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PubMed1997

S-phase function of Drosophila cyclin A and its downregulation in G1 phase.

BACKGROUND: Cyclin E is the normal inducer of S phase in G1 cells of Drosophila embryos. Stable G1 quiescence requires the downregulation both of cyclin E and of other factors that can bypass the normal regulation of cell cycle progression. RESULTS: High-level expression of cyclin A triggered the G1/S transition in wild-type embryos and in mutant embryos lacking cyclin E. Three types of control downregulated this activity of cyclin A. First, cyclin destruction limited the accumulation of cyclin A protein in G1. Second, inhibitory phosphorylation of cdc2, the kinase partner of cyclin A, reduced the S-phase promoting activity of cyclin A in G1. Third, rux, a protein with unknown biochemical function, limited cyclin A function in G1. Overexpression of rux blocked S phase induction by coexpressed cyclin A and promoted the degradation of cyclin A. Rux also prevented a stable cyclin A mutant from inducing S phase, indicating that inhibition does not require cyclin destruction, and drove the nuclear localization of cyclin A. CONCLUSIONS: Cyclin A can drive the G1/S transition, but this function is suppressed by three types of control: cyclin A destruction, inhibitory phosphorylation of cdc2, and inhibition by rux. The partly redundant contributions of these three inhibitory mechanisms safeguard the stability of G1 quiescence until the induction of cyclin E. The action of rux during G1 resembles the action of inhibitors of mitotic kinases present during G1 in yeast, although no obvious sequence similarity exists.

Animals↗
PubMed2002

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos.

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.

Animals↗
PubMed1999

UCN-01-mediated G1 arrest in normal but not tumor breast cells is pRb-dependent and p53-independent.

In this study we investigated the growth inhibitory effects of UCN-01 in several normal and tumor-derived human breast epithelial cells. We found that while normal mammary epithelial cells w were very sensitive to UCN-01 with an IC(50) of 10nM tumor cells displayed little to no inhibition of growth with any measurable IC(50) at low UCN-01 concentrations (i.e. 0-80 nM). The UCN-01 treated normal cells arrested in G1 phase and displayed decreased expression of most key cell cycle regulators examined, resulting in inhibition of CDK2 activity due to increased binding of p27 to CDK2. Tumor cells on the other hand displayed no change in any cell cycle distribution or expression of cell cycle regulators. Examination of E6- and E7-derived strains of normal cells revealed that pRb and not p53 function is essential for UCN-01-mediated G1 arrest. Lastly, treatment of normal and tumor cells with high doses of UCN-01 (i.e. 300 nM) revealed a necessary role for a functional G1 checkpoint in mediating growth arrest. Normal cells, which have a functional G1 checkpoint, always arrest in G1 even at very high concentrations of UCN-01. Tumor cells on the other hand have a defective G1 checkpoint and only arrest in S phase with high concentrations of UCN-01. The effect of UCN-01 on the cell cycle is thus quite different from staurosporine, a structural analogue of UCN-01, which arrests normal cells in both G1 and G2, while tumor cells arrest only in the G2 phase of the cell cycle. Our results show the different sensitivity to UCN-01 of normal compared to tumor cells is dependent on a functional pRb and a regulated G1 checkpoint.

Alkaloids↗
PubMed2002

Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase.

Cell cycle progression is tightly regulated by cyclins, cyclin-dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D-type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up-regulated in the mid-G1 phase (4-6 h) while cyclin D3 expression emerged only in late G1 (9-12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid-G1 phase. Exposure of C6 glioma to VPA induced a marked up-regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid-G1 phase (3-5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA-induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase.

Active Transport, Cell Nucleus↗
PubMed1998

An important role for the retinoblastoma protein in staurosporine-induced G1 arrest in murine embryonic fibroblasts.

In this study, we investigated the molecular basis of the ability of staurosporine to induce G1 arrest in murine embryonic fibroblasts (MEFs). We used MEFs from transgenic mice lacking several negative regulators of the G1/S phase transition including cells from mice lacking p53, p21, retinoblastoma (Rb), or p16 genes. We found that p53 function was not essential for staurosporine-induced G1 arrest. In contrast, MEFs from mice lacking Rb genes showed approximately a 70% reduced capacity to arrest in the G1 phase following staurosporine treatment. In support of a role for Rb in staurosporine-induced G1 arrest, rat embryonic fibroblasts stably overexpressing cyclin D1/Cdk4(R24C) exhibited approximately a 50% reduced G1 arrest response to staurosporine. The role of Rb in determining the degree of staurosporine-induced G1 arrest did not depend on the function of the cyclin-dependent kinase inhibitors p16 or p21 because MEFs lacking either of these genes were still capable of undergoing G1 arrest following staurosporine exposure. Our studies provide evidence of an important role for the Rb protein in determining the degree of staurosporine-induced G1 arrest in the first cell cycle.

