Protein Of DNA Polymerase Delta Research Articles

Comparative Analysis of Cluster of Differentiation 57 and Proliferating Cell Nuclear Antigen Expression in Different Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The immune defense against tumor cells is mainly mediated by the natural killer (NK) cells. Cluster of differentiation 57 (CD57) is a 110-kd glycoprotein, typically expressed by the NK cells, attacks the cancer cells and inhibits tumor development. Proliferating cell nuclear antigen (PCNA) is a 36-kd auxiliary protein for DNA polymerase delta that correlates with cell proliferation and DNA synthesis. It is an essential component of DNA replication, DNA recombination, and DNA repair. The uncoordinated proliferation of PCNA protein characterizes the biological behavior of malignant lesions. The aim of the present study is to compare and correlate the expression of CD57 and PCNAin different grades of oral squamous cell carcinoma (OSCC) by immunohistochemistry. This retrospective analysis comprises 30 samples of various grades of OSCCsand10 samples of healthy mucosa.Sections of 4-5 µm thickness were done and stained with monoclonal anti-PCNA and anti-CD57 antibodies.The statistical package for social science (SPSS) version 16.0 software (IBM Corp., Armonk, NY) was used to analyze the data in this study. The expression of CD57 and PCNA was compared and correlated between the groups using analysis of variance (ANOVA) post hoc, Dunnet t-test, and Pearson's correlation coefficient test.For statistical significance, a p-value of 0.05 or less was used. A significant decrease in CD57 labeling index was seen from well-differentiated squamous cell carcinoma (16.63 ± 2.33) to poorly differentiated squamous cell carcinoma (5.53 ± 1.20) whereas the significant increase in PCNA labeling index was noted from well-differentiatedsquamous cell carcinoma (45.88 ± 2.20), followed by moderately differentiated and poorly differentiated squamous cell carcinoma (72.77 ± 4.35). The combination of CD57 and PCNA biomarkers appears to be good indicators of the immune status of the patient and the aggressiveness of the lesion.

PCNA and GSK3β interact with each other to regulate H1299 lung adenocarcinoma cells apoptosis.

Glycogen synthase kinase beta (GSK3β) is considered as a promising target for lung cancer treatment and its inhibitor lithium chloride (LiCl) is widely regarded as having potent anti-proliferative and apoptosis-modulating activities. Proliferating cell nuclear antigen (PCNA), as an auxiliary protein for DNA polymerase delta, which regulates DNA replication and repair, has been reported to play an important role in regulating apoptosis. Here, we showed that GSK3β interacted with PCNA in H1299 lung adenocarcinoma cells using GST pull-down and co-immunoprecipitation experiments. We discovered that their interaction can be enhanced within the first 3 h after UVC irradiation and decreased gradually with time. Overexpression of PCNA protein decreased GSK3β Ser9 phosphorylation, whereas knockdown of PCNA using small interfering RNA (siRNA) increased Ser9 phosphorylated GSK3β, which was attenuated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 after UVC irradiation, indicating the involvement of the PI3K-AKT pathway. Functional analyses suggested that downregulation of PCNA sensitized H1299 cells to LiCl-induced apoptosis. Thus, our results unraveled a novel regulatory of GSK3β by PCNA and provided a promising direction for treatment of lung cancer.

Current methods of studying of the proliferative activity in experiment

Current methods of studying of the proliferative activity in experiment

Immunohistochemical study of p53 and proliferating cell nuclear antigen expression in odontogenic keratocyst and periapical cyst

