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MCF-7 breast cancer cells increase their rate of proliferation, as indicated by incorporation of tritiated thymidine into DNA, when exposed to estrogen. In confirmation of other studies, 10 nM 17beta-estradiol (E2) increased proliferation by 2.8-fold after 6 days of exposure. As indicated by trypan blue exclusion and TUNEL assays, cell survival was increased and apoptosis decreased by the presence of E2. The estradiol metabolite 2-methoxyestradiol (2-ME) when present in the culture medium in concentrations greater than 1 microM for three days, dose-dependently reduced the effectiveness of E2 on cell proliferation by increasing the rate of apoptosis. To examine a mechanism for the increase in apoptosis, expression of p34cdc2 and cyclin B1 protein levels were monitored by examination of immunoblots of their proteins. E2 increased p34cdc2 and cyclin B1 protein levels significantly after 6 days of exposure. This effect was inhibited significantly by the presence of 2-ME. The results indicate that up-regulation of p34cdc2 and cyclin B1 is closely associated with increased survivability and lack of apoptosis in estrogen-induced proliferation of MCF-7 cells. Further, anti-estrogenic effects of 2-ME in these cells can be accounted for by its activation of apoptotic functions, which are correlated with reductions in expression of p34cdc2 and cyclin B1 genes.

Cellular proliferation is regulated by several kinasic complexes associating cyclins and their catalytic subunits cyclin-dependent kinases (CDKs). In order to gain insight into the mechanisms underlying proliferation in non-Hodgkin's lymphoma (NHL), we examined the expression of certain cell cycle regulatory proteins normally expressed in lymphoid cells, cyclins A, B, D3 and E and cdk1, 2, 4, and 6. In 70 patients presenting a previously untreated lymphoma, cyclins and CDKs were studied by Western blotting and quantified by densitometry. Flow cytometry study of DNA content was carried out for all patients in order to study cell proliferation and level of ploidy. The results were analysed according to the histological types, the immunological phenotypes of the lymphomas and the outcome of the patients. Cdkl and cyclin A were correlated with the percentage of cells in S and S+G2/M phases, and significantly different according to the grade of malignancy, with the lowest expression in low-, and the highest in high-grade NHL according to the Working Formulation. In B-NHLs, cdk1, cyclin A, as well as cdk2, cyclin D3 and E expression was higher in the aneuploid than in the euploid group. Our results point to some particularities of cell cycle regulation in two lymphoma sub-types: 1) a low expression of cyclin D3 and cdk6 in mantle cell lymphomas and 2) a discrepancy between the high proliferative activity and the level of protein expression in Burkitt's lymphomas. CDK1 and cyclin A showed a significant prognostic value for achievement of complete remission (Cdk 1) and for both disease free (cyclin A) and overall survival (cyclin A and cdk1): low protein level was associated with the best prognosis in B-NHLs. Our results show that differential cell cycle regulating protein expression may be associated with different biological and clinical behaviour of NHLs and confirm the usefulness of the study of cell cycle regulation as a tool for understanding lymphoid malignancies.

Objective: The effects of the gonadotropin releasing hormone (GnRH) agonist (D-Trp(6)) were examined in two human ovarian cancer cell lines and in severe combined immune deficiency (SCID) mice to evaluate its potential as a cytocidal, cytostatic, or differentiating antitumor agent., Methods: We treated the human ovarian cancer cell lines OVCAR-3 and SKOV-3 for 5 or 7 days and sex-matched SCID mice with GnRH agonist for 29 days. The antitumor effect of GnRH agonist were studied in various aspects. To confirm the antiproliferative effect, we used 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide colorimetric assay, in vitro, and a serial measurement of tumor growth in vivo. The disturbances of progression in the cell cycle and the changes of cyclin-dependent kinase 1 following treatment with GnRH agonist were evaluated with flow cytometric analysis in vitro. The induction of apoptosis following treatment with GnRH agonist was studied using in situ terminal deoxyribonucleotidyl transferase (Tdt) and further quantitated with ELISA in vitro. The presence of telomerase activity following treatment with GnRH agonist was measured by PCR-based telomeric repeat amplification protocol and ELISA detection in cell lines and xenografts in vitro and in vivo., Results: Continuous exposure of cell lines and xenografts to GnRH agonist resulted in growth inhibition of cancer cells in a dose- and time-dependent manner. In cultured cells, the GnRH agonist blocked cell cycle progression in G0/G1 phase and thus reduced the number of cells in S and G2/M phases. The phenomenon of apoptosis was documented in cultured cells treated with GnRH agonist by in situ Tdt assay. The frequency of apoptotic cells in the in situ Tdt assay was 5-6% compared with control, 4-5%. Apoptosis quantified by ELISA revealed a high incidence in cultured cells treated with GnRH agonist. The activities of telomerase in cell lines and xenografts were not decreased by GnRH agonist. There were not any significant changes of expression of CA-125 by flow cytometry and of the cellular morphology observed with light microscopy., Conclusions: Our results indicate that the antiproliferative effect of GnRH agonist in epithelial ovarian cancer cells may be mainly attributed to cytostatic activities resulting in blocking of cell cycle progression in the G0/G1 phase and minimally related to the induction of apoptosis., (Copyright 1999 Academic Press.)

