P53 represents a key player in apoptosis-induction in cancers including colorectal cancer (CRC) that ranks third worldwide in cancer prevalence as well as mortality statistics. Although a pro-apoptotic effect of resveratrol has been repeatedly proven in CRC cells, its pathway mechanisms are not completely understood, as there are controversial statements in the literature regarding its activation or inhibition of the counteracting proteins Sirt-1 and p53.

CRC cells as wild-type (HCT-116 WT) or p53-deficient (HCT-116 p53-/-) were cultured using multicellular tumor microenvironment (TME) cultures containing T-lymphocytes and fibroblasts to elucidate the role of p53/Sirt-1 modulation in resveratrol's concentration-dependent, pro-apoptotic, and thus anti-cancer effects.

Resveratrol dose-dependently inhibited viability, proliferation, plasticity as well as migration, and induced apoptosis in HCT-116 WT more effectively than in HCT-116 p53-/- cells. Moreover, resveratrol stimulated Sirt-1 expression when administered at low concentrations (<5µM) but suppressed it when added at high concentrations (>10µM) to CRC-TME. In parallel, similar to the knockdown of Sirt-1 at the mRNA level, treatment with high-concentration resveratrol boosted the acetylation of p53, the expression of p21, Bax, cytochrome C, caspase-3, and ultimately induced apoptosis in CRC WT but not in CRC p53-/- cells. Notably, increasing concentrations of resveratrol were found to promote hyperacetylation of p53 and FOXO3a as post-translational substrates of Sirt-1, indicating a negative regulatory loop between Sirt-1 and p53.

These results demonstrate for the first time, a negative reciprocal crosstalk between the regulatory circuits of p53 and Sirt-1, consequently, apoptosis induction by higher resveratrol concentrations in CRC-TME.

An imbalance between apoptotic and proliferative processes is believed to underlie colorectal neoplasia. We evaluated the expression of bcl-2, p53, mdm2 proteins, and apoptosis in colorectal neoplasms, as well as their correlation with clinico-pathological parameters, using image analysis. Biopsies from 46 colorectal cancers, 121 adenomas, and 25 controls were studied using monoclonal antibodies against p53, bcl-2, mdm2 and the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) method for apoptosis. P53 and bcl2 protein expression was higher in adenomas >or=1 cm (P < 0.03) and tubulovillous-villous adenomas (P < 0.03), and correlated with dysplasia (P < 0.03). In Cox regression analysis, Dukes' stage was the most significant independent prognostic indicator of a worse survival (P < 0.019), whereas when stage was eliminated, bcl-2 expression was also a powerful predictor for bad prognosis (P = 0.02). In conclusion, both bcl-2 and p53 immunohistochemical profiles may be useful adjuncts in detecting adenomas with a malignant potential, whereas bcl-2 could be used in combination with Dukes' stage as a predictor of prognosis in colorectal cancer.

The present study was designed to investigate the clinical/prognostic relevance of immunohistochemical expression of p53-targeted genes mdm-2, p21WAF1 and bcl-2 alone and in combination with p53 for the indirect assessment of p53 gene status in breast cancer. 141 archival breast carcinomas were immunostained, and the putative mutational status of the p53 gene was defined in 21 of them, as a control for immunohistochemistry, using the polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) analysis. Genetic changes of p53 correlated significantly with p53 protein overexpression (p = 0.01) but did not do so with any of the related molecules. Immunohistochemical p53 status was directly correlated with mdm-2 (p = 0.0001), p21 (p = 0.0004) and inversely with bcl-2 (p = 0.005) expression. bcl-2 proved to be an independent marker of prognosis, p53 only in the group of node-positive carcinomas, whereas bcl-2-/p53+ tumours revealed the worst prognosis. Mdm-2 and p21 expression was of prognostic significance neither alone nor in combination. We conclude that the detection of down-stream regulators of p53 does not increase the efficacy of immunohistochemistry in assessing the functional status of p53 in breast cancer; however, their combined analysis may help to select subgroups of patients at the extremes of risk for recurrence, or those with greater chances for survival.

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen- and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regions- associated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.

To investigate the expression of bcl-2 in colorectal carcinoma and to examine its association with mediators of apoptosis (p53 and mdm-2), clinicopathological features and long-term outcome.

