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Results for “Resting Phase, Cell Cycle”

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PubMed2004

Phorbol myristate induces apoptosis of taxol-resistant sarcoma cells in vitro.

Taxol was found to induce polyploidization and apoptosis in cultured methylcholanthrene-induced sarcoma cells (Meth-A cells), but some of the cells in G1 phase were not affected. We refer to these cells as taxol-resistant cells. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) regulator, was used to test the taxol-resistant cells. Many of the taxol-resistant cells disappeared after treatment with taxol in the presence of PMA. To explore the mechanism of this effect, we employed flow cytometry to determine levels of p53, p21, bcl-2 and caspase proteins in the taxol-resistant cells, and found that the expression of the bcl-2 protein was markedly decreased and the expression of the caspase protein markedly increased after treatment with taxol in the presence of PMA. These findings suggest that PMA enhances the sensitivity of taxol-resistant cells to taxol, and taxol treatment in the presence of PMA induces the apoptosis of taxol-resistant cells by inhibiting the expression of the bcl-2 protein and increasing the expression of the caspase protein.

Animals↗

PubMed1993

Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues.

Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.

Animals↗

PubMed2005

NK cells inhibit T cell proliferation via p21-mediated cell cycle arrest.

NK cells have been shown to influence immune responses via direct interaction with cells of the adaptive immune system, such as dendritic cells, B cells, and T cells. A role for NK cells in down-regulation of T cell responses has been implicated in several studies; however, the underlying mechanism of this suppression has remained elusive. In this study we show that dark Agouti rat NK cells inhibit syngeneic T cell proliferation via up-regulation of the cell cycle inhibitor, p21, resulting in a G0/G1 stage cell cycle arrest. The inhibition is cell-cell contact dependent, reversible, and Ag nonspecific. Interestingly, NK cells do not inhibit IL-2 secretion or IL-2R up-regulation and do not induce T cell death. Thus, our results show that NK cells do not affect early T cell activation events, but specifically inhibit T cell proliferation by direct interaction with T cells. Our findings suggest that NK cells may play an important role in maintaining immune homeostasis by directly regulating clonal expansion of activated T cells. This novel mechanism of T cell regulation by NK cells provides insight into NK cell-mediated regulation of adaptive immunity and provides a mechanistic link between NK cell function and suppression of T cell responses.

Animals↗

PubMed2006

Lamina-associated polypeptide 2alpha regulates cell cycle progression and differentiation via the retinoblastoma-E2F pathway.

Lamina-associated polypeptide (LAP) 2alpha is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2alpha in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2alpha by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2alpha-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2alpha COOH terminus directly bound Rb, and overexpressed LAP2alpha inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2alpha associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2alpha in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2alpha on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.

3T3 Cells↗

PubMed1991

[Cytogenetic adaptive response induced by low dose radiation].

Human and rabbit peripheral blood lymphocytes were irradiated in vitro with 1.5 Gy x-rays after exposure to 0.01 Gy of x-rays (dose rate: 0.01 Gy/min). The frequency of chromosome aberration in the cells pre-exposed to 0.01 Gy of x-rays at Go, G1, S and G2 phases of cell cycle respectively was markedly lower than that in the cells irradiated with 1.5 Gy alone (P less than 0.01). This adaptive response would last for 3 cell-cycles, beyond which the damage of the chromosome following the second large dose occurred with the same magnitude as that without a previous conditioning exposure. However, when the low dose was repeated after the third cell cycle the adaptive response could be revived. Meanwhile, it was found that the adaptive response could be also induced in vivo in the bone marrow cells and germ cells of mice by low dose irradiation. In addition, the effect of dose rate of the first irradiation on the induced adaptive response was also studied.

Adaptation, Physiological↗

PubMed1992

Internucleosomal DNA fragmentation during phorbol ester-induced monocytic differentiation and G0/G1 arrest.

