 (54%) of 93 cases. Forty-two (84%) of the 50 cases were accompanied by p53 mobility shifts, which indicated mutations. Interestingly, statistical analysis revealed an almost significant correlation between the carcinomas with mdm-2/p53 coexpression and lymph node disease (P = 0.058), which indicated a possible "gain of function" phenotype. In addition, absence of reactivity for both proteins was statistically more frequent in the patients without lymph node disease (P = 0.006). The mdm-2-positive/p53-negative immunohistochemical profile was more often seen in adenocarcinomas (P = 0.003), especially in well-differentiated ones (P = 0.02), than in other histologic types of lung cancer, which suggested a p53-independent pathway of mdm-2 overexpression. Molecular analysis showed that mdm-2 overexpression was a consequence of increased transcription rather than of mdm-2 gene amplification. The smoking history of the patients was strongly related to p53 (P = 10(-4)) even in the group of adenocarcinomas (P = 0.012). No correlation was observed between cigarette consumption and mdm-2 immunoreactivity.

Prognostic Significance of Immunohistochemically Detected p53 Protein Expression in Stage IIIB Squamous Cell Carcinoma of the Uterine Cervix Treated with Radiation Therapy Alone

Immunohistochemical studies were performed to investigate the prognostic significance of p53 protein expression in 46 patients with stage IIIB squamous cell carcinoma of the uterine cervix who were treated with radiation therapy alone. Tumor cells showed p53 protein expression in 29 of 46 patients. The 5-year overall actuarial and cause-specific survival rates for the patients with p53-positive tumor were 52.9 and 70.4%, respectively. The corresponding rates for the patients with p53-negative tumor were 58.2 and 69.0%, respectively. There was no significant difference of local control rates and of distant metastases rates between the two groups. In conclusion, no relationship was observed between radiation treatment outcome and the immunohistochemically detected p53 protein expression in the patients with stage IIIB squamous cell carcinoma of the uterine cervix.

Concordance between p53 protein overexpression and gene mutation in a large series of common human carcinomas

Immunohistochemical (IHC) detection of p53 protein was compared with the presence of p53 gene mutation in many colorectal ( n = 100), breast ( n = 92), endometrial ( n = 122), and gastric ( n = 116) carcinomas. Two commercially available antibodies, D07 and CMl, were used for IHC analysis of paraffin-embedded tissue sections. Screening for gene mutations in frozen and paraffin-embedded tumor samples was carried out using polymerase chain reaction—single-strand conformation polymorphism (PCR-SSCP). The frequency of nuclear staining with D07 or CMl for each tumor type, respectively, was colorectal (36%, 23%); breast (15%, 19%); endometrial (21%, 33%); and gastric (23%,-). Overall correlation between the two antibodies for nuclear staining was 90% for the 314 tumors analyzed. Cytoplasmic staining was observed with D07 in 7% of breast and 5% of gastric carcinomas and with CMl in 17% of breast and 54% of endometrial carcinomas. p53 gene mutation was found in 39% of colorectal, 28% of breast, 13% of endometrial, and 25% of gastric cancers. The concordance between p53 nuclear overexpression and gene mutation (both positive or both negative) was 68% for colorectal, 79% for breast, 76% for endometrial, and 73% for gastric carcinomas. This study provides further evidence that IHC detection of p53 protein accumulation does not always indicate the presence of a gene mutation and vice versa. Discordant results were observed in approximately 20% to 30% of the tumors studied, highlighting the need for careful characterization of both p53 gene and protein alterations when assessing the relationship between p53 status and tumor behavior.

