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At least 19 recordsLinked to original sources
PubMed1995

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells.

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.

Base Sequence↗
PubMed1997

Different mechanisms for inhibition of cell proliferation via cell cycle proteins in PC12 cells by nerve growth factor and staurosporine.

PC12 cells have previously been shown to cease cell division during nerve growth-factor (NGF)-induced differentiation by affecting specific cell cycle proteins. Staurosporine, a protein kinase inhibitor, also causes PC12 cell differentiation, independently of neurotrophins or plasma membrane receptors. We have investigated the relationship of the tumor suppressor protein, p53, and other cell cycle proteins to the antiproliferative effects of NGF and staurosporine in PC12 cells. NGF treatment of PC12 cells stimulated an increase of p53 protein in the nucleus and, more slowly, an increase in total cellular p53 protein. Levels of the cyclin-kinase inhibitor p21/WAF1, cyclin D1, and cyclin G, all downstream transcriptional targets of p53, increased after short times of NGF treatment. Cessation of replication and differentiation occurred more rapidly in defined medium (2 days) than in serum medium (6 days), in correspondence with the more rapid changes in both p53 and p21/WAF1 levels in defined medium (1 hour) than in serum (1 day). Levels of p34cdc2 and p33cdk2 kinase dropped after 6 to 10 days treatment with NGF in serum, close to the time of terminal differentiation. Staurosporine, on the other hand, inhibited DNA replication of PC12 cells in a time- and dose-dependent fashion by affecting cyclin-dependent kinases. Staurosporine had no effect on the protein levels of p53, p21/WAF1, or cyclin G. The kinase activity of both p34cdc2 and p33cdk2 were inhibited in vitro with IC50 values of 20 nM and 75 nM, respectively. In vivo p34cdc2 kinase activity was inhibited within 1 day, before the decrease in the levels of p34cdc2 protein at days 2 to 3. In contrast, in vivo p33cdk2 kinase activity only decreased in concert with protein levels. Although both NGF and staurosporine inhibit DNA replication concomitant with induction of differentiation by affecting the activity of p34cdc2 and p33cdk2, the mechanism of the two agents is quite different. NGF achieves inhibition of activity of these cyclin-dependent kinases by signalling through the TrkA receptor to the tumor suppressor protein p53 and then to p21/WAF1. In contrast, staurosporine directly inhibits the activity of p34cdc2 and p33cdk2 by binding to them and also indirectly by alteration of their phosphorylation through other regulatory kinases.

Animals↗
PubMed1998

Analysis of cell death in myeloid cells inducibly expressing the cell cycle protein p55Cdc.

p55Cdc, a cell cycle protein is expressed in cycling mammalian cells and is required for normal cell division. Expression of this protein is regulated during the cell cycle, peaking in late G1 and S. We have previously shown that constitutive expression of p55Cdc results in inhibition of granulocyte differentiation. Degradation of p55Cdc is also required for apoptosis in growth factor and serum starved cells. In the present study we prepared stably transfected cells conditionally expressing p55Cdc in response to zinc stimulation to investigate the role of inducible p55Cdc expression in apoptosis of myeloid cells. We report that inducible expression of p55Cdc in the myeloid leukemic cell line 32Dc13 resulted in increased cell death. p55Cdc overexpression led to a statistically significant decrease in the viability of 32Dc13 cells compared with that of control cells. Furthermore, cell staining and flow cytometry analysis revealed that p55Cdc-overexpressing 32Dc13 cells progressed to apoptosis much earlier than uninduced cells. These results suggest that inducible expression of p55Cdc leads to earlier increases in cell death in the absence of growth factor and serum in myeloid leukemic cells.

Animals↗
PubMed2006

The unfolded protein response affects neuronal cell cycle protein expression: implications for Alzheimer's disease pathogenesis.

