S-phase Transition Research Articles

Sustained Survivin Expression from OX40 Costimulatory Signals Drives T Cell Clonal Expansion

Sustained signaling from the T cell receptor (TCR) and costimulatory molecules is thought necessary for generating high numbers of effector T cells. Here, we show that Survivin is controlled in peripheral T cells by OX40 cosignaling via sustained PI3k and PKB activation. Survivin is induced by OX40 independent of mitotic progression in late G1, and blocking Survivin suppresses S-phase transition and division of T cells and leads to apoptosis. Moreover, Survivin expression alone is sufficient to restore proliferation and to antagonize apoptosis in costimulation-deficient T cells and can rescue T cell expansion in vivo. Survivin allows effector T cells to accumulate in large numbers, but Bcl-2 family proteins are required for T cell survival after the phase of active division. Thus, sustained Survivin expression from costimulatory signaling maintains T cell division over time and regulates the extent of clonal expansion.

Flavin-containing oxidases regulate progression from G 1 to S phase of the cell cycle in normal human diploid fibroblasts

The effects of flavin-containing oxidase enzymes on cellular progression from G 1 to S phase of the cell cycle were examined in normal human diploid fibroblasts by use of the cumulative labeling index method. Significant transient arrests in G 1 occurred when quiescent cell populations incubated with diphenyliodonium (DPI) for a 20 h period at concentrations as low as 0.05 μM were stimulated to proliferate by subculturing to low density in control growth medium. Quiescent cells pre-treated with DPI and subcultured in DPI-containing growth medium were permanently arrested in G 1. Induction of this G 1 checkpoint was associated with activation of the p53/p21 Waf1 signaling pathway. Incubation of G 0/G 1 phase cells with DPI (0.05–0.2 μM) resulted in significant decreases in the expression levels of cyclin D1 and p21 Waf1; no effect was detected on cyclin A expression. Collectively, these results suggest that DPI-sensitive signaling primarily regulates early G 1 events that lead to S-phase transition. Furthermore, they support a critical role for membrane originating oxidants in the regulation of cellular proliferation pathways.

Mechanistic and Predictive Profiling of 5-Fluorouracil Resistance in Human Cancer Cells

Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor kappaB (NFkappaB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFkappaB DNA-binding activity. Cotransfection of NFkappaB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFkappaB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G(1)-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G(1), and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.

Ingestion of an isothiocyanate metabolite from cruciferous vegetables inhibits growth of human prostate cancer cell xenografts by apoptosis and cell cycle arrest.

Epidemiological surveys indicate that intake of cruciferous vegetables is inversely related to prostate cancer incidence, although the responsible dietary factors have not been identified. Our studies demonstrated that exposure of human prostate cancer cells in culture to the N-acetylcysteine (NAC) conjugate of phenethyl isothiocyanate (PEITC-NAC), the major metabolite of PEITC that is abundant in watercress, inhibited proliferation and tumorigenesis. The PEITC-NAC is known to mediate cytoprotection at initiation of carcinogenesis. The relevance of PEITC-NAC in diets on the growth of prostate tumor cells has been evaluated in immunodeficient mice with xenografted tumors of human prostate cancer PC-3 cells. The daily PEITC-NAC (8 micromol/g) supplemented diet group showed a significant reduction in tumor size in 100% of the mice during the 9-week treatment period. Tumor weight at autopsy was reduced by 50% compared with mice on the diet without PEITC-NAC (P = 0.05). Mitosis and in vivo 5-bromo-2'-deoxyuridine labeled proliferating cells were reduced in these tumors. The PEITC-NAC diet up-regulated the inhibitors of cyclin-dependent kinases p21WAF-1/Cip-1 and p27Kip1, and reduced the expression of cyclins D and E, indicating they were potential molecular targets. As a result, phosphorylated Rb was significantly decreased and the G1- to S-phase transition retarded. The treated tumors also showed a significant increase in apoptosis as determined by in situ end-labeling, and by poly ADP-ribose polymerase cleavage. This study demonstrates the first in vivo evidence of dietary PEITC-NAC inhibiting tumorigenesis of prostate cancer cells. PEITC-NAC may prevent initiation of carcinogenesis and modulate the post-initiation phase by targeting cell cycle regulators and apoptosis induction.