We have previously shown that Actinobacillus actinomycetecomitans produces an immunosuppressive factor (ISF) capable of impairing human lymphocyte function by perturbing cell cycle progression. We now report that ISF is the product of the cdtB gene, one of three genes encoding the family of cytolethal distending toxins (Cdt). The ISF polypeptide exhibits >/=95% identity with Hemophilus ducreyi CdtB protein and

Background: Abnormalities of the cyclin-dependent protein kinase (CDK) machinery have been linked to cancer development. Hyperphosphorylation of the retinoblastoma (Rb) protein results in release from inhibition of progression through the G1 phase of the cell cycle. Hyperexpression of CDK1 and CDK2 may enable progression through late G1, S and the G2 phases of the cell cycle., Methods/results: To investigate tumor-associated protein kinase activities, control and tumor samples were fractionated by MonoS chromatography and tested for their ability to phosphorylate histone H1. Two major peaks of histone H1 phosphotransferase activity were resolved. The first appeared in the flow through fractions, and occasionally showed enhanced activity in the tumor samples, whilst the second was consistently increased and eluted at approximately 0.4 M NaCl. Western immunoblotting with CDK1 and PSTAIRE antibodies confirmed the co-elution of CDK1 and CDK2 with the second peak. Next, the phosphotransferase activities (following specific immunoprecipitation) and protein levels of CDK1, 2, 4, and 6 were determined in human colon cancer samples and their respective controls. CDK4 activity was elevated in only 3 of 7 tumor samples (range 40-160%) relative to control samples from the same patients, whereas a significant increase in CDK6 activity was observed in the same group (p < 0.05). This contrasted sharply with the universal activations of CDK1 (up to 18-fold, p < 0.01, n = 12) and CDK2 (up to 17-fold, p < 0.05) in the same groups., Conclusions: CDK1 especially, and to a lesser extent CDK2 and CDK6 demonstrate the most consistent biochemical activation in human colon cancer and may represent targets for pharmacological intervention. Cellular proliferation as gauged by MIB1 was not directly correlated with the amplitude of activation.

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.

Despite the repeatedly observed impaired proliferative response of T lymphocytes from aged donors, the precise molecular basis underlying such a defect is still poorly understood. The aim of this study was to determine whether cyclin-dependent kinase 1 (cdk1), a serine-threonine kinase required for entry into mitosis, is implicated in this age-associated dysregulation of the cell cycle. T lymphocytes derived from young and elderly donors were blocked in S phase by hydroxyurea after a 48-h activation by anti-CD3 Abs. Under these experimental conditions, only the cells that were already located beyond the S phase were able to complete the cell cycle, decreasing their DNA content from 4n to 2n chromosomes. Using this procedure, a delay in the accomplishment of mitosis could be observed in cells from elderly individuals, as evidenced by propidium iodide staining. In this age group, only a minimal cdk1 activity could be immunoprecipitated from cells sorted in G2/M after nocodazole block. The decrease in cdk1 activity observed in T lymphocytes from aged donors could be accounted for by at least three mechanisms: 1) a failure of these cells to express a sufficient amount of cdk1, 2) a reduced level of the associated cyclin B1, and 3) an incomplete dephosphorylation of the kinase on tyrosine. This low cdk1 activity is likely to postpone the progression through the G2/M transition and participates in the dysfunction of the cell cycle during the process of aging.

Platelet-derived growth factor AB (PDGF-AB) has to be permanently present in the culture medium to achieve full proliferation (>90%) of AKR-2B fibroblasts. Upon removal after 1 h incubation time, only a small number of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis. The PDGF-receptor autophosphorylation reached a maximum after 30 min incubation with PDGF-AB. Tyrosine phosphorylation was no longer detectable after 2-4 h. The clustering of receptors into coated pits, analyzed by indirect immunofluorescence using a specific antibody against PDGF-beta-receptor, showed in contrast to autophosphorylation a biphasic kinetic. A first maximum was reached after 30 min, followed by a complete disappearance of coated pits, which regenerated in a second phase after 3 h and were long lasting. If PDGF-AB was removed after 1 h, the second phase was obliterated. The involvement of two different signalling pathways in these two phases was investigated in detail: (1) The ras-raf-MAP-kinase pathway and (2) the PI-3-kinase/p70(S6)-kinase pathway. PDGF-AB addition caused a fast (10 min) activation of MAP-kinase, which returned to background level after 1 h without any further activation later on. In contrast PDGF-AB led to a rapid (15-30 min) activation of the p70(S6)-kinase that persisted for 8-12 h just prior to the entry of the cells into S-phase. If PDGF-AB was removed after 1 h, the activation of this kinase ceased 3 h later. PDGF-AA, which is unable to promote division of AKR-2B cells, induced only a shortlasting p70(S6)-kinase activation. These observations add further evidence for the involvement of the p70(S6)-kinase pathway in the proliferation control of AKR-2B fibroblasts in the late G1 phase (4-8 h after growth factor addition). On the other hand, if the p70(S6)-kinase activation was prevented by the addition of 10 nM rapamycin, the cell division was not inhibited but only delayed by 4 h. Similar kinetics were observed when the PI-3-kinase was inhibited by 400 nM wortmannin. It is suggested that a regulatory element exists upstream of the p70(S6)-kinase and the PI-3-kinase. This regulatory element should be responsible for the transmission of late signals required for the progression through the cell cycle. This element is not involved in the immediate responses after PDGF-AB addition but must be stimulated within a second later phase of PDGF activation., (Copyright 1998 Academic Press.)

A protein-independent fibrosarcoma, Gc-4 PF, grows exponentially in a protein-free medium. The doubling time (approximately 26 h) was similar to that of the serum-dependent parental clone, Gc-4 SD cultivated in the presence of fetal calf serum (FCS). We demonstrated here that the protein-free cultivation of Gc-4 PF cells concomitantly activates apoptotic phenotypes (one third of total cell population), including typical morphology, high uptake of Hoechst 33342 dye, and cleavage of DNA to large fragments, as observed in protein-deprived Gc-4 SD cell previously. Gc-4 SD cells arrested in the G0/G1-phase in response to the protein-free condition. In contrast, Gc-4 PF cells did not reach G0/G1 arrest in the protein-free condition; instead the durations of both G0/G1 and G2-phases were markedly reduced. The estimation of one cell cycle duration revealed that the cell division cycle was accelerated to 1.7 (27 h/15.4 h)-fold. Then the growth kinetics was able to be verified quantitatively by both the cell division rate and apoptotic cell loss. Protein-free cultivation resulted in slight down-regulation of c-myc protein in both cell types, while the down-regulation of p34cdc2, shown clearly in Gc-4 SD cells, was avoided in Gc-4 PF cells. Interestingly, while the expression of p53 was not affected in Gc-4 SD cells in response to the protein-free condition, the suppressor gene product expression was suppressed markedly in Gc-4 PF cells. These results suggest that Gc-4 PF cells may have acquired an ability to accelerate cell division by shortening the cell cycle duration to maintain a proper growth rate in response to intrinsic apoptosis activation with, at least in part, a suppression of p53 expression as well as an escape of down-regulation of p34cdc2.