Alzheimer's disease (AD) is characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Previously, we have shown that the UPR is activated in AD neurons. In actively dividing cells, activation of the UPR is accompanied by decreased cell cycle protein expression and an arrest in the G1 phase of the cell cycle. Aberrant expression of cell cycle proteins has been observed in post mitotic neurons in AD and is suggested to be involved in neurodegeneration. In this study we show that the protein levels of BiP/GRP78, an ER-stress marker, is increased in Braak stages B and C for amyloid deposits. This is in contrast to the levels of cell cycle markers cyclin D1, cyclin E and phosphorylated retinoblastoma protein (ppRb) which are decreased in Braak stage C compared to Braak stage A for amyloid deposits. In addition, we report a negative correlation between neuronal expression of ppRb and expression levels of BiP/GRP78 in control and AD cases. Activation of the UPR in neuronal cells induces changes in cell cycle protein expression similar to these observed in AD brain. ER stress inducers tunicamycin and thapsigargin down-regulate cell cycle proteins ppRb and cyclin D1 in differentiated neuroblastoma cells. In contrast, protein levels of p27, a cyclin dependent kinase inhibitor, are increased after induction of ER-stress using tunicamycin. These data suggest that activation of the UPR affects cell cycle protein expression in neurons during neurodegeneration in AD.

Alzheimer Disease↗
PubMed2006

Induction of apoptosis and down regulation of cell cycle proteins in mantle cell lymphoma by flavopiridol treatment.

Typical mantle cell lymphoma (MCL) is a distinct B-cell non-Hodgkin's lymphoma associated with over-expression of cyclin D1 related to translocation between the IgH and BCL-1 genes. Due to the important functional interaction between cyclin D1 and cyclin dependent kinases, cyclin dependent kinase inhibitors such as flavopiridol are under consideration for treatment of patients with MCL. The present study investigated the in vitro effects of flavopiridol on the MCL cell line (JeKo-1). Flavopiridol at a dose of 10nmol/L induced apoptosis by 6h of treatment as noted by flow cytometric analysis, morphologic examination and Western blotting. The cleavage of procaspase-3 and PARP and the decrease of flavopiridol-induced apoptosis by pan-caspase inhibition suggested that the caspase pathway serves an important role in the apoptotic process. Furthermore, MCL cells exposed to flavopiridol showed down regulation of key cell cycle proteins acting at the restriction point control between the G1 and S phases. The onset of flavopiridol-induced apoptosis also coincided with the down regulation of Mcl-1, anti-apoptotic protein. Collectively, our data indicates that flavopiridol may have significant therapeutic potential in the context of MCL.

Apoptosis↗
PubMed2001

Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and mammalian target of rapamycin.

Cell cycle progression represents a key event in vascular proliferative diseases, one that depends on an increased rate of protein synthesis. An increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity is associated with vascular smooth muscle cell proliferation, and rapamycin, which blocks the activity of the mammalian target of rapamycin, inhibits this proliferation in vitro and in vivo. We hypothesized that these 2 molecules converge on a critical pathway of translational regulation that is essential for successful upregulation of cell cycle-regulatory proteins in activated smooth muscle cells. p70(S6) kinase, a target of PI 3-kinase and the mammalian target of rapamycin, was rapidly activated on growth factor stimulation of quiescent coronary artery smooth muscle cells and after balloon injury of rat carotid arteries. The translational repressor protein 4E-binding protein 1 was similarly hyperphosphorylated under these conditions. These events were associated with increases in the protein levels of cyclin B1, cyclin D1, cyclin E, cyclin-dependent kinase 1, cyclin-dependent kinase 2, proliferating cell nuclear antigen, and p21(Cip1) in vivo and in vitro, whereas inhibition of the PI 3-kinase signaling pathway with either rapamycin or wortmannin blocked the upregulation of these cell cycle proteins, but not mRNA, and arrested the cells in vitro before S phase. In contrast to findings in other cell types, growth factor- or balloon injury-induced downregulation of the cell cycle inhibitor p27(Kip1) was not affected by rapamycin treatment. These data suggest that cell cycle progression in vascular cells in vitro and in vivo depends on the integrity of the PI 3-kinase signaling pathway in allowing posttranscriptional accumulation of cell cycle proteins.

Angioplasty, Balloon↗
PubMed2004

Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4.