Conjugated Linoleic Acid Inhibits Cell Proliferation through a p53-Dependent Mechanism: Effects on the Expression of G1-Restriction Points in Breast and Colon Cancer Cells

Previous reports have documented the antiproliferative properties of a mixture of conjugated isomers (CLA) of linoleic acid [LA (18:2)]. In this study, we investigated the mechanisms of CLA action on cell cycle progression in breast and colon cancer cells. Treatment with CLA inhibited cell proliferation in breast cancer MCF-7 cells containing wild-type p53 (p53(+/+)). At cytostatic concentrations, CLA elicited cell cycle arrest in G1 and induced the accumulation of the tumor suppressors p53, p27 and p21 protein. Conversely, CLA reduced the expression of factors required for G1 to S-phase transition including cyclins D1 and E, and hyperphoshorylated retinoblastoma Rb protein. In contrast, the overexpression of mutant p53 (175Arg to His) in MFC-7 cells prevented the CLA-dependent accumulation of p21 and the reduction of cyclin E levels suggesting that the expression of wild-type p53 is required for CLA-mediated activation of the G1 restriction point. To further elucidate the role of p53, the effects of CLA in colon cancer HCT116 cells (p53(+/+)) and p53-deficient (p53(-/-)) HCT116 cells (HCTKO) were examined. The treatment of HCT116 cells with CLA increased the levels of p53, p21, p27 and hypophosphorylated (pRb) protein and reduced the expression of cyclin E, whereas these effects were not seen in p53-deficient HCTKO cells. The t10,c12-CLA isomer was more effective than c9,t11-CLA in inhibiting cell proliferation of MCF-7 breast cancer cells and enhancing the accumulation of p53 and pRb. We conclude that the antiproliferative properties of CLA appear to be a function, at least in part, of the relative content of specific isomers and their ability to elicit a p53 response that leads to the accumulation of pRb and cell growth arrest.

Expression of an active p110 catalytic subunit of phosphatidylinositol 3-kinase alters the proliferative capacity of interleukin-2 receptor signals.

Activation of phosphatidylinositol 3-kinase (PI3K) is an early and essential step in interleukin-2 receptor (IL-2R) signalling, and plays an important role in regulating both cell survival and cellular proliferation. In the present study, we utilized Baf-B03 cells expressing mutated IL-2R to examine the contribution of PI3K to proliferative capacity. In this model IL-2-mediated induction of the downstream PI3K-dependent signalling molecule p70 S6 kinase was detected, but there was no proliferative response. Increasing the level of PI3K activity by transfection of an active form of the catalytic subunit, p110*, enabled the proliferative capacity of the mutated receptor. Whereas, in cells without p110*, IL-2 lacked the capacity to induce c-myc and to overcome an S-phase checkpoint, S-phase transition was restored by transfection of p110*, and this was accompanied by an increase in the c-myc response. Despite the presence of p110*, activity cells still required IL-2R-derived signals for proliferation, and IL-2Rbeta truncated at amino acid 350 were sufficient to provide this signalling activity. The data support a model in which the level of available PI3K can determine the cellular response to IL-2.

