Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21^{WAF 1/CIP1} protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

A tumour-associated cell-surface glycoprotein accompanying p53 overexpression and higher growth potential for gastric cancer.

Tumour-associated cell-surface glycoprotein is associated with tumour progression in gastric cancer. We investigated the biological significance of tumour-associated cell-surface glycoprotein, determined by the binding of Helix pomatia agglutinin (HPA), with regard to survival time and to the malignant potential of cancer cells in serosally invasive gastric cancer in 119 patients. HPA was positively stained in 75 of 119 patients (63.0%) with gastric cancer with serosal invasion. In patients with HPA-positive tissue, the tumour was larger than in HPA-negative cases and was frequently located in the middle third of the stomach. The incidence of lymph node metastasis was higher than in patients with HPA-negative tissue. There were no differences between the cases staining negatively and positively with HPA with respect to the other factors examined. Gastric cancer tissues with HPA-positive staining revealed a higher positive rate of abnormal p53 staining and a higher concentration of proliferating cell nuclear antigen (PCNA) labelling. The survival time of the patients with HPA positive staining was shorter than for those whose tissues were HPA negative. Thus, tumour-associated cell-surface glycoprotein is apparently closely related to the malignant potential of serosally invasive gastric cancer.     

Differential gene expression profiles of scirrhous gastric cancer cells with high metastatic potential to peritoneum or lymph nodes

Scirrhous gastric cancer is often accompanied by metastasis to the peritoneum and/or lymph nodes, resulting in the highest mortality rate among gastric cancers. Mechanisms involved in gastric cancer metastasis are not fully clarified because metastasis involves multiple steps and requires the accumulation of altered expression of many different genes. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of gastric cancer metastasis. In this study, we performed global analysis on differential gene expression of a scirrhous gastric cancer cell line (OCUM-2M) and its derivative sublines with high potential for metastasis to the peritoneal cavity (OCUM-2MD3) and lymph nodes (OCUM-2MLN) in a nude mice model. By applying a high-density oligonucleotide array method, expression of approximately 6800 genes was analyzed, and selected genes were confirmed by the Northern blot method. In our observations in OCUM-2MD3 cells, 12 genes were up-regulated, and 20 genes were down-regulated. In OCUM-2MLN cells, five genes were up-regulated, and five genes were down-regulated. The analysis revealed two functional gene clusters with altered expression: (a) down-regulation of a cluster of squamous cell differentiation marker genes such as small proline-rich proteins [SPRRs (SPRR1A, SPRR1B, and SPRR2A], annexin A1, epithelial membrane protein 1, cellular retinoic acid-binding protein 2, and mesothelin in OCUM-2MD3 cells; and (b) up-regulation of a cluster of antigen-presenting genes such as MHC class II (DP, DR, and DM) and invariant chain (II) in OCUM-2MLN cells through up-regulation of CIITA (MHC class II transactivator). We then analyzed six gastric cancer cell lines by Northern blot and observed preferential up-regulation of trefoil factor 1, alpha-1-antitrypsin, and galectin 4 and down-regulation of cytidine deaminase in cells prone to peritoneal dissemination. Genes highly correlated with invasion or peritoneal dissemination of gastric cancer, such as E-cadherin or integrin beta4, were down-regulated in both of the derivative cell lines analyzed in this study. This is the first demonstration of global gene expression analysis of gastric cancer cells with different metastatic potentials, and these results provide a new insight in the study of human gastric cancer metastasis.     

TP53 mutation and p53 overexpression for prediction of response to neoadjuvant treatment in breast cancer patients.