Introduction:p53 protein is a product of p53 gene, which is now classified as a tumor suppressor gene. The gene is a frequent target for mutation, being seen as a common step in the pathogenesis of many human cancers. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and plays a critical role in initiation of cell proliferation.Aim:The aim of this study is to assess and compare the expression of p53 and PCNA in lining epithelium of odontogenic keratocyst (OKC) and periapical cyst (PA).Materials and Methods:A total of 20 cases comprising 10 OKC and 10 PA were included in retrospective study. Three paraffin section of 4 μm were cut, one was used for routine hematoxylin and eosin stain, while the other two were used for immunohistochemistry. Statistical analysis was performed using Chi-square test.Results:The level of staining and intensity were assessed in all these cases. OKC showed PCNA expression in all cases (100%), whereas in perapical cyst only 60% of cases exhibited PCNA staining. (1) OKC showed p53 expression in 6 cases (60%) whereas in PA only 10% of the cases exhibited p53 staining. Chi-square test showed PCNA staining intensity was more significant than p53 in OKC. (2) The staining intensity of PA using p53, PCNA revealed that PCNA stating intensity was more significant than p53.Conclusion:OKC shows significant proliferative activity than PA using PCNA and p53. PCNA staining was more intense when compared with p53 in both OKC and PA.

Ozone Exposure Suppresses Proliferative Response in Mice Skin

Ozone Exposure Suppresses Proliferative Response in Mice Skin

Proliferating Cell Nuclear Antigen as a Prognostic Factor After Total Mesorectal Excision of Stage II–III Rectal Cancer

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta that is tightly associated with sites of DNA replication; whether PCNA is a definite prognostic factor remains controversial. This study determined the clinicopathological factors associated with the long-term oncological outcome after radical resection of stage II-III rectal cancer, focusing on PCNA. We retrospectively reviewed 135 consecutive patients who underwent curative surgery for stage II-III rectal cancer between August 2001 and April 2004. Prognostic factors including immunohistochemical PCNA expression and the clinical outcome were evaluated. The PCNA index correlated with lymph node metastasis (P = 0.010). A multivariate analysis identified two independent factors that significantly affected both disease-free and overall survival: lymph node metastasis and PCNA index. With a median follow-up period of 60 months (range 8-87 months), both 5-year disease-free and overall survival of the low PCNA group were significantly higher than those of the high PCNA group (71.8% versus 32.0%, P < 0.001; 83.9% versus 50.0%, P < 0.001, respectively). For patients undergoing curative resection for rectal cancer, pathological N stage and high PCNA expression can provide valuable prognostic information about survival. This study suggests that the PCNA index may be used as an independent prognostic factor in stage II-III rectal cancer patients.

Short-interfering RNA-mediated silencing of proliferating cell nuclear antigen inhibit proliferation and induce apoptosis in HeLa cells

Proliferating cell nuclear antigen (PCNA) is an important protein for DNA polymerase delta in the nucleus, and shown to have a fundamental role in cellular proliferation. It is overexpressed to support cell growth in cervical carcinoma. To study its role in stress response, we design and use short hairpin RNA (shRNA) to inhibit PCNA expression in HeLa cells and validate its effect on cell proliferation. In this study, three PCNA-shRNA expression vectors are constructed and introduced into HeLa cells, and the cell cycle is analyzed by flow cytometry. Apoptotic cell is detected by single cell gel electrophoresis assay (comet assay), and caspase cleavage is studied also. Expression of PCNA is assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it is found that expression of PCNA decreased in shRNA-transfected cells, downregulation of PCNA inhibit cell growth and induce apoptosis in HeLa cells. PCNA downregulation also increase cell population in the G0-G1 phase. In conclusion, our findings demonstrate that shRNA can inhibit the DNA replication and induce apoptosis in HeLa cells effectively and, therefore, could be used as a new potential anticancer tool for therapy of human cervical carcinoma.

Secondary neurogenesis in the brain of the African clawed frog, Xenopus laevis, as revealed by PCNA, Delta‐1, Neurogenin‐related‐1, and NeuroD expression

After primary neurogenesis in the Xenopus laevis embryo, a massive new surge of neurogenesis and related neurogenic and proneural gene expression occurs in the spinal cord at the beginning of the larval period (starting at Stage 46), which corresponds to well-documented secondary neurogenesis in larval zebrafish central nervous system development. Here, we document related neural proliferation and gene expression patterns in the brain of Xenopus, in various embryonic and larval stages, showing the distribution of proliferative cells (immunostaining of cells containing the proliferating cell nuclear antigen; the auxiliary protein of DNA polymerase delta; PCNA), and the activity of some critical genes expressed during neurogenesis (i.e., Delta-1, Neurogenin-related-1, NeuroD). This study reveals that the early larval stage in Xenopus (Stage 48) displays patterns of proliferation (PCNA), as well as of neurogenic (Delta-1) and proneural (Ngnr-1; NeuroD) gene expression that are qualitatively almost identical to those seen in the 3-day postembryonic zebrafish or the 12.5/13.5-day embryonic mouse. Furthermore, a comparable bauplan of early proliferation zones (including their neuromeric organization) as described in the postembryonic zebrafish apparently exists in tetrapods (Xenopus). Altogether, the data presented suggest a common brain bauplan on the level of early proliferation patterns and neurogenic/proneural gene activity in anamniotes, if not vertebrates.