Cytogenetic and molecular findings related to rhabdomyosarcoma. An analysis of seven cases

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Histologically, it is subdivided histologically into two main subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is characterized by t(2;13)(q35;q14) or its variant t(1;13)(p36;q14), which fuse PAX3 and PAX7, respectively, with FKHR to produce chimeric genes. ERMS is frequently associated with loss of heterozygosity of 11p15.5. We investigated seven RMS (three ARMS and four ERMS) by means of cytogenetic, fluorescence in situ hybridization, and molecular analyses, including the study of the main genes implicated in the G1- to S-phase cell cycle transition, and correlated these studies with pathologic findings and clinical outcome. All tumors showed clonal, numerical, and structural chromosomal abnormalities. Two ARMS had the t(2;13)(q35;q14) and the third a PAX7/ FKHR fusion, a cryptic t(1;13)(p36;q14), undetected by cytogenetic techniques, but revealed by reverse transcriptase polymerase chain reaction. One ERMS showed a der(11)t(3;11)(p21;p15) as a sole structural anomaly. Gene amplification was seen in four tumors, as double minutes or in the form of homogeneously staining regions. Overexpression of MYCN oncogene was found in two ARMS; N-myc DNA probe detected oncogene amplification located on the double minutes of these cases. Analysis of the regulatory genes responsible for G1- to S-phase transition showed a homozygous deletion of the 9p21 locus genes in a spindle-cell ERMS.

NPAT expression is regulated by E2F and is essential for cell cycle progression.

NPAT is an in vivo substrate of cyclin E-Cdk2 kinase and is thought to play a critical role in coordinated transcriptional activation of histone genes during the G(1)/S-phase transition and in S-phase entry in mammalian cells. Here we show that NPAT transcription is up-regulated at the G(1)/S-phase boundary in growth-stimulated cells and that the NPAT promoter responds to activation by E2F proteins. We demonstrate that endogenous E2F proteins interact with the promoter of the NPAT gene in vivo and that induced expression of E2F1 stimulates NPAT mRNA expression, supporting the idea that the expression of NPAT is regulated by E2F. Consistently, we find that the E2F sites in the NPAT promoter are required for its activation during the G(1)/S-phase transition. Moreover, we show that the expression of NPAT accelerates S-phase entry in cells released from quiescence. The inhibition of NPAT expression by small interfering RNA duplexes impedes cell cycle progression and histone gene expression in tissue culture cells. Thus, NPAT is an important E2F target that is required for cell cycle progression in mammalian cells. As NPAT is involved in the regulation of S-phase-specific histone gene transcription, our findings indicate that NPAT links E2F to the activation of S-phase-specific histone gene transcription.

Loss of p27(Kip1) cooperates with cyclin E in T-cell lymphomagenesis.

Cyclin E and p27(Kip1) are key regulators for cyclin-dependent kinases (Cdks) acting at the G1-/S-phase transition of the cell cycle. Whereas cyclin E is required for the activation of Cdk2, p27(Kip1) is a specific Cdk inhibitor and can block cell division. High levels of cyclin E and low levels of p27(Kip1) expression have been associated with malignant lymphomas in humans; the level of p27(Kip1) is even considered of prognostic significance. However, mice that lack p27(Kip1) do not develop any malignant lymphomas despite a pronounced lymphoid hyperplasia in thymus and spleen. We have previously described transgenic mice that carry a construct in which the cyclin E cDNA is under the control of the CD2 promoter/enhancer region and thus express high levels of cyclin E in the T-cell compartment (CD2-cyclin E). These animals are not predisposed for T-cell lymphomas in the absence of other cooperating events. Here we show that T-cells from CD2-cyclin E mice that at the same time are deficient for p27(Kip1) show a significantly higher Cdk2 activity than cells from wild-type or single mutant animals. Accordingly, a higher percentage of T cells in S/G2/M phase is found in CD2-cyclin E/p27(Kip1-/-) mice. After a long latency period of over 200 days, these animals develop spontaneous monoclonal T cell lymphoma whereas none of the single CD2-cyclin E transgenic or the p27(Kip1)-deficient mice showed any sign of lymphoid malignancies. Our findings demonstrate that a deregulation of control mechanisms at the G1/S transition by the combination of high cyclin E levels in the absence of p27(Kip1) is sufficient to predispose mice to develop lymphoid malignancies and further support a role of p27(Kip1) as a tumor suppressor and of cyclin E as a dominant oncogene.

Hormone-dependent repression of the E2F-1 gene by thyroid hormone receptors.