The treatment of human myeloid leukemia cell lines with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), is associated with loss of proliferative capacity and induction of monocytic differentiation. The present results demonstrate that treatment of asynchronous human U-937 leukemia cells with 10 nM TPA is also associated with oligonucleosomal DNA cleavage. This pattern of DNA fragmentation, which is observed in programmed cell death, was detectable in populations of TPA-treated cells that had entered a nonproliferative G0/G1 phase. Similar findings were obtained after TPA treatment of a synchronous population of G1 cells. These cells progressed through S and G2/M phases before undergoing internucleosomal DNA cleavage during G0/G1 arrest. These G0/G1 cells displayed characteristics of monocytic differentiation, including down-regulation of c-myc expression and induction of c-fms transcripts. DNA fragmentation was also studied in cells treated with 5 nM TPA for 48 h and then monitored in drug-free long-term culture. Endonucleolytic cleavage was similarly observed in the differentiated G0/G1 population. However, longer periods of culture were associated with a decrease in DNA fragmentation to undetectable levels. This effect was followed by retrodifferentiation and reentry of cells into cycle. Taken together, these findings demonstrate that internucleosomal DNA fragmentation occurs during induction of monocytic differentiation, and that both of these events are detectable in G0/G1 cells.

Cell Differentiation↗

PubMed2000

Cyclin A down-regulation in TGFbeta1-arrested follicular lymphoma cells.

Transforming growth factor beta1 (TGFbeta1) induces growth arrest in many cell types, including B lymphocytes. We examined the effect of TGF on cell cycle progression of a non-Hodgkin lymphoma cell line of follicular lymphoma subtype (FL). After 48 h of TGFbeta1 (10 ng/ml) treatment, a significantly increased number of DoHH2 cells was retained in G(0)/G(1) phase. We examined the level of cell cycle components, cyclins, cyclin-dependent kinases (cdk), and their inhibitors. We found that the expression of cyclin A and p21(WAF1) molecules was primarily modulated by TGFbeta1 treatment while the expression of other regulatory components, like cyclins D, cyclin E, cdk2, cdk4, and cdk6 or p15(INK4B), p16(INK4A), and p27(KIP1) was not significantly affected. We further examined expression and activity of CREB/ATF family members to examine their roles in cyclin A inhibition. The binding activity of CREB-1 and ATF-2 to the CRE region of the cyclin A promoter was almost completely abolished due to the treatment. The total level of CREB-1, ATF-2, and ATF-3 was notably reduced. Moreover, CREB-1 was dephosphorylated due to the treatment as revealed by immunoblotting. We assume that down-regulation of cyclin A was mediated by the absence of CREB/ATF activation dimers. The profound effect on the ATF family of transcription factors indicates the complexity of TGFbeta1 action on FL B malignant cells.

Cell Cycle↗

PubMed2004

m-Calpain implication in cell cycle during muscle precursor cell activation.

Milli-calpain, a member of the ubiquitous cysteine protease family, is known to control late events of cell-cell fusion in skeletal muscle tissue through its involvement in cell membrane and cytoskeleton component reorganization. In this report, we describe the characterization of m-calpain compartmentalization and activation during the initial steps of muscle precursor cell recruitment and differentiation. By immunofluorescence analysis, we show that m-calpain is present throughout the cell cycle in the nucleus of proliferating myoblast C2 cells. However, when myoblasts enter a quiescent/G0 stage, m-calpain staining is detected only in the cytoplasm. Moreover, comparison of healthy and injured muscle shows distinct m-calpain localization in satellite stem cells. Indeed, m-calpain is not found in quiescent satellite cells, but following muscle injury, when satellite cells start to proliferate, m-calpain appears in the nucleus. To determine the implication of m-calpain during the cell cycle progression, quiescent myoblasts were forced to re-enter the cell cycle in the presence or not of the specific calpain inhibitor MDL 28170. We demonstrate that this calpain inhibitor blocks the cell cycle, prevents accumulation of MyoD in the G1 phase and enhances Myf5 expression. These data support an important new role for m-calpain in the control of muscle precursor cell activation and thus suggest its possible implication during the initial events of muscle regeneration.

Animals↗

PubMed1999

Chromosomes exhibit preferential positioning in nuclei of quiescent human cells.

