Relationship of p53 overexpression to other cell cycle regulatory proteins in oral squamous cell carcinoma

Aberrations of the p53 gene and the overexpression of its protein are described in a variety of neoplasms, including oral and other head and neck cancers. Here we report the association of p53 (over)expression with a downstream cell cycle inhibitor p21/waf 1 in oral squamous cell carcinoma (SCC). The loss of expression of p16 and p27, two other cyclin-dependent kinase (cdk) inhibitors, was also examined. In this panel of tumours, 10/24 carcinomas were p53-immunopositive. Heterogeneous expression of p21 and p27 was seen in 10/24 SCC and 9/16 SCC, respectively, and this was not correlated to p53 status. The expression of p21 and p27 in these SCCs suggests the existence of mechanisms by which some growing tumour cells may tolerate these cell cycle inhibitors; eight SCCs lacked expression of both inhibitors but only two of these cancers overexpressed p53, suggesting that accumulation of p21/p27 can be independent of the functional status of the p53 gene. Data do not support a clear example of a phenotype that shows an overexpression of p53 with downregulation of p21 or p27 leading to cell cycle alterations. Furthermore, only three SCCs were p16-negative and p53-positive. This suggests that these two tumour suppressors may act in separate pathways.     

A non-random deletion in the p53 gene in oral squamous cell carcinoma

In a retrospective study of the mutational spectrum of the p53 gene in oral squamous cell carcinoma, 80 primary tumours diagnosed in 1980-90 were included. Using polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis 47 mutations were found distributed in 39 of the tumours (49%). Unexpectedly, the majority of the mutations (29/47; 62%) were found in exon 8, and at sequencing 17 of them showed a 14 bp deletion in codons 287-292, causing formation of a stop codon and accordingly a truncated protein lacking the C-terminal. The majority of the patients with the 14 bp deletion were women (13/17), and it seemed as though certain potential risk factors for carcinoma of the head and neck were less common in this group.     

Prognostic significance of p53 gene mutations in squamous cell carcinoma of the lung

The aim of the present study was to analyze the prevalence and clinical importance of p53 gene mutations in surgically treated squamous cell lung carcinoma. Sixty patients were included. Fifty-one patients in stages I to IIIa were submitted to radical resection. Twenty-five samples tested positive for the p53 immunohistochemistry assay, and were analyzed for p53 gene mutations. Eleven mutations were found. Patients harboring p53 gene mutations suffered a higher incidence of recurrence and a higher mortality rate. Disease-free interval and overall survival were shorter for patients with mutated p53 gene (p=0.03 and p=0.005, respectively).     

bcl-2, p53 immunophenotypes and apoptosis in squamous cell carcinoma of the oesophagus

To establish baseline information on neopterin concentrations in livestock, companion and laboratory animals and identify the factors that may influence these concentrations, blood samples were taken from normal dairy cattle, horses, llamas, dogs, cats and rats of varying ages and sexes. In addition, neopterin concentrations in normal, adult equines were compared with those found in racing Thoroughbreds. There were no differences due to sex, sexual maturity, pregnancy, castration, or age. For all ages and sexes combined, mean neopterin concentrations were significantly lower in llamas (2.27+/-0.33 nmol litre(-1)) and rats (2.13+/-0.21 nmol litre(-1)) when compared with the other species tested. Racing Thoroughbreds demonstrated higher neopterin values than adult equines not in training (3.54+/-0.64 vs 3.13+/-1.02 nmol litre(-1)).     

Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control

Chemoresistance is a major concern in cancer erradication; it involves various mechanisms, including defects in the apoptosis program induced by anticancer drugs. In order to further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin treatment, we established an in vitro model, mimicking a clinical protocol of administration of cisplatin. Therefore, IGROV1 ovarian carcinoma cells were exposed for 2 hr to the drug and allowed to recover for several weeks; this way of exposure was reiterated with escalating doses. We followed changes in cytotoxicity of the drug, cell cycle kinetics and long-term survival of cells after cisplatin treatment, and found that resistance to cisplatin was not associated with altered apoptosis pathway, since both cisplatin sensitive and resistant cells underwent apoptosis in a similar way. Acquisition of resistance to cisplatin was associated with the ability of the treated cells to progress through the cell cycle beyond the G1/S checkpoint; although most cells died by apoptosis, a few surviving cells proliferated and recolonized the cultures. Compared to sensitive cells, the chemoresistant variants were able to override the G1/S checkpoint whatever the dose, and the recurrent cells recolonized the cultures much faster. Analysis of alterations in gene expression suggests that the defect in cell cycle regulation could take place at the level of the cdk inhibitor p21(CIP1/WAF1).     

p53 antibodies in the saliva of patients with squamous cell carcinoma of the oral cavity

A significant age dependence of the risk of complete heart block complicating radiofrequency ablation of the AV nodal slow pathway was noticed, with no patients <45 years of age experiencing this complication.     