Cyclin dependent kinases propel the cell cycle in collaboration with cyclins. We have examined the expression of cdk2/cdc2 in adenoma and focal carcinoma in adenomatous tissue to explore their role in tumorigenesis of colorectum. Immunohistochemical study revealed that cdk2/cdc2 was overexpressed in a subsets of adenoma (14/50; 28.0%) but this overexpression was much more obvious in focal carcinoma (13/15; 86.7%). These results suggest that cdk2/cdc2 is remarkably upregulated together with a malignant change. In an effort to demonstrate a significant role for cdk2/cdc2 in colon cancer, we investigated growth and apoptosis with butyrolactone I, a specific inhibitor for cdk2/cdc2, using 4 colon carcinoma cell lines (HCT116, LoVo, HT29, Colo 320DM). Butyrolactone I inhibited proliferation of all colon carcinoma cell lines at 100 microM and it induced apoptosis in LoVo cell line with induction of p53. Our findings suggest that inhibition of cdk2/cdc2 may be a useful strategy against colon cancer.

Our study was designed to compare cellular kinetic parameters of classical Hodgkin's disease (HD) with those of anaplastic large cell lymphomas (ALCL-C, common type; and ALCL-HL, Hodgkin's like), with a particular focus on the G2/M transition. These disorders share some phenotypic properties, e.g., CD30 positivity of putative neoplastic cells. The percentages of cells expressing p34cdc2 (p34) and cyclin B-1 (cyclin-B), which form a complex (maturation/mitosis promoting factor, MPF) regulating the G2-M phases of the cell cycle, were also registered. Highly significant differences between HD and ALCL-C were recognized: a) in HD, evidence for abortive mitosis (i.e., difficulty to proceed beyond the metaphase stage) and consequent multinucleation and/or deletion of CD30+ cells was prominent, in contrast to ALCL-C. This was associated with a markedly lower fraction of large atypical cells (LAC) expressing cyclin-B in the cytoplasm and the nucleus (C + N) in HD than in ALCL-C; b) the extent of multinucleation of CD30+ cells in HD, but not in ALCL-C, was correlated with the %p34+ LAC; c) the proportions of LAC expressing p34 and/or cyclin-B (C) were positively related to the percentages of cyclin-B (C + N)+ LAC in ALCL-C but not in HD; d) in HD, in contrast to ALCL-C, the size of the fraction of cyclin-B (C + N)+ LAC did not correlate with the ana/telophase indices (ATI, reflecting successful completion of mitosis) and the magnitude of cell loss; e) in ALCL-C, the percentages of p34+ LAC were positively correlated with ATI or the degree of CD30+ cell deletion, but inversely in HD. With regard to all parameters mentioned above, ALCL-HL tended to take an intermediate position between HD and ALCL-C, but sided more with the latter. In conclusion, our present results suggest a derangement of MPF kinetics and functions that is more profound in HD than in ALCL-C.

Cyclin C, a putative G1 cyclin, was originally isolated through its ability to complement a Saccharomyces cerevisiae strain lacking the G1 cyclin gene CLN1-3. Unlike cyclins D1 and E, the other two G1 cyclins obtained by the same approach and subsequently shown to play important roles during the G1/S transition, there is thus far no evidence to support the hypothesis that cyclin C is indeed critical for the promotion of cell cycle progression. In BAF-B03 cells, an interleukin 3 (IL-3)-dependent murine pro-B-cell line, cyclin C gene mRNA was induced at the G1/S phase upon IL-3 stimulation and reached a maximal level in the S phase. Enforced expression of exogenous cyclin C in this cell line failed to alter its growth properties. In the present study, we examined whether cyclin C is capable of cooperating with the cytokine-responsive immediate-early gene products c-Myc and c-Fos in the promotion of cell proliferation. We found that cyclin C is able to cooperate functionally with c-Myc, but not c-Fos, to induce both BAF-B03 cell proliferation in a cytokine-independent fashion and the formation of cell clusters. Furthermore, cyclin C was primarily responsible for the induction of cdc2 gene expression. Our data define a novel role for cyclin C in the regulation of both the G1/S and G2/M phases of the cell cycle, and this effect appears to be independent of the activity of CDK8 in the control of transcription.

Expression of the cdk1 (p34cdc2) gene is enhanced 5-10 fold as cells re-enter the cell cycle from quiescence in response to serum-refeeding or following exposure to the protein phosphatase 1/2A inhibitor okadaic acid. Transient transfection analysis of nested deletions of the human cdk1 promoter identified regions that confer sensitivity to okadaic acid on a CAT-reporter gene. Putative okadaic acid response elements (OARE) were located between nt -942 to -763 (Site I) and nt -416 to -186 (Site II) before transcription start. The Site I element has enhancer-like characteristics as activity is independent of sequence orientation. Mobility shift analysis of Site I revealed the presence of 2 high molecular weight complexes, one of which was enhanced in the presence of okadaic acid-treated cell extracts. Site I contained several sequence motifs with conserved homology to heat shock response element core sequences and homeobox protein binding sites. Site II contained a myb-binding site, a G1/S phase enhancer, and 2 retinoblastoma response elements flanking an E2F binding site. Enhancement of cdk1 expression appears dependent on 2 nonhomologous okadaic acid-sensitive promoter regions.

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the invitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277-1289; N. Sinha et al., 1993 Genes Dev. 7: 787-797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro.