The relative spatial positioning of chromosomes 7, 8, 16, X and Y was examined in nuclei of quiescent (noncycling) diploid and triploid human fibroblasts using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes and digital imaging. In quiescent diploid cells, interhomolog distances and chromosome homolog position maps revealed a nonrandom, preferential topology for chromosomes 7, 8 and 16, whereas chromosome X approximated a more random distribution. Variations in the orientation of nuclei on the culture substratum tended to hinder detection of an ordered chromosome topology at interphase by biasing homolog position maps towards random distributions. Using two chromosome X homologs as reference points in triploid cells (karyotype = 69, XXY), the intranuclear location of chromosome Y was found to be predictable within remarkably narrow spatial limits. Dual-FISH with various combinations of chromosome-specific DNA probes and contrasting fluorochromes was used to identify adjacent chromosomes in mitotic rosettes and test whether they are similarly positioned in interphase nuclei. From among the combinations tested, chromosomes 8 and 11 were found to be closely apposed in most mitotic rosettes and interphase nuclei. Overall, results suggest the existence of an ordered interphase chromosome topology in quiescent human cells in which at least some chromosome homologs exhibit a preferred relative intranuclear location that may correspond to the observed spatial order of chromosomes in rosettes of mitotic cells.

Cell Cycle↗

PubMed1999

Effects of butyrate and propionate on the adhesion, growth, cell cycle kinetics, and protein synthesis of cultured human gingival fibroblasts.

BACKGROUND: Various periodontal and root canal pathogens, such as the Bacteroides species, can produce significant amounts of short chain fatty acids (SCFA). The roles of SCFA in the pathogenesis of periodontal disease are still not fully understood. METHODS: We therefore investigated 2 main SCFA, butyrate and propionate, on the functional behavior of cultured human gingival fibroblasts (GF) such as cell growth, protein synthesis, cell adhesion capacity, and cell cycle progression. RESULTS: Butyrate and propionate inhibited the growth of healthy (HGF) and inflamed gingival fibroblasts (IGF) in a dose dependent manner. At concentrations of 4, 8, and 16 mM, butyrate suppressed the cell growth by 11 to 58%, 16 to 60%, and 50 to 71%, respectively. The response of cultured gingival fibroblasts to SCFA showed individual differences. Morphologically, GF became larger and more flattened in appearance following exposure to butyrate (>8 mM) and propionate (>24 mM) for 5 days. Inhibitory effects of butyrate (>2 mM) and propionate (>8 mM) on the growth of GF were due possibly to their inhibition of cell-cycle progression. At concentrations of 2 and 8 mM, butyrate led to G0/G1 arrest. Elevation of the exposure concentration to 8 to 24 mM further result in G2/M phase arrest of GF. On the other hand, propionate, at concentrations ranging from 4 to 24 mM, led to G0/G1 arrest. Butyrate (>2 mM) inhibited the proline-rich protein synthesis of GF. At concentrations of 4, 8, 16, and 24 mM, butyrate inhibited the protein synthesis of HGF-1 by 42%, 43%, 51%, and 54%, respectively. In all strains of cultured GF, the suppressive effect of propionate is less than that of butyrate. At concentration range of 4 to 24 mM, propionate suppressed the protein synthesis of HGF-1 by 23 to 43%. However, both butyrate and propionate (4 to 48 mM) exerted little effects on the adhesion of GF to type I collagen within 3 hours of incubation. CONCLUSIONS: These results suggested that SCFA released by pathogenic microorganisms can contribute to the gingival tissue dysfunction and breakdown through their actions on specific biological functions of GF.

Butyrates↗

PubMedDate not supplied

Sodium phenylacetate (NaPa) induces modifications of the proliferation, the adhesion and the cell cycle of tumoral epithelial breast cells.

Sodium phenylacetate (NaPa), a physiological product of phenylalanine metabolism, present in micromolar concentrations in human plasma, has been shown to induce in vivo and in vitro cytostatic antiproliferative effects at millimolar concentrations. Cadherin molecules are powerful invasion suppressor molecules and the reduction of E-cadherin expression plays an important role in the invasion and metastasis of human breast cancer. In this study, we demonstrated, on one hand, that NaPa stimulated aggregation by increasing the expression of E-cadherin at the surface of breast cancer MCF-7ras cells transformed by Ha-ras oncogene and inhibited its expression in MCF-7 cells. We demonstrated that NaPa increased the formation of MCF-7ras cell aggregates and did not alter the formation of MCF-7 cell aggregates. By Northern blot, we demonstrated that the E-cadherin expression was not regulated at the transcriptional level. On the other hand, we analyzed the cell cycle of these 2 cell lines after NaPa treatment and showed that NaPa induced arrest at the G1/S phase in both MCF-7 and MCF-7ras cells. bFGF increased the growth of MCF-7 cells, but inhibited MCF-7ras cell proliferation. NaPa treatment suppressed the stimulation of MCF-7 cell proliferation and increased MCF-7ras cell growth inhibition. We have demonstrated a new target of NaPa action in blocking the cell cycle of tumor cells in G0/G1. We suggest that the anti-proliferative effect of NaPa associated to the restoration of the cadherin function in human mammary carcinoma cells indicates that NaPa could be a novel therapeutic agent in breast cancer.

