Cell Cycle-dependent Expression Research Articles

Cell cycle-dependent expression of the CHO2 antigen, a minus-end directed kinesin-like motor in mammalian cells

A 69 kDa protein present in the interphase centrosome and mitotic spindle/pole was identified with the CHO2 monoclonal antibodies raised against mitotic spindles isolated from Chinese hamster ovary (CHO) cells. Isolation and characterization of antigen-specific cDNAs and recombinant proteins demonstrated that the protein is a minus-end-directed microtubule motor with the motor domain located at the C terminus. Affinity-purified polyclonal antibodies prepared to bacterially expressed fusion proteins revealed the presence of the antigen in interphase nuclei, and the degree of nuclear immunostaining intensity varied among cells at different cell cycle stages. In order to examine the change of antigen expression during the cell cycle, we prepared synchronized populations of CHO cells, double stained with CHO2 and PCNA (proliferating cell nuclear antigen) antibodies, and quantitated the amounts of nuclear fluorescence using the MetaMorpho image analysis software package. Cells right after cell division contained nuclei with the lowest level of CHO2 immunofluorescence. The immunofluorescence intensity progressively increases through G1 to S, reaching a maximum level by the end of G2. The antibody uniformly stained the entire nuclear region, and the total amount of fluorescence detected in G2 cells was greater than three times that of G1 cells. Cell cycle dependent accumulation of the CHO2 antigen was further confirmed by immunoblot analysis of the protein included in whole cell extracts and nuclei isolated from synchronized CHO cells. Northern blot analysis showed that, although the CHO2-transcript accumulated during later stages of the cell cycle, its abundance declined through G1 to S, and was lowest in cells at the early S phase. The difference in the expression pattern of the antigen protein and its transcript may suggest the presence of multiple mechanisms controlling the level of CHO2 antigen during the course of the cell cycle.

Cell cycle--dependent expression of L- and T-type Ca2+ currents in rat aortic smooth muscle cells in primary culture.

The expression of L- and T-type Ca2+ channels has been reported to change during various biological events, including cellular differentiation and proliferation. The present study aimed to examine whether or not the expression of L- and T-type Ca2+ channels depends on the cell cycle in rat aortic smooth muscle cells in primary culture. Both the phase of the cell cycle and the functional expression of Ca2+ channels were determined in the same single cell, using an immunocytochemical analysis of cell cycle-specific nuclear antigens and a whole-cell voltage-clamp method, respectively. In the G0 (n = 130) and M (n = 75) phases, all cells showed only L-type Ca2+ currents. The cells showing a T-type Ca2+ current appeared in the G1 phase (37%, n = 85) and increased in the S phase (90%, n = 21). For L-type Ca2+ channels, the current density was significantly greater in the G1 phase than in the G0 and M phases. However, either the voltage-dependent properties or the dose-response relationships of Bay K 8644- and second messenger-induced modulations of L-type Ca2+ current did not differ in the four phases of the cell cycle. These findings thus indicate that the expression of L- and T-type Ca2+ channels depends on the cell cycle, whereas the characteristics of L-type Ca2+ channels do not differ between the phases of the cell cycle.

Transcriptional control of the cell cycle

Although a significant amount of evidence has demonstrated that there are intimate connections between transcriptional controls and cell cycle regulation, the precise mechanisms underlying these connections remain largely obscure. A number of recent advances have helped to define how critical cell cycle regulators, such as the retinoblastoma family of tumor suppressor proteins and the cyclin-dependent kinases, might function on a biochemical level and how such mechanisms of action have been conserved not only in the regulation of transcription by all three RNA polymerases but also across species lines. In addition, the use of in vivo techniques has begun to explain how the activity of the E2F transcription factor family is tied to the cell cycle dependent expression of target genes.

Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells. The Rad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA. By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Differential expression of D-type G1 cyclins during mouse development and liver regeneration in vivo.

