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Results for “Resting Phase, Cell Cycle”

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At least 451 records · Page 25Linked to original sources

PubMed2001

Protective function of p27(KIP1) against apoptosis in small cell lung cancer cells in unfavorable microenvironments.

A previous study of ours unexpectedly found that in contrast to frequent reductions in non-small cell lung cancer, high expression of the p27(KIP1) cyclin-dependent kinase (CDK) inhibitor was retained in virtually all small cell lung cancers (SCLCs), suggesting the possibility of high expression of nonfunctional p27(KIP1) in this virulent tumor. The study presented here, however, shows that p27(KIP1) in SCLC biochemically functions as a CDK inhibitor, clearly showing induction apparently associated with G(1)/G(0) arrest and efficient binding to and inhibition of the cyclin E-CDK2 complex. Interestingly, induction of p27(KIP1) seems to confer on SCLC cells the ability to survive under culture conditions unfavorable for cell growth such as a lack of nutrients and hypoxia. Subsequent experiments manipulating p27(KIP1) levels by using a sense p27(KIP1) expression construct or an antisense oligonucleotide supported this notion. These observations suggest that high expression of p27(KIP1) in vivo may favor the survival of SCLC by preventing apoptosis in a microenvironment unfavorable for cell proliferation.

Animals↗

PubMed2004

beta-Carotene exacerbates DNA oxidative damage and modifies p53-related pathways of cell proliferation and apoptosis in cultured cells exposed to tobacco smoke condensate.

Human intervention trials have suggested that supplemental beta-carotene resulted in more cancer in smokers, whereas it was protective in non-smokers. However, the mechanisms underlying these effects are still unknown. The aim of this study was to evaluate the effects of an association of cigarette smoke condensate (tar) and beta-carotene on DNA oxidative damage and molecular pathways involved in cell cycle progression and apoptosis in cultured cells. In RAT-1 fibroblasts, tar caused increased levels of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and this effect was enhanced by the concomitant presence of beta-carotene (0.5-4.0 microM) in a dose- and time-dependent manner. In contrast, beta-carotene alone did not significantly modify it. Fibroblasts treated with tar alone decreased their cell growth with respect to control cells through an arrest of cell cycle progression in the G0/G1 phase and an induction of apoptosis. These effects were accompanied by an increased expression of p53, p21 and Bax and by a decreased expression of cyclin D1. In contrast, fibroblasts treated with tar and beta-carotene, after an initial arrest of cell growth at 12 h, re-entered in cell cycle and were unable to undergo apoptosis at 36 h. Concomitantly, their p53 expression, after an increase at 12 h, progressively returned at basal levels at 36 h by a mechanism independent of Mdm2. Such a decrease was followed by a decrease in p21 and Bax expression and by an increase in cyclin D1 expression. Moreover, the presence of the carotenoid remarkably enhanced cyclooxygenase-2 expression induced by tar. During tar treatment, a depletion of beta-carotene was observed in fibroblasts. The effects of tar and beta-carotene on 8-OHdG levels, cell growth and apoptosis were also observed in Mv1Lu lung, MCF-7 mammary, Hep-2 larynx and LS-174 colon cancer cells. This study supports the evidence for potential detrimental effects of an association between beta-carotene and cigarette smoke condensate.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

E2F-dependent mitogenic stimulation of the splicing of transcripts from an S phase-regulated gene.

There is one class of genes whose expression increases at the G1/S transition of the cell cycle. One of these genes codes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), an enzyme that controls glycolysis. The cell-cycle regulation of the PFK-2 gene depends on a binding site for the transcription factor E2F located at the 5'end of the first exon and involves not only a transcriptional, but also a post-transcriptional, mechanism. We have investigated this mechanism by studying in Rat-1 fibroblasts mature and immature mRNAs from the endogenous PFK-2 gene and from stably expressed transgenes containing PFK-2 gene regions. An increase in precursor mRNA half-life and processing took place at the G1/S transition. Transgenes with a mutated E2F binding site or with mutated splice sites lost the regulation by serum, indicating that both an intact E2F binding site and an efficient splicing reaction are necessary for proper mitogenic stimulation. In quiescent cells, the transgene lacking the E2F binding site was more efficiently spliced than the wild-type construct. These results indicate that, in the wild-type gene, precursor mRNA splicing is blocked in G0and that this block requires the E2F binding site. The data provide evidence for a coupling between stimulation of promoter activity and increased mRNA splicing in the mitogenic regulation of S phase-regulated genes.