Adenocarcinoma↗

PubMedDate not supplied

The neurotrophin receptor p75NTR is a tumor suppressor in human prostate cancer.

The loss of tumor suppressor gene function contributes to the transformation of human prostate epithelial cells to a malignant pathology. Previous studies have demonstrated a loss of protein expression of the neurotrophin receptor, the p75NTR, from the pathological human prostate. Through cell cycle analysis, we demonstrate that a dose-dependent increase in the expression of p75NTR protein induces increased quiescence of a series of prostate tumor cells in vitro. When the same series of tumor cells were injected into the flanks of SCID mice, the growth of prostate tumors was suppressed in proportion to increased p75NTR expression. Tumor analysis showed that a dose-dependent increase in p75NTR expression was associated with a dose-dependent increase in the proportion of apoptotic cells, as determined by TUNEL assay, and a dose-dependent decrease in the proportion of cells committed to proliferation, as determined by proliferating cell nuclear antigen assay. These results demonstrate that the neurotrophin receptor p75NTR is a tumor suppressor of prostate growth by enhancing cell cycle quiescence, reducing proliferation and increasing apoptosis of the tumor cells.

Animals↗

PubMed1995

Somatostatin-14 and its analog octreotide exert a cytostatic effect on GH3 rat pituitary tumor cell proliferation via a transient G0/G1 cell cycle block.

Somatostatin (SRIF) and its analogs exert potent inhibitory effects on hormonal hypersecretion. In addition, they have been demonstrated to inhibit the proliferation of various cell lines as well as the growth of some endocrine tumors in vivo. To evaluate the action of SRIF and its analog octreotide on the proliferation and cell cycle kinetics of endocrine cells, we investigated their effect on GH3 rat pituitary tumor cells, a GH-producing cell line. Using flow cytometric DNA analysis with propidium iodide staining, we found that octreotide inhibits the proliferation of synchronized GH3 cells, achieving a maximal reduction, compared to controls, of 19.4 +/- 5.3% and 22.4 +/- 5.1% with 100 ng/ml and 1000 ng/ml octreotide, respectively (P < 0.05). This effect was demonstrated to be due to a block in progression from the G0/G1 phase to the S phase of the cell cycle. This was most evident after 24 h of exposure to 100 ng/ml octreotide, at which time there was a 7.1 +/- 1.4% increase in cells in G0/G1 (P < 0.01) and a 6.6 +/- 1.3% decrease in cells in S phase (P < 0.01). However, unless octreotide was replenished, this effect was transient and overcome by 36-48 h. No apoptosis was seen, and trypan blue studies confirmed that cell death by necrosis did not occur. A single exposure to native SRIF-14 had little effect, but a G0/G1 cell cycle block and inhibition of proliferation were seen if SRIF was regularly replenished. We conclude that SRIF and octreotide exert a cytostatic effect on GH3 cells by causing a partial G0/G1 cell cycle block. These findings suggest that the actions of SRIF and octreotide occur through signal transduction pathways that act predominantly on downstream regulators.

Animals↗

PubMed1995

Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis.

Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed when cytoskeletal stiffness was measured directly in living cells using magnetic twisting cytometry. These results emphasize the importance of matrix-dependent changes in cell and nuclear shape as well as higher order structural interactions between different cytoskeletal filament systems for control of capillary cell growth during angiogenesis.

Actin Cytoskeleton↗

PubMed2006

Preparation, characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents.