D-type G1 cyclins are the primary cell cycle regulators of G1/S transition in eukaryotic cells, and are differentially expressed in a variety of cell lines in vitro. Little is known, however, about the expression patterns of D-type G1 cyclins in normal mouse in vivo. Thus, in the present study, tissue-specific expressions of cyclin D1 and D3 genes were examined in several tissues derived from adult male mice, and stage-specific expression of cyclin genes was studied in brain, liver, and kidney of developing mice from embryonic day 13 to postnatal day 11. Cell cycle-dependent expression of cyclins was also examined in regenerating livers following partial hepatectomy. Our results indicate that (1) cyclins D1 and D3 are expressed in a tissue-specific manner, with cyclin D1 being highly expressed in kidney and D3 in thymus; (2) cyclin D3 mRNA is abundantly expressed in young proliferating tissues and is gradually reduced during development, whereas cyclin D1 mRNA fluctuates during development; and (3) compensatory regeneration of liver induces cyclin D1 gene expression 12 hr after partial hepatectomy, and cyclin D3 gene expression from 36 to 42 hr (at the time of G1/S transition). In conclusion, this study indicates that cyclin D1 and D3 genes are differentially expressed in vivo in a tissue-specific, developmental stage-dependent, and cell cycle-dependent manner.

Cell cycle-dependent expression of the mouse

Cell cycle-dependent expression of the mouse

Substrate Specificity and Cell Cycle Regulation of the Nek2 Protein Kinase, a Potential Human Homolog of the Mitotic Regulator NIMA of Aspergillus nidulans

The human Nek2 protein kinase is the closest known mammalian relative of the mitotic regulator NIMA of Aspergillus nidulans. The two kinases share 47% sequence identity over their catalytic domains and display a similar cell cycle-dependent expression peaking at the G2 to M phase transition. Hence, it is attractive to speculate that human Nek2 and fungal NIMA may carry out similar functions at the onset of mitosis. To study the biochemical properties and substrate specificity of human Nek2 and compare them to those reported previously for other NIMA-related protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protein kinase and may undergo autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and beta-casein was found to be a suitable substrate for assaying Nek2 in vitro. By exploiting these findings, we have studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G1 but high during S and G2 phase. Taken together, our results suggest that human Nek2 resembles fungal NIMA in its primary structure, cell cycle regulation of expression, and substrate specificity, but that Nek2 may function earlier in the cell cycle than NIMA.

Rat genomic structure of amidophosphoribosyltransferase, cDNA sequence of aminoimidazole ribonucleotide carboxylase, and cell cycle-dependent expression of these two physically linked genes

Genomic structure of rat amidophosphoribosyltransferase (ATase; EC 2.4.2.14), which catalyzes the first committed step in de novo purine nucleotide synthesis, was determined by polymerase chain reaction (PCR)-based methods. There are 11 exons and all exon-intron boundaries were conserved among rat, human, and chicken ATase genes. A rat aminoimidazole ribonucleotide carboxylase (AIRC) cDNA encoding a bifunctional enzyme of AIRC (EC 4.1.1.21) at step 6 and SAICAR synthetase (EC 6.3.2.6) at step 7 in de novo purine nucleotide synthesis was cloned and sequenced. The size of the cloned rat AIRC cDNA was 1329 bp, and amino acid identity with human and chicken AIRC was 96 and 85%, respectively. The intergenic sequence using a phage clone and the PCR product disclosed that ATase and AIRC genes are physically linked with the 736 bp sequence between the translation start sites, and the determination of the transcriptional start sites by the primer extension assay for these genes disclosed that distance between the two major transcriptional start sites is 585 bp. The amount of mRNAs of both genes showed approx. 5–6-fold increase in G 1 S phase of the cell cycle over those in G 0 phase in synchronized rat 3Y1 fibroblasts.

Cell cycle-dependent modulation of alpha-interferon-inducible gene expression and activation of signaling components in Daudi cells.

