Modulate Cell Cycle Progression Research Articles

PDK1-dependent activation of atypical PKC leads to degradation of the p21 tumour modifier protein.

p21(WAF1/CIP1) Contributes to positive and negative growth control on multiple levels. We previously mapped phosphorylation sites within the C-terminal domain of p21 that regulate proliferating cell nuclear antigen binding. In the current study, a kinase has been fractionated from mammalian cells that stoichiometrically phosphorylates p21 at the Ser146 site, and the enzyme has been identified as an insulin-responsive atypical protein kinase C (aPKC). Expression of PKCzeta or activation of the endogenous kinase by 3-phosphoinositide dependent protein kinase-1 (PDK1) decreased the half-life of p21. Conversely, dnPKCzeta or dnPDK1 increased p21 protein half-life, and a PDK1-dependent increase in the rate of p21 degradation was mediated by aPKC. Insulin stimulation gave a biphasic response with a rapid transient decrease in p21 protein levels during the initial signalling phase that was dependent on phosphatidylinositol 3- kinase, PKC and proteasome activity. Thus, aPKC provides a physiological signal for the degradation of p21. The rapid degradation of p21 protein during the signalling phase of insulin stimulation identifies a novel link between energy metabolism and a key modulator of cell cycle progression.

Cyclin D1 and cyclin E are co-localized with cyclo-oxygenase 2 (COX-2) in pyramidal neurons in Alzheimer disease temporal cortex.

Regular use of non-steroidal anti-inflammatory drugs (NSAIDs) seems to reduce the progression of several diseases, including colon cancer, lung cancer, breast cancer and Alzheimer disease (AD). Several studies have shown that NSAIDs can modulate cell cycle progression, especially in the G0/G1 phase. The main target of most NSAIDs is the enzyme cyclo-oxygenase (COX), which occurs in 2 isoforms, COX-1 and COX-2. In AD and non-demented control brain, COX-2 is expressed in neuronal cells. In this study the expression of COX-2, cyclin D1, and cyclin E was investigated at the immunohistochemical level in AD and non-demented control temporal cortex. COX-2, cyclin D1, and cyclin E expression was detected in pyramidal neurons in both AD and control patients. The number of COX-2-immunoreactive neurons positively correlated with the number of cyclin E- and cyclin D1-immunoreactive neurons. Moreover, immunostaining of sequential tissue sections and double immunofluorescence labeling revealed co-expression of COX-2 and cyclin D1 and E in neuronal cells. In addition, an inverse correlation was observed between the neuronal expression of COX-2 and cyclin E and the Braak score for amyloid beta deposits. Our findings suggest a relationship between the neuronal expression of COX-2 and cell cycle markers, which may be involved early in AD pathology.

Calcitriol (1,25-Dihydroxycholecalciferol) Potentiates Activity of Mitoxantrone/Dexamethasone in an Androgen Independent Prostate Cancer Model

Mitoxantrone combined with glucocorticoids is widely used for androgen independent prostate cancer. It is well tolerated, reduces prostate specific antigen, diminishes pain and improves quality of life. Calcitriol (1,25-dihydroxycholecalciferol) inhibits proliferation, modulates cell cycle progression, induces apoptosis and potentiates the cytotoxic effects of a number of agents. Glucocorticoids potentiate the antitumor effects of calcitriol and blunt calcitriol induced hypercalcemia. Therefore, we investigated the effect of calcitriol on the antitumor efficacy of mitoxantrone and dexamethasone or mitoxantrone/dexamethasone in the PC-3 androgen independent prostate cancer model. We treated PC-3 cells in vitro with various concentrations of mitoxantrone/dexamethasone with and without calcitriol, and assessed growth inhibition by crystal violet assays. We similarly treated mice bearing PC-3 xenografts and performed excision clonogenic assays and tumor outgrowth studies to assess antitumor activity. Calcitriol significantly increased mitoxantrone/dexamethasone mediated growth inhibition in PC-3 cells (p <0.05). Median dose effect analysis indicated that calcitriol is synergistic with mitoxantrone. Adding calcitriol to mitoxantrone/dexamethasone significantly reduced the surviving fraction per gm. tumor compared with mitoxantrone/dexamethasone or untreated controls (p <0.03). Calcitriol plus mitoxantrone/dexamethasone also caused significantly greater tumor regression in PC-3 xenografts compared with treatment with mitoxantrone/dexamethasone or untreated controls (p <0.02). These preclinical data demonstrate that calcitriol increases the antitumor activity of mitoxantrone/dexamethasone in the PC-3 model system. This combination may be efficacious for prostate cancer.