We determined by immunohistochemistry the expression of bcl-2 in 102 colorectal carcinomas with 10-year follow-up. In 66 of these cases in which we had previously assessed p53 status, no correlation was seen between bcl-2 and p53. The mdm-2 protein was not detected in any of the 66 cases. Cytoplasmic staining of the bcl-2 gene product was seen in the tumour cells of 22 cases (22%). Using a polymerase chain reaction technique we showed that overexpression of bcl-2 was not due to rearrangement of the bcl-2 gene. Expression of bcl-2 protein was related to tumour grade but was unrelated to patient age, sex, tumour site, tumour size or Dukes' stage. There was a trend towards increased survival in those whose tumours expressed bcl-2 protein (P = 0.055). When entered into a multivariate analysis, this survival difference was independent of tumour stage (P = 0.05).

These results suggest that bcl-2 expression in colorectal carcinoma is associated with a better long-term prognosis.

The expression of p53 protein was examined in a series of 111 colorectal cancer adenocarcinomas with a long follow-up. A quantitative luminometric immunoassay (LIA) was used for the measurement of wild-type and mutant p53 protein in extracts from colorectal tumour cytosols, p53 being detected in 42% of the samples (range 0.0-52 ng (mg-1)). Using an arbitrary cut-off value of 2.7 ng mg(-1), 25% of the tumours were classified as manifesting high p53 levels. There was no association of p53 expression with patient age, sex, serum preoperative carcinoembryonic antigen (CEA) levels, tumour site and size, nodal status or TNM stage. Significant and independent correlation was found to exist between high p53 levels and prolonged disease-free survival (P = 0.05) at a median follow-up of 60 months. This survival advantage was most apparent among stage III cancer patients. The results from this study would suggest that expression of high p53 levels appear to be useful in selecting a group of colorectal cancer patients with a better prognosis.

The aim of our study was to investigate the expression of p53 and mdm2 mRNA and protein in colorectal adenocarcinoma. For the detection of mRNA, 60 fresh frozen human tumour samples and 12 samples of corresponding normal tissue were examined. After total RNA extraction, reverse transcription (RT) was performed followed by cDNA amplification with specific primers using RT-polymerase chain reaction (PCR). Immunohistochemical detection of protein was examined in 81 formalin-fixed and paraffin-embedded human tumour specimens as well as 15 samples of adjacent normal colorectal tissue. p53 mRNA was detected in 80% (48/60) of the tumours and in 67% (8/12) of normal tissue samples; 87% (52/60) of tumours had mdm2 mRNA in contrast to only 17% (2/12) of normal tissue specimens. Nuclear p53 protein expression was observed in 52% (42/81) of the tumour specimens and in none of the 15 normal specimens, whereas mdm2 protein was found in the nucleus (31%, 25/81) and also in the cytoplasm (86%, 70/81) of tumour samples. In normal tissue, mdm2 protein expression was only observed in the cytoplasm (13%, 2/15) and not in the nucleus. There was a significant correlation between coexpression of p53 and mdm2 protein and the occurrence of lymph node metastases (P = 0.03) as well as between p53 protein expression and the occurrence of distant metastases (P = 0.007). Additionally, significant associations were found between p53 mRNA and p53 protein, p53 mRNA and mdm2 mRNA or protein, and also between mdm2 mRNA and mdm2 protein expression, supporting the existence of a regulatory mechanism involving p53 and mdm2.

To offer more tailored treatment to individual patients with squamous cell carcinoma of the vulva, more accurate prediction of lymph node metastases is required. As p53 and mdm2 are genes known to be involved in the development of other tumours, we studied expression of p53 and mdm2 in carcinogenesis of squamous cell carcinoma of the vulva and their clinical relevance. Archival material of 141 T1 and T2 vulvar tumours were used. Of the 141 primary tumours, the corresponding 39 lymph node metastases (LNM) were studied, and in 90 cases the pre-existent epithelia adjacent to the tumour (EAT) and in 14 cases vulvar intraepithelial neoplasia adjacent to the tumour (VIN) was also investigated. Detection of p53 and mdm2 protein was immunohistochemically performed. Scoring categories were: negative (1); weakly positive (2); moderately to markedly positive (3); and markedly positive (4). Overexpression of p53 was seen in 56% of the LNM, 39% of the primary tumours, 21% of the VIN lesions and 0% in the group of EAT. No relation was found between overexpression of p53 in the primary tumour and LNM. Expression of mdm2 was seen in 14% of the primary tumours, of which four cases were marked positive. In the group of LNM no mdm2-positive staining was observed. In the group of EAT, 25% was mdm2-positive, of which six cases were marked positive. In the group of VIN, 36% showed moderate (score 3) mdm2 expression. No relation was found between expression of mdm2 and LNM. In squamous cell carcinoma, overexpression of p53 is a late event in carcinogenesis. Marked expression of mdm2 is rarely seen in vulvar carcinomas, indicating that aberrant p53 cannot induce mdm2 expression. LNM cannot be predicted by detection of these proteins.