To investigate cell cycle-dependent expression of interferon (IFN)-induced genes and signaling pathways, we examined the modulation of IFN-inducible gene expression and p91 activation, and JAK-1 and Tyk-2 kinases' activation in human Burkitt's lymphoma Daudi cells which were synchronized at different points in the cell cycle progression. We observed that each one of 100-, 67-, and 46-kDa 2'-5'-oligoadenylate synthetase (2-5(A)-synthetase) proteins had a unique expression pattern in different phases of the cell cycle which was different from the expression of 2-5(A)-synthetase proteins in exponentially growing Daudi cells. The cell cycle-dependent changes in the overall levels of expression of 2-5(A)-synthetase proteins correlated well with the induction of expression of 2-5(A)-synthetase mRNA as well as the appearance of IFN stimulatory gene factor-3 and expression and activation of p91 in cells released from the G1/S block in the absence of exogenous IFN-alpha. In addition, cells from the G1/S phase exhibited increased tyrosine phosphorylation of JAK-1 and Tyk-2 kinases and p91 by IFN-alpha as compared with asynchronized cells. Enhanced stimulation of activation of JAK-1, Tyk-2, and p91 by IFN-alpha was progressively reduced from G1/S to G2/M to pass G2/M. These findings demonstrate that the levels of constitutive and inducible expression of IFN-alpha-inducible genes, activation of p91, and tyrosine phosphorylation of JAK-1 and Tyk-2 kinases are differentially influenced by the state of the cell cycle.

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells.

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Cyclin D1 expression is regulated by the retinoblastoma protein.

The product of the retinoblastoma susceptibility gene, pRb, acts as a tumor suppressor and loss of its function is involved in the development of various types of cancer. DNA tumor viruses are supposed to disturb the normal regulation of the cell cycle by inactivating pRb. However, a direct function of pRb in regulation of the cell cycle has hitherto not been shown. We demonstrate here that the cell cycle-dependent expression of one of the G1-phase cyclins, cyclin D1, is dependent on the presence of a functional Rb protein. Rb-deficient tumor cell lines as well as cells expressing viral oncoproteins (large tumor antigen of simian virus 40, early region 1A of adenovirus, early region 7 of papillomavirus) have low or barely detectable levels of cyclin D1. Expression of cyclin D1, but not of cyclins A and E, is induced by transfection of the Rb gene into Rb-deficient tumor cells. Cotransfection of a reporter gene under the control of the D1 promoter, together with the Rb gene, into Rb-deficient cell lines demonstrates stimulation of the D1 promoter by Rb, which parallels the stimulation of endogenous cyclin D1 gene expression. Our finding that pRb stimulates expression of a key component of cell cycle control, cyclin D1, suggests the existence of a regulatory loop between pRb and cyclin D1 and extends existing models of tumor suppressor function.

Cell cycle-dependent expression of cyclin D1 and a 45 kD protein in human A549 lung carcinoma cells.

Cyclin D1, which is suggested to have a role in G1 control during the cell cycle, is genetically linked to BCL-1 and is widely overexpressed in parathyroid, breast, and squamous cancer cells. We postulated that cyclin D1 regulation may also be important in lung cancer. Therefore, we characterized the cell cycle-dependent expression of cyclin D1 at both mRNA and protein levels in synchronized human A549 lung carcinoma cells. Monospecific anti-cyclin D1 C-terminal peptide antibodies recognized both p36cyclinD1 and an as-yet uncharacterized 45 kD protein (p45). A549 cells were synchronized with well-studied drugs. Cyclin D1 mRNA expression remained relatively constant, with less than a twofold fluctuation during the cell cycle and with a minor peak at M phase. However, the p36cyclinD1 protein fluctuated during the A549 cell cycle and was expressed at very low levels in late G1 and at the G1/S boundary, but then increased in S phase and peaked at M phase. In contrast, p45 protein was expressed at relatively high levels in late G1 and reached maximal levels at the G1/S boundary, was expressed at decreased levels in S phase, and then had disappeared by M phase. Moreover, p45 was highly expressed only in transformed alveolar epithelial cells, but not in normal rat alveolar epithelial cells or fetal rat lung fibroblasts in primary cultures. In mink Mv1Lu cells, the expression of p45 was totally blocked by transforming growth factor-beta 1 treatment or contact inhibition. p45 protein was phosphorylated on serine, threonine, and tyrosine residues in A549 cells in culture. The phosphorylation of the p45 protein was cell cycle-regulated and reached its maximal levels at G2/M phase. The p45 protein had a different peptide map from p36cyclinD1 after cleavage with N-chlorosuccinimide. Immunoprecipitation studies showed that p45 was also anti-ubiquitin immunoreactive during the cell cycle. We conclude that p36cyclinD1 and the p45 protein are differentially regulated in a cell cycle-dependent manner in A549 cells. Although p45 is antigenically related to p36cyclinD1, it is probably not a closely cyclin-related protein. We speculate that p45 may be associated with malignant transformation and may play a distinct role from p36cyclinD1 in regulation of the cell cycle in A549 cells.