P53 Protein Overexpression in Early Stage Endometrial Cancer

Overexpression of p53 protein has been reported to correlate with a poor prognosis in endometrial cancer. Most endometrial adenocarcinomas are clinical stage I at the time of diagnosis and the majority of women to die of this neoplasm had stage I disease at initial presentation. The aim of our study was to evaluate the prognostic significance of p53 overexpression in early stage endometrial carcinoma. Ninety-two patients with surgically treated endometrial adenocarcinoma FIGO stage I were examined for overexpression of immunohistochemically detected mutant p53 protein. Follow-up time ranged from 0.4 to 137.8 months (mean, 34.8). Thirteen women died of their tumor. A nuclear staining reaction for p53 was observed in eight cases. Women with p53 protein overexpression showed a significant poorer overall survival in univariate analysis (relative risk, 4.78; 95% confidence interval, 1.56–14.61;P= 0.006, Wald test) and also in multiple analysis adjusted for grading (relative risk, 4.39; 95% confidence interval, 1.39–13.90;P= 0.01, Wald test). Histological grading and histologic stage did not correlate with p53 protein overexpression (P= 0.26,P= 1.0, respectively, exact χ2test). Immunohistochemically detected p53 protein overexpression in early stage endometrial adenocarcinoma could aid in predicting prognosis and subsequently have some impact on adjuvant therapy.

Immunostaining of p53 protein in ovarian carcinoma: correlation with histopathological data and clinical outcome.

The objective of this study was to analyze the incidence of immunohistochemically detectable p53 protein accumulation in epithelial ovarian carcinomas and to correlate these data with the clinical outcome so as to clarify further the role of p53 mutations in prognosis with these patients. Tumor tissues from 179 patients with epithelial ovarian carcinoma were used for immuno-histochemical analysis with monoclonal antibody DO1 and BP 53-12-1 on formalin-fixed, paraffin-embedded tissue. A total of 78 cases (44%) showed positive nuclear p53 staining. The p53-positive cases were found in all histological types of epithelial ovarian tumors. p53 staining was found in tumors of all stages with a higher percentage of positive cases in stage IV ovarian carcinomas (not significant). Poorly differentiated carcinomas showed a significantly higher percentage of p53 protein expression than did highly differentiated tumors (P = 0.0002). Clinical follow-up of up to 14 years (median 25 months) showed a slightly but not significantly shortened disease-free and overall survival time for patients with p53-positive epithelial ovarian carcinomas. We conclude from our data that p53 expression in ovarian carcinoma is associated with poor differentiation but not with the disease being in an advanced stage. There was a tendency for shortened disease-free and overall survival for patients with p53-positive tumors.

Detection of p53 protein in oropharyngeal carcinoma. Prognostic implications.

To demonstrate how the detection of p53 protein in formaldehyde-fixed, paraffin-embedded oropharyngeal carcinoma may be used as a factor in estimating prognosis. University medical centers. Validation cohort. Formaldehyde-fixed, paraffin-embedded squamous cell carcinoma of the oropharynx tissues from 106 patients who underwent surgical therapy between 1975 and 1988 were immunostained by using M-7001 antibody (IgG class). Overexpression of p53 was observed in 46 tumors (43.4%). The detection of nuclear p53 was significantly associated with an increased risk of recurrence of oropharyngeal carcinoma (P = .05). Similar results were obtained when the presence or absence of p53 in the nuclei of the tumor cells was studied in relation to overall survival (P < .001). In a multivariate analysis stratified according to grade, pathological stage, and lymph node status, nuclear p53 status was an independent predictor of overall survival (P < .001). In patients with squamous cell carcinoma of the oropharynx, an accumulation of p53 in the tumor cell nuclei detected by immunohistochemical methods predicts a significantly increased risk of death, independent of tumor grade, stage, and lymph node status. The p53 overexpression appears to be a useful prognostic factor.

Cellular content of p53 protein in rat skin after exposure to the space environment.

The effects of stress in space, microgravity and space radiation, on living organisms are still unknown. We have examined the cellular content of p53 protein, a tumor-suppressor gene product, in skin from rats by the Western blot method using a blotting-amplification system. Three groups of rats were used. The first group was kept on Earth normally and showed hardly any detectable p53 protein. The second group made a 14-day flight into space on the second Spacelab Life Sciences-2 mission (F). The last group was subjected to the same kinds of stress as the rats in the second group except for spaceflight (SC). The F and the SC rats were killed on day zero (F-0 and SC-0 groups) and day nine (F-9 and SC-9 groups) after return. F-0 rats showed marked accumulation of p53 protein, whereas SC-0 rats showed a slight decrease. F-9 and SC-9 rats showed almost the same amount of p53 protein, but F-9 rats showed a slightly higher expression. From these results, it is suggested that the accumulation of cellular p53 protein is induced in rat skin cells by exposure to the space environment.