Thyroid hormone induces differentiation of many different tissues in mammals, birds, and amphibians. The different tissues all differentiate from proliferating precursor cells, and the normal cell cycle is suspended while cells undergo differentiation. We have investigated how thyroid hormone affects the expression of the E2F-1 protein, a key transcription factor that controls G1- to S-phase transition. We show that during thyroid hormone-induced differentiation of embryonic carcinoma cells and of oligodendrocyte precursor cells, the levels of E2F-1 mRNA and E2F-1 protein decrease. This is caused by the thyroid hormone receptor (TR) regulating the transcription of the E2F-1 gene. The TR binds directly to a negative thyroid hormone response element, called the Z-element, in the E2F-1 promoter. When bound, the TR activates transcription in the absence of ligand but represses transcription in the presence of ligand. In addition, liganded TR represses transcription of the S-phase-specific DNA polymerase alpha, thymidine kinase, and dihydropholate reductase genes. These results suggest that thyroid hormone-induced withdrawal from the cell cycle takes place through the repression of S-phase genes. We suggest that this is an initial and crucial step in thyroid hormone-induced differentiation of precursor cells.

Impairment in S-phase entry of splenocytes of Parp-1 knockout mice.

One immediate cellular response to DNA damage is the polyADP-ribosylation reaction by poly(ADP-ribose) polymerase-1 (Parp-1). The importance of Parp-1 has been established in many cellular processes, such as the maintenance of genomic stability, DNA repair and cell-death induction. Here, we established Parp-1-/- mice of C57BL/6J congenic strain and characterized the role of Parp-1 in cell-cycle progression. In this study, we also improved a method to observe G0/G1 to S-phase transition of splenocytes and bone marrow cells prepared from mice. The cells were cultured and stimulated with mitogens (50 μM ionomycin/1 μM phorbol 12, 13-dibutyrate). We found that addition of a commercially available growth supportive reagent, BM Condimed RH1, greatly enhanced the transition of G0/G1 to the S-phase, which was determined by bromodeoxyuridine (BrdU) incorporation to DNA. Using this method, G0/G1 to the S-phase entry was measured using splenocytes derived from Parp-1-/-, Parp-1+/- and wild-type (Parp-1+/+) mice. DNA synthesis in Parp-1+/+ and Parp-1+/- splenocytes started from day 1 after addition of mitogens, whereas that in Parp-1-/- cells started from day 2. The peak of the S-phase was at day 2 in all genotypes and notably DNA synthesis in Parp-1-/- cells was approximately halved compared to Parp-1+/+ cells on day 2, 3 and 4. These results suggested that Parp-1 is involved in positive regulation of S-phase entry in quiescent mouse splenocytes.(Contributed by Takashi SUGIMURA, M. J. A., Oct. 14, 2003)

Modulation of estrogen receptor-dependent reporter construct activation and G0/G1-S-phase transition by polycyclic aromatic hydrocarbons in human breast carcinoma MCF-7 cells.

It has been suggested that the estrogenicity of PAHs could contribute to their carcinogenic effects via increased tissue-specific cell proliferation. Both benzo[a]pyrene (BaP) and benz[a]anthracene (BaA) are known to weakly activate estrogen receptor (ER)-dependent reporter constructs. In this study, several other PAHs, including fluorene, fluoranthene, pyrene, chrysene, phenanthrene and anthracene, were found to act as very weak inducers of ER-mediated activity in the MCF-7 cell line stably transfected with a luciferase reporter gene. The effects of PAHs were time-dependent and they were not completely inhibited by antiestrogen ICI 182,780. In addition, BaP and BaA, as well as weakly estrogenic fluoranthene, significantly potentiated the maximum ER-mediated activity of 17beta-estradiol. Therefore, the effects of inhibitors of several types of protein kinases known to activate ERalpha in a ligand-independent manner were investigated. However, neither inhibitors nor inducers of extracellular signal-regulated kinases 1 and 2 (ERK1/2), phosphatidylinositol-3 kinase, protein kinase C, c-Src, or protein kinase A modified ER-mediated activity in this model. Neither estradiol nor BaA activated ERK1/2, two kinases suggested to play significant roles in ER signaling, suggesting that another kinase is involved in the observed phosphorylation of ERalpha. Similar to 17beta-estradiol, BaA stimulated G(0)/G(1)-S-phase transition in MCF-7 cells, which was fully suppressed by ICI 182,780. In conclusion, some PAHs can potentiate 17beta-estradiol-induced ER activation and stimulate cell cycle entry in vitro. However, their exact mode(s) of action and whether this phenomenon is of in vivo relevance remains to be elucidated.