Analysis of p53 serum antibodies in patients with head and neck squamous cell carcinoma

BACKGROUND: Mutation of the p53 tumor suppressor gene (also known as TP53) often leads to the synthesis of p53 protein that has a longer than normal half-life. Mutant p53 protein that accumulates in tumor cell nuclei can be detected by means of immunohistochemical staining techniques. Serum antibodies directed against p53 protein (p53-Abs) have been detected in some cancer patients. PURPOSE: We assayed serum samples from 80 patients with head and neck squamous cell carcinoma (HNSCC) for the presence of p53-Abs, and we evaluated potential associations between the presence of these antibodies and other histopathologic and clinical features. METHODS: Serum was collected from each patient at the time of diagnosis. In addition, tumor biopsy specimens were obtained before the initiation of treatment. An enzyme-linked immunosorbent assay was used to detect p53-Abs. The accumulation of p53 protein in tumor cell nuclei was assessed immunohistochemically by use of the anti-p53 monoclonal antibody DO7. Patient treatment consisted of radiotherapy alone, primary chemotherapy followed by radiotherapy, or surgery and postoperative radiotherapy. Relapse-free and overall survival from the beginning of treatment were estimated by use of the Kaplan-Meier method; survival comparisons were made by use of the logrank statistic. Univariate and multivariate analyses were conducted to identify factors associated with survival. Reported P values are two-sided. RESULTS: Fifteen (18.8%) of the 80 patients had p53-Abs. Tumor cell nuclei in 43 (58.9%) of 73 assessable biopsy specimens exhibited strong p53 immunostaining. Patient treatment method and the accumulation of p53 protein in tumor cell nuclei were not associated with increased risks of relapse or death. In univariate analyses, advanced tumor stage (> T1 [TNM classification]) and the presence of p53-Abs were significantly associated with an increased risk of death (P for trend = .007 and P = .002, respectively), whereas advanced tumor stage, substantial regional lymph node involvement (> N1), and the presence of p53-Abs were associated with an increased risk of relapse (P for trend = .002, P = .02, and P < .0001, respectively). In multivariate analyses, advanced tumor stage and the presence of p53-Abs were significantly associated with increased risks of relapse (p for trend = .04 and P = .003, respectively) and death (P for trend = .04 and P = .03, respectively). At 2 years of follow-up, the overall survival proportion was 63% (95% confidence interval [CI] = 47%-80%) when no p53-Abs were detected compared with 29% (95% CI = 4%-54%) when p53-Abs were detected. Relapse-free survival at 2 years was 62% (95% CI = 49%-76%) if no p53-Abs were detected compared with 13% (95% CI = 0%-31%) if p53-Abs were detected. CONCLUSIONS AND IMPLICATIONS: The proportion of patients with HNSCC who have serum p53-Abs is smaller than that of patients exhibiting tumor cell accumulation of p53 protein. The presence of p53-Abs is significantly associated with increased risks of relapse and death.     

Loss of heterozygosity and mutation analysis of the p16 (9p21) and p53 (17p13) genes in squamous cell carcinoma of the head and neck

We analyzed allelic loss at the p53 gene (17p13) and at chromosome region 9p21 in 35 primary head and neck squamous cell carcinomas. Loss of heterozygosity (LOH) at p53 and 9p21 was found in 50 and 75% of informative cases, respectively. LOH at the p53 gene did not increase significantly with tumor stage, but was more frequent in moderately and poorly differentiated tumors than in well-differentiated tumors. LOH plus mutation or homozygous deletion of p53 was limited to advanced stage and poorly differentiated tumors. Allelic loss at 9p21 is frequent in early stage head and neck squamous cell carcinoma and is not significantly associated with LOH at p53. The second exon of the p16/MTS1/CDKN2 gene was found to be homozygously deleted in 1 of 19 cases showing LOH at 9p21, but direct sequencing did not show mutations in the remaining 18 cases. This suggests that p16 plays a limited role in the development of head and neck squamous cell carcinoma.     