Cell cycle regulation: Mechanisms and therapeutics

Cyclin-dependent kinase 4 (Cdk4) is an important regulator of G1/S cell cycle progression of mammalian cells. To understand the role of Cdk4in vivo we generated strains of mice that either lack Cdk4 or express an activated form of this enzyme (Cdk4R24C), which cannot interact with the cycli kinase inhibitor, p16INK4A. Cdk4R24C mutations that abolish the interaction of pl6INK4A and CDK predispose humans to familial melanoma. Cdk4-/- mutant mice are viable but express defects in growth, fertility and are diabetic due to loss of insulin-producing islet cells. In contrast, CDK4 (R24C/R24C) mice exhibit growth advantage and their fibroblasts have a shorter cycle with high proliferative rates. The Cdk4 (R24C/R24C) fibroblasts fail to undergo senescence and are capable long-term growth in culture. Moreover, approximate 80% of Cdk4 (R24C/R24C) mice developed spontaneous tumors of varying cellular origin such as breast, pituitary, brain, skin, pancreas, liver, lung, testes, ovarian and hematopoietic tumors by 12 month. In addition, cdk4(R24C/R24C) mice are highly susceptible to TPA or DMBA carcinogen-induced tumors and Cdk4(R24C/R24C) mice developed skin tumors with average latency of 10–12 weeks. In contrast, wild type mice had a reduced frequency and latency of tumor formation. These studies indicated that the cdk4(R24C) mutation could exacerbate de-regulated Ras-pathways and accelerate tumorigenesisIn addition, Cdk4 (R24C/R24C) mice exhibit severe hyperplasia of the immune system and are highly susceptible to the development of hematopoietic malignancies. These results suggest that Cdk4 is an important modulator of cell cycle progression and abnormalities associated with this gene results in the development of abnormalities in multiple organs including the hematopoietic compartment.

Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest.

The intermediate filament cytoskeleton is composed of keratins in all epithelial cells and imparts mechanical integrity to these cells. However, beyond this shared function, the functional significance of the carefully regulated tissue- and differentiation-specific expression of the large keratin family of cytoskeletal proteins remains unclear. We recently demonstrated that expression of keratin K10 or K16 may regulate the phosphorylation of the retinoblastoma protein (pRb), inhibiting (K10) or stimulating (K16) cell proliferation (J. M. Paramio, M. L. Casanova, C. Segrelles, S. Mittnacht, E. B. Lane, and J. L. Jorcano, Mol. Cell. Biol. 19:3086-3094, 1999). Here we show that keratin K10 function as a negative modulator of cell cycle progression involves changes in the phosphoinositide 3-kinase (PI-3K) signal transduction pathway. Physical interaction of K10 with Akt (protein kinase B [PKB]) and atypical PKCzeta causes sequestration of these kinases within the cytoskeleton and inhibits their intracellular translocation. As a consequence, the expression of K10 impairs the activation of PKB and PKCzeta. We also demonstrate that this inhibition impedes pRb phosphorylation and reduces the expression of cyclins D1 and E. Functional and biochemical data also demonstrate that the interaction between K10 and these kinases involves the non-alpha-helical amino domain of K10 (NTerm). Together, these results suggest new and essential roles for the keratins as modulators of specific signal transduction pathways.

Induction of p53-independent apoptosis by simian virus 40 small t antigen.