Functional analysis of CDK inhibitor p21WAF1.

p21WAF1 was originally identified as a protein that binds and inhibits cyclin-dependent kinases (CDKs). p21WAF1 is recognized to have at least two separate roles-first as a CDK inhibitor, and second as an inhibitor of PCNA, an accessory protein of DNA polymerase delta. p21WAF1 plays a critical role in the cellular response to DNA damage. Additionally, p21WAF1 plays a role in DNA repair, apoptosis, cellular senescence, terminal differentiation, and cell cycle arrest upon extracellular signaling. p21WAF1 protein levels are regulated both by transcriptional control by p53 and by factors other than p53, as well as by posttranscriptional regulation. Although the role of p21WAF1 has been explained so far only by its interaction with CDKs and with PCNA, it has several other binding partners. The ability of p21WAF1 to participate in several cellular functions has been widely studied by transfection of cells with p21WAF1 vectors. We describe here procedures for analysis of p21WAF1 function in mammalian cells after transfection of p21 plasmids. The procedures include inhibition of DNA synthesis, cellular localization, association with binding partners, and half-life measurements.

Obstructive jaundice alters proliferating cell nuclear antigen expression in rat small intestine.

Translocation of bacteria and endotoxin has long been documented in obstructive jaundice, and altered intestinal barrier function is considered to be one of the important mechanisms for this phenomenon. Proliferating cell nuclear antigen (PCNA), also known as cyclin, is an auxiliary protein of DNA polymerase-delta, and the level of synthesis correlates directly with rates of cellular proliferation and DNA synthesis. This study was designed with the aim of evaluating the effect of obstructive jaundice on PCNA expression in small bowel epithelium. Male Sprague-Dawley rats were randomized to four groups. Group A (n = 10, control group) underwent a sham operation. Group B (n = 9, obstructive jaundice group for 1 week) underwent common bile duct ligation. Group C (n = 8, obstructive jaundice group for 2 weeks) underwent common bile duct ligation. Group D (n = 8, obstructive jaundice group for 2 weeks) underwent common bile duct ligation with oral glutamine intake. After periods of 7 days and 2 weeks, segments of small bowel were harvested from groups A & B and groups C & D, respectively. Nuclear immunohistochemical expression of PCNA in small bowel was evaluated. The PCNA-labeling index [(PCNA-positive cells/500 cells) x 100] was quantified. Comparisons among the four groups were performed. The PCNA-labeling index in small bowel of group B was significantly higher than that of group A (29.0% vs 21.2%, p = 0.001). After 2 weeks of common bile duct ligation, the PCNA-labeling index in small bowel of group C was significantly lower than that of group A (19.4% vs 21.2%, p = 0.045). With oral glutamine intake daily, the PCNA-labeling index in small bowel of Group D was restored and was significantly higher than that of group A (24.5% vs 21.2%, p = 0.002). Obstructive jaundice for 1 week upgraded PCNA expression in rat small intestine. PCNA expression in rat small intestine later became depressed after obstructive jaundice for 2 weeks. Oral glutamine intake daily could effectively restore the PCNA expression in small bowel of rats subjected to obstructive jaundice for 2 weeks.

Nipradilol prevents L-NAME-exacerbated nephrosclerosis with decreasing of caspase-3 expression in SHR.