Endothelial cell proliferation in response to VEGF plays an important role in physiological and pathological angiogenesis. The role of PDE2 and PDE4 in VEGF-induced proliferation in HUVEC was investigated: 1) VEGF increased cAMP-hydrolytic activity by up-regulating the expression of PDE2 and PDE4 isozymes; 2) VEGF increased progression in cell cycle with an increase in p42/p44 MAP kinase, cyclin A and cyclin D1 expressions and with a decrease in p21 waf1/cip1 and p27 kip1 expressions; 3) EHNA (20 micro M), a selective PDE2 inhibitor, RP73401 (10 micro M), a selective PDE4 inhibitor blocked the VEGF-induced increase in p42/p44 MAP kinase expression; 4) RP73401, but not EHNA, blocked the VEGF-induced increase in cyclin A and decrease in p27 kip1 expressions; 5) EHNA, contrary to RP73401, enhanced the VEGF-induced increase of cyclin A and decrease of p27 kip1. 6) EHNA and RP73401 together blocked the VEGF-induced increase in cyclin D1 and decrease in p21 waf1/cip1 expressions; 7) Inhibition of VEGF-upregulated PDE2 and PDE4 reversed the VEGF-induced alterations in cell cycle protein expression, bringing back endothelial cells to a non-proliferating status. Consequently, PDE2 and PDE4 inhibitions were able to inhibit VEGF-induced endothelial cell proliferation by restoring cell cycle key protein expression, and might thus be useful in excessive angiogenesis. Furthermore, the differences between PDE2 and PDE4 effects may suggest compartmentalized effects.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Characterization of keratin and cell cycle protein expression in cell lines from squamous intraepithelial lesions progressing towards a malignant phenotype.

Two cell lines derived from vaginal intraepithelial neoplasias (VAINs) expressing human papillomavirus (HPV) 33 (VAIN I, UT-DEC-1) and 16 (VAIN II, UT-DEC-2) E6-E7 mRNA were studied in organotypic culture for their keratins and cell cycle regulatory proteins in relation to replicative aging. Early-passage UT-DEC-1 and UT-DEC-2 cells reproduced epithelial patterns consistent with VAIN. Cells from later passages resembled full-thickness intraepithelial neoplasia (UT-DEC-1) and microinvasive cancer (UT-DEC-2). The morphological changes were compatible with these cell lines' ability for anchorage-independent growth at later passages. Simple epithelial keratins were aberrantly expressed in both cell lines. K18 (absent in normal vaginal keratinocytes) and K17 expression increased in UT-DEC-1 and UT-DEC-2 cells at late passages. No marked differences in expression of p53 (wild type in both cell lines), mdm-2 or PCNA were detected in parallel with progression. The expression of p21WAF1/cip1 localized mostly to the upper half of the epithelium at early passage and was more intense in the HPV 16-positive UT-DEC-2 cell line expressing K10. In Northern blot analyses, the transcription pattern of the HPV 33 E6-E7 of the UT-DEC-1 cell line changed during later passages, whereas that of the HPV 16 E6-E7 of the UT-DEC-2 cell line remained unaltered. The present characterization of the phenotype of these cell lines derived from natural squamous intraepithelial lesions shows an association between simple epithelial-type keratin expression and progressive changes in growth and morphology, but fails to demonstrate consistent changes in the expression of cell cycle regulatory proteins studied in parallel with progression.

Carcinoma in Situ↗
PubMed2005

Expression of cyclin A1 and cell cycle proteins in hematopoietic cells and acute myeloid leukemia and links to patient outcome.

Abnormal expression of several key regulators essential for G1/S transitions has been implicated in tumorigenesis. A critical role of cyclin A1 in the development of acute myeloid leukemia (AML) has previously been demonstrated in transgenic mice. Our present study focused on the expression and prognostic significance of cyclin A1 and a panel of cell cycle regulatory proteins including cyclin A2, cyclin B1, cyclin E, CDK1, CDK2, p21 and p27 in bone marrow samples from 40 patients with AML. Freshly isolated CD34+ hematopoietic cells and bone marrow samples from 10 healthy donors were also assessed for cell type- and subcellular-specific expression of the cell cycle regulatory proteins. The level of cyclin A1 expression was the only factor that showed a significant correlation with patient outcome. In log-rank test stratified by levels of cyclin A1 expression, patients with high levels of cyclin A1 had significantly worse overall survival (OS) (P = 0.012) compared to those with low levels. Further, patients with high levels of cyclin A1 had significantly lower disease-free survival (DFS) (P = 0.028). Multivariate analysis indicated that cyclin A1 protein expression was an independent prognostic factor for predicting DFS (P = 0.035) and OS (P = 0.045). No correlation between cyclin A1 expression and age was found. However, expression of cyclin A2, cyclin B1, cyclin E, CDK1, CDK2, p21 and p27 did not show prognostic significance in these AML patients.

