PCNA Activity Research Articles

Complexity of the Mechanisms of Initiation and Maintenance of DNA Damage-Induced G2-Phase Arrest and Subsequent G1-Phase Arrest: TP53-Dependent and TP53-Independent Roles

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.

Homocysteine induces vascular expression of TIMP-1, PCNA and gelatinolytic activity in vivo and in vitro

Homocysteine induces vascular expression of TIMP-1, PCNA and gelatinolytic activity in vivo and in vitro

Resistance to UV-induced cell-killing in nucleophosmin/B23 over-expressed NIH 3T3 fibroblasts: enhancement of DNA repair and up-regulation of PCNA in association with nucleophosmin/B23 over-expression

Nucleophosmin/B23 was rapidly up-regulated after UV irradiation as p53, PCNA and c-Jun. UV induction of nucleophosmin/B23 was evidently increased at 3 h post-irradiation, and reached a maximum at 12 h, and remained high for at least 24 h. Over-expression of nucleophosmin/B23 made cells more resistant to UV-induced cell growth inhibition and death as compared with control vector-transfected cells through three main observations: cell growth/death percentage determination; clonogenic survival assay; and flow cytometric analysis. Moreover, nucleophosmin/B23 over-expressed cells had a greater capacity to repair UV-damaged reporter plasmid, indicating a higher nucleotide excision repair (NER) activity. Furthermore, PCNA, an essential component for DNA repair machinery, was correlated with nucleophosmin/B23 expression. Both protein level and promoter activity of PCNA were higher in nucleophosmin/B23 over-expressed cells than in control vector-transfected cells. On the other hand, treatment of cells with nucleophosmin/B23 antisense oligonucleotides decreased nucleophosmin/B23 and PCNA proteins, and potentiated the UV-induced cell killing. The effect of PCNA up-regulation may be one of the reasons that nucleophosmin/B23 over-expression made cells resistant to UV-induced growth inhibition and cell-killing.

Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase 2 activity to disrupt PCNA function in S phase.

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-mediated G(1) inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.

Unilateral Testicular Torsion: Evaluation of bcl-2, p-53 and PCNA Expression in Contralateral Testes

Objectives and Methods: The present study aims to evaluate the histologic as well as apoptotic changes indicated by PCNA, p-53 and bcl-2 expression in the contralateral testes after a period of unilateral testicular torsion of 4 h. Results: Regarding the histologic changes, while some significant alterations such as complete or incomplete spermatocytic arrest as well as sertoli cell only formation have to some extent been noted after the detorsion procedure, reasonably better preserved histology could be demonstrated following the orchiectomy procedure both in the early and late follow-up. Thus, the orchiectomy procedure was found to be limiting enough on the severity of the histologic changes in the contralateral testes after a certain period of time following the torsion procedure. On the other hand, however, in relation to the apoptotic events indicated by the three markers, while PCNA activity was found to be significantly different depending on the procedure performed (detorsion or orchiectomy), p-53 and bcl-2 positivity did not exhibit any difference in this aspect. Increased PCNA activity (especially after the detorsion procedure) together with marked positivity of p-53 both in the early and late follow-up indicated the increased cell turnover in the contralateral testes, which in turn may be accepted as a sign of increased apoptosis in these testes. In addition to these findings, bcl-2 expression has been found to be consistently negative in all specimens evaluated both in the early and late follow-up. Conclusion: Taking into account the strong inhibitory effect of bcl-2 during apoptotic events, these findings again support the likelihood of increased apoptosis in the contralateral testes.

Assessment of proliferation indicators in residual prostatic adenocarcinoma cells after radical external beam radiotherapy.

Fifty-five patients who had received radical external beam radiotherapy (RRT) for clinically staged, localized prostatic adenocarcinoma were examined with systematic sextant mapping with the help of transrectal ultrasound-guided core biopsies. The average follow-up after RRT was 6.8 years. Residual cancer was found histopathologically in 67% (37/55) of the patients investigated. In the present report, the viability of these tumor cells was studied using immunohistochemical staining methods with the monoclonal antibodies MIB1 and PC10, which are specific for the proliferation-associated antigens Ki-67 and PCNA. Available data concerning proliferating activity in the pretreatment situation in 12 of these patients revealed that proliferating activity was substantially reduced in the majority of cases after RRT. Post-RRT, Ki-67, and PCNA activity was nonetheless found to varying degrees in 64% and 94% of the 36 evaluable tumor-harboring specimens, respectively. In the majority of cases, the proportion of proliferating cells, designated "score", was low. However, Ki-67 scores of up to 8% and PCNA scores of up to 50% were obtained in 35% (8/23) and 97% (33/34) of the specimens, respectively, with proliferative activity. There was no significant correlation between tumor grade and proliferative score in the follow-up biopsies. In the 36 cases investigated, endocrine treatment did not influence the proliferation results. The present study demonstrates a proliferative capacity in residual tumor cells in a long-term follow-up after RRT, suggesting that these cancer cells might have a biological significance.

Rectal cancer: The influence of tumor proliferation on response to preoperative irradiation

Purpose : Regression of rectal carcinoma after preoperative irradiation is variable, likely reflecting differences in the physical and biologic properties of these tumors. This study examines the assocation between the pathologic response of rectal cancer after irradiation and its pretreatment proliferative state as assayed by the activity of the proliferative dependent antigens (Ki-67, PCNA) and mitotic counts. Methods and Materials : One hundred and twenty-two patients with locally advanced rectal cancer received preoperative irradiation followed by surgery. Pretreatment tumor biopsies were scored for the extent of Ki-67 and PCNa immunostaining and the number of mitoses per 10 high-powered fields. Postirradiation surgical specimens were examined for extent of residual disease. Results : The tumors of 38 of 122 patients (31%) exhibited marked pathologic downstaging (no residual tumor or cancer confined to teh rectal wall) after preoperative irradiation. Two features were associated with the likelihood of marked pathologic regression after preoperative irradiation: tumor proliferative activity and lesion size. When stratified by lesion size, marked tumor regression occurred most frequently in smaller tumors with high Ki-67, PCNA, and mitotic activity compared to larger tumors with lower Ki-67, PCNA, and mitotic activity. Intermediate downstaging rates were seen for small or large tumors with moderate Ki-67, PCNA, and mitotic activity. Conclusion : Tumor Ki-67, PCNA, and mitotic activity predicts the likelihood of response to irradiation, which may aid in formulating treatment policies for patients with rectal cancer.

PCNA activity in benign and primary and metastatic melanocytic lesions

PCNA activity in benign and primary and metastatic melanocytic lesions

Localization of Proliferating Cell Nuclear Antigen in Aural Cholesteatoma.

Middle ear cholesteatoma is not a genuine tumor but has a remarkable proliferative activity which causes serious destruction of the mastoid bone. In the present study, we used immunohistochemistry with antiproliferating cell nuclear antigen (PCNA) antibody on cholesteatomatous tissues to evaluate the localization of PCNA, as it has been said that PCNA is a very available protein for showing cell proliferative activity. Moderately concentrated PCNA was demonstrated within the germinal basal layer cells of the cholesteatomatous epithelium in three of eight surgical specimens. Furthermore, in one case of very active osteolytic cholesteatoma, PCNA activity was demonstrated in the mesenchymal cells, probably fibroblast-like cells, in direct contact to the destroying mastoid bone lesions. Although the etiology and histopathology of the invasive and proliferative activity of middle ear cholesteatomatous tissues are unclear, this observation suggests that immunohistopathological examination using PCNA antibody might be a useful tool for evaluating bone resorption activity and for establishing the prognosis of various types of cholesteatoma.