The proliferative cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be required for both DNA synthesis and repair. Previously, we showed that prolonged NO synthase (NOS) inhibition produced severe nephrosclerosis with an increase of glomerular cell DNA fragmentation (apoptosis), glomerular ischemia and hypertension in spontaneously hypertensive rats (SHR). The objective of the present study was to investigate the effects of the vasodilating, nonselective, NO-releasing beta-adrenoceptor blocker nipradilol on DNA fragmentation and synthesis/repair of glomerular cells in this prolonged NOS blockaded SHR. Twenty-week-old SHR were administered an NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 80 mg/l in drinking water) or co-treated with the same dose of L-NAME and nipradilol (20 mg/kg/day) for 3 weeks. After this treatment, expression of apoptosis was histologically examined using caspase-3, an apoptosis inducer, in addition PCNA (DNA synthesis/repair), and examination of glomerular morphometric changes, including cell number and tuft area. Nipradilol reduced blood pressure and preserved creatinine clearance reduction in L-NAME/SHR. These effects were associated with normalization of the glomerular cell apoptosis index and caspase-3 score, an increase in PCNA index, and increases in glomerular cell numbers and glomerular tuft area, resulting in a decreased glomerular injury score. Thus, in SHR administered an NOS inhibitor, nipradilol improved nephrosclerosis in association with a decrease in apoptosis and an increase in DNA synthesis/repair of glomerular cells. These findings may provide important insights into DNA repair/repair and apoptosis in nephrosclerosis.

The Response of Proliferating Cell Nuclear Antigen to Ionizing Radiation in Human Lymphoblastoid Cell Lines Is Dependent on p53

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and epsilon involved in DNA replication and nucleotide excision repair. There are two intranuclear fractions: a detergent-extractable, soluble fraction and a tightly DNA-bound fraction. To function, PCNA forms a trimeric sliding clamp which is loaded onto DNA. To better understand the role of the p53/p21 pathway in the regulation of PCNA after irradiation, we studied three closely related human lymphoblastoid cell lines, WTK1, TK6 and TK6E6, an HPV16 E6-transfected line, that differ in p53 status, radiosensitivity and susceptibility to radiation-induced apoptosis. Time-dependent changes in PCNA levels were measured in the different nuclear fractions by Western blot analysis after protein crosslinking. The results were compared to those for human diploid fibroblasts studied under different growth conditions. There was no change in total cellular levels of PCNA after irradiation, consistent with predominantly post-translational regulation. Changes in intranuclear distribution and complex formation occurred in a p53/p21-dependent manner. The loading of PCNA onto DNA was increased in cells with low p21 levels. A disruption of PCNA trimers was observed in exponentially growing p53+ cells in the soluble fraction. Thus the p53/p21 signal transduction pathway appears to play a significant role in the regulation of the response of PCNA to radiation.

O-12-178 - The biological behaviour of the pilocytic astrocytoma by Ki-67-and p53-immunostaining related to neuro-imaging

Information about a tissue's proliferative activity can be obtained from the immunocytochemical investigation of proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta expressed by cycling cells. To determine whether a relationship exists between morphology and PCNA expression in normal, regenerative, and malignant neoplastic hepatocytes, this study was undertaken on 48 fine-needle aspiration cytology (FNAC) cell blocks from eight normal livers, eight cirrhotic livers, and 32 hepatocellular carcinomas (HCCs), as well as on 41 needle or wedge biopsy specimens from 10 normal livers, 13 cirrhotic livers, one focal nodular hyperplastic liver, and 17 HCCs. Anti-PCNA monoclonal antibody PC10 was applied to formalin-fixed, paraffin-embedded tissue using the avidin-biotin method. Proliferating cell nuclear antigen immunoreactivity was evaluated as follows: absent; minimal, less than 5% positive nuclei; grade 1, 5% to 25% positive nuclei; grade 2, 26% to 50% positive nuclei; grade 3, 51% to 75% positive nuclei; and grade 4, 76% to 100% positive nuclei. In both the FNAC and biopsy series normal and regenerative lines were either completely negative or minimally immunoreactive (under 5% positive nuclei). In contrast, all well-differentiated HCC cases exhibited over 15% positive nuclei. Most well-differentiated HCCs were grade 1 (85.7% in the FNAC series and 76.92% in the biopsy series) and the majority of moderately differentiated HCCs were grade 3 (63.63% in the FNAC series, but only 50% in the biopsy series). Therefore, absent or minimal PCNA immunoreactivity seems to be a useful adjuvant to discriminate normal/regenerative liver from HCC, whose degree of differentiation tends to correlate with the level of PCNA expression. These observations apply to both the FNAC and biopsy series, which yielded very similar data.

Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control.

The proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase delta and epsilon, is involved in DNA replication and repair. This protein forms a homotrimeric structure which, encircling DNA, loads the polymerase on the DNA template. A role for PCNA in the cell cycle control is recognised on the basis of the interaction with cyclins, cyclin-dependent kinases (cdks) and the cdk-inhibitor p21 waf1/cip1/sdi1 protein. Association with the growth-arrest and DNA-damage inducible proteins gadd45 and MyD118, further demonstrates the role of PCNA as a component of the cell cycle control apparatus.

Association of tumor cell proliferation with lymph node metastasis in early gastric cancer.

Recently, many studies have reported on the relationship between malignant potential of neoplasms and cell proliferation. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and is considered to correlate with the cell's proliferative state. In this study, we investigated the correlation between tumor cell proliferation and nodal status of early gastric carcinoma. One hundred two endoscopically biopsied specimens from patients with early gastric carcinoma prior to operation were investigated by an immunohistochemical study, using anti-PCNA monoclonal antibody. Correlation of PCNA labeling index (percentage of positive cells per over 500 tumor cells) with nodal status were studied. PCNA labeling index in patients with lymph node metastasis was significantly higher in those without metastasis. Moreover, multivariate analysis indicated that PCNA labeling index is an independent significant factor for lymph node metastasis. As a result of this study, PCNA labeling index was suggested to be effective as one of the predictors of lymph node metastasis.

Temporal and histologic relationships of proliferating cell nuclear antigen and human papillomavirus type 11 in the mouse xenograft system.

Proliferating cell nuclear antigen (PCNA) is an accessory protein of DNA polymerase delta. This protein is associated with cell cycle progression and can be detected in the replicating cells of normal tissues. Condylomata acuminata are benign epithelial tumors caused by infection with human papillomaviruses and are characterized by abnormal cell proliferation. The athymic mouse xenograft model of HPV 11 infection was used to test the hypothesis that PCNA is induced early in the course of HPV 11 infection and to study the temporal and histologic relationships between detection of PCNA and HPV DNA. Human foreskin tissue was infected with HPV 11 and implanted under the renal capsules of 10 athymic mice. Pairs of mice were sacrificed every week beginning four weeks after implantation. HPV DNA was detected in sections of foreskin implants by in situ hybridization. PCNA was as or more abundant in implants removed at earlier time points than at later time points, whereas HPV DNA became increasingly more abundant with time. PCNA was detected only in basal cells in areas of histologically normal epithelium that were also negative for HPV DNA. In contrast, PCNA was present throughout the epithelium in regions that were HPV DNA-positive. HPV DNA was detected only in differentiated epithelial cells in implants removed at all five time points, but in HPV DNA-positive regions, PCNA was detected with equal intensity in differentiated and undifferentiated cells. The foci of PCNA-positive cells were well demarcated and were larger than, but included, the foci of HPV DNA-positive cells. PCNA may be induced maximally in differentiated epithelium by HPV 11 prior to significant HPV DNA replication.

The C-terminal Region of p21SDI1/WAF1/CIP1 Is Involved in Proliferating Cell Nuclear Antigen Binding but Does Not Appear to Be Required for Growth Inhibition

The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.