Axillary buds of pea (Pisum sativum L. cv. Alaska) do not grow on intact plants. Dormant axillary buds can be stimulated to grow rapidly after decapitation. Here, we isolated cDNAs of PCNA, cyclinB, cyclinD, and cdc2 from pea. The mRNA expression levels of these genes were very low in dormant axillary buds, whereas they remarkably increased after decapitation. Based on the mRNA accumulation patterns of these genes, we found that most cells in dormant axillary buds are arrested at the G1 phase in the cell cycle. There are four buds at the second node on pea seedlings. After decapitation, mRNAs became abundant in the large and small buds and were kept during the following 3 d. After 4 d, mRNAs were still present in the large bud, but not in the small bud. However, after removal of the large bud, the mRNA levels started to increase again in the small bud. These mRNA accumulation patterns were the same as those after the first decapitation. These results suggested that most cells in axillary buds at the second node are arrested at the G1 phase again and have the capacity to undergo multiple cycles of dormancy and growth. Moreover, in situ hybridization analyses demonstrated that PCNA mRNA increased in all parts of the axillary buds after decapitation.

Dramatic morphological, biochemical and cytological changes occur in parotid glands of rats and mice which have been treated with the beta-adrenergic receptor agonist isoproterenol (Ipr). Changes include glandular hypertrophy, induction of tissue-specific proline-rich proteins (PRPs), increases in cAMP, and occurrence of polyploidy. Similar changes also occur after feeding mice polyphenolic compounds commonly referred to as tannins. Data are presented which show that changes in cell cycle proteins are due to stimulation of the beta-adrenergic receptor by either isoproterenol or tannin treatment of mice. Both p34cdc2 mRNA and protein levels were elevated dramatically after mice were treated. Induction of p34cdc2 occurred within 24 hrs. and was transient during treatment. The beta1-adrenergic receptor antagonist atenolol blocked both tissue-specific expression of proline-rich proteins and induction of p34cdc2. Coincident with the increase in p34cdc2, cyclin-dependent kinase complexes containing cyclins A and B increased forty- and ten-fold, respectively. These results show that in mouse parotid glands activation of the beta1-adrenergic receptor by either the administration of isoproterenol or ingestion of dietary tannins induces synthesis of p34cdc2 and most likely contributes to the occurrence of polyploidy.

The cdc25C, cyclin A and cdc2 genes are regulated during the cell cycle through two contiguous repressor binding sites, the CDE and CHR, located in the region of transcription initiation and interacting with a factor termed CDF-1. The target of this repression seems to be transcriptional activation of these promoters by transcription factors bound upstream. The majority of these factors falls into the class of glutamine-rich activators, suggesting that CDF-1-mediated repression might be activation domain specific. In the present study we have used chimeric promoter constructs to demonstrate that the cdc25C UAS, but not the core promoter, is crucial for repression. In addition, we show that only specific transcription factors and activation domains are responsive to CDE-CHR-mediated cell cycle regulation. These observations clearly indicate that CDF-1 interferes with activation of transcription by a specific subset of transactivators. The repressible activation domains belong to the same class of glutamine-rich activators, pointing to specific interactions of CDF-1 with components of the transcription machinery. In agreement with this conclusion we find that a simple inversion of the CDF-CHR module completely abrogates cell cycle-regulated repression.

Numerous malignant neoplasias are found to contain varying proportions of high-ploidy cells. Although the role they play in the tumor is poorly understood, several lines of evidence suggest that these cells could be especially resistant to various aggressions, a possibility of great interest in cancer treatment. In the present study, we tested this hypothesis through the analysis of the presence of high-ploidy cells following the administration of the chemotherapeutic agent methotrexate. We also determined the expression of two proliferation markers, PCNA and CDK1, after methotrexate-treatment. Cultured cells from the murine melanoma B16F10 were treated with high doses of methotrexate for seven days prior to determination of DNA content and proliferation markers. Our results showed an obvious increase in the mean ploidy of this population. Specifically, there was a dramatic reduction in the proportion of tetraploid cells (predominant in the original population), and an increase in the proportion of cells with higher ploidies, particularly those whose DNA content was greater than 8c, including some cells with ploidies greater than 16c. Furthermore, there was a reduction in the number of PCNA-expressing cells and the reduction was much more marked in the case of CDK1 that was almost absent in the modal-ploidy treated cells. These alterations concerning ploidy and expression of proliferation markers had completely reverted two weeks after withdrawal of the drug. Our results indicate that methotrexate at a high dosage selects a cell population heterogeneous concerning its ploidy level, composed of one subpopulation of high-ploidy cells and another of modal-ploidy cells that, considering its lack of CDK1 expression, would remain in a latent state to evade the effects of the drug.

The cdc25C , cyclin A and cdc2 genes are regulated during the cell cycle through two contiguous repressor binding sites, the CDE and CHR, located in the region of transcription initiation and interacting with a factor termed CDF-1. The target of this repression seems to be transcriptional activation of these promoters by transcription factors bound upstream. The majority of these factors falls into the class of glutamine-rich activators, suggesting that CDF-1-mediated repression might be activation domain specific. In the present study we have used chimeric promoter constructs to demonstrate that the cdc25C UAS, but not the core promoter, is crucial for repression. In addition, we show that only specific transcription factors and activation domains are responsive to CDE-CHR-mediated cell cycle regulation. These observations clearly indicate that CDF-1 interferes with activation of transcription by a specific subset of transactivators. The repressible activation domains belong to the same class of glutamine-rich activators, pointing to specific interactions of CDF-1 with components of the transcription machinery. In agreement with this conclusion we find that a simple inversion of the CDE-CHR module completely abrogates cell cycle-regulated repression.

The cdc25C , cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study we have identified a factor, CDF-1, which interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro , in a manner similar to that seen by genomic footprinting. In agreement with in vivo binding data and its putative function as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avidly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B- myb promoter. Conversely, E2F complexes do not recognize the cdc25C CDE-CHR and CDF-1 is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at different stages during the cell cycle. While E2F-mediated repression seems to be associated with genes that are up-regulated early (around mid G1), such as B- myb , CDE-CHR-controlled genes, such as cdc25C , cdc2 and cyclin A , become derepressed later. Finally, the fractionation of native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF-1 and E2F.