Animals↗
PubMed2005

ERK activation in arsenite-treated G1-enriched CL3 cells contributes to survival, DNA repair inhibition, and micronucleus formation.

Arsenite is known to induce chromosomal damage and extracellular signal-regulated kinases 1/2 (ERK) signaling transduction pathway. Arsenite also perturbs mitotic spindle and induces G2/M prolongation, leading to genomic instability. However, little is known concerning whether G1 phase is susceptible to arsenite in causing genomic instability and ERK activation. In this study, we investigate the roles of ERK activation in survival, micronucleus formation, and nucleotide excision repair (NER) synthesis in arsenite-treated G1-enriched CL3 human non-small-cell lung carcinoma cells. We found that G1 was the most insensitive phase to arsenite cytotoxicity, yet it was highly susceptible to arsenite in micronucleus induction. After arsenite exposure, the G1 cells exhibited a marked retard in the formation of binucleated cells when they were cultured in cytochalasin B, an inhibitor of cytokinesis, suggesting that arsenite delays the cell cycle progression. Arsenite activated sustained-ERK signal in G1 cells whose suppression further decreased cell proliferation and survival and could lower the micronucleus induction. The NER synthesis activity of G1 cells was inhibited by arsenite as a function of the extent of ERK activation. Intriguingly, blockage of ERK activation recovered NER synthesis activity in the arsenite-treated G1 cells. Together, these results suggest that ERK activation in arsenite-treated G1 cells counteracts cytotoxicity and contributes to genomic instability via NER synthesis inhibition and micronucleus induction.

Arsenites↗
PubMed1994

The fraction of cells in G1 with bound retinoblastoma protein increases with the duration of the cell cycle.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein with growth-suppressing effects. During early G1 phase, pRB is underphosphorylated and bound in the nucleus. The association between the duration of the cell cycle/G1 phase and the fraction of cells in G1 with bound pRB was studied in the human pre-B cell line Reh. The cell-cycle duration was varied by growing cells at different concentrations (25, 10, 2, 0.5 and 0%) of fetal calf serum (FCS); pRB binding was studied by flow cytometry. The culture doubling time increased from 21 h in 25% FCS to 54 h in 0.5% FCS. Cell death occurred in the absence of FCS, and the culture doubling time therefore could not be defined. The fraction of cells in G1 did not change significantly with decreasing FCS concentration (0.47 in 25% FCS, 0.52 in 0% FCS). In contrast, the fraction of G1 cells with bound pRB increased from 0.12 in 25% FCS to 0.65 in 0% FCS. Continuous labelling with bromodeoxyuridine demonstrated that the growth fraction was close to unity at all FCS concentrations down to 0.5%, hence, the duration of the cell cycle was equal to the culture doubling time under these conditions. The duration of early G1 phase (where pRB is underphosphorylated and bound) increased 10-fold, while the duration of late G1 phase increased twofold, for Reh cells grown in 0.5% FCS compared with cells grown in 25% FCS. The increase in the duration of late G1, and the increased S and G2+M phase transit times, indicate that other factors, in addition to pRB kinase activity, regulate the duration of G1 and the cell cycle of serum-deprived Reh cells.

Animals↗
PubMed1995

Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth.

The proliferation rate of mammalian cells is regulated normally in the G1 phase of the cell cycle. During this phase, it is convenient to assign positive and negative roles to the molecular programs that regulate the duration of G1 and the phase transition from G1 to S phase. Density-dependent inhibition of cellular proliferation results in an increase in the duration of G1. This form of regulation is due to both secreted factors and cell-cell contact. Serum is mitogenic to a variety of mammalian cell types. Because quiescent cells enter S phase as a result of serum addition to culture media, serum is usually regarded as a source of positive regulatory growth factors. We have measured the length of the G1, S and G2+M phases of NIH 3T3 cells during exponential growth as a function of cell density and serum concentration. The G1 length increases during exponential growth as a function of density while S and G2+M are relatively constant. Further, this increase in G1 phase time, or density mediated negative regulation, is inhibited by increasing serum concentration. This phenotype is saturable between 10% to 20% serum. Serum concentrations above 2.5% are able to increase the rate of cell cycling (decrease the G1 phase time) by inhibiting density dependent negative regulation of NIH 3T3.

3T3 Cells↗
PubMed2005

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum.

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.

Animals↗
PubMed1995

Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1.