Animals↗

PubMed2004

N-methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA.

BACKGROUND: Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce senescence in the HCT-116 colon cancer cell line. RESULTS: Cells treated with lower concentrations of MNNG (0-25 microM) for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50-100 microM) showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of beta-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and mRNA levels of APC were drastically reduced in cells treated with higher concentrations of MNNG. The loss of APC expression might lead to chromosomal instability as well as microtubular disorganization through its dissociation with tubulin. In fact, the protein level of alpha-tubulin was also drastically decreased in senescence-like arrested cells treated with higher concentrations of MNNG. The levels of telomeric DNA also decreased in cells treated with higher concentrations of MNNG. CONCLUSIONS: These results suggest that in response to DNA alkylation damage the senescence-like arrest of HCT-116 cells was associated with decreased levels of APC protein, microtubular organization, and telomeric DNA.

Adenomatous Polyposis Coli Protein↗

PubMed2000

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence.

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.

Breast Neoplasms↗

PubMed2000

Identification of E2F-3B, an alternative form of E2F-3 lacking a conserved N-terminal region.

We have identified a novel form of the full-length E2F-3 protein that we term E2F-3B. In contrast to full-length E2F-3, which is expressed only at the G1/S boundary, E2F-3B is detected throughout the cell cycle with peak levels in GO where it is associated with Rb. Transfection and in vitro translation experiments demonstrate that a protein identical to E2F-3B in size and iso-electric point is produced from the E2F-3 mRNA via the use of an alternative translational start site. This alternative initiation codon was mapped by mutagenesis to codon 102, an ACG codon. Mutation of the ACG codon at position 102 abolished E2F-3B expression, whereas the conversion of ACG 102 to a consensus ATG led to the expression of a protein indistinguishable from E2F-3B. Given these results, E2F-3B is missing 101 N-terminal amino acids relative to full-length E2F-3. This region includes a moderately conserved sequence of unknown function that is present only in the growth-promoting E2F family members, including E2F-1, 2 and full-length E2F-3. These observations make E2F-3B the first example of an E2F gene giving rise to two different protein species and also suggest that E2F-3 and E2F-3B may have opposing roles in cell cycle control.

3T3 Cells↗

PubMed2005

SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway.

The genome of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains four structural genes that are homologous to genes found in other coronaviruses, and also contains six subgroup-specific open reading frames (ORFs). Expression of one of these subgroup-specific genes, ORF7a, resulted in apoptosis via a caspase-dependent pathway. Here, we observed that transient expression of ORF7a protein fused with myc or GFP tags at its N or C terminus inhibited cell growth and prevented BrdU incorporation in different cultural cells, suggesting that ORF7a expression may regulate cell cycle progression. Analysis by flow cytometry demonstrated that ORF7a expression was associated with blockage of cell cycle progression at G0/G1 phase in HEK 293 cells after 24 to 60 h post-transfection. Similar results were observed in COS-7 and Vero cells. Mutation analysis of ORF7a revealed that the domain spanning aa 44-82 of 7a protein was essential for its cytoplasmic localization and for induction of the cell cycle arrest. After analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that ORF7a expression was correlated with a significant reduction of cyclin D3 level of mRNA transcription and expression, and phosphorylation of retinoblastoma (Rb) protein at ser795 and ser809/811, not with the expression of cyclin D1, D2, cdk4 and cdk6 in HEK 293 cells. These results suggest that the insufficient expression of cyclin D3 may cause a decreased activity of cyclin D/cdk4/6, resulting in the inhibition of Rb phosphorylation. Accumulation of hypo- or non-phosphorylated pRb thus prevents cell cycle progression at G0/G1 phase.

Animals↗

PubMed2000

Transient deactivation of ERK signalling is sufficient for stable entry into G0 in primary avian fibroblasts.