A naturally occurring point substitution in Cdc25A, and not Fv2/Stk, is associated with altered cell-cycle status of early erythroid progenitor cells

The Friend virus susceptibility gene 2 (Fv2) controls the polyclonal expansion of infected cells that occurs early during Friend erythroleukemia virus infection. Fv2 has recently been shown to encode a truncated form of the Stk receptor tyrosine kinase (Sf-Stk). This observation, coupled with earlier work, suggested that Sf-Stk drives the expansion of infected cells by forming a complex with the Friend virus envelope glycoprotein, gp55, and the erythropoietin receptor. Fv2 has also been implicated in the control of cell cycling in early erythroid progenitors (erythroid blast-forming units [BFU-Es]). Mouse strains that are homozygous for the resistant allele of Fv2 (Fv2(rr)) have few actively cycling BFU-Es. In this report, we demonstrate that the control of BFU-E cycling is encoded by a gene linked to, but distinct from, Fv2, and suggest that this gene is the dual-specific protein phosphatase Cdc25A, which regulates the G1- to S-phase transition of the cell cycle. We show that a naturally occurring allele of Cdc25A, which increases Cdc25A phosphatase activity and promotes cell-cycle progression, segregates in mouse strains that exhibit high levels of BFU-E cell cycling. In wild-type mice, this allele of Cdc25A does not overtly affect erythropoiesis; however, when this allele is combined with a mutation of the Kit receptor (Kit(WV)), the anemia of the mice is enhanced. Furthermore, overexpression of Cdc25A in bone marrow cells causes a defect in the BFU-E colony formation. These results suggest that proper regulation of the cell cycle through Cdc25A is required for normal erythropoiesis.

Ultraviolet light-induced apoptosis is associated with S-phase in primary human fibroblasts

Transcription-coupled nucleotide excision repair (tcNER)-deficient human fibroblasts are extremely sensitive to the induction of apoptosis in response to low doses of ultraviolet light (UV light), but are less sensitive to the induction of apoptosis following exposure to high doses [J. Invest. Dermatol. 117 (2001) 1162]. This seemingly paradoxical observation led us to re-evaluate the relationship between UV dose and the induction of apoptosis. Here we report that the reduction in the extent of UV-induced apoptosis in tcNER-deficient strains following exposure to elevated doses of UV light does not result from impaired gene expression alone because neither inhibitors of transcription nor inhibitors of translation blocked UV-induced apoptosis. Furthermore, UV-induced apoptosis was greatly reduced by inhibiting S-phase progression with either mimosine or serum withdrawal. Importantly, DNA synthesis following UV-irradiation occurred only at doses that induced apoptosis in these cell lines and the apoptotic cells contained nascent DNA. Moreover, deregulation of G 1- to S-phase transition by expression of human papillomavirus E7 sensitized cells to UV-induced apoptosis. Taken together these results suggest that the induction of apoptosis requires S-phase progression following UV-irradiation.

Contribution of double-stranded RNA-activated protein kinase toward antiproliferative actions of heparin on vascular smooth muscle cells.