Regulation of Asialoglycoprotein Receptor Expression in Rat Hepatocytes Cultured under Proliferative Conditions

The expression of many glycoproteins on the surface of hepatocytes has been related to the state of differentiation of the liver. In particular, the asialoglycoprotein receptor (ASGP-R) is modulated in many physiological and pathological conditions in which hepatocyte proliferative activity is modified. We studied ASGP-R expression in primary monolayer cultures of rat hepatocytes stimulated to proliferate by the addition of epidermal growth factor and insulin. In proliferating hepatocytes the receptor concentration on the cell surface was lowered, with no modifications in its distribution; similarly, both the binding activity and the amount of specific transcripts were decreased. The decreases were related to the peak of cellular growth, as judged by [ 3H]thymidine incorporation into DNA. The results suggest the presence of a cell cycle-dependent ASGP-R expression also in in vitro systems; this control could be exerted by a decreased availability of receptor-specific mRNA molecules or on the stability of transcripts.

Proximal promoter region of the wheat histone H3 gene confers S phase-specific gene expression in transformed rice cells.

The cis-regulatory elements that confer cell cycle-dependent expression to the wheat histone H3 gene were investigated in rice cells (Oc strain) transformed with H3/GUS chimeric genes. 5' deletion mutants of the H3 promoter region (from -1711, -908 or -185 to +57 relative to the transcription start site) were joined to the coding sequence of the bacterial beta-glucuronidase (GUS) gene then introduced stably into rice cells. S1 analyses of the RNA from transformed rice cells whose cell cycles had been synchronized by treatment with aphidicolin showed that the steady-state levels of the transcripts from chimeric genes were altered with the change in DNA synthesis and the content of rice H3 mRNA throughout the cell cycle. Even though H3 promoter activity decreased as 5' deletion proceeded, transcripts from the chimeric genes showed increases, as much as 10-fold 1 h after release from the aphidicolin block, which were rapidly lost over the next 4 h. The results suggest that the 242 bp sequence from -185 to +57, which contains the basal promoter region, confers the S phase-specific expression of the H3 gene and that the upstream sequence from position -186 is required for the full activity of this promoter.

Coordinate gene expression of five subclass histones and the putative transcription factors, HBP-1a and HBP-1b, of histone genes in wheat.

The expression of genes encoding five histones (H1, H2A, H2B, H3 and H4) and the putative transcription factors HBP-1a (17) and HBP-1b (c38) was examined during early germination and in various tissues of young wheat seedlings. The steady-state levels of core histone (H2A, H2B, H3 and H4) mRNAs were coordinately cell cycle-dependent and paralleled the rate of DNA synthesis during early germination, whereas the expression pattern of the linker histone (H1) genes differed. The five subclass histone genes were actively expressed in the meristematic tissues of young seedlings. Moreover, H1 genes were expressed in leaves that consist mostly of non-proliferating cells, in which core histone genes showed little expression. Quantitative alterations to the mRNAs of the putative transcription factors HBP-1a (17) and HBP-1b (c38) of wheat histone genes were similar to those of the core histone mRNAs, suggesting that both factors function in the cell cycle-dependent expression of wheat core histone genes.