Re-entry into the cell cycle from quiescence requires the activation of mitogen-activated protein (MAP) kinases of the extracellular-signal-regulated kinase (ERK) family [1,2]. The relationship between ERK and cell-cycle control is, however, complex, as ERK activation can also lead to terminal differentiation [3] or a senescence-like growth arrest [4]. Here, we report that reversible cell-cycle exit induced by serum withdrawal in primary avian fibroblasts is associated with rapid deactivation of ERK, but ERK activity is subsequently regenerated and sustained at high levels in fully quiescent (G0) cells. As in proliferating cells, ERK activation during G0 required the MAPkinase kinase MEK and was partially dependent on cell adhesion. Active, phosphorylated ERK was concentrated in the nucleus in cycling cells, but was largely confined to the cytoplasm during G0. This was unexpected, as activatory phosphorylation mediated by MEK is thought to play an important role in promoting nuclear translocation [5,6]. These results indicate that transient deactivation of ERK signalling can be sufficient for stable cell-cycle exit, and that MEK-mediated phosphorylation is not sufficient for nuclear translocation of active ERK in G0. Cytoplasmic sequestration may prevent active ERK from accessing critical nuclear cell-cycle targets, thus allowing quiescent or post-mitotic cells to retain ERK activity for other physiological functions.

3T3 Cells↗

PubMed2002

Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression.

The TCL1 gene at 14q32.1 is involved in chromosomal translocations and inversions in mature T cell leukemias. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. In transgenic animals, the deregulated expression of TCL1 leads to mature T cell leukemia, demonstrating the role of TCL1 in the initiation of malignant transformation in T cell neoplasia. Expression of high levels of Tcl1 have also been found in a variety of human tumor-derived B cell lines ranging from pre-B cell to mature B cell. Here we describe the phenotype of transgenic mice, E mu-TCL1, established with TCL1 under the control of a V(H) promoter-Ig(H)-E mu enhancer to target TCL1 expression to immature and mature B cells. Flow cytometric analysis reveals a markedly expanded CD5(+) population in the peritoneal cavity of E mu-TCL1 mice starting at 2 mo of age that becomes evident in the spleen by 3-5 mo and in the bone marrow by 5-8 mo. Analysis of Ig gene rearrangements indicates monoclonality or oligoclonality in these populations, suggesting a preneoplastic expansion of CD5(+) B cell clones, with the elder mice eventually developing a chronic lymphocytic leukemia (CLL)-like disorder resembling human B-CLL. Our findings provide an animal model for CLL, the most common human leukemia, and demonstrate that deregulation of the Tcl1 pathway plays a crucial role in CLL pathogenesis.

Aging↗

PubMed2006

PAI-1 transcriptional regulation during the G0 --> G1 transition in human epidermal keratinocytes.

Plasminogen activator inhibitor type-1 (PAI-1) is the major negative regulator of the plasmin-dependent pericellular proteolytic cascade. PAI-1 gene expression is normally growth state regulated but frequently elevated in chronic fibroproliferative and neoplastic diseases affecting both stromal restructuring and cellular migratory activities. Kinetic modeling of cell cycle transit in synchronized human keratinocytes (HaCaT cells) indicated that PAI-1 transcription occurred early after serum stimulation of quiescent (G0) cells and prior to entry into a cycling G1 condition. PAI-1 repression (in G0) was associated with upstream stimulatory factor-1 (USF-1) occupancy of two consensus E box motifs (5'-CACGTG-3') at the PE1 and PE2 domains in the PF1 region (nucleotides -794 to -532) of the PAI-1 promoter. Chromatin immunoprecipitation (ChIP) analysis established that the PE1 and PE2 site E boxes were occupied by USF-1 in quiescent cells and by USF-2 in serum-activated, PAI-1-expressing keratinocytes. This reciprocal and growth state-dependent residence of USF family members (USF-1 vs. USF-2) at PE1/PE2 region chromatin characterized the G0 --> G1 transition period and the transcriptional status of the PAI-1 gene. A consensus E box motif was required for USF/E box interactions, as a CG --> AT substitution at the two central nucleotides inhibited formation of USF/probe complexes. The 5' flanking sites (AAT or AGAC) in the PE2 segment were not necessary for USF binding. USF recognition of the PE1/PE2 region E box sites required phosphorylation with several potential involved residues, including T153, maping to the USF-specific region (USR). A T153A substitution in USF-1 did not repress serum-induced PAI-1 expression whereas the T153D mutant was an effective suppressor. As anticipated from the ChIP results, transfection of wild-type USF-2 failed to inhibit PAI-1 induction. Collectively, these data suggest that USF family members are important regulators of PAI-1 gene control during serum-stimulated recruitment of quiescent human epithelial cells into the growth cycle.