Study of the function and regulation of the important cell cycle regulator cdc25 phosphatase has been hampered by the lack of a sensitive and specific substrate and assay. Here we report the production of a specific and sensitive substrate for the cdc25 phosphatase. The substrate is human cyclin B1/cdc2 phosphorylated on the inhibitory Thr14 and Tyr15 residues and activating Thr161 on cdc2, and is relatively simple to produce from readily available materials. The assay is based on the cdc25-specific dephosphorylation and activation of the phosphorylated cyclin B1/cdc2 substrate (PY15), using the increased histone H1 kinase activity of the activated PY15 as a read-out of cdc25 activity.

The mammalian cellular response to ionizing radiation results in delays in progression through the cell cycle at several checkpoints and includes alterations in the activity of cyclin-dependent kinases. The product of the CDC2 gene is a key kinase involved in cell cycle progression. The signaling events that regulate its expression after exposure to DNA-damaging agents are not known. We show that cdc2 mRNA and protein are down-regulated after irradiation of normal human and mouse fibroblasts with doses as low as 0.5 Gy. This down-regulation is preceded by induction of p53 and p21Waf1 proteins. In human cells in which p53 was nonfunctional and in p53-/- or p21-/- mouse embryo fibroblasts, no effect of ionizing radiation on p34cdc2 expression levels was observed. These findings indicate that CDC2 down-regulation after irradiation is p53-dependent and involves the cyclin-dependent kinase inhibitor p21Waf1 as a negative factor in the control of CDC2 expression. Correspondence between the delay in initiation of DNA synthesis in irradiated cells and the down-regulation of CDC2 is described.

Injections of parathyroid hormone (PTH) have been reported to stimulate skeletal accretion through increased bone formation in several species, and osteoblast proliferation is a critical component of bone formation. However, the biological mechanisms of PTH-stimulated bone cell proliferation are largely unknown. In this study, we demonstrated that PTH stimulates proliferation of the osteoblast precursor cell line, TE-85, in association with increasing cdc2 protein levels and its kinase activity. cdc2 antisense oligonucleotides blocked PTH-induced DNA synthesis and cell cycle progression. Analysis of the time course of PTH-stimulated cdc2 message levels demonstrated that cdc2 mRNA levels were increased 1.5- to 4-fold between 3-18 h following release from cell synchronization. Transfections of TE-85 cells with a series of cdc2 promoter-luciferase deletion constructs revealed PTH stimulation of the cdc2 promoter. Promoter constructs containing a mutation in the E2F binding site were not stimulated by PTH. Gel mobility shift assays demonstrated increased free E2F levels in TE-85 nuclear extracts in response to PTH. Furthermore, the ratios of hyperphosphorylated to hypophosphorylated forms of Rb protein were increased by PTH treatment. These results demonstrate that PTH-stimulated cdc2 expression was associated with TE-85 cell proliferation and that the mechanism of stimulating cdc2 gene expression involved increasing the levels of free E2F.

Objective: To investigate the role of expression of p34cdc2 protein kinase in normal, benign, and malignant ovarian epithelium., Material and Methods: Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34cdc2 protein kinase, followed by detection with use of a standard peroxidase-labeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed., Results: Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity was not associated with the histologic grade or surgical stage., Conclusion: Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in carcinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdc2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34cdc2 protein kinase in other malignant lesions suggests a common mechanism.

Full-grown mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment. By contrast, growing oocytes are not competent to resume meiosis; the molecular basis of meiotic competence is not known. Entry into M phase of the eukaryotic cell cycle is controlled by MPF, a catalytically active complex comprising p34cdc2 kinase and cyclin B. Incompetent oocytes contain levels of cyclin B comparable to those in competent oocytes, while their level of p34cdc2 is markedly lower; p34cdc2 accumulates abruptly at the end of oocyte growth, at the time of meiotic competence acquisition. We show here that this change in p34cdc2 concentration is not secondary to a corresponding change in the concentration of the cognate mRNA, indicating that translational control may be involved. Microinjection of translatable p34cdc2 mRNA into incompetent oocytes yielded high levels of the protein, but it did not lead to resumption of meiosis. Similarly, microinjection of cyclin B1 mRNA resulted in accumulation of the protein, but not in the acquisition of meiotic competence. By contrast, the microinjection of both p34cdc2 and cyclin B1 mRNAs in incompetent oocytes induced histone H1 and MAP kinase activation, germinal vesicle breakdown, and entry into M-phase including the translational activation of a dormant mRNA. Thus, endogenous cyclin B1 in incompetent oocytes is not available for interaction with p34cdc2, suggesting that a posttranslational event must occur to achieve meiotic competence. Microinjection of either p34cdc2 or cyclin B1 mRNAs accelerated meiotic reinitiation of okadaic acid-treated incompetent oocytes. Taken together, these results suggest that acquisition of meiotic competence by mouse oocytes is regulated at both translational and posttranslational levels.

After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Here we have used quail neuroretina as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become postmitotic between E7 and E8. To acquire a better understanding of the molecular events leading to quiescence in NR cells, we have analyzed the expression of cdc2 and of two activators of p34(cdc2): cyclin A and cyclin B2 in the developing neuroretina. We report that these three proteins are downregulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. We also report, using an immunohistochemical approach, that p34(cdc2) downregulation is correlated with the appearance of the microtubule-associated protein tau. These results strongly suggest that inhibition of cdc2 gene expression is closely linked to the achievement of terminal differentiation in neurons. However, we also show that postmitotic ganglion cells precursors begin to synthesize the early neuronal differentiation marker beta3-tubulin while p34(cdc2) is still detectable in these cells, suggesting that p34(cdc2) or a closely related kinase could play a role in some "young" postmitotic neurons.

