Cell Cycle-specific Expression Research Articles

The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae.

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.

N(6)-methyladenosine methylation-regulated polo-like kinase 1 cell cycle homeostasis as a potential target of radiotherapy in pancreatic adenocarcinoma

In pancreatic cancer, methyltransferase-like 3 (METTL3), a N(6)-methyladenosine (m6A) methyltransferase, has a favorable effect on tumors and is a risk factor for patients’ prognosis. However, the details of what genes are regulated by METTL3 remain unknown. Several RNAs are methylated, and what genes are favored in pancreatic cancer remains unclear. By epitranscriptomic analysis, we report that polo-like kinase 1 (PLK1) is an important hub gene defining patient prognosis in pancreatic cancer and that RNA methylation is involved in regulating its cell cycle-specific expression. We found that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) binds to m6A of PLK1 3′ untranslated region and is involved in upregulating PLK1 expression and that demethylation of this site activates the ataxia telangiectasia and Rad3-related protein pathway by replicating stress and increasing mitotic catastrophe, resulting in increased radiosensitivity. This suggests that PLK1 methylation is essential for cell cycle maintenance in pancreatic cancer and is a new therapeutic target.

Contradictory mRNA and protein misexpression of EEF1A1 in ductal breast carcinoma due to cell cycle regulation and cellular stress

Encoded by EEF1A1, the eukaryotic translation elongation factor eEF1α1 strongly promotes the heat shock response, which protects cancer cells from proteotoxic stress, following for instance oxidative stress, hypoxia or aneuploidy. Unexpectedly, therefore, we find that EEF1A1 mRNA levels are reduced in virtually all breast cancers, in particular in ductal carcinomas. Univariate and multivariate analyses indicate that EEF1A1 mRNA underexpression independently predicts poor patient prognosis for estrogen receptor-positive (ER+) cancers. EEF1A1 mRNA levels are lowest in the most invasive, lymph node-positive, advanced stage and postmenopausal tumors. In sharp contrast, immunohistochemistry on 100 ductal breast carcinomas revealed that at the protein level eEF1α1 is ubiquitously overexpressed, especially in ER+ , progesterone receptor-positive and lymph node-negative tumors. Explaining this paradox, we find that EEF1A1 mRNA levels in breast carcinomas are low due to EEF1A1 allelic copy number loss, found in 27% of tumors, and cell cycle-specific expression, because mRNA levels are high in G1 and low in proliferating cells. This also links estrogen-induced cell proliferation to clinical observations. In contrast, high eEF1α1 protein levels protect tumor cells from stress-induced cell death. These observations suggest that, by obviating EEF1A1 transcription, cancer cells can rapidly induce the heat shock response following proteotoxic stress, and survive.

Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments

Gene expression in multiple individual cells from a tissue or culture sample varies according to cell-cycle, genetic, epigenetic and stochastic differences between the cells. However, single-cell differences have been largely neglected in the analysis of the functional consequences of genetic variation. Here we measure the expression of 92 genes affected by Wnt signaling in 1,440 single cells from 15 individuals to associate single-nucleotide polymorphisms (SNPs) with gene-expression phenotypes, while accounting for stochastic and cell-cycle differences between cells. We provide evidence that many heritable variations in gene function--such as burst size, burst frequency, cell cycle-specific expression and expression correlation/noise between cells--are masked when expression is averaged over many cells. Our results demonstrate how single-cell analyses provide insights into the mechanistic and network effects of genetic variability, with improved statistical power to model these effects on gene expression.

Molecular mechanism of Aurora kinase inhibitor PHA739358 in inhibited proliferation and induced apoptosis of breast cancer cells