We have created fibroblast cell lines that express cyclin A under the control of a tetracycline-repressible promoter. When stimulated to reenter the cell cycle after serum withdrawal, these cells were advanced prematurely into S phase by induction of cyclin A. In an asynchronous population, induction of cyclin A caused a decrease in the percentage of cells in G1. These results demonstrate that expression of cyclin A is rate limiting for the G1-to-S transition and suggest that cyclin A can function as a G1 cyclin. Although the level of exogenous cyclin A was constant throughout the cell cycle, its associated kinase activity increased as cells approached S phase. Low kinase activity in early G1 was found to correlate with the presence of p27Kip1 in cyclin A-associated complexes, while high kinase activity in late G1 was correlated with its absence. These results suggest that a function of p27Kip1 in G1 is to prevent premature activation of cyclin A-associated kinase. Cyclin A expression in early G1 led to phosphorylation of the product of the retinoblastoma susceptibility gene (pRb). Thus, cyclin A expression can be rate limiting for pRb phosphorylation, implicating pRb as a physiological substrate of the cyclin A-dependent kinase. Taken together, these results demonstrate that deregulated expression of cyclin A can perturb the normal regulation of the G1-to-S transition.

Animals↗
PubMed1997

Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation.

BACKGROUND: The contemporary treatment of coronary athero-occlusive disease by percutaneous transluminal coronary angioplasty is hampered by maladaptive wound healing, resulting in significant failure rates. Morbid sequelae include smooth muscle cell (SMC) hyperplasia and restenosis due to vascular neointima formation. METHODS AND RESULTS: In this study, we examined the inhibitory effects of a concentrated retroviral vector bearing an antisense cyclin G1 gene on aortic SMC proliferation in vitro and on neointima formation in vivo in a rat carotid injury model of restenosis. Retroviral vectors bearing an antisense cyclin G1 construct inhibited the proliferation of transduced aortic SMCs in 2- to 6-day cultures, concomitant with down-regulation of cyclin G1 protein expression and decreased [3H]thymidine incorporation into DNA. Morphological examination showed evidence of cytolysis, giant syncytia formation, and apoptotic changes evidenced by overt cell shrinkage, nuclear fragmentation, and specific immunostaining of nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation. Pronounced "bystander effects" including neighboring cells were noted in aortic SMCs transduced with the antisense cyclin G1 vector, as determined by quantitative assays and fluorescent labeling of nontransduced cells. In an in vitro tissue injury model, the proliferation and migration of antisense cyclin G1 vector-transduced aortic SMCs were inhibited. Moreover, in vivo delivery of high-titer antisense cyclin G1 vector supernatant to the balloon-injured rat carotid artery in vivo resulted in a significant reduction in neointima formation. CONCLUSIONS: These findings represent the first demonstration of the inhibitory effects of an antisense cyclin G1 retroviral vector on nonneoplastic cell proliferation. Taken together, these data affirm the potential utility of antisense cyclin G1 constructs in the development of novel gene therapy approaches to vascular restenosis.

Animals↗
PubMed2006

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs.

The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.

Animals↗
PubMed1995

The three-dimensional organization of ribosomal genes and the architecture of the nucleoli vary with G1, S and G2 phases.

The three-dimensional (3-D) organization of the nucleolus, a defined nuclear territory containing tandem repeats of the ribosomal genes (rDNA), was investigated in PtK1 cells. Identification of the interphase stages was performed in single cells using DNA quantification by cytometry for the G1 and G2 phases while the S phase was identified by immunolabelling of the proliferating cell nuclear antigen (PCNA). The 3-D organization of the rDNA in the nucleolus was analyzed by fluorescence in situ hybridization using confocal microscopy. All the rDNA was located inside the nucleolar structures during all stages and the two rDNA loci were orthogonal. The rDNA was heterogeneously distributed in each nucleolus during G1, S and G2, with alternate sites of clustered genes (spots) and of genes in more extended configurations. The number of spots (4 to 6 in G1) increased during S phase (7 to 12) and their 3-D organization was progressively relaxed from G1 to G2. Double spots in G2 could reflect a similar gene organization of two chromatids. During mid-S phase, PCNA co-localized with some clustered genes (spots), indicating that rDNA replication occurs inside nucleoli and at different sites of the same locus simultaneously. The evaluation of the rDNA transcription units in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-treated cells indicated a mean of 16 units per G1 nucleus and 25 units per G2 nucleus. For G1 and G2, the fine 3-D structure of nucleoli was reconstructed using ultrathin serial sections after specific contrast of DNA and RNA, digitization of the serial section images and computer-assisted 3-D architecture. Fibrillar centers (FCs) formed discrete structures (about 10 in G1 and 20 in G2) connected by a network of the dense fibrillar component. The 3-D arrangement of the FCs in G1 and G2 are similar to the rDNA spots. In conclusion, the architecture of the nucleoli during interphase reflects the distribution of the rDNA that is characterized by alternation of clustered and extended genes.

Animals↗
PubMed2004

Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle.

We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.

Animals↗
PubMed1999

Induction of S phase by G1 regulatory factors.