Two of three promoter elements identified in a rice gene for proliferating cell nuclear antigen are essential for meristematic tissue‐specific expression

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta, and it is highly conserved among eukaryotes. The rice PCNA promoter, truncated to position -263, was previously shown to confer meristematic tissue-specific expression in transgenic plants. By DNasel footprinting and gel retardation analysis, three cis-acting elements were defined in this truncated promoter, which designated site I (-201 to -194, CCAGGTGG), site IIa (-197 to -188, TGGGCCCGT) and site IIb (-178 to -169, TGGTCCCAC). Site I resembles the G-box, and sites IIa and IIb resemble the conserved motif (T/GGTCCCAT) that is found in promoter regions of auxin-regulated genes. Functional analysis of expression of a PCNA-GUS gene fusion in transgenic tobacco plants revealed that a mutation in site I in the full-length 2.0 kb promoter had no significant effect on the activity. However, the mutation in site I in the truncated -263 promoter, which had 14% of the activity of the full-length promoter, caused a considerable decrease in the activity, suggesting that site I contributes in part to transcriptional activation. Simultaneous disruption of sites IIa and IIb in the full-length promoter caused about 80-85% loss of promoter activity, while separate disruption of site IIa or site IIb resulted in no marked change on the activity. These observations suggest that site IIa and site IIb play an important role in the meristematic tissue-specific expression of the rice PCNA gene, presumably by mediating putative enhancer activities dependent on the far-upstream region.

Association of Proliferating Cell Nuclear Antigen With Cyclin-Dependent Kinases and Cyclins in Normal and Transformed Human T Lymphocytes

Abstract The proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be needed for both DNA synthesis and DNA repair. It is present in low amount in resting normal human T lymphocytes and, upon mitogenic stimulation with phorbol dibutyrate and ionomycin, begins to increase in mid-G1 phase, approximately 12 to 15 hours before entry into S phase. PCNA continues to increase in amount throughout the cell cycle and remains high in proliferating cultures. PCNA was extracted from activated normal T cells and from the transformed T-lymphoblastoid cell line Jurkat by a method that recovered approximately 98% of total cellular PCNA but yet retained its associations with other proteins. PCNA immunoprecipitates possessed H1 histone kinase activity, which increased in parallel with increasing cellular content of PCNA. Both the cdc2 and cdk2 kinases were found associated with PCNA in normal T cells, in amounts consistent with detected kinase activity. The results indicate that PCNA is not an inhibitory molecule of cdk/cyclin activity. Both normal and transformed T cells contained PCNA in association with cdk2, cdk4, cdk5, and cdk6, with the amount of PCNA associated with these molecules increasing in the order listed. Relatively high amounts of PCNA were also found associated with cyclins D2 and D3, the major cyclin partners of cdk6 in T cells. Though detected in normal cells, PCNA/cdc2 complexes were present in exceedingly low amount, if at all, in Jurkat cells. This cell line appeared to contain more of nearly all of the cdk and cyclin molecules analyzed, but there seemed to be little difference in the patterns of association of these molecules with PCNA in the cell line as compared with normal human T cells.

Association of proliferating cell nuclear antigen with cyclin-dependent kinases and cyclins in normal and transformed human T lymphocytes

The proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be needed for both DNA synthesis and DNA repair. It is present in low amount in resting normal human T lymphocytes and, upon mitogenic stimulation with phorbol dibutyrate and ionomycin, begins to increase in mid-G1 phase, approximately 12 to 15 hours before entry into S phase. PCNA continues to increase in amount throughout the cell cycle and remains high in proliferating cultures. PCNA was extracted from activated normal T cells and from the transformed T-lymphoblastoid cell line Jurkat by a method that recovered approximately 98% of total cellular PCNA but yet retained its associations with other proteins. PCNA immunoprecipitates possessed H1 histone kinase activity, which increased in parallel with increasing cellular content of PCNA. Both the cdc2 and cdk2 kinases were found associated with PCNA in normal T cells, in amounts consistent with detected kinase activity. The results indicate that PCNA is not an inhibitory molecule of cdk/cyclin activity. Both normal and transformed T cells contained PCNA in association with cdk2, cdk4, cdk5, and cdk6, with the amount of PCNA associated with these molecules increasing in the order listed. Relatively high amounts of PCNA were also found associated with cyclins D2 and D3, the major cyclin partners of cdk6 in T cells. Though detected in normal cells, PCNA/cdc2 complexes were present in exceedingly low amount, if at all, in Jurkat cells. This cell line appeared to contain more of nearly all of the cdk and cyclin molecules analyzed, but there seemed to be little difference in the patterns of association of these molecules with PCNA in the cell line as compared with normal human T cells.