p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation

BACKGROUND: The complications associated with anterior craniofacial resections for benign and malignant tumors were reviewed in 104 patients treated between January 1981 and June 1996. METHODS: Information regarding patient characteristics, histologic type, history of prior therapy, extent of the disease, extent of surgical procedure, and type of reconstruction were entered in a microcomputer database. To better understand and stage postoperative complications, we divided them into early (<14 days) and late (>14 days) according to the time of presentation, into major and minor depending on the morbidity potential of complication, and into local and systemic ones. Comparison between risk factors associated with complications was made using chi-square analysis with Yates' correction for continuity. Survival analysis was performed using the Kaplan-Meier product limit method. RESULTS: There were 8 (7.6%) postoperative deaths, with only 1 occurring from systemic complications. Complications occurred in 53 (48.6%) patients. Local major complications occurred in 49 (45%) patients, local minor in 29 (26.6%), and systemic in 11 (10%). Early complications occurred in 40 (38.5%) patients and late complications in 13 (12.5%) patients. These complications developed during a period ranging from 1 day to 5 months. More than one complication occurred in a number of patients. Bacterial contamination leading to local septic complications was the principal cause of morbidity, accounting for 54.7% (29/53) of complications. Major complications included meningitis in 8 patients associated with cerebrospinal fluid leak in 7, cerebral abscess in 2, sepsis in 1, and subdural hemorrhage in 1, all of which resulted in death except for one case. The extent of the craniofacial resection (p = .011) was the most important factor associated with major complications. Invasion of the dura and the type of reconstruction of the anterior skull base were the most important factors related to cerebrospinal fluid leakage (p = .048 and p = .032) and meningitis (p = .011). CONCLUSION: Contemporary surgical approaches and methods of reconstruction have enabled skull base surgeons to extend their cranial base resections and increase the 5-year survival rates of patients. Nevertheless, significant complications persist. Knowledge and high index of suspicion together with early recognition of these complications are essential for effective management of patients undergoing craniofacial resection. The factors related to major complications found in this study stressed the need to develop more effective methods to prevent contamination of intracranial structures.     

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.     

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 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.     

Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.     

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.     

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.     

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.     

Proliferative activity of gastric cancer assessed by immunostaining for proliferating cell nuclear antigen

The growth activity of 107 gastric carcinomas was assessed by immunohistochemical staining for formalin-fixed, paraffin-embedded tissue with a monoclonal antibody against proliferating cell nuclear antigen (PCNA). When the tumor doubling times (Tds) of 10 patients were estimated from the serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9, there was an inverse correlation between the Tds and PCNA labeling index (LI) at P = 0.055. Flow-cytometric analysis was carried out by double staining for PCNA and DNA using fresh materials from 14 patients. The PCNA-positive cell fraction revealed by flow cytometry showed a good linear correlation with PCNA LI in routinely stained tissue. The LI of well-differentiated adenocarcinoma was significantly higher than that of the poorly differentiated type. When the LI was analyzed in well- or poorly differentiated adenocarcinoma, the value was significantly higher in the well-differentiated type with hepatic metastasis and in the poorly differentiated type with lymph node metastasis.     

Analysis of proliferative activity of the parathyroid glands using proliferating cell nuclear antigen in patients with hyperparathyroidism

To elucidate the cellular proliferative kinetics of the parathyroidal gland in patients with hyperparathyroidism, we investigated the expression of proliferating cell nuclear antigen (PCNA) in parathyroidal tissues using an immunohistochemical procedure. The PCNA labeling index (LI; maximum LI, maximal stained area; average LI, evenly distributed stained area) indicating cellular proliferative activity was defined as the number of PCNA-positive cells per 1000 parathyroid cells in the region of interest. We used these indexes to compare and investigate the proliferative activity of parathyroid cells under various conditions. The specimens used for the study were 42 parathyroid glands from 21 patients with primary hyperparathyroidism (19 cases of adenoma and 2 cases of primary hyperplasia due to multiple endocrine neoplasia type 1) and 129 parathyroid glands from 32 patients with secondary hyperparathyroidism. An additional 40 parathyroid glands resected during thyroid surgery of 30 normocalcemic patients were used as normal controls. In normally functioning parathyroids, a small number of cells in the growth phase were found. In primary hyperparathyroidism, proliferative activity was highest in the adenoma followed by primary hyperplasia. In contrast, the PCNA LIs showed a low value in the normal rim of the adenoma and normal glands resected as biopsy specimens from adenoma patients. We, therefore, assumed that proliferative activity was suppressed in these cells compared with that in normally functioning glands. In secondary hyperparathyroidism, when the cell component of the parathyroid tissues was divided into five types, PCNA immunoreactivity was lowest in the dark chief cells. Proliferative activity in cells of the oxyphil series was the same or higher than that in the clear chief cells or vacuolated chief cells. When classified according to the structure of the parathyroid glands, cell proliferation was significantly higher in the nodular type than in the diffuse type (maximum LI, 176 +/- 231 vs. 38.3 +/- 55.7; average LI, 120 +/- 188 vs. 24.8 +/- 43.5; mean +/- SD; P < 0.001). More PCNA-immunoreactive cells were found in autotransplanted glands with recurrence than in glands resected during the initial surgery. To summarize the PCNA expression classified according to the pathological types of hyperparathyroidism, the PCNA LIs were highest in secondary hyperplasia (maximum LI, 144 +/- 212; average LI, 96.0 +/- 169) and adenoma (maximum LI, 102 +/- 81.7; average LI, 67.5 +/- 67.7), followed by primary hyperplasia (maximum LI, 25.0 +/- 25.4; average LI, 19.2 +/- 22.2) and normal glands (maximum LI, 13.6 +/- 23.9; average LI, 4.40 +/- 8.90). These findings suggest that the cellular proliferative kinetics of the parathyroid gland differ depending on the type of hyperparathyroidism, glandular structure, and cell components. As the detection method of intranuclear expression of PCNA in cells is too sensitive, we should be careful not to overestimate the number of cells in the proliferative cycle. However, these results could not have been obtained using a conventional method such as DNA analysis by flow cytometry.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

Molecular genetic and biochemical analysis of Brassica napus proliferating cell nuclear antigen function

A cDNA encoding the proliferating cell nuclear antigen (PCNA) from Brassica napus (oilseed rape) was shown to complement the lethal deletion mutation in the PCNA gene (delta POL30) of Saccharomyces cerevisiae. We provide unequivocal evidence that the B. napus PCNA can perform all the essential functions of the yeast PCNA in DNA replication, although some species-specific differences may exist. In addition, the B. napus PCNA expressed as a fusion polypeptide with glutathione S-transferase (GST) was shown to stimulate the activity and processivity of two delta-like DNA polymerases from wheat in vitro. These experiments provide direct biochemical evidence that the B. napus PCNA may function as an auxiliary factor in plant cell DNA replication.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.     

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.