We have shown previously that M-phase phospho-epitopes accumulate in neuronal tau proteins incorporated into the hallmark neurofibrillary tangles (NFT) of Alzheimer's disease (AD). In M phase, the epitopes are produced by cdc2/cyclin B1 kinase by a highly conserved mechanism believed to be quiescent in terminally differentiated neurons of adult brain. To determine whether an M-phase mechanism is possible in AD neurons, we first investigated the presence of cdc2 and cyclin B1 in AD. Both proteins were enriched in neurons with NFT and in neurons susceptible to NFT. An antibody specific for catalytically active cdc2 stained numerous NFT-containing neurons in AD but did not react with normal neurons. Double-labeling studies showed that active cdc2 and cyclin B1 coexist in AD neurons and co-localize with AD-specific mitotic phospho-epitopes. Mitotic kinase purified from AD and normal brain, using the yeast p13suc1 protein as affinity ligand, showed higher histone H1 phosphorylation activity in AD. Accordingly, the levels of cdc2 and cyclin B1 in p13suc1 fractions from AD were higher than normal. Consistent with a physiological relationship between NFT and mitotic kinase, NFT proteins co-purified with and became phosphorylated by the p13suc1-bound kinase in vitro. Furthermore, cdc2/cyclin B1 is the only one of several proline-directed kinases that created the TG/MC mitotic phospho-epitopes in recombinant tau in vitro. These findings suggest that aberrantly reexpressed cdc2/cyclin B1 in NFT-bearing neurons in AD brain contributes to the generation of M-phase phospho-epitopes in NFT.

Many agents have been known to induce the differentiation of HL-60 cells. However, only a small number of reports on the basophilic differentiation of this cell line are known. In this study we show that the exposure of HL-60 cells to chloroquine induces to differentiate into basophils. This chloroquine-induced change suggests that the increase in intracellular pH and the upregulation of p21 with subsequent downregulation of cdc2 kinase are triggers for basophilic differentiation of this cell line.

By interacting with key regulatory proteins such as the pRb family, cyclins, cyclin-dependent kinases and p300/CBP of host cells, adenoviral E1A interferes with various cellular processes to provide a suitable environment for the replication of viruses. E1A may promote DNA synthesis and cell cycle progression, immortalize rodent cells in culture and transform cultured cells in cooperation with E1B, Ras, or other oncoproteins. Both extreme N terminus and conserved region 1 of E1A are required for the immortalization and the transformation of rodent cells, transcriptional repression and specific induction of the expression of cellular genes such as the proliferating cell nuclear antigen (PCNA) and heat shock protein 70 (HSP70). Although the molecular mechanisms of these functions of E1A are not fully understood, it is believed that protein-protein interactions may play essential roles. In this communication, we report that a new set of cellular proteins with apparent molecular weight of 200, 90, 45, 30, and 28 specifically associate with the extreme N terminus of E1A. Further analysis demonstrate that these associations do not depend on E1A's association with p300 or pRB. Neither the 30 kDa nor the 28 kDa polypeptide is identical to Cdc2 or Cdk2. The region of E1A required for the protein interaction is also required for the recently identified N-terminal transactivation activity of E1A. Our observations suggest that in addition to p300/CBP, the new set of cellular proteins may be involved in the functional complexity of the N terminus of E1A, thus predicting a p300/CBP independent pathway.

We investigated the in vivo expression of cyclin B1 and Cdc2 (key molecules for G2-M transition during the cell cycle) in nonmalignant and cancerous human breast lesions using immunohistochemistry and quantitative proliferative index (PI) analysis. Breast epithelial cells co-expressed cyclin B1 and Cdc2 in their cytoplasm in the G2 phase and in their nuclei in the M phase. Cyclin B1, but not Cdc2, immunostaining rapidly disappeared from the nuclei during the mitotic metaphase to anaphase transition. Static image analysis revealed the mean proliferative index for cyclin B1/cdc2 for each type of lesion to be as follows: normal glands (n = 20), 2.0/2.5%; benign lesions, including typical ductal hyperplasia (n = 76), 2.5/5.8%; atypical ductal hyperplasia (n = 21), 3.0/6.6%; carcinomas in situ (n = 70), 7.4/14.0%; and invasive carcinomas (n = 58), 10.0/22.9%. Proliferative index data for atypical hyperplasia were virtually identical to those for benign lesions and were significantly lower than those for breast cancer, suggesting that expression levels of cyclin B1 and Cdc2 may be used to distinguish premalignant human breast lesions from advanced disease. Furthermore, the proliferative index for cyclin B1 for comedo-type ductal carcinomas in situ agreed with that for invasive ductal carcinomas (mean, 10.1% versus 9.5%), apparently explaining the clinicopathological aggressiveness of this tumor at the molecular level.

Root-knot and cyst nematodes are plant parasites that induce large multinucleated feeding cells in the roots of their hosts. Cytological observations have shown that root-knot nematodes induce giant cells by cycles of mitosis without cytokinesis whereas cyst nematodes provoke cell wall degradation leading to the formation of a large syncytium. This study was intended to characterize and compare the ability of both types of nematodes to induce progression through the cell cycle. For this purpose, the expression, upon nematode infection, of two cell cycle markers was followed: a marker for division competence, the cyclin-dependent kinase cdc2a and a marker for the G2 phase, the mitotic cyclin cyc1At. For both types of nematodes, transcriptional activation of these markers was correlated with early phases of feeding cell development. Using molecular markers, it was thus possible to confirm and extend the observations of repeated mitosis in root-knot nematode-induced giant cells. Surprisingly, promoter activation of both cdc2a and cyc1At markers was also found upon cyst nematode infection, in feeding cells in which mitosis has not been clearly reported. Incorporation of tritiated thymidine in these syncytia confirms that they progress through the S phase of the cell cycle. One possibility is that cyst nematodes induce cycles of DNA endoreduplication shunting the M phase. Despite obvious differences in ontogeny, common molecular mechanisms, involving cycles of DNA endoreduplication and cdc2a and cyc1At expression, might thus be involved in the formation of a giant cell or a syncytium.