To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells. The in vitro cultured T47D cells in logarithmic growth phase were used. The effects of PHA739358 on cell proliferation were examined by MTT (methyl thiazolyl tetrazolium) assay. The variety of nuclear and spindle morphologies was examined by immunofluorescence. And G2/M arrest was determined by flow cytometry. The morphological changes of apoptotic cells were observed by fluorescent microscope. The levels of Aurora kinase relative protein expression phosphonate-AuroraA (p-AuroraA), AuroraA, phosphonate-histone H3 (p-histone H3), histone H3, cell cycle-specific protein expression CyclinB1, cell cycle regulative protein expression Cdc2, Cdc25c, phosphonate-Cdc2 (p-Cdc2), phosphonate-Cdc25c (p-Cdc25c) and apoptosis relative protein expression PARP, Bcl-2 and Bax were detected by Western blot. The apoptotic rate was examined by flow cytometer. PHA739358 obviously inhibited the proliferation of T47D cells after a 24 h or 48 h treatment in a dose-dependent and time-dependent manner. Their IC50 values were (3.44 ± 0.54) and (0.21 ± 0.67) µmol/L respectively. Flow cytometry showed that G2/M arrested in a dose-dependent manner. PHA739358 dose-dependently and time-dependently promoted the dissection of PARP (poly (ADP-ribose) polymerase); down-regulated the expressions of Bcl-2, p-AuroraA, p-histone H3, CyclinB1 and up-regulated the levels of Bax, p-Cdc25c, p-Cdc2, P21 and P53 protein. PHA739358 had no significant effects on the expressions of AuroraA and histone H3. Flow cytometry and fluorescence microscope showed that PHA739358 significantly induced apoptosis. Flow cytometry showed the rate of apoptosis significantly increased from 0.31% ± 0.03% to 40.6% ± 0.81%. The proliferation-inhibiting and apoptosis-inducing effects of PHA739358 may provide new therapeutic approaches of breast cancer.

Phosphorylation of HOX11/TLX1 on Threonine-247 during mitosis modulates expression of cyclin B1

BackgroundThe HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11.ResultsCell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis.ConclusionsOur results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint.

Bimodal expression of Sprouty2 during the cell cycle is mediated by phase-specific Ras/MAPK and c-Cbl activities.

Sprouty2 is an important inhibitor of cell proliferation and signal transduction. In this study, we found a bimodal expression of Sprouty2 protein during cell cycle progression after exit from quiescence, whereas elevated Sprouty4 expression in the G1 phase stayed high throughout the rest of the cell cycle. Induction of the mitogen-activated protein kinase via activated Ras was crucial for increased Sprouty2 expression at the G0/G1 transition. Following the first peak, accelerated proteasomal protein degradation caused a transient attenuation of Sprouty2 abundance during late G1. Since the decline in its expression was abolished by dominant negative c-Cbl and the timely restricted interaction between Sprouty2 and c-Cbl disappeared at the second peak of Sprouty2 expression, we conclude that the second phase in the cell cycle-specific expression profile of Sprouty2 is solely dependent on ubiquitination by c-Cbl. Our results suggest that Sprouty2 abundance is the result of strictly coordinated activities of Ras and c-Cbl.

Cell cycle specific expression and nucleolar localization of human J-domain containing co-chaperon Mrj

J-domain containing co-chaperone Mrj (mammalian relative to DnaJ) has been implicated in diverse cellular functions including placental development and inhibition of Huntingtin mediated cytotoxicity. It has also been shown to interact with keratin intermediate filaments. Since keratins undergo extensive reorganization during cell division, its interactor Mrj might also play an important role in the regulation of cell cycle. In support of this hypothesis, we report the up-regulation of Mrj protein in M-phase of HeLa cells implicating its role in mitosis related activities. The protein is dispersed throughout the cell during late mitosis and is localized in nucleolus during interphase, confirming that the activity of Mrj is regulated by its cell cycle specific expression together with its differential subcellular localization.

Self-Organizing Maps with Statistical Phase Synchronization (SOMPS) for Analyzing Cell Cycle-Specific Gene Expression Data

Based on previous studies related to the yeast cell cycle, it is well known that the underlying cellular network in yeast consists of many interactions between genes that have periodic expression patterns during the cell division cycle. In this study, it is proposed that cell cycle-specific gene expression can be understood as a phenomenon of collective synchronization or, in other words, an ensemble of non-identical oscillating response signals from different systems. Therefore, we aimed to apply the theory of statistical multivariate phase synchronization to understand the cell's cyclic transcriptome as a phenomenon of collective synchronization. To this end, a novel algorithm called Self-Organizing Maps with statistical Phase Synchronization (SOMPS) is proposed and evaluated using yeast cell cycle-specific gene expression data. From the evaluation experiments, we draw the following conclusions: 1) It is possible to find groups of genes that have biological interactions with each other and significantly share gene ontology slim terms of biological processes using the theory of multivariate phase synchronization with cell cycle-specific gene expression signals; 2) Among all output clusters of SOMPS, a relatively large cluster with high periodicity with respect to its trained mean field can be considered a prominent cluster; 3) For each gene, it is possible to identify the degree of the strength of its biological interactions with other genes using the coupling strength of synchronization with its trained mean field; and 4) It is feasible to understand cell cycle-specific expression patterns as a phenomenon of collective synchronization.

