Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.     

Clinical relevance of p53 index and expression of proliferating cell nuclear antigen and Ki-67 in gastric cancer

To improve the therapeutic outcome for inoperable non-small-cell lung cancer, we applied definitive thoracic radiotherapy combined with concurrent administration of carboplatin and etoposide. We retrospectively analyzed 55 eligible patients with Stage III disease. The one-year rate of overall survival (OAS) and distant metastasis-free survival (DMFS) of the total group were 46.1% and 36.1%, respectively. Twenty-nine patients developed thoracic failures (52.7%) and 23 (41.8%) distant failures. Using univariate and multivariate analyses, radiation dose, performance status and LDH were revealed as significant prognostic factors of OAS, and LDH had a strong adverse effect on DMFS. Leucopenia of Grade 3 or higher was noted in 75.9%, anemia in 55.6%, thrombocytopenia in 59.3%, esophagitis in 20.4%, and lung injury in 10.9%. Sufficient gain was not obtained by our strategy, and higher morbidity, especially of lung, was noted than was expected. It was suspected that simultaneous use of oral etoposide might increase radiation pneumonitis, so one should take special care of unexpected toxicity in concurrent chemoradiotherapy. Both the hyperfractionated technique of radiotherapy and the time-dose modification of anti-tumor drugs should be considered in further steps.     

Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression

BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.     

p53 alteration, proliferating cell nuclear antigen, and nucleolar organizer regions in thymic epithelial tumors

We examined p53 protein expression, p53 gene mutation, proliferating cell nuclear antigen (PCNA), and argyrophilic nuclear organizer regions (AgNOR), in 30 patients with surgically-treated thymic tumors (26 thymoma and 4 thymic carcinoma cases). p53 expression ratio with DO-1 was divided as p53 negative (0% positivity), low expressor (<10% positivity), high expressor (>10% positivity). The incidence of p53 low and high expressor in thymoma were 19% (5/26) and 8% (2/26), respectively. p53 immunopositivity in thymoma was significantly correlated with PCNA labeling index (LI). p53 expression ratio in invasive thymoma (33%) tended to be higher than that in non-invasive thymoma (18%). p53 expression was detected in one of the thymic carcinoma. There were no p53 gene mutations in 15 invasive thymoma, although one of four (25%) thymic carcinomas showed two point mutations. p53 gene alterations seem to be associated with malignant activity of tumor cells, and therefore detection of p53 gene mutations is useful as a diagnostic factor.     

Proliferative potential of meningiomas evaluated by proliferating cell nuclear antigen expression

Meningiomas are principally benign in nature. Some meningiomas, however, grow fast or recur even after total removal. The biological behavior of meningiomas often can not be predicted from conventional histopathological studies. A monoclonal antibody against proliferating cell nuclear antigen (PCNA) was used to investigate the usefulness of the PCNA index as a parameter to estimate the proliferative activity of meningiomas. Fifty-two meningiomas were examined. The mean PCNA index of recurrent meningiomas (3.37 +/- 0.92%) was significantly higher than that of non-recurrent meningiomas (1.12 +/- 0.51%) (p < 0.005). The PCNA indices of recurrent cases were all higher than 2.0%. A semilog linear regression analysis between tumor doubling time and PCNA index showed a significant correlation (r = 0.90, p < 0.05). An inverse linear correlation between PCNA index and interval to recurrence was observed (r = 0.62, p < 0.05). A good linear correlation was also shown between PCNA index and BUdR labeling index (r = 0.88, p < 0.01). The results of this study suggest that, providing the methods of tissue processing, immunostaining and counting of positive nuclei are unified, the PCNA index is a useful parameter for estimating the biological behavior of meningiomas.     

p53 and c-jun expression in urinary bladder transitional cell carcinoma: correlation with proliferating cell nuclear antigen (PCNA) histological grade and clinical stage