The value of p53 to predict the cytotoxic effect of two commonly used chemotherapy regimens was assessed in patients with advanced breast cancer. Response to a DNA-damaging combination therapy [fluorouracil, epirubicin, cyclophosphamide (FEC] considered to induce p53-dependent apoptosis was compared with a microtubule stabilizing therapy (paclitaxel) expected to be independent of p53 function. The p53 status of the patients' breast tumors was assessed using both immunohistochemistry (IHC) and direct sequencing of the entire p53 gene. p53 findings were correlated with treatment response, and linkage between p53 function and cellular response was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. In a series of 67 breast tumors, 19% had TP53 gene mutations, 40% had a positive p53 IHC, and 12% had both. In the FEC group, treatment failure was related to both the presence of TP53 gene mutations (P = 0.0029) and a positive IHC (P < 0.0001). Apoptosis was almost exclusively found in tumors having normal p53 in both parameters (P < 0.0001). In the paclitaxel group, treatment response was neither related to apoptosis nor to normal p53. Combination of sequencing and IHC results revealed a significant association between abnormal p53 and response to paclitaxel (P = 0.011). We found TP53 mutations, as well as p53 protein overexpression, to be associated with response to chemotherapy. Whereas clinical response to FEC was found to be dependent on normal p53, the cytotoxicity of paclitaxel was related to defective p53. The efficiency of paclitaxel during mitosis might be supported by lack of G1 arrest due to p53 deficiency. Therefore, patients with p53-deficient tumors may benefit from paclitaxel.     

bcl-2 overexpression combined with p53 protein accumulation correlates with hormone-refractory prostate cancer.

Seventy-seven men with histologically proven and newly diagnosed prostate cancer we investigated for the presence of bcl-2 protein overexpression and p53 protein accumulation 1 immunohistochemistry. Forty-five men had evidence of locally advanced and metastatic disease and we treated by means of hormone manipulation. Twenty-eight patients either failed to respond to initial hormone manipulation or relapsed within 37 months from diagnosis (median 20 months). Of the 77 cancers, 37 (48% showed bcl-2 overexpression at diagnosis. Twenty-seven of those were treated with androgen ablation and 2 (74%) had hormone-refractory disease (P = 0.0128). Twenty-three of 77 men (29.8%) had nuclear staining for p53 protein. Twenty-one of those were treated with hormone manipulation and 14 (66.6%) showed hormone resistance (P = 0.0012). Seventeen patients had both bcl-2 overexpression and p53 protein accumulation, 16 of whom were hormonally treated, with 13 (81.2%) having hormone-refractory disease (P < 0.0001). These findings suggest that the combined detection of p53 protein accumulation and bcl-2 overexpression may be useful in predicting hormone resistance in prostate cancer. By deregulating programmed cell death, alteration in these genes may prevent patients from responding to androgen ablation, or allow them to escape hormonal control of the disease.     

p53 overexpression is associated with cytoreduction and response to chemotherapy in ovarian cancer

The aim of this study was to assess the association of p53 status with primary cytoreduction, response to chemotherapy and outcome in stage III-IV primary ovarian cancer patients. Immunohistochemical analysis of p53 was performed on formalin-fixed, paraffin-embedded specimens from 168 primary ovarian carcinomas by using the DO-7 monoclonal antibody. p53 nuclear positivity was found in 84 out of 162 (52%) malignant tumours. A higher percentage of p53 nuclear positivity was observed in patients with advanced stage of disease than in stage I-II (57% vs 23% respectively; P = 0.0022) and in poorly differentiated versus well/moderately differentiated tumours (59% vs 32% respectively; P = 0.0038). The multivariate analysis aimed to investigate the association of FIGO stage, grade and p53 status with primary cytoreduction in 136 stage III-IV patients showed that stage IV disease may influence the possibility to perform primary cytoreduction in ovarian cancer patients. p53-positivity also maintained a trend to be associated with poor chance of cytoreduction. In patients who underwent pathologic assessment of response, cases who did not respond to chemotherapy were much more frequently p53-positive than p53-negative (86% vs 14% respectively; P = 0.012). Moreover, patients with stage III disease and < 2-cm residual tumour were more likely to respond to treatment. In multivariate analysis, FIGO stage and p53 expression were independently correlated with pathologic response to chemotherapy. Time to progression and survival rates were shown not to be different in p53-positive versus p53-negative patients.     