In oesophageal squamous cell carcinoma vascular endothelial growth factor is associated with p53 mutation, advanced stage and poor prognosis

Vascular endothelial growth factor (VEGF) affects malignant tumours by promoting angiogenesis. The tumour-suppressor gene p53 has been thought to regulate VEGF. We investigated the effect of VEGF on oesophageal carcinoma and the connection between VEGF and p53. One hundred and nine resected oesophageal squamous cell carcinomas were examined. VEGF expression was analysed by immunohistochemical staining. Sixty-five tumours (59.6%, 65 out of 109) were classified as VEGF positive. A significant correlation was found between the VEGF expression and both the depth of invasion (P = 0.0001) and lymph node metastasis (P < 0.0001). With regard to p53, we compared the expression of VEGF with the mutation of p53, examined using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing in tumour samples obtained from 36 patients who we have reported previously. The VEGF expression was significantly correlated to p53 mutation (P = 0.0291). To evaluate the angiogenesis, microvascular density (MVD) was counted, and endothelial cells were stained immunohistochemically using anti-CD34 monoclonal antibody against 29 cases with invasion limited to the submucosal layer. The average MVD had a tendency to correlate to VEGF expression (P = 0.1626). The prognoses of patients with VEGF-positive primary tumours were significantly worse than for those with VEGF-negative primary tumours (P = 0.0077). We have assumed that VEGF contributes to aggressive characteristics in oesophageal carcinomas and that VEGF expression might be affected by p53 status.     

Wild-type p53 and a p53 temperature-sensitive mutant suppress human soft tissue sarcoma by enhancing cell cycle control

Soft-tissue sarcomas are a heterogeneous group of tumors that are putatively mesenchymal in origin. Therapeutic advances in this disease have been limited over the past several decades. Approximately one-half of all patients will ultimately succumb, usually to uncontrollable pulmonary metastases. Although little is known about the underlying molecular determinants driving soft-tissue sarcoma inception, proliferation, and metastasis, mutation of the p53 gene is the most frequently detected molecular alteration in this disease. Accordingly, we were interested in determining whether transduction of wild-type (wt) p53 into soft-tissue sarcomas bearing mutated p53 genes might alter the malignant phenotype. SKLMS-1 is a human-derived leiomyosarcoma cell line with a codon 245 p53 point mutation. Cationic liposome was used to transfect wt p53 or 143Ala temperature-sensitive mutant p53 into this cell line. SKLMS-1 stable transfectants expressing wt p53 had decreased cell proliferation in vitro, decreased in vitro colony formation in soft agar, and decreased tumorigenicity in severe combined immunodeficient mice in vivo. Flow cytometric analysis of cell cycle components demonstrated markedly increased G1 cell cycle arrest and decreased entry into S phase, which corresponded to the induction of p21cip1 protein in the transfectants. Using SKLMS-1 stable transfectants expressing the 143Ala p53 temperature-sensitive mutant, we demonstrated the kinetics of and the causal relationship between wt p53 expression, the wt p53-dependent induction of cell cycle inhibitor p21cip1, and inhibition of cell cycle progression in p53-transfected SKLMS-1 cells. The ability to restore wt p53 growth-regulatory functions in soft-tissue sarcoma may ultimately be useful as a future therapy in patients with soft-tissue sarcomas.     

p53 gene alternation in squamous cell carcinoma of the esophagus detected by PCR-cold SSCP analysis

Mutations of the p53 tumor suppressor gene play an important role in the development of common human malignancies. Previous reports revealed that the frequencies of p53 alternations in esophageal carcinoma varied from 26% to 87%. The clinical significance of p53 alternations is still disputed. In the present study, we used polymerase chain reaction--"cold" single-strand conformation polymorphism (PCR-"cold" SSCP)--to investigate p53 genetic alternations in 63 surgical specimens of esophageal squamous cell carcinoma (ESC). Our experiments showed that the optimum buffer temperature for "cold" SSCP analysis was 14 degrees C while the PCR products were around 200-300 bp in size. Among 63 tumorous samples, p53 alternations was detected in 47 tumors, or an incidence rate of 74.6%. For nontumorous mucosal samples, the incidence of p53 alternations was 55.5% (35/63 samples). Additionally, p53 alternations occurred most frequently at exon 6 (50.8%), followed by exon 7 (33.3%), exon 8 (17.5%) and exon 5 (12.7%). Multiple genetic alternations (> or = 2 exons) between p53 exons 5-8 in the same examined samples were found in 21 (33.3%) of 63 tumors, and in 8 (12.7%) of 63 nontumorous mucosal specimens. Our results further showed that p53 alternations did not correlate with age, depth of tumor invasion, lymph node metastasis, tumor stage, cell differentiation or lymphovascular invasion in ESC (P > 0.05). Moreover, the survival rate in patients with p53 alternations was similar to that in patients without p53 alternations (P > 0.05). In conclusion, PCR-"cold" SSCP is a rapid and sensitive method for identifying p53 genetic alternations. p53 genetic alternations occur with a relatively high incidence for ESC, but p53 abnormality has no impact on prognosis.     

Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma

PURPOSE: Standard therapies of head and neck squamous cell carcinoma (HNSCC) often cause profound morbidity and have not significantly improved survival over the last 30 years. Preclinical studies showed that adenoviral vector delivery of the wild-type p53 gene reduced tumor growth in mouse xenograft models. Our purpose was to ascertain the safety and therapeutic potential of adenoviral (Ad)-p53 in advanced HNSCC. PATIENTS AND METHODS: Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of Ad-p53, either with or without tumor resection. Patients were monitored for adverse events and antiadenoviral antibodies, tumors were monitored for response and p53 expression, and body fluids were analyzed for Ad-p53. RESULTS: Tumors of 33 patients were injected with doses of up to 1 x 10(11) plaque-forming units (pfu). No dose-limiting toxicity or serious adverse events were noted. p53 expression was detected in tumor biopsies despite antibody responses after Ad-p53 injections. Clinical efficacy could be evaluated in 17 patients with nonresectable tumors: two patients showed objective tumor regressions of greater than 50%, six patients showed stable disease for up to 3.5 months, and nine patients showed progressive disease. One resectable patient was considered a complete pathologic response. Ad-p53 was detected in blood and urine in a dose-dependent fashion, and in sputum. CONCLUSION: Patients were safely injected intratumorally with Ad-p53. Objective antitumor activity was detected in several patients. The infectious Ad-p53 in body fluids was asymptomatic, and suggests that systemic or regional treatment may be tolerable. These results suggest the further investigation of Ad-p53 as a therapeutic agent for patients with HNSCC.     

The influence of p53 and associated factors on the outcome of patients with oral squamous cell carcinoma

Radiation synovectomy is efficacious in controlling the symptoms of rheumatoid arthritis. However, the procedure is not widely used because of concerns about leakage of radiopharmaceuticals from the treated joints. Leakage can be minimized by selecting particles of an appropriate size. In this study, we labelled microspheres with 188Re and analysed its biodistribution after intra-articular injection in rabbits with antigen-induced arthritis. Gamma camera imaging was performed to quantify the mean retention of 188Re in the knees. The mean retention of 188Re was 98.7, 94.6 and 93.6% at 1, 24 and 48 h, respectively. The biodistribution data revealed very low radioactivity in all organs at different times, which suggests the leakage of radiotracer from the knee was negligible. Our preliminary results indicate that 188Re microspheres are a potentially effective radiopharmaceutical for radiation synovectomy.     

Alterations of p53 gene and Ha-ras gene are independent events in solar keratosis and squamous cell carcinoma

In order to clarity the multiple-step progression from solar keratosis to squamous cell carcinoma, aberrations of the p53 gene (exons 2-11) and ras genes (exons 1 and 2) in solar keratosis and squamous cell carcinoma were investigated by polymerase chain reaction and single-strand conformation polymorphism analysis. In a series of Japanese patients, eight of 27 (30%) samples of solar keratosis and three of six (50%) samples of squamous cell carcinoma showed structural abnormalities in the p53 gene. Only one solar keratosis (4%) showed a point mutation in the Ha-ras gene but not in the p53 gene. Among these cases, no mutation of ras genes could be detected in squamous cell carcinoma. Simultaneous mutation of ras genes and the p53 gene was not detected in any cases of either solar keratosis or squamous cell carcinoma. It is concluded that aberrations of the p53 gene and ras genes are induced through independent processes of ultraviolet irradiation in the course of carcinogenic change from solar keratosis to squamous cell carcinoma.     