OBJECTIVE: To investigate p53 and c-jun oncoproteins and proliferating cell nuclear antigen (PCNA) in transitional cell urinary bladder carcinomas (TCCs) and to determine their relationships to tumour grade, stage and survival. MATERIALS AND METHODS: The expression of p53, c-jun and PCNA was studied using immunohistochemistry in formalin-fixed, paraffin-embedded tissues in a series of 110 TCCs. RESULTS: 58% of our cases were positive for p53 and 88% for c-jun. A statistically very significant correlation (p < 0.0001) was observed between p53 and c-jun (r = 0.781), p53 and PCNA (r = 0.772), c-jun and PCNA (r = 0.831) as well as between each of the two oncoproteins and the histological grade and clinical stage (p < 0.001). There was no correlation of either p53, PCNA or c-jun with clinical outcome in terms of patients survival. CONCLUSION: p53 and c-jun proteins' overexpression are strongly related to rapid tumour cell proliferation and hence with aggressive growth in urinary bladder TCC. PCNA score remains an important prognostic index in transitional cell carcinoma of the bladder.     

A unique form of proliferating cell nuclear antigen is present in malignant breast cells

Despite extensive research efforts to identify unique molecular alterations in breast cancer, to date, no characteristic has emerged that correlates exclusively with malignancy. Recently, it has been shown that the multiprotein DNA replication complex (synthesome) from breast cancer cells has a significantly decreased replication fidelity compared to that of nonmalignant breast cells. Proliferating cell nuclear antigen (PCNA) functions in DNA replication and DNA repair and is a component of the synthesome. Using two-dimensional PAGE analysis, we have identified a novel form of PCNA in malignant breast cells. This unique form is not the result of a genetic alteration, as demonstrated by DNA sequence analysis of the PCNA gene from malignant and nonmalignant breast cells. This novel form is most likely the result of an alteration in the post-translational modification of PCNA in malignant breast cells. These findings are significant in that it is now possible to link changes in the fidelity of DNA replication with a specific alteration of a component of the DNA synthetic apparatus of breast cancer cells. The novel form of PCNA may prove to be a new signature for malignant breast cells.     

Prognostic implications of proliferating cell nuclear antigen (PCNA), AgNORs and P53 in non-Hodgkin's lymphomas

We investigated the prognostic value of proliferating cell nuclear antigen (PCNA) and p53 oncoprotein expression and of nucleolar organiser region (NOR) scoring, in relation to classic clinicopathological parameters, in a series of non-Hodgkin's lymphomas (NHL). Paraffin embedded tissue from 91 patients with NHL was stained immunohistochemically with the monoclonal antibodies PC-10 (PCNA) and DO-1 (p53) and histochemically with the AgNOR technique. The median follow-up was 48 (4 to 193) months. The impact of PCNA and p53 expression and of AgNOR number on survival was tested using univariate as well as multivariate analysis, in order to circumvent the heterogeneity in histologic grade, type and therapy. Univariate analysis identified seven variables related to overall survival: histologic type and grade, clinical stage, chemotherapy, p53 labelling index (LI), PCNA LI and AgNOR score, whereas only one parameter i.e. histologic grade influenced disease-free survival. In multivariate analysis stage, PCNA LI and AgNOR score predicted independently overall survival. PCNA was also the only independent predictor of post-relapse survival and histologic grade the most important indicator of disease-free survival. In conclusion, PCNA expression and AgNOR number may be better predictors of overall and post-relapse survival than histologic grade. The latter remains the most valuable prognostic indicator of disease-free survival.     

Erythroid precursor cells in a tissue culture from subjects exposed to ionizing radiation

Cloning efficiency of erythroid progenitor cells in gel diffusion chambers from patients irradiated as a result of the Chernobyl accident was studied. Stimulation of erythroid progenitor cells, activation of enzymatic activity in peripheral blood cells and an increase of 2,3-D Ph G were detected. These data supplement the notion about the development of adaptative processes in the erythron system as a response to effects of radiation.     