Ras-p21,p53在阴茎癌腹股沟淋巴结中的表达及临床意义

Objective: The aim of this study was to investigate the expression and clinical significance of ras-p21 and p53 proteins in inguinal lymph nodes with penis carcinoma. Methods: The clinical data of 44 patients of penis (squamous) carcinoma and 40 non-tumor patients from 1990 to 2002 in our hospital were added to our research, 84 inguinal lymph nodes were got by lymph node biopsy from each patient at random. Pathological examination showed that 18 cases of cancer group were metastatic carcinoma as group A, th...     

乳腺癌PCNA、p53及HER-2的表达与淋巴结转移相关性的临床研究

目的　探讨乳腺癌细胞PCNA、p5 3及HER - 2的表达与淋巴结转移的相互关系。方法　采用S-P法检测 89例乳腺癌中的PCNA、p5 3及HER - 2的表达。结果　PCNA、p5 3及HER - 2表达分别为6 0 .7% (5 4/ 89)、43.8% (39/ 89)及 39.9% (35 / 89) ,与淋巴结转移有明显相关性 (P <0 .0 5 )。结论　PCNA、p5 3及HER - 2的表达可能是评估乳腺癌预后的有用指标。     

p53 immunohistochemical analysis of the lymph nodes in node-negative breast cancer patients who had subsequent relapse

We performed a genetic dignosis using p53 immunohistochemistry for the detection of lymph node micrometastasis. Immunohistochemical analysis was performed in 12 node-negative patients with breast cancer who subsequently showed relapse. There was no p53 immunostaining in 197 lymph nodes from 12 patients, while 2 were positive in the 12 primary lesions. p53 immunohistochemistry is not an alternative method for detecting lymph node micrometastasis in node-negative breast cancer.     

p53 overexpression as a prognostic factor for advanced stage bladder cancer

Overexpression of the TP53 gene protein detected by immunohistochemistry appears to identify those patients with superficial bladder cancer at risk of the development of muscle invasive or metastatic disease. However, the role of p53 overexpression in patients with advanced or metastatic bladder cancer is not yet well established. In the present study, 44 specimens from 44 patients with advanced stage bladder tumours (T₂–T₄) undergoing radical cystectomy were investigated for different biological and clinical characteristics as possible prognostic factors: sex, age, depth of tumour infiltration, T-stage, histological grade, lymph node status, application of adjuvant systemic chemotherapy (MVAC), proliferative activity (staining for proliferating cell nuclear antigen (PCNA) by monoclonal antibody (PC10) as well as overexpression of the p53 oncoprotein (monoclonal antibody pAb 1801)). After a median follow-up of 22 months, 16 of the 23 patients (70%) with more than 40% of tumour cells stained positively for p53 (Group B) died from tumour progression compared with 7 of the 21 patients (33%) with less than 40% of tumour cells positive for p53. During univariate analysis, p53 overexpression (P = 0.008), staining for PCNA (80% of cells positive) (P = 0.01) and tumour stage (P = 0.01) were significant prognostic factors for survival, among which p53 overexpression (P = 0.023) as well as T-stage (P = 0.012) remained independent significant predictors during multivariate analysis. Prospective studies are needed to confirm the independent prognostic potential of p53 overexpression in patients with advanced bladder cancer. The availability of more refined prognostic factors should assist decision making regarding the value of more aggressive treatment options, such as adjuvant or neoadjuvant chemotherapy, for prognostically defined subgroups of patients.     

The relationship between smoking exposure and p53 overexpression in colorectal cancer.