The products of bcl-2 and p53 genes are involved in the regulation of apoptosis and proliferation and have been associated with prognosis in several malignancies, including colorectal adenocarcinoma. Although 2 European studies have reported a prognostic significance of Bcl-2 expression in colorectal adenocarcinomas, a study from the United States did not observe such an association. Therefore, we used immunohistochemistry to evaluate the prognostic significance of Bcl-2 expression, p53 nuclear accumulation and their concomitant expression in 134 US patients with colorectal adenocarcinoma. Antigen retrieval was required for adequate detection of Bcl-2 expression. Fifty percent of the colorectal tumors were classified as expressing Bcl-2, and Bcl-2 expression was associated with longer patient survival. Antigen retrieval was not necessary for detecting nuclear accumulation of p53 by immunohistochemistry. Nuclear accumulation of p53 was detected in 44% of colorectal adenocarcinomas and was associated with decreased patient survival. Tumors that did not express detectable levels of Bcl-2 but exhibited nuclear accumulation of p53 were associated with the shortest patient survival (log rank, p = 0.001). Multivariate Cox regression analysis demonstrated that Bcl-2 expression (p = 0.018), p53 nuclear accumulation (p = 0.024) and regional lymph-node metastasis (p = 0.005) were independent prognostic factors. Although a trend toward an inverse correlation between Bcl-2 and p53 expression was observed, the prognostic value of Bcl-2 expression was independent of p53 status. Thus, assessment of both Bcl-2 and p53 status may be valuable in predicting the prognosis of patients with colorectal adenocarcinomas.

The aim of the present study was to assess a possible role of the tumour suppressor gene p53 in neuroendocrine (Merkel cell) carcinoma of the skin with regard to tumour development and tumour progression. p53 was investigated in a series of routinely processed Merkel cell carcinomas, with application of four different p53 antibodies (CM-1, PAb1801, DO7, and PAb240) to 25 carcinomas and screening for p53 mutations of exons 4-8 by single-strand conformation polymorphism (SSCP) analysis in 9 cases. All 25 tumours in the present series showed the characteristic microscopic and immunohistochemical features of Merkel cell carcinoma of the skin. In 5 of the 25 Merkel cell carcinomas investigated 5-10% of tumour cell nuclei showed a positive p53 reaction with at least one anti-p53 antibody. A few scattered p53 positive nuclei were found in an additional 9 cases. The remaining 11 cases completely lacked p53 immunostaining. SSCP analysis of exons 4-8 revealed no significant alterations in the mobility shift of the single strand DNAs in the five cases with 5-10% p53-immunoreactive tumour nuclei or in five cases lacking p53 accumulation significant. Our results suggest that alterations of the p53 gene play only a minor part in the development or progression of Merkel cell carcinoma of the skin.

MDM2 is an oncoprotein that inhibits p53 tumour-suppressor protein. Amplification of the MDM2 gene and overexpression of its protein have been observed in some human malignancies, and these abnormalities have a role in tumorigenesis through inactivation of p53 function. To determine the clinicopathological and prognostic value of MDM2 abnormalities in non-small-cell lung cancer (NSCLC), MDM2 gene amplification and its protein expression status were analysed in surgically resected materials. MDM2 gene amplification was detected in only 2 (7%) of the 30 tested patients. MDM2 protein was found immunohistochemically in a total of 48 (24%) of the 201 patients. MDM2 protein was slightly frequently observed in patients with adenocarcinoma, but its presence or absence was not associated with clinicopathological factors such as T-factor, N-factor, stage, tumour size, differentiation or p53 protein status. Overall, MDM2-positive patients tended to have a better prognosis (P = 0.062). In particular, among immunohistochemically p53-negative patients (n = 110), those with positive MDM2 protein expression showed significantly better prognosis (P = 0.039) and, in a multivariate analysis, MDM2 protein status was a favourable prognostic factor (P = 0.037). In contrast, among p53-positive patients (n = 91), there was no difference in prognosis depending on MDM2 protein status. Thus, in the NSCLC patients studied, MDM2 gene amplification was a minor event, but expression of its protein, which was often observed immunohistochemically, was a favourable prognostic marker, especially among patients without p53 protein accumulation. Further study is needed to determine how MDM2 protein expression results in a better prognosis.

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 "insular" carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2-11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.