The first gap phase (G1) in the mammalian cell cycle plays a pivotal role in determining whether or not cells are to initiate DNA replication. Progression through G1 phase and transition into S phase are positively and negatively regulated by a series of factors, collectively termed G1 regulators. Among them, D-type G1 cyclins and a Cdk inhibitor, p27Kip1, function as the target of growth factors to integrate extracellular signals into cell cycle regulators. Another G1 cyclin, cyclin E, and a transcription factor E2F are situated the furthest downstream of known G1 regulators and seem to be directly involved in the initiation of chromosomal DNA replication. Alterations in G1 regulator genes are often present in human tumors, indicating that G1 regulators participate in tumor suppressive mechanisms as well as in cell proliferation.

Animals↗
PubMed2006

The role of MYCN in the failure of MYCN amplified neuroblastoma cell lines to G1 arrest after DNA damage.

We previously reported that 3 p53 wild type (wt) MYCN amplified (MNA) neuroblastoma cell lines failed to G1 arrest after DNA damage despite induction of p53, p21(WAF1) and MDM2. We hypothesised that this was due to high MYCN expression. p53 responses to DNA damage were examined in an additional 13 p53 wt neuroblastoma cell lines. MNA was significantly associated with a failure to G1 arrest after DNA damage (p < 0.001) and higher levels of apoptosis after irradiation (p < 0.05). p21(WAF1) and hypophosphorylated (hypo) RB accumulation post irradiation were significantly lower in cell lines that failed to G1 arrest (p < 0.05). Conditional MYCN expression in non-MNA SHEP Tet21N cells did not affect the G1 arrest after irradiation. MYCN knockdown using siRNA in 3 p53 wt MNA cell lines did not restore a G1 arrest after irradiation, but increased the baseline G1 population, p21(WAF1) and hypo RB expression. MYCN siRNA also caused a G1 arrest in a p53 mutant MNA cell line. This study is the first to determine that MNA correlates with a failure to G1 arrest and attenuated p21(WAF1) induction; however MYCN expression alone is not causally responsible.

Apoptosis↗
PubMed2003

Cyclin G1 has growth inhibitory activity linked to the ARF-Mdm2-p53 and pRb tumor suppressor pathways.

Cyclin G1 is a p53-responsive gene that is induced in alternative reading frame (ARF)-arrested cells, yet its role in growth control is unclear. We tested its effects on growth and involvement in the ARF-Mdm2-p53 tumor suppressor pathway. We show that cyclin G1 interacts with ARF, Mdm2, and p53 in vitro and in vivo. At high levels, cyclin G1 induces a G(1)-phase arrest in mammalian cells that coincides with p53 activation. Conversely, lower levels of cyclin G1 lack intrinsic growth inhibitory effects yet potentiate ARF-mediated growth arrest. Notably, cyclin G1 is down-regulated by Mdm2 through proteasome-mediated degradation. These data suggest that cyclin G1 is a positive feedback regulator of p53 whose expression is restrained by Mdm2. Interestingly, growth inhibition by cyclin G1 does not require p53 but instead exhibits partial retinoblastoma protein (pRb) dependence. These findings reveal that cyclin G1 has growth inhibitory activity that is mechanistically linked to ARF-p53 and pRb tumor suppressor pathways.

3T3 Cells↗
PubMed1996

Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells.

Progression of mammalian cells through G1 is controlled by the concerted action of protein kinases, the activities of which are modulated in both positive (cyclins) and negative [cyclin-dependent kinase inhibitors (CDIs)] manners by families of regulatory proteins. In differentiation of leukemia cells, a G1 arrest is a common, if not invariable, occurence and takes place after the appearance of markers of monocytic differentiation in human leukemia HL60 cells treated with 1,25 dihydroxyvitamin D3 (1,25D3) at low to moderately high concentrations (F. Zhang et al., Cell Proliferation 27: 643-654, 1994). In the present study, we investigated the protein levels of several G1 regulatory proteins that are potential mediators of the 1,25D3-induced G1 block. During the first 24 h of exposure to a high concentration (4 x 10(-7) M) of 1,25D3, no increase was noted in the immunodetectable levels of cyclins D1 or E, or CDIs p16Ink4, p21Cip1/Waf1, or p27Kip1, even though monocytic differentiation markers were evident, and a prolongation of G1 was noted. After 48 h of exposure 4 x 10(-7) M to 1,25D3, a G1 to S-phase block progressively increased in parallel with the abundance of the p27Kip1 CDI. A transient increase in p21Cip1/Waf1 was noted only at 48 hr. The increase in p27Kip1 protein level was dependent on the concentration of 1,25D3 and was accompanied by an increase in cyclin D and E proteins, which normally peak in mid-G1 and at the G1 to S-phase transition, respectively. These results indicate that p27Kip1 protein is a strong candidate for the cell cycle regulator that blocks the entry into the S-phase in 1,25D3-treated HL60 cells.

Animals↗