Amino Acid Substitution↗

PubMed1995

cycMs3, a novel B-type alfalfa cyclin gene, is induced in the G0-to-G1 transition of the cell cycle.

Cyclins are key regulators of the cell cycle in all eukaryotes. We have previously isolated two B-type cyclin genes, cycMs1 and cycMs2, from alfalfa that are primarily expressed during the G2-to-M phase transition and are most likely mitotic cyclin genes. Here, we report the isolation of a novel alfalfa cyclin gene, termed cycMs3 (for cyclin Medicago sativa), by selecting for mating type alpha-pheromone-induced cell cycle arrest suppression in yeast. The central region of the predicted amino acid sequence of the cycMs3 gene is most similar to the cyclin box of yeast B-type and mammalian A- and B-type cyclins. In situ hybridization showed that cycMs3 mRNA can be detected only in proliferating cells and not in differentiated alfalfa cells. When differentiated G0-arrested cells were induced to reenter the cell cycle in the G1 phase and resume cell division by treatment with plant hormones, cycMs3 transcript levels increased long before the onset of DNA synthesis. In contrast, histone H3-1 mRNA and cycMs2 transcripts were not observed before DNA replication and mitosis, respectively. In addition, cycMs3 mRNA was found in all stages of the cell cycle in synchronously dividing cells, whereas the cycMs2 and histone H3-1 genes showed a G2-to-M phase- or S phase-specific transcription pattern, respectively. These data suggest that the role of cyclin CycMs3 differs from that of CycMs1 and CycMs2. We propose that CycMs3 helps control reentry of quiescent G0-arrested cells into the G1 phase of the cell cycle.

Amino Acid Sequence↗

PubMed2006

Isolation of rat hepatocytes.

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined. Although applicable to the liver of various species, including human, the practical aspects of the method are explained here for rat liver. Critical parameters for the successful isolation of primary rat hepatocytes are highlighted and a troubleshooting guide is provided. In addition, a new development based on the inhibition of histone deacetylase activity is presented. This approach allows inhibition of cell-cycle reentry during hepatocyte isolation, a process known to underlie the dedifferentiation process of cultured hepatocytes.

Animals↗

PubMed1998

U.V.C.-induction of p53 activation and accumulation is dependent on cell cycle and pathways involving protein synthesis and phosphorylation.

Transcriptional activation and stabilization of p53 is a major response of mammalian cells to U.V.-light induced genetic damages, and possibly responsible for cell damage control. We have studied here by gel mobility shift and immunoblotting assays the activation and accumulation of p53 by U.V.C. and its dependency on cell cycle, protein synthesis and protein phosphorylation. In G0/G1 synchronized cells U.V.C.-induced p53 DNA-binding activity, but not its accumulation, whereas both events took place in G1/S and S-phase cells. The kinetics of p53 activation by U.V.C. were slow requiring at least 1 h and slowly increasing thereafter with full activation observed at 6 h. Treatment of cells with cycloheximide (CHX) prevented the activation of p53 in all phases of the cell cycle and its accumulation in G1/S and S. However, removing CHX-block allowed full activation and accumulation of p53 with fast kinetics even if 4 h had lapsed since the initial U.V.C. insult. This suggests that the protein synthesis-dependent signal initiating p53 activation by U.V.C. remains continuous in the cells. The requirement of protein phosphorylation as mediator of p53 activation by U.V.C. was studied by using chemical protein kinase inhibitors. Of the tested inhibitors, only staurosporine, a known inhibitor of protein kinase C (PKC) and various other kinases, inhibited both p53 activation and accumulation, whereas specific PKC inhibitors, tyrosine kinase inhibitors and a serine/threonine kinase inhibitor did not. PKC-mediation of the p53 U.V.-response was further ruled out by the reactivity of the activated p53 to C-terminal antibody PAb 421. Kinetic studies showed that staurosporine-mediated inhibition of p53 function is an early event in cell damage response. Thus dual, kinetically different events, de novo protein synthesis and staurosporine-inhibited protein phosphorylation are required for p53 activation and accumulation in all phases of the cell cycle. Notably, in the absence of U.V.-induced accumulation in G0/G1 cells, p53 activation is still subject to inhibition of protein synthesis.