Computerized determination of proliferating cell nuclear antigen expression in meningiomas. A comparison with non-automated method

Proliferating cell nuclear antigen (PCNA) expression has been proven to be a significant marker of cell proliferation in meningiomas, which correlates with growth rate and, as shown by several authors, possibly provides prognostic information concerning biologic behavior. However, the current method for determining PCNA labeling index (LI) is tedious and time consuming like all the nonautomated methods for evaluating cell kinetics, presenting high interobserver and interlaboratory variability and low reproducibility. In the present study, we introduce a semi-automated computer-assisted image analysis method for determining PCNA LI in 38 meningiomas, in parallel with the current nonautomated method. Image analysis technique permits unbiased cell counting, standardizes the degree of staining intensity and provides instant results. By calculating coefficient of variability, the method proved to be highly reproducible. The correlation between the results provided by the nonautomated and the semiautomated image analysis method showed a high agreement between them, with a correlation coefficient, r, of 0.82. In conclusion, we consider that image analysis contributes to the accuracy, reproducibility, and practicality of PCNA LI determination so that along with other useful parameters this significant marker may serve to predict the clinical behavior in meningiomas.     

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen

We identified an expansion of the CAG trinucleotide repeat in the coding region of the Machado-Joseph disease gene in 7 of 24 American families diagnosed with autosomal dominant ataxia. All affected individuals were heterozygous for an expanded allele that ranged from 67 to more than 200 CAG repeats, whereas the normal allele had 14 to 33 repeats. In contrast to the Azorean-Portuguese origins of Machado-Joseph disease, the two largest American families were of German and Dutch-African descent. Clinical, pathologic, and genetic evaluations suggest that American families with spinocerebellar ataxia type 3 differ from those with Machado-Joseph disease by their ethnic origins, predominant spinopontine atrophy, lack of dystonic features, and larger CAG repeat expansion.     

The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.     

Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma

Uterine leiomyoma is the most common smooth muscle cell tumor of the myometrium. Estrogen and progesterone (P4) are believed to be physiological regulators of leiomyoma growth. We recently showed that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to its expression in the normal myometrium and that Bcl-2 protein expression in cultured leiomyoma cells was up-regulated by P4, but down-regulated by 17 beta-estradiol (E2). To further characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined the effect of menstrual phase on proliferating cell nuclear antigen (PCNA) expression in leiomyoma and investigated whether sex steroids could influence PCNA expression in leiomyoma cells cultured under serum-free conditions by immunoblot and immunohistochemical analyses. As epidermal growth factor (EGF) has been shown to mediate estrogen action and to play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on the expression of EGF and EGF receptor (EGF-R) in cultured leiomyoma cells. The PCNA labeling index in leiomyomas was much greater in the secretory, P4-dominated, phase than in the proliferative phase of the menstrual cycle and was significantly higher than that in the adjacent normal myometrium throughout the menstrual cycle. In monolayer cultures of leiomyoma cells, the addition of either E2 (10 ng/mL) or P4 (100 ng/mL) resulted in an increase in PCNA expression in the cells compared to that in control cultures, whereas in monolayer cultures of myometrial cells, the addition of E2 augmented PCNA expression in the cells, but P4 did not. Immunoblot analysis of proteins extracted from cultured leiomyoma cells revealed that leiomyoma cells contained immunoreactive EGF with a molecular mass of 133 kDa and that the addition of P4 resulted in a remarkable increase in the expression of 133- and 71-kDa immunoreactive EGF in the cells compared to that in control cultures, whereas the addition of E2 resulted in a somewhat lower expression of immunoreactive EGF in the cells. Furthermore, immunocytochemical analysis with a monoclonal antibody to human EGF-R demonstrated that the treatment with E2 augmented EGF-R expression in the cells compared to that in untreated cells, but P4 did not. The concentrations of sex steroids used were within the physiological tissue concentrations found in leiomyomas and myometria. These results indicate that P4 up-regulates the expression of PCNA and immunoreactive EGF in leiomyoma cells, whereas E2 up-regulates the expression of PCNA and EGF-R in those cells. As it is evident that EGF plays a crucial role as a local factor in regulating leiomyoma growth, the P4-induced increase in PCNA expression in leiomyoma cells may be mediated by P4-induced enhanced expression of EGF-like proteins in the cells, whereas the E2-induced increase in PCNA expression in leiomyoma cells may be mediated by E2-induced enhanced expression of EGF-R in those cells. It is, therefore, conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF-like proteins and EGF-R expression in uterine leiomyoma.     

Activation of the ATM kinase by ionizing radiation and phosphorylation of p53

The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.