PRAD1/lCyclin D1 proto‐oncogene: Genomic organization, 5′ dna sequence, and sequence of a tumor‐specific rearrangement breakpoint

PRAD1 (previously D11S287) is a putative proto-oncogene at 11q13, activated by overexpression through gene rearrangement or gene amplification in several types of human tumors including parathyroid adenomas, centrocytic lymphomas and other B-cell tumors with t(11;14), and breast cancers. PRAD1 (also CCND1) encodes cyclin D1, which may regulate the G1-S phase transition in the cell cycle. Here, we report the cloning and characterization of the chromosomal PRAD1/cyclin D1 gene and the sequence of its promoter region. The gene spans about 15 kb and has 5 exons; its promoter region has Sp1 binding sites and no obvious TATA box, characteristics of housekeeping genes and growth-regulating genes. Furthermore, an E2F binding motif present close to the major transcription start site may be involved in cell cycle-dependent expression of this gene. We also report the sequence of DNAs spanning joining regions of a reciprocal parathyroid hormone/PRAD1 gene rearrangement in a parathyroid adenoma. Comparison with normal sequences suggests that the rearrangement was not a simple break-and-ligate event, but rather involved multiple steps, including two microdeletions and a microinversion. Very short sequences conserved near the breakpoints and symmetrical elements in the eventually inverted DNA segment might have played a role in this illegitimate complex recombination, which may have similarities with a constitutional translocation in Duchenne muscular dystrophy.

Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells.

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G1 at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G1 and enter S phase. As data presented here show that mRNA synthesis is needed for 2-3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G1 arrest protocol to analyze gene expression in late G1. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G1, genes normally expressed in late G1 were expressed only after release from the V point. The expression of late G1 genes in cells released from the V point was temporally similar, in respect to G1 location, as was seen in stimulation of quiescent G0 cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G1 and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G1, is required for BALB/c-3T3 fibroblasts to traverse late G1 and initiate DNA synthesis.

Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gene transcription and alternative RNA splicing in a model of developing intestinal epithelium.

Transcriptional and post-transcriptional regulation of CFTR (cystic fibrosis transmembrane conductance regulator) gene expression was studied in HT29 cells. It is known that the abundance of CFTR mRNA increases during differentiation of pluripotent HT29-18 cells and is maintained at high levels in the stably differentiated HT29-18-C1 subclone. Nuclear run-on assays suggest that increased transcription of the CFTR gene explains the increased abundance of total CFTR mRNA in differentiated HT29 cells. The increased transcription cannot be ascribed to cell cycle-dependent expression of the CFTR gene or to changes in CFTR gene copy number between subcloned cells. Similar to native tissue cells, differentiated HT29 cells contain low copy numbers of CFTR transcripts (1-5/cell), and a portion of the CFTR transcripts are alternatively spliced to remove exon 9 (and make 9-mRNA). During differentiation of HT29-18 cells, the absolute amount of full-length CFTR mRNA increases 8-fold, whereas the amount of 9- mRNA increases 18-fold. The fraction of 9- mRNA in the CFTR mRNA pool is increased in differentiated HT29 cells. The results show that gene transcription regulates the abundance of CFTR transcripts and that regulatory control of alternative RNA splicing may also be a cellular mechanism to modulate CFTR function.

Downregulation of histone H4 gene transcription during postnatal development in transgenic mice and at the onset of differentiation in transgenically derived calvarial osteoblast cultures.

In vivo regulation of cell cycle dependent human histone gene expression was examined in transgenic mice using a fusion construct containing 6.5 kB of a human H4 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transcriptional control of histone gene expression, as a function of proliferative activity, was determined. We established the relationship between DNA replication dependent H4 mRNA levels (Northern blot analysis) and H4 promoter activity (CAT assay) during postnatal development in a broad spectrum of tissues. In most tissues sampled in adult animals, the cellular representation of H4 gene transcripts declined in parallel with promoter activity. This result is consistent with transcriptional control of H4 gene expression at the cessation of proliferation. Interestingly, while H4 mRNA was detectable at very low levels post-proliferatively in brain, promoter activity persisted in adult brain, where most of the cells are terminally differentiated. This dissociation between histone gene promoter activity and histone mRNA accumulation points to the possibility of post-transcriptional regulation of histone gene expression in brain. Cultures of osteoblasts were prepared from calvaria of transgenic mice carrying the H4 promoter/CAT reporter construct. In contrast to the brain, in these bone-derived cells, we established by immunohistochemistry that the transition to the quiescent, differentiated state is associated with a transcriptionally mediated downregulation of histone gene expression at the single cell level.