Simian virus 40 small t antigen (st) is required for optimal transformation and replication properties of the virus. We find that in certain cell types, such as the human osteosarcoma cell line U2OS, st is capable of inducing apoptosis, as evidenced by a fragmented nuclear morphology and positive terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining of transfected cells. The cell death can be p53 independent, since it also occurs in p53-deficient H1299 cells. Genetic analysis indicates that two specific mutants affect apoptosis induction. One of these (C103S) has been frequently used as a PP2A binding mutant. The second mutant (TR4) lacks the final four amino acids of st, which have been reported to be unimportant for PP2A binding in vitro. However, TR4 unexpectedly fails to bind PP2A in vivo. Furthermore, a long-term colony assay reveals a potent colony inhibition upon st expression, and the behavior of st mutants in this assay reflects the relative frequency of nuclear fragmentation observed in transfections using the same mutants. Notably, either Bcl-2 coexpression or broad caspase inhibitor treatment could restore normal nuclear morphology. Finally, fluorescence-activated cell sorting analysis suggests a correlation between the ability of st to modulate cell cycle progression and apoptosis. Taken together, these observations underscore that st does not always promote proliferation but may, depending on conditions and cell type, effect a cell death response.

Inhibitory Effects of Sodium Butyrate on Proliferation and Invasiveness of Human Glioma Cells

OBJECTIVE Sodium butyrate (SB), a differentiation-inducing agent, has been demonstrated to inhibit cellular proliferation in a number of human cell lines. Its precise mechanisms remain to be clarified, however. We investigated molecular mechanisms of SB-induced growth suppression as well as the effects of SB on the invasiveness of human glioma cells. METHODS Human glioma U87MG and U251MG cells were treated with 1 or 2 mmol/L SB for 48 hours, and the inhibition of cell growth was assessed by spectrophotometric analysis. Cell cycle analysis was carried out by the 5-bromo-2′-deoxyuridine incorporation method, and expression of cell cycle-regulatory proteins was determined by immunoblotting. In addition, invasiveness was assessed using a Transwell chamber (Iwaki, Tokyo, Japan) with extracellular matrix substrate fibronectin or laminin (Iwaki). RESULTS SB treatment resulted in significantly suppressed proliferation of both U87MG and U251MG cells in a dose-dependent manner. It inhibited the G1-S transition, which was associated with increased expression of p21 and cyclin D1 and reduced pRb phosphorylation. Treatment with antisense oligonucleotide for Rb abrogated SB-induced G1 arrest. p21 up-regulation was independent of the p53 status of the glioma cells. SB treatment also inhibited invasiveness on fibronectin and laminin. CONCLUSION Our results indicate that SB may suppress the growth of human glioma cells through modulation of cell cycle progression and also may affect their invasiveness on extracellular matrix substrates, which suggests that SB may be a useful therapeutic agent in treating multiple aspects of malignant gliomas.

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Inhibitory effects of sodium butyrate on proliferation and invasiveness of human glioma cells.

Sodium butyrate (SB), a differentiation-inducing agent, has been demonstrated to inhibit cellular proliferation in a number of human cell lines. Its precise mechanisms remain to be clarified, however. We investigated molecular mechanisms of SB-induced growth suppression as well as the effects of SB on the invasiveness of human glioma cells. Human glioma U87MG and U251MG cells were treated with 1 or 2 mmol/L SB for 48 hours, and the inhibition of cell growth was assessed by spectrophotometric analysis. Cell cycle analysis was carried out by the 5-bromo-2'-deoxyuridine incorporation method, and expression of cell cycle-regulatory proteins was determined by immunoblotting. In addition, invasiveness was assessed using a Transwell chamber (Iwaki, Tokyo, Japan) with extracellular matrix substrate fibronectin or laminin (Iwaki). SB treatment resulted in significantly suppressed proliferation of both U87MG and U251MG cells in a dose-dependent manner. It inhibited the G1-S transition, which was associated with increased expression of p21 and cyclin D1 and reduced pRb phosphorylation. Treatment with antisense oligonucleotide for Rb abrogated SB-induced G1 arrest. p21 up-regulation was independent of the p53 status of the glioma cells. SB treatment also inhibited invasiveness on fibronectin and laminin. Our results indicate that SB may suppress the growth of human glioma cells through modulation of cell cycle progression and also may affect their invasiveness on extracellular matrix substrates, which suggests that SB may be a useful therapeutic agent in treating multiple aspects of malignant gliomas.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that γ-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to γ-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