Acute Disease↗
PubMed1999

Cell cycle proteins and the development of oral squamous cell carcinoma.

Expression of cell cycle regulatory proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of the cell cycle may be altered in the development of oral squamous cell carcinoma. Archived paraffin-embedded specimens (n = 90) from 25 patients with recurrent or persistent lesions were evaluated in immunohistochemically stained sections for cell cycle regulatory proteins p53, Rb, Cyclin D1, p27, and p21. The cell cycle was also evaluated by expression of nuclear protein Ki 67. Sections were graded semiquantitatively using a 0-3 + scale to indicate the percentage of positively stained cells. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty-three of 90 specimens showed positive p53 expression, 11 of which were dysplasias. Eighty-nine of 90 specimens, from all stages of disease, showed positive Rb expression. Twenty-three of 90 specimens showed positive Cyclin D1 expression, typically in the later stages (carcinoma) of a patient's disease. Eighty-four of 90 specimens showed positive p21 expression; while 55 of 90 specimens were positive for p27. In control mucosa, p27 was highly expressed, while Rb and p21 proteins were expressed at relatively low levels; p53 and Cyclin D1 proteins were largely absent. Generally, staining of p53, Rb, p21, and Ki 67 increased with time in serial biopsies, while p27 showed decreased staining with disease progression. These data show that cell cycle regulatory proteins are altered in both premalignant and malignant disease, and that protein phenotypes are heterogeneous. P53 expression is seen early, and Cyclin D1 expression is seen late in the development of oral premalignant and malignant disease. Expression of p53, Rb, p21 and Ki67 increased, while p27 decreased, with disease progression.

Adult↗
PubMed2004

Oxidized low-density lipoprotein retards the growth of proliferating cells by inhibiting nuclear translocation of cell cycle proteins.

OBJECTIVE: Our study tested the hypothesis that the mitogenic effect of oxidized low-density lipoprotein (oxLDL) on vascular cells may be further enhanced by the presence of cytokines and growth factors known to be present in the atherosclerotic environment. METHODS AND RESULTS: Quiescent fibroblasts and vascular smooth muscle cells were treated with 10 or 50 microg/mL minimally-oxidized LDL in combination with serum for 24 or 48 hours. Surprisingly, these cells showed inhibited release from growth arrest and a significant reduction in the number of cells completing the cell cycle when compared with cells treated with serum alone. This was not due to an induction of apoptosis. The antiproliferative effects were not closely associated with changes in the expression of cell cycle proteins. Instead, oxLDL inhibited the translocation of cell cycle proteins cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, Cdk 4, Cyclin A, Cyclin B1, Cyclin D1, and proliferative cell nuclear antigen (PCNA) into the nucleus, as compared with separate treatments with serum alone. Kinase activation associated with specific cell cycle proteins was also inhibited by oxLDL. CONCLUSIONS: oxLDL, in the presence of serum, has a surprising inhibitory effect on cell proliferation that occurs through an inhibition of import of cell cycle proteins into the cell nucleus.

Active Transport, Cell Nucleus↗
PubMed2005

NMR and mass spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6 with cell differentiation proteins MyoD and ID-2.

Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.

Animals↗
PubMed2001

Expression of cell cycle proteins in ovarian carcinoma cells in serous effusions-biological and prognostic implications.

OBJECTIVE: The aim of this study was to investigate the expression of cell cycle proteins in ovarian carcinoma cells in serous effusions and respective solid tumors. METHODS: Fifty-five malignant effusions and 38 tumors (20 primary, 18 metastatic) were immunohistochemically stained for cyclin A, p27(kip1), and Ki-67. Staining extent (0-100% cells) and intensity (0-3 scale) were scored. Cyclin A and p27(kip1) expression was additionally studied in 29 malignant effusions using immunoblotting. Immunohistochemistry results in effusions were evaluated for possible association with clinicopathologic parameters. RESULTS: Nuclear immunoreactivity for all markers was detected on carcinoma cells in the majority of effusions using immunohistochemistry. Similarly, immunoblotting showed the presence of cyclin A and p27(kip1) in 29/29 and 25/29 specimens, respectively. Intense (3) immunoreactivity for Ki-67 was detected more often in peritoneal effusions, compared with those of pleural location (P = 0.036). Staining in primary and metastatic lesions was generally comparable to that of tumor cells in effusions. Staining for p27(kip1) was more diffuse in effusion specimens obtained prior to the institution of chemotherapy (P = 0.042). In an analysis of all effusions, an association was observed between the number of cells that were immunoreactive for Ki-67, cyclin A, and p27(kip1) (cyclin A-Ki-67: P = 0.008; p27(kip1)-Ki-67: P = 0.019; cyclin A-p27(kip1): P = 0.032). In survival analysis, the presence of more diffuse (P = 0.042) and intense (P = 0.019) staining for cyclin A correlated with prolonged overall survival. CONCLUSIONS: The expression of the studied cell cycle markers does not differ markedly between ovarian carcinoma cells in the pleural and peritoneal cavity, supporting our previous studies of several metastasis-associated molecules. The presence of cyclin-A-positive cell populations is associated with a more favorable disease outcome, possibly due to the targeting of proliferating cells by chemotherapeutic agents. However, the decline in the fraction of p27(kip1)-positive cells in posttreatment specimens may point to additional mechanisms involved in this selection.