Normalization of EGFR mRNA levels following restoration of wild-type p53 in a head and neck squamous cell carcinoma cell line

Hapalotrema mehrai Rao, 1976 and Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchidae) are redescribed from the heart and pulmonary arteries of the green turtle, Chelonia mydas, from Moreton Bay in south-eastern Queensland. Hapalotrema pambanensis Gupta and Mehrotra, 1981 from C. mydas in India is made a synonym of H. mehrai. Hapalotrema dorsopora Dailey, Fast and Balazs, 1993 from C. mydas from Hawaii was described with a dorsally opening uterine pore, but this is found to be the opening of Laurer's canal; therefore H. dorsopora is also made a synonym of H. mehrai. In addition to differences in the numbers of testes and general dimensions, H. mehrai and H. postorchis differ in the development of Laurer's canal and in the absence of a canalicular seminal receptacle in H. postorchis.     

Expression of p53 and p21 proteins in oral squamous cell carcinoma: correlation with lymph node metastasis and response to chemoradiotherapy

p53 protein, a product of the p53 cancer suppressor gene, and p21 protein, a cyclin-dependent kinase inhibitor, were immunohistochemically investigated in 150 oral squamous cell carcinomas (SCCs) and the relationship between their expression and clinicopathological findings were evaluated. The positivity for p53 and p21 proteins was not correlated with the T-stage, mode of tumor cell invasion or tumor cell differentiation. However, the expression of p53 and p21 proteins was correlated with lymph node metastasis. Of 62 SCCs with regional lymph node metastasis, 45 SCCs (72.6%) were positive for p53 while 45 (52.9%) of 88 SCCs without metastasis expressed p53 protein (p < 0.02). In addition, p21 protein was observed in 25 (38.5%) and 18 (21.2%) SCCs with and without metastasis, respectively (p < 0.05). Furthermore, p53 protein was inversely correlated with the histopathological effect of inductive chemoradiotherapy; the rate of chemoradiotherapy-induced lethal degeneration (56.7%) in p53-negative SCCs was significantly higher than that (28.9%) in p53-positive SCCs (p < 0.005). However, no clear difference in the effect was observed between p21-positive and p21-negative SCCs. Finally, the 5-year-survival rate was highest in p53(-)-p21(+) (80.0%) followed by 76.3% in p53(-)-p21(-), 65.9% in p53(+)-p21(+) and 65.4% in p53(+)-21p(-) SCCs. These results indicate that although the expression of p21 protein is only weakly correlated with the clinico-histopathological findings, p53 protein is a useful prognostic marker and that inductive chemoradiotherapy can be successfully planned by immunohistochemical examination of p53 protein.     

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines

We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The first intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.     

Growth inhibitory concentrations of EGF induce p21 (WAF1/Cip1) and alter cell cycle control in squamous carcinoma cells

Previous studies have reported inhibition of A431 squamous carcinoma cell growth by nanomolar concentrations of epidermal growth factor (EGF), a potent mitogen for cells of epithelial origin. In this study, we examined potential mechanisms through which inhibition of keratinocyte growth mediated by EGF might occur by analysing components of the cell cycle regulatory machinery in A431, HN6 and HN30 keratinocytes in the presence of growth inhibitory or growth stimulatory doses of EGF. Treatment of cells with 25 pM EGF produced an increase in [3H]thymidine incorporation in A431, HN6 and HN30 cells, with respect to control cultures. Exposure to 2.5 nM EGF reduced [3H]thymidine incorporation in A431 cells and HN6 cells to 11% and 70% of control levels, respectively, whereas HN30 cells continued to proliferate in the presence of EGF. [3H]thymidine incorporation assays carried out over 24 h revealed repression of DNA synthesis in A431 cells after 12 h exposure to 2.5 nM EGF compared to untreated cells. Flow cytometry studies demonstrated accumulation of cells in G0/G1 after addition of 2.5 nM, but not 25 pM EGF. Western blot analysis revealed elevation of p21 (WAF1/CIP1/SDI1) protein levels in A431 and HN6 cells under growth-inhibitory conditions. Stimulatory doses of EGF did not induce p21 in these cells. Northern blot hybridization demonstrated elevated levels of p21 mRNA within 4 h of exposure of A431 cells to 2.5 nM EGF, which remained elevated above basal levels at 24 h. In vitro kinase assays demonstrated temporal differences in CDK2 and CDK6 activities which were related to EGF concentration. Immunocomplex Western blotting demonstrated increased association of p21 with CDK2 and CDK6 in A431 cells treated with 2.5 nm EGF. Furthermore, temporal alterations in the association of PCNA with p21 and with CDK6 were observed. The data indicate that p21 is a likely mediator of EGF-induced growth-inhibition, probably through mechanisms involving sequestration of PCNA and inhibition of CDK activity.