Although epidemiological studies of the relationship between cigarette smoking and colorectal cancer risk have been equivocal, a positive association is consistently found for colorectal adenoma development. We performed an epidemiological study to determine whether p53 protein overexpression, in tumours obtained at the time of resection, is associated with cigarette exposure in colorectal cancer. A total of 163 colorectal cancer cases and 326 healthy controls responded to a standardised questionnaire on colorectal cancer risk factors including detailed information on their history of cigarette smoking. All patients'' tumours were analysed immunohistochemically for p53 overexpression using an avidin-biotin immunoperoxidase procedure and polyclonal anti-p53 antibody CM1. Comparison of colorectal cases with controls revealed an elevated risk for ex-smokers (OR = 1.34, 95% CI 0.85-2.12) and current smokers (OR = 1.13, 95% CI 0.63-2.02) when compared with non-smokers. No dose-response relationship was found for total pack-years of smoking (trend test: P = 0.19). However, a trend for total pack-years of smoking was found when p53-positive cases were compared with p53-negative cases suggesting aetiological, heterogeneity (trend test: P = 0.06). Estimating the individual relative risk of developing a p53-positive tumour relative to controls showed no associations for smoking status or total pack-years of smoking. However, when p53-negative cases were compared with controls, an elevated risk was found for ex-smokers (OR = 1.84, 95% CI 1.00-3.37) and current years of smoking (trend test: P = 0.03). Colorectal tumours developing through p53-positive dependent pathways were not associated with smoking exposure. A significant increase in risk was observed for the p53-negative independent pathway with smoking. p53 overexpression appears to be associated with smoking exposure in colorectal cancer.     

Micrometastatic p53-positive cells in the lymph nodes of early stage epithelial ovarian cancer: prognostic significance

We used immunohistochemical staining of p53 protein to detect micrometastasis in regional lymph nodes that were judged tumor-free by conventional histopathological methods in 58 patients with stage I or II (pT1 or 2, N0) epithelial ovarian cancer. Overexpression of p53 protein in the primary lesions of ovarian cancer was observed in 31 patients (53%), and p53 protein-positive cells were detected in the regional lymph nodes (micrometastasis-positive) in 19 of 31 patients with p53 protein overexpression (61%). In patients with micrometastasis, the prognosis was significantly poorer than that in those without micrometastasis (p < 0.05). Detection of micrometastasis of the regional lymph nodes of ovarian cancer by immunohistochemical staining of p53 protein may be useful in predicting the prognosis of patients with stage I or II epithelial ovarian cancer.     

启东肝癌组织p53过表达的免疫组化检测

目的　了解p5 3突变在启东地区的肝癌发生中所起的作用。方法　应用免疫组化 (IHC)的方法对 90例肝癌和 75例癌旁组织切片进行p5 3蛋白检测。结果　在 90例肝癌中 ,有 4 0例 (44 .4 % )在核中表达为强阳性 ,2 5例 (2 7.8% )在核中表达为弱阳性 ;在 75例癌旁中 ,没有细胞核强阳性表达 ,仅 1例 (1.3% )有核弱阳性表达 ;肝癌的p5 3核表达显著高于癌旁 (P =0 .0 0 0 )。另外 ,在肝癌中有 9例 (10 % )为细胞质p5 3表达阳性 ,在癌旁中有 17例 (2 2 .7% )为细胞质表达阳性 ,肝癌的胞质p5 3表达显著低于癌旁 (P =0 .0 2 6 )。结论　p5 3基因突变和p5 3的功能失活在启东的肝癌发展中有着重要意义。     

Gastric cancer with para-aortic lymph nodes metastasis: curable dissection possible but be cautiously selected

This is a reply letter to Dr. M Daniele et al. entitled as “gastric cancer with para-aortic lymph nodes metastasis: do not miss a chance of cure!”. Para-aortic lymph node metastasis has been regarded as a pattern of distant metastasis in gastric cancer. Even patients with PAN metastasis undergo extensive lymph nodes, the prognosis is unsatisfactory and the overall survival is not better than the patients receiving palliative chemotherapy. However, for the patients who were responded to the preoperative chemotherapy, subsequent radical gastrectomy performed prolonged survival. Meanwhile, patients who were resistant to the previous chemotherapy were not optimal candidates for curative resection in advanced gastric cancer patients with PAN metastasis. How large with lymph nodes dissection for the patients with initially PAN metastasis after effective neo-chemotherapy is still a controversial issue and needs further large-scale randomized trials.     