Curcumin is pharmaceutically active in many ways, having properties including anticoagulation, anti-proliferation, anti-inflammatory, and may be used to fabricate drug-eluting stents to treat in-stent restenosis after stent implantation. Here we describe our investigations of curcumin-eluting PLGA coatings formed using the biodegradable polymer PLGA (polylactic acid-co-glycolic acid) as drug carrier and uniformly fabricated on the surface of 316L stainless steel stents by an ultrasonic spray method. Three doses were explored--low dose ( approximately 140 microg per stent or 115 microg/cm(2)), moderate dose ( approximately 280 microg per stent or 230 microg/cm(2)), and high dose ( approximately 490 microg per stent or 408 microg/cm(2)). Pre- and post-expansion morphologies of the stent coating were examined by optical microscopy (OM) and scanning electron microscopy (SEM), indicating that the coating not only was very smooth and uniform but also had the ability to withstand the compressive and tensile strains imparted without cracking from the stent during the expansion process. Atomic force microscopy (AFM) images indicated the topography of the PLGA-only and moderate dose curcumin-eluting stent that showed an average roughness below 1 nm; no drug particles could be seen on the stent surface, indicating that curcumin can be mixed with PLGA at the molecular level using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier Transform Infrared (FTIR) spectroscopy and X-ray electron spectroscopy (XPS), with results suggesting that there was no chemical reaction between curcumin and the drug. The results of in vitro measurements of drug release from curcumin-eluting stents showed that all the curcumin-eluting stents studied exhibited a nearly linear sustained-release profile with no significant burst releases within the measurement period. The in vitro anticoagulation behavior of curcumin-eluting stents was investigated by static platelet adhesion and APTT (activated partial thromboplastin time) tests, revealing that the anticoagulation properties of curcumin-eluting stents are superior to those for stainless steel stents and PLGA-only-coated stents. The anticoagulation behavior of curcumin stents improved significantly as the drug dose was increased.

Anticoagulants↗

PubMed2003

Heme oxygenase-1 expression levels are cell cycle dependent.

Heme oxygenase-1 (HO-1) is a stress protein, which has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as angiotensin II (Ang II). The purpose of the present study was to examine the role of human HO-1 in cell-cycle progression. We investigated the effect of Ang II on HO-1 gene expression in serum-deprived media to drive human endothelial cells into G(0)/G(1) (1% FBS) compared to exponentially grown cells (10% FBS). The addition of Ang II (100 ng/ml) to endothelial cells increased HO-1 protein and activity in G(0)/G(1) in a time-dependent manner, reaching a maximum HO-1 level at 16 h. Real-time RT-PCR demonstrated that Ang II increased the levels of HO-1 mRNA in G(0)/G(1) as early as 1 h. The rate of HO-1 induction in response to Ang II was several-fold higher in serum-starved cells compared to cells cultured in continuous 10% FBS. The addition of Ang II increased the generation of 8-epi-isoprostane PGF(2 alpha). Inhibition of HO-1, by Stannis mesoporphyrin (SnMP), potentiated Ang II-mediated DNA damage and generation of 8-epi-isoprostane PGF(2 alpha). These results imply that expression of HO-1 in G(0)/G(1), in the presence of Ang II, may be a key player in attenuating DNA damage during cell-cycle progression. Thus, exposure of endothelial cells to Ang II causes a complex response involving generation of superoxide anion, which may be involved in DNA damage. Upregulation of HO-1 ensures the generation of bilirubin and carbon monoxide (CO) in G(0)/G(1) phase to counteract Ang II-mediated oxidative DNA damage. Inducibility of HO-1 in G(0)/G(1) phase is essential and probably regulated by a complex system involving oxygen species to assure controlled cell growth.

Anions↗

PubMed2003

An E2F site in the 5'-promoter region contributes to serum-dependent up-regulation of the human proliferating cell nuclear antigen gene.

The synthesis of proliferating cell nuclear antigen (PCNA) is strictly regulated during the cell cycle. To investigate the contribution of the promoter region to the up-regulation of human PCNA expression at the onset of S phase, we have examined 17 putative elements with reporter assays in quiescent L-O2 cells and following serum stimulation. The E2F-like sequence 5'-TTCCCCGCAA-3' located at -84 to -75 is required for the serum-induced transactivation. In electrophoretic mobility shift assays, nuclear extracts from asynchronous L-O2 cells exhibit two binding activities toward the -75 E2F oligonucleotide, and the minor band, whose formation could be interfered with by E2F-1 antibody, represents an S phase-specific complex. This is the first demonstration of the E2F site in the human PCNA 5' promoter as a serum-responsive element.

Cell Cycle Proteins↗

PubMed1996

Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1.

The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements.

Animals↗