CAMP Protection of Pancreatic Cancer Cells against Apoptosis Induced by ERK Inhibition

Large increases in cAMP concentration inside the cell are generally growth inhibitory for most cell lines of mesenchymal and epithelial origin. Moreover, recent data suggest a role of cAMP in survival of different cell types. Herein, the ability of forskolin (an adenylyl cyclase activator) and IBMX (3-isobutyl-1-methylxanthine) (a phosphodiesterase inhibitor) to modulate cell cycle progression and survival of human pancreatic cancer cells was evaluated. We showed that forskolin + IBMX inhibited serum-induced ERK activities, Rb hyperphosphorylation, Cdk2 activity, and p27Kip1 downregulation and caused G1 arrest in MIA PaCa-2 cells. Furthermore, forskolin + IBMX protected pancreatic cells against apoptosis induced by prolonged inhibition of ERK activities by preventing Bcl-XL downregulation, activation of caspases 3, 6, 8, and 9, and PARP cleavage and by inducing Bad phosphorylation (ser112). Taken together, our data demonstrate for the first time that cAMP is an inhibitor of cell cycle progression and apoptosis in human pancreatic cancer cells.

Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation.

Ebp1, an ErbB-3 binding protein, inhibits the proliferation and induces the differentiation of human breast cancer cells. The mechanisms of these effects are unknown. Rb, the product of the retinoblastoma gene, is an important modulator of cell cycle progression and cellular differentiation. We report that Rb is a binding target for Ebp1. Ebp1 was localized to both the nucleus and the cytoplasm of logarithmically growing AU565 breast cancer cells and HeLa cells as determined by confocal immunofluorescent microscopy. Ebp1 was present in Rb immunoprecipitates derived from AU565 breast cancer cells. GST-Rb also bound endogenous Ebp1. Using GST-Ebp1 constructs, we determined that the 72 C-terminal amino acids of Ebp1 were sufficient to bind Rb. Dephosphorylation of Ebp1 enhanced the interaction of Ebp1 with Rb. The overexpression of Ebp1 in MCF-7 and AU565 (Rb(+)) cells inhibited the activity of the E2F1 regulated cyclin-E promoter. Ebp1 bound E2F1 indirectly via Rb in lysates of MCF-7 cells. The interaction of Ebp1 with Rb may prove to be an important mechanism of Ebp1 induced changes in cell proliferation and differentiation.

A Human BRCA2 Complex Containing a Structural DNA Binding Component Influences Cell Cycle Progression

Germline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BR CA2-A ssociated F actor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression.

Modulation of apoptosis, proliferation, and p27 expression in a porcine coronary angioplasty model

Smooth muscle cell (SMC) proliferation is a prominent feature of intimal hyperplasia after percutaneous coronary interventions. p27 is a critical regulator of cell proliferation. Our aims were to analyze the time course of p27 expression, Ki67 proliferative index, and apoptosis after angioplasty in the porcine coronary artery. We also investigated the effects of rapamycin — an antiproliferative drug — on these events. The expression of p27 and Ki67, and apoptosis were determined in porcine coronary arteries harvested at timed intervals from 1 h to 28 days after angioplasty. A gradual increase in p27 expression was observed from 7 to 28 days. Ki67 expression peaked by 7–14 days after angioplasty. By 21–28 days, Ki67 expression decreased, while p27 reached maximal levels. An early apoptotic response was found by 6 h, followed by a gradual return to baseline. Rapamycin induced a reduction in Ki67 proliferative index (2±0.5%) and an increase in apoptosis (7±1%) versus untreated animals at the 28-day time point (5±1 and 1±0.5%, respectively; P<0.05). In summary, coronary angioplasty induced a rapid apoptotic response, followed by a progressive increase in proliferation. Later on, as p27 expression increased in the vessel wall, cell proliferation decreased. Modulation of cell cycle progression may be a useful therapeutic approach in the treatment of intimal hyperplasia after angioplasty.