A cell-free system derived from Xenopus eggs was used to identify the 'destruction box' of the Schizosaccharomyces pombe B-type cyclin, Cdc13, as residues 59-67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N-terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box-mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N-terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs). Arabidopsis thaliana contains two cdk genes, cdc2aAt and cdc2bAt. This paper compares the developmental and cell cycle phase-dependent transcription of both cdk genes. In situ hybridizations revealed that cdc2bAt steady-state mRNAs, much like cdc2aAt, are found both in meristematic cells and cells with a high proliferative competence. Cdc2aAt is expressed in every meristematic cell whereas cdc2bAt is found to be expressed in a patchy pattern. An even smaller number of meristematic cells express the mitotic cyc1At. These data indicate that cdc2bAt and cyc1At mRNAs accumulate in a particular cell cycle phase in agreement with evidence provided by hybridization experiments of flow cytometrysorted nuclei and the use of cell cycle blockers on roots. The data indicate that cdc2bAt is preferentially expressed in S and G2 phases whereas cdc2aAt expression is constitutive throughout the cell cycle, as shown previously. The existence of two distinct CDK classes in plants is proposed: (i) constitutively expressed CDKs containing a PSTAIRE motif (e.g. cdc2aAt) and (ii) CDKs with divergent motifs which are expressed during a limited interval of the cell cycle (e.g. cdc2bAt).

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107-deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb-deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.

We have isolated a Leishmania mexicana homologue of the fission yeast suc1 gene using PCR with oligonucleotides designed to conserved regions of cdc2 kinase subunits (cks). The product of cks1 is a 12 kDa polypeptide, which has 70% identity with human p9cks1 and 44% identity with fission yeast p13suc1.p12cks1 was detected in the three life-cycle stages of L. mexicana by immunoblotting. Recombinant p12cks1 (p12cks1his) bound to agarose beads was used as a matrix to affinity-select histone H1 kinase complexes from Leishmania, yeast and bovine extracts. Immunoblotting showed that yeast and bovine cdc2 kinase bound to p12cks1his, thus demonstrating functional homology between L. mexicana p12cks1 and yeast p13suc1. Histone H1 kinase activity was found at a high level in the proliferative promastigote and amastigote forms of L. mexicana, but at a low level in the non-dividing metacyclic form. These activities are likely to be the same as the leishmanial p13suc1 binding kinase (SBCRK) described previously [Mottram, Kinnaird, Shiels, Tait and Barry (1993) J. Biol. Chem. 268, 21044-21051]. A distinct cdc2-related kinase, L. mexicana CRK1, was also found to associate with p12cks1his but affinity-depletion experiments showed that CRK1 was not responsible for the histone H1 kinase activity associating with p12cks1his in promastigote cell extracts. The finding that p12cks1 associates with at least two cdc2-related kinases, SBCRK and CRK1, is consistent with the presence of a large gene family of cdc2-related kinases in trypanosomatids, a situation thought to be more similar to higher eukaryotes than yeast.

Cyclin-dependent protein kinases (Cdks) play a key role in the cell division cycle of eukaryotic cells. Cdc2, the first mammalian Cdk that was discovered, is expressed in S phase and functions in the G2 to M phase transition. By transfecting segments of the human cdc2 promoter linked to a reporter gene into monkey kidney (CV-1) cells, we identified the region containing the Sp1, E2F, and two CCAAT box binding sites as essential and sufficient for basal transcription. SV40 large T antigen (SV40-LT) is a viral oncoprotein that transactivates viral and cellular promoters and induces DNA synthesis in quiescent cells. SV40-LT transactivated wild-type cdc2 promoter/reporter constructs in a dose-dependent manner, coinciding with an increase in endogenous cdc2 mRNA. A mutant promoter from which the two CCAAT box motifs were deleted was 8-fold less sensitive to SV40-LT. Activation by SV40-LT did not require its ability to bind the retinoblastoma or p53 tumor suppressor proteins. SV40-LT induced a specific CCAAT box-binding factor (CBF) in CV-1 and COS-7 cells, as judged by gel shift and Southwestern analyses. Similar results were obtained in human fibroblasts expressing a conditional SV40-LT. The SV40-LT-inducible CBF appears to be novel and differs from the CBF that activates heat shock protein 70 gene expression.

An adenocarcinoma cell line which has the ability to arrest in G0 phase under exhausted culture conditions was established. Using the cell line, we investigated the expression of cell cycle-associated proteins including cdc2, cdk2, cyclin A, cyclin Dl, and Rb during entry into or withdrawal from the cell cycle. MAP kinase expression was also investigated as one of the most downstream proteins of the signal transduction of growth factors. The cells in the quiescent state did not express cdc2. In contrast, cdk2 was expressed weakly, and cyclin A and cyclin D1 were strongly expressed in the quiescent cells. The expression of cdk2 and cyclin D1 in the quiescent cells was reduced after stimulation by renewal of the medium and then increased, accompanied by Rb phosphorylation and cdc2 expression around the G1/S transition. Cdc2 and the hyperphosphorylated form of Rb disappeared as the cells became quiescent. MAP kinase expression was unchanged throughout all the phases analyzed. The results indicate that down-regulation of neither cdk2, cyclin A, cyclin D1, nor MAP kinase is necessary to arrest cells in G0, but that only Rb dephosphorylation and down-regulation of cdc2 are accompanied by an arrest of cell proliferation in G0 in the cell line.