EGFR, c-erbB2 and p53 protein in the primary lesions and paired metastatic regional lymph nodes in breast cancer.

AIMS: Various biological parameters are now being evaluated as predictors for the response of chemohormonal therapy for breast cancer. Few studies compare these parameters between the primary lesions and metastatic regional lymph nodes of breast cancer. METHODS: Immunohistochemical analyses for epidermal growth factor receptor (EGFR), c-erbB2 and p53 protein were performed on the primary lesions and matching metastatic regional lymph nodes of 107 breast cancers. The intensity of the immunoreactivity was graded for heterogeneous or 10-50% staining, and diffuse or >50% staining. RESULTS: EGFR, c-erbB2 and p53 protein showed a concordance between the primary lesions and matching regional lymph nodes in terms of a negative or positive finding (+ and ++) in 98 (92%) of 106 cases, 76 (100%) of 76 cases and 79 (93%) of 85 cases respectively, while EGFR, c-erbB2 and p53 protein also showed a concordance in the intensity of the immunoreactivity in 24 (89%) of 27 cases 14 (74%) of 19 cases and 30 (94%) of 32 cases respectively. In 21 of 24 cases which showed a disconcordance in the positivity or the intensity of the positivity of EGFR, c-erbB2 and p53 protein, one of the primary lesions and matching regional lymph nodes showed heterogeneous or 10-50% immunostaining. CONCLUSIONS: The immunoreactivity of EGFR, c-erbB2 and p53 protein shows a concordance between the primary lesions and matching metastatic regional lymph nodes in a majority of breast cancers.     

c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes.

The c-erbB-2 gene is overexpressed in about 20% of human breast cancers. Four hundred and eighty-three cases previously examined by immunohistochemical staining for c-erbB-2 expression were analysed to assess the risk associated with the elevated protein expression. Oncoprotein expression was correlated with increasing tumour grade but not with oestrogen receptor status, nodal involvement, tumour size or age. There was an increased risk of relapse and death associated with c-erbB-2 expression irrespective of nodal involvement. This marker thus appears to be a significant prognostic factor in the early as well as the late stages of breast cancer.     

Cooccurrence of reduced expression of alpha-catenin and overexpression of p53 is a predictor of lymph node metastasis in early gastric cancer.

BACKGROUND AND OBJECTIVES: Even though the pathological background contributes to lymph node metastasis, the biological characteristics of tumors have also gained wide attention. In this study, the expression of the cadherin-catenin complex and p53 was studied in early gastric cancer. Their correlation with lymph node metastasis and the predictability of lymph node metastases, by combining these factors, were also discussed. METHODS: One hundred and one specimens obtained from surgery were studied by immunohistochemistry using monoclonal anti-E-cadherin, anti-alpha-catenin and anti-p53 antibodies. RESULTS: Expression of E-cadherin and alpha-catenin was reduced in 50.5 and 64.4%, respectively. p53 protein staining was positive in 29.7%. There was a significant correlation between E-cadherin and alpha-catenin expression, but no correlation was found between p53 expression and E-cadherin or alpha-catenin expression. A reduction in alpha-catenin expression and p53 overexpression correlated to lymph node metastases, respectively. Multivariate analysis showed that cooccurrence of reduced expression of alpha-catenin and overexpression of p53 was an independent factor indicating lymph node metastases. CONCLUSION: A study of both alpha-catenin and p53 expression may be helpful to predict lymph node metastases in early gastric cancer.     

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.