Ascitic Fluid↗
PubMed1998

Interaction between high glucose and TGF-beta in cell cycle protein regulations in MDCK cells.

Transforming growth factor-beta (TGF-beta) may mediate high glucose effects in renal cells. Thus, Madin-Darby canine kidney cells were studied for the modulation of cell cycle regulatory proteins by high glucose (27.5 mM) and TGF-beta1. We showed that unlike other renal cells, TGF-beta1 mRNA and its bioactivity were not induced by high-glucose culture. Furthermore, high glucose per se increased cellular proliferation without alterations in cell size. High glucose also increased the percentage of cells in the G2/M phase while decreasing cells in the G0/G1 phase of the cell cycle. In contrast, TGF-beta1 dose dependently (1 to 4 ng/ml) decreased cellular mitogenesis while increasing hypertrophy in the cells, especially in the presence of high glucose. TGF-beta1 also increased the percentage of cells arrested in the G0/G1 phase while decreasing cells in the G2/M phase of the cell cycle. Regarding two of the cell cycle regulatory proteins, high glucose increased cdc2 kinase activity and retinoblastoma protein (pRb) phosphorylation. In contrast, TGF-beta1 decreased cdc2 kinase activity and pRb phosphorylation, especially in the presence of high glucose. Additionally, glucose dose dependently (5.5, 16.5, 27.5, and 38.5 mM) increased type I and II TGF-beta receptor protein expression. In conclusion, changes in cdc2 kinase activity and pRb phosphorylation were correlated with high glucose and TGF-beta1-induced growth effects in a cell cycle-dependent manner in the Madin-Darby canine kidney cells. Furthermore, high glucose may potentiate TGF-beta1-induced effects by enhancing TGF-beta receptor protein expression.

Animals↗
PubMed1996

Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells.

Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.

CDC2-CDC28 Kinases↗
PubMed1997

Identification of DNA replication and cell cycle proteins that interact with PCNA.

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.

Animals↗
PubMed2005

Cell cycle protein profile of the hepatic stellate cells(HSCs)in dimethylnitrosamine-induced rat hepatic fibrosis.

Cell cycle regulating proteins are known to have close relation with the proliferation of the mammalian cells. In injured liver, the number of HSCs is increased from proliferation. However, the expression of cell cycle proteins of HSCs during proliferation remains unevaluated. Therefore, cell cycle protein profiles of HSCs were studied in dimethyl-nitrosamine (DMN)-induced rat liver fibrosis model. Sprague-Dawley rats were intraperitoneally injected of DMN and the animals were sacrificed every week up to 4 weeks. HSCs were separated and the number of the cells in S phase was counted to evaluate the cell proliferation by flow cytometry. The expression of cyclin A, cyclin B, cyclin D1, cdk2, cdk4, cdc2, proliferating cell nuclear antigen (PCNA), p21(Cip/WAF1), and p27 was examined with immunoblotting analysis. Portion of S-phase cells peaked 7days after DMN injection. At that time, cyclin A, and PCNA showed significant increase in HSCs compared to untreated HSCs (114% and 116%, respectively, P<0.001). p21(Cip/WAF1) was decreased significantly in DMN-treated HSCs compared to control cells (88%, P<0.001). The increase of cyclin A, and PCNA and the decrease of p21(Cip/WAF1) seem to play important roles in the proliferation of HSCs during the early period of DMN treatment.

Animals↗