Detecting biological associations between genes based on the theory of phase synchronization

This study presents a novel approach to detect biological associations for gene pairs with cell cycle-specific expression profiles. Previous studies have shown that periodic transcription is commonly regulated by transcription factors that are also periodically transcribed, and there is a growing number of examples where cell cycle regulated genes are conserved in yeast and mammalian cells. Some genes have periodicity for their oscillatory activity throughout cell division. These cell cycle-specific oscillatory activities could be explained by a biological phenomenon in terms of efficiency and logical order. In the yeast data used in this study, about 13% of genes behave in this manner based on a previous yeast study. Microarrays have been applied to determine genome-wide expression patterns during the cell cycle of a number of different cells. Moreover, several previous studies have shown that many pairs of genes, which have linearly correlated expression profiles, have similar cellular roles or physical interactions. Based on this point of view, the traditional clustering methods have focused on similar expression profiles based on the premise that genes with similar expression profiles have similar biological functions or relevant biological interactions. However, there are a number of previous studies indicating that the expression of some genes may be delayed compared to others due to a time lag in their transcriptional control. Therefore, we propose a novel clustering method, named as phase-synchronization clustering, based on the theory of phase synchronization for detecting biological associations using cell cycle-specific expression profiles. We evaluate phase-synchronization clustering here using Saccharomyces cerevisiae microarray data. Phase-synchronization clustering is able to detect biologically associated gene pairs that have linearly correlated (simultaneous and inverted) as well as time-delayed expression profiles. The performance of phase-synchronization clustering is compared with other conventional clustering methods. The likelihood of finding relevant biological associations by phase-synchronization clustering is significantly higher than other clustering methods. Therefore, phase-synchronization clustering is more efficient for detecting known biological interactions for gene pairs than other conventional clustering methods for analyzing cell cycle-specific expression data. The evaluation analysis of the results by phase-synchronization clustering also suggests that the cellular activities during the cell division process could be understood as a phenomenon of collective synchronization.

CAMP regulates vegetative growth and cell cycle in Candida albicans

We demonstrate here the regulatory role of cAMP in cell cycle of Candida albicans. cAMP was found to be a positive signal for growth and morphogenesis. Phosphodiesterase inhibitor aminophylline exhibited significant effects, i.e., increased growth, as well as induced morphogenesis. Atropine and trifluoperazine negatively regulated (inhibited) growth and did not induce morphogenesis. These changes were attributed to increase in cAMP levels and protein kinase A (PKA) activity in presence of aminophylline, while reduction was observed in atropine and trifluoperazine (TFP) grown cells. Alteration in cAMP signaling pathway affected the cell cycle progression in Candida albicans. Increased cAMP levels in aminophylline grown cells reduced the duration of cell cycle by inciting the cell cycle-specific expression of G1 cyclins (CLN1 and CLN2). However atropine and trifluoperazine delayed the expression of G1 cyclins and hence prolonged the cell cycle. Implication of cAMP signaling pathway in both the cell cycle and morphogenesis further opened the channels to explore the potential of this pathway to serve as a target for development of new antifungal drugs.

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

Cell Cycle–Specific and Cell Type–Specific Expression of Rb in the Developing Human Retina

To define the pattern of Rb expression relative to cell cycle position and cell type in the developing human retina. Cryosections of fetal week 11-18 retinas were immunostained for Rb and cell cycle- or cell type-specific markers. Rb was prominent in retinal progenitor cells (RPCs) expressing the cyclin D1, cyclin A, and cytoplasmic cyclin B markers of G1, S, and early to mid G2 phases, but not in RPCs expressing the phosphohistone H3 marker of late G2 and M. Rb was not detected in the earliest postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrating away from the ventricular layer, but was detected as such cells underwent further differentiation. Among photoreceptors, Rb was not detected in the earliest RXRgamma(+) cone precursors or in the earliest Nrl(+) rod precursors, but subsequently rose to high levels in cones and to low levels in rods. Rb was prominent at the time when Müller glia exit the cell cycle and was generally expressed in a pattern complementary to p27(Kip1). Rb exhibits cell cycle-specific expression in RPCs, with loss in late G2-M and restoration in G1. Rb is re-expressed after postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrate away from the ventricular layer; after the appearance of early cone and rod markers; but coinciding with Müller glia cell cycle withdrawal. The results suggest that Rb does not mediate the initial proliferative arrest of retinal neurons, but may indirectly induce arrest in RPCs or maintain an arrest in postmitotic precursors.

Proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in the marine dinoflagellate Prorocentrum donghaiense Lu and the green alga Dunaliella salina Teodoresco

Proliferating cell nuclear antigen (PCNA) was detected in Prorocentrum donghaiense Lu and Dunaliella salina Teodoresco by enhanced chemiluminescence techniques with one mono-antibody. The observed band detected on western blots of P. donghaiense and D. salina had a molecular weight of 36‐33 kDa rather than a PCNA-like protein with a size of 55 kDa reported in the dinoflagellates Crypthecodinium cohnii Biecheler and Gymnodinium catenatum Brav. The abundance of PCNA proteins was growth-stage dependent. Whole-cell immunoflurescence labeling showed that the PCNA antibody specifically stained the target proteins in P. donghaiense and D. salina, and PCNA is only present in the nucleus during the cell cycle. Synchronized cells of P. donghaiense show a cell cycle specific expression pattern with the highest expression in S phase and little expression in the G1 and G2/M phases. The results demonstrated that the PCNA-like proteins could be a marker for the estimation of marine phytoplankton growth rates. The different sizes of the PCNA-like proteins observed in dinoflagellates could be related to the variety of dinoflagellate chromosomal structure.

Mutant MyoD lacking Cdc2 phosphorylation sites delays M-phase entry.

The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G(2)/M transition by controlling the expression of p21(Waf1/Cip1) (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G(2). In growing myoblasts, MyoD reaccumulates during G(2) concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G(2) and delays M-phase entry. This G(2) arrest is not observed in p21(-/-) cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G(2)/M transition.

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells.

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.

Nuclear Localization and Cell Cycle-specific Expression of CtIP, a Protein That Associates with the BRCA1 Tumor Suppressor

The BRCA1 tumor suppressor has been implicated in a diverse spectrum of cellular processes, including transcriptional regulation, DNA repair, and cell cycle checkpoint control. CtIP was recently identified as a protein that associates with BRCA1 and two other nuclear factors, CtBP1 and Rb1. To understand the functions of CtIP, we have evaluated its biological properties with respect to those of BRCA1. Our results show that CtIP, like its associated factors, is predominantly a nuclear protein. A subset of the endogenous pool of CtIP polypeptides exists in a protein complex that includes both BRCA1 and the BRCA1-associated RING domain protein (BARD1). At the protein level, CtIP expression varies with cell cycle progression in a pattern identical to that of BRCA1. Thus, the steady-state levels of CtIP polypeptides, which remain low in resting cells and G(1) cycling cells, increase dramatically as dividing cells traverse the G(1)/S boundary. In contrast to BRCA1, however, the G(1)/S induction of CtIP expression is mediated primarily by post-transcriptional mechanisms. Finally, the interaction between CtIP and BRCA1 is shown to be stable in the face of genotoxic stress elicited by treatment with UV light, adriamycin, or hydrogen peroxide. Together, these results indicate that CtIP can potentially modulate the functions ascribed to BRCA1 in transcriptional regulation, DNA repair, and/or cell cycle checkpoint control.

A G1 cyclin is necessary for maintenance of filamentous growth in Candida albicans.

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.

Activation of Heat-Shock Transcription Factor 1 in Heated Chinese Hamster Ovary Cells Is Dependent on the Cell Cycle and Is Inhibited by Sodium Vanadate

Inducible heat-shock protein 70 (HSP72) is expressed in a cell cycle-specific manner in Chinese hamster ovary (CHO) cells after heating for 15 min at 45.0 degrees C, with the highest level in S-phase cells. Since heat shock induces the transcription of heat-shock proteins through the transactivation of heat-shock elements (HSEs) by heat-shock factor HSF1, we wished to determine whether the cell cycle-specific expression of HSP72 was regulated at the level of transcription. The levels of HSF1 did not vary through the cell cycle, as measured by polyclonal antibodies and flow cytometry. The binding of HSF1 to the heat-shock element was measured with the gel mobility shift assay using cell extracts from Hoechst 33342-labeled heated cells sorted from G1, S and G2/M phases. The HSF1-HSE binding activity was twofold higher in S phase than in G1 or G2/M phase. When CHO cells were exposed to 10 microM sodium vanadate, an inhibitor of tyrosine phosphatase, for 24 h before heat shock, the binding of HSF1 to HSE was reduced by a factor of 2 and the level of HSP72 was greatly reduced. The HSF1 binding to HSE was completely eliminated by using anti-HSF1 antibody in the gel mobility shift assays. Antibodies against HSP73 did not reduce the HSF1-HSE binding activity, but antibodies against HSP40 actually increased the binding activity. These results support the hypothesis that cell cycle-dependent binding of HSF1 to HSE is the cause of the cell cycle-specific expression of HSP72 in heated CHO cells and is regulated by phosphorylation.

The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells.

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.