We have shown that normal and human papillomavirus (HPV) type 16 immortalized human foreskin keratinocytes are growth inhibited by tumor necrosis factor alpha (TNF-alpha), whereas HPV-18- and SV40-immortalized keratinocytes are resistant to this cytokine (1). In this report, we investigated the expression of mitotic regulatory proteins, such as cyclin A, cyclin B, and p34cdc2. After exposure to TNF-alpha, normal and HPV-16-immortalized cells exhibited a dramatic decrease in the expression of these proteins. In contrast, no alteration in the levels of these proteins was observed after treatment of the resistant cell lines, as well as two HPV-positive cervical carcinoma cell lines. Expression of cyclin E does not seem to be modulated by TNF-alpha in any of the cells tested. On the other hand, cyclin D1, expression is slightly increased in normal keratinocytes and in the HPV-16-immortalized cells, whereas no alteration was observed in the HPV-18-transfected cells. The phosphorylation state of pRb correlated with cell growth; sensitive cells, which accumulate in G0-G1, after exposure to TNF-alpha, exhibited an accumulation of hypophosphorylated pRb, whereas no effect on pRb phosphorylation was observed for HPV-18-immortalized cells. These results clearly correlate with TNF-alpha-induced growth arrest in G0-G1.

The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H-ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest.

SV40 T antigen induces karyotype instability soon after it is expressed in human diploid fibroblasts and ultimately promotes cell immortalization and tumorigenesis. Protein levels and activities of mitotic cell cycle proteins have been shown to be elevated in several immortal cell lines relative to their normal parental cells, suggesting a possible role for the aberrant regulation of mitosis in karyotype instability. We show here that IMR-90 human diploid lung fibroblasts expressing the SV40 tumor antigens display increased protein levels and associated enzymatic activities of cyclin A, cyclin B, and p34cdc2 long before crisis and immortalization. These elevations cannot be explained by faster cell growth or altered cell cycle distributions. Increased protein levels were not totally accounted for by elevated levels of the corresponding mRNA, indicating that T antigen modulates expression at least partially by posttranscriptional mechanisms. These results indicate that perturbation of mitotic regulatory proteins precedes crisis, and imply that altered mitotic control is a direct consequence of T antigen expression rather than an outcome of secondary events associated with immortalization.

Immunocytochemical and autoradiographic techniques were used to examine proliferating cells in the adult rat brain along with the expression of specific growth factor receptors and cell cycle proteins. Two hours following an injection of [3H]thymidine ([3H]Thy), dividing cells were detected in the subgranular region of the dentate gyrus and in the subependymal region (SER) extending into the olfactory bulb. Many cells continued to divide over the next 24 h as demonstrated by the ability for thymidine-labeled cells to incorporate bromodeoxyuridine (BrdU); however, the results of BrdU, PSTAIR, and vimentin staining suggest that the majority of the progeny cells detectable by [3H]Thy autoradiography at 3 days and 1 week after injection are postmitotic and at least partially differentiated. Significant numbers of thymidine-labeled cells detected 2 h following thymidine injection in the subgranular region of the dentate gyrus and in the SER of the lateral ventricle stained positively for epidermal growth factor receptor-, vimentin-, and PSTAIR-like immunoreactivity. Significant numbers of thymidine-labeled cells in the SER also stained positively for the low-affinity neurotrophin receptor p75NGFR. No [3H]Thy/p75NGFR-labeled cells were detected in the dentate gyrus. In addition, very few [3H]Thy/PSTAIR- or [3H]Thy/ vimentin-labeled cells were detected in region CA4. These data suggest that proliferating cells located in different regions of the adult brain may not be homogeneous and may be subject to different growth factor regulation.

Mizoribine has been used to prevent rejection of organ allografts in humans and in animal models. Recent clinical trials have demonstrated its efficacy in rheumatoid arthritis and lupus nephritis, in which abnormalities of B cell functions are also involved. We therefore examined the effects of mizoribine on the in vitro function of human B cells. IgM production was induced from highly purified B cells obtained from healthy donors by stimulation with Staphylococcus aureus Cowan I (SA) plus IL-2. Mizoribine suppressed the production of IgM at its pharmacologically attainable concentrations (1 to 3 micrograms/ml) in a dose-dependent manner. Mizoribine had to be present within the first 96 h after the initiation of cultures to exert its suppressive effects on B cell responses. Cell cycle analysis disclosed that mizoribine significantly decreased the numbers of B cells in S + G2 + M phases. Mizoribine did not decrease GTP levels in SA-stimulated B cells, whereas mizoribine led to a decrease in GTP levels in activated T cells, which was reversed by addition of GMP. Consistently, the suppressive effects of mizoribine on the IgM production of SA-stimulated B cells was not reversed by the addition of GMP as much as 40 microM, which overcame the inhibitory effects of mizoribine on the proliferation of anti-CD3-stimulated T cells. Although mizoribine did not suppress the expression of CD25 and cdc2 kinase, mizoribine markedly suppressed the expression of cyclin A in SA-activated B cells. These results indicate that mizoribine directly suppresses the function of human B cells without interfering with the initial phase of activation. Moreover, the data demonstrate that mizoribine interferes with the cell cycle progression of activated B cells by suppressing the expression of cyclin A by a mechanism distinct from guanine ribonucleotide depletion.

The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley leaves.

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of cells to reenter the cell cycle results in induction of cdc2 expression, beginning at the G1-to-S transition and reaching maximum levels during late S and G2 phases. To investigate cdc2 transcriptional regulation throughout cell cycle progression, we monitored protein-DNA interactions by in vivo footprinting along 800 bp of the human cdc2 promoter in quiescent fibroblasts and at different time points following serum stimulation. We found 11 in vivo protein-binding sites, but no protein binding was observed at a high-affinity E2F site that had previously been implicated in cdc2 regulation. Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Sp1 sites, and one ets-2 site) bind transcription factors constitutively throughout the cell cycle. However, at two elements located at positions -60 and -20 relative to the transcription start site, the binding pattern changes significantly as the cells are entering S phase. A G0- and G1-specific protein complex disappears at the -20 element at the beginning of S phase. This sequence deviates at one base position from known E2F consensus binding sites. We found that the major E2F activity in human fibroblasts contains E2F-4 and p130. The -20 element of the cdc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G0 cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutant cdc2 promoter constructs indicate that the -20 element is involved in suppressing cdc2 activity in quiescent cells. We suggest that the presence of the p130-E2F-4 complex in G0/G1 blocks access of components of the basal transcription machinery or prevents transaction by the constitutively bound upstream activator proteins.