Functional Implication of Human Serine/Threonine Kinase, hAIK, in Cell Cycle Progression

Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines. The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm). During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes. In addition, overexpression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive. Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures.

Functional implication of human serine/threonine kinase, hAIK, in cell cycle progression

Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines. The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm). During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes. In addition, overexpression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive. Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures.

Sustained activation of Erk1/2 MAPK and cell growth suppression by the insert-negative, but not the insert-positive isoform of the human calcitonin receptor.

Recently we reported that calcitonin (CT) induces growth arrest at the G2 stage of the cell cycle in HEK-293 cell lines expressing the most abundant, insert-negative, isoform of the human CT receptor (insert -ve hCTR). The present study investigates the involvement of the MAPK signalling pathway in the anti-proliferative actions of CT and compares the activity of an isoform of the hCTR that contains a 16 amino acid insert in the first putative intracellular loop (insert +ve hCTR). Comparison of HEK-293 cells stably transfected with the insert -ve or the insert +ve hCTR, showed that accumulation of cAMP and intracellular free calcium in response to CT were specific for the insert -ve receptor isoform. However, a novel acidification of the extracellular medium was mediated by both isoforms. Treatment with CT of cells expressing the insert -ve hCTR, caused a decrease in cell growth associated with an induction of p21(WAF1/CIP1). Analysis by fluorescence-activated cell scanning showed that growth inhibition was associated with an accumulation of cells in G2. CT treatment of cells expressing the insert -ve, but not insert +ve hCTR, induced the phosphorylation of Erk1/2 MAPK, which persisted for at least 72 h. Treatment of cells expressing the insert -ve hCTR with the MAPK kinase (MEK) inhibitor, PD-98059, inhibited the phosphorylation of Erk1/2 and abrogated the growth inhibitory effects of salmon CT, the accumulation of cells in G2, and the associated induction of p21(WAF1/CIP1). These data suggest that activation of Erk1/2 are downstream effectors of the insert -ve hCTR in modulating cell cycle progression.

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro.

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10(-5) M, 10(-6) M or 10(-7) M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P < 0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10(-5) M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G0/G1 phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P < 0.05), 13.9% at 24 h (P < 0.001), and 76.5% at 48 h (P < 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P < 0.005) and 12 h (P < 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G1/S transition by prolonging G2/M and through decreasing the DNA synthesis rate.

Modulation of cell-cycle progression after irradition of human carcinoma cells in vitro

Modulation of cell-cycle progression after irradition of human carcinoma cells in vitro

CTGF modulates cell cycle progression in cAMP‐arrested NRK fibroblasts

Connective tissue growth factor (CTGF) is a 38-kDa cysteine-rich peptide, whose synthesis and secretion are selectively induced by transforming growth factor beta (TGF-β) in connective tissue cells. Previous studies have demonstrated that CTGF functions as a downstream mediator of TGF-β mitogenic activity, where it controls cell cycle progression through late G1 and S-phase entry of NRK fibroblast suspension cultures. Here we report that CTGF induces this S-phase entry by upregulating cyclin A levels. The molecular mechanism for cyclin A induction appears to be via reduction of p27Kip1 levels, which results in hyperphosphorylation of pRb and release of E2F, a known modulator of cyclin A gene transcription. These data indicate that CTGF acts as a mediator of TGF-β–induced fibroblast proliferation in suspension cultures by regulating cdk activities. J. Cell. Physiol. 182:119–126, 2000. © 2000 Wiley-Liss, Inc.