3T3 Cells↗

PubMed1998

RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells.

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 microM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells. Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.

3T3 Cells↗

PubMed1995

Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content.

Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G0 cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G0-G1 transition in unperturbed and drug-treated cell populations.

3T3 Cells↗

PubMed2005

Molecular mechanisms of econazole-induced toxicity on human colon cancer cells: G0/G1 cell cycle arrest and caspase 8-independent apoptotic signaling pathways.

Econazole (Eco), a potent broad-spectrum anti-fungal agent, has been used in the treatment of superficial mycosis. Eco is a store-operated Ca2+ channel antagonist which induces cytotoxic cell death of leukemia. However, little is known about its cytotoxic effect upon solid tumor cells. The purpose of this study is to investigate both the in vitro and in vivo molecular mechanisms of Eco-induced toxicity on colon cancer cells. We used COLO 205 cell line and nude mice xenograft model to investigate the cytotoxic effect of Eco. We demonstrated that lower doses Eco (5-20 microM) arrested human colon cancer cells at the G0/G1 phase of the cell cycle. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated while CDK2 and CDK4 kinase activity were significantly suppressed by Eco treatment in COLO 205 cells. At higher doses (40-60 microM), Eco induced COLO 205 cells apoptosis evidenced by ladder formation in DNA fragmentation assay and sub-G1 peak in flow cytometry analysis. Western blot analysis showed that caspases 3, 9 but not 8 were activated by high dose Eco treatment to the COLO 205 cells accompanied with cytochrome c and apoptosis-inducing factor (AIF) translocation. Significant anti-tumorigenesis effect was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with Eco 50 mg/kg intraperitoneally. Our findings highlight the molecular mechanisms underlying the Eco-induced toxicity on colon cancer cells.

Animals↗

PubMed1996

C-myc is required for the G0/G1-S transition of primary hepatocytes stimulated with a deleted form of hepatocyte growth factor.

Primary rat hepatocytes stimulated in vitro with the addition of a deleted form of hepatocyte growth factor (dHGF) enter the S-phase 48 h after addition of the growth factor. The c-myc gene is believed to play a role in a variety of cellular stages, such as proliferation, differentiation and cell death. In primary hepatocytes c-myc was expressed constitutively at both mRNA and protein levels, independently of the growth conditions. dHGF induced significant c-myc expression at times correlated with the long-lasting pre-S phase, and no induction was observed at the G0/G1 traverse compared with the unstimulated hepatocytes. An antisense construct coding for all three exons of c-myc was imported into hepatocytes by using the transferrin receptor-mediated endocytosis methodology (transferrinfection). Expression of the antisense construct inhibited the biosynthesis of the c-Myc protein, however it did not interfere with the expression of c-met, encoding the receptor for HGF/dHGF. Continuous expression of the antisense construct inhibited entry of the hepatocytes into the S-phase. Regulated induction of the antisense c-myc by dexamethasone for up to 6 h in culture, did not interfere with the entry of hepatocytes into the S-phase. c-myc expression was shown to be required between 6 and 12 h in dHGF-stimulated hepatocytes, and inhibition of its expression during this time by the antisense myc construct did not allow these cells to enter the S-phase. Inhibition of c-myc biosynthesis between 24 and 48 h hours slightly affected the DNA synthetic response. It is proposed that the expression of c-Myc protein interferes with the "priming' of hepatocytes to become responsive to growth-factor stimuli, or in the absence of such stimuli it interferes with the maintenance of a non-proliferating phenotype and subsequent in vitro de-differentiation.

Animals↗

PubMed2003

Trihydrophobin 1 is a new negative regulator of A-Raf kinase.

Our previous work indicated that instead of binding to B-Raf or C-Raf, trihydrophobin 1 (TH1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1-372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase-dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G(0)/G(1) phase-arrested. Taken together, our data provide a clue to understanding the cellular function of TH1 on Raf isoform-specific regulation.

Animals↗