The proliferation of vascular smooth muscle cells (VSMCs) in blood vessels after endothelial injury contributes to the onset of atherosclerosis. Heparin is a potent antiproliferative agent for VSMCs in vivo and in vitro. Although heparin has shown promise in suppressing VSMC proliferation after invasive procedures in laboratory animals, the mechanism of its antiproliferative actions is largely unknown. Here, we present evidence for the first time that the antiproliferative action of heparin is in part mediated by its ability to activate double-stranded RNA-activated protein kinase (PKR), an interferon-induced protein kinase. We have analyzed the VSMC proliferation by cell-cycle analysis and correlated it to the kinase activity of PKR in the presence of heparin. Heparin treatment of VSMCs results in activation of PKR by direct binding and results in a block in G1- to S-phase transition. PKR-null cells are largely insensitive to the antiproliferative actions of heparin, and inhibition of PKR in VSMCs results in a partial abrogation of the antiproliferative effects of heparin. These results invoke the involvement of novel PKR-dependent regulatory pathways in mediating the antiproliferative actions of heparin.

Transcriptional coregulation by the cell integrity mitogen-activated protein kinase Slt2 and the cell cycle regulator Swi4.

In Saccharomyces cerevisiae, the heterodimeric transcription factor SBF (for SCB binding factor) is composed of Swi4 and Swi6 and activates gene expression at the G(1)/S-phase transition of the mitotic cell cycle. Cell cycle commitment is associated not only with major alterations in gene expression but also with highly polarized cell growth; the mitogen-activated protein kinase (MAPK) Slt2 is required to maintain cell wall integrity during periods of polarized growth and cell wall stress. We describe experiments aimed at defining the regulatory pathway involving the cell cycle transcription factor SBF and Slt2-MAPK. Gene expression assays and chromatin immunoprecipitation experiments revealed Slt2-dependent recruitment of SBF to the promoters of the G(1) cyclins PCL1 and PCL2 after activation of the Slt2-MAPK pathway. We performed DNA microarray analysis and identified other genes whose expression was reduced in both SLT2 and SWI4 deletion strains. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated and reveal a role for Swi4, the DNA-binding component of SBF, which is independent of the regulatory subunit Swi6. Some of the Swi4- and Slt2-dependent genes do not require Swi6 for either their expression or for Swi4 localization to their promoters. Consistent with these results, we found a direct interaction between Swi4 and Slt2. Our results establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond its prominent role in controlling cell cycle transcription.

Acetylation can regulate cell-cycle progression

The retinoblastoma tumour suppressor (pRb) is crucial for the negative control of cell proliferation. The protein has a major role in G1- to S-phase transition by controlling key transcription factors such as the E2F family. In tumour cells, pRb is sequestered by viral oncoproteins (E1A) and regulated by phosphorylation through G1 cyclin-dependent kinases (CDKs),primarily cyclinD/CDK4 and cyclinE/Cdk2,which sequentially phosphorylate pRb as the cells move towards S-phase.In a new study, Chan et al. 1xAcetylation control of the retinoblastoma tumour-suppressor protein. Chan, H.M. et al. Nat. Cell Biol. 2001; 3: 667–674CrossRef | PubMed | Scopus (194)See all References1 have identified acetylation as a new type of regulator of pRb function. The acetylation is mediated by p300 through adenovirus E1A recruitment of pRb and p300/CREB binding protein (CBP) in a multimeric complex. Both pRb and p300/CBP are targets for viral oncoproteins, such as the adenovirus E1A.In an elegant series of experiments, the authors tested the acetylation of pRb by the histone acetyl transferase (HAT) activity of p300 using a glutathione S-transferase (GST)–pRb fusion protein in vitro. This is influenced by the integrity of a pocket domain at the C-terminus of the pRb protein. Two pocket mutants, isolated from tumour cells, showed reduced acetylation. To establish whether this also happened in vivo, the authors used an antibody against acetylated lysines in pRB and showed the presence of endogenous acetylated pRb. Under similar circumstances, the pocket mutant was not acetylated. Use of mutant derivatives showed that the main site of acetylation was located between amino-acid residues 794 and 880. There are eight lysine residues in this region, and Chan et al. focused on the five lysines between residues 830 and 884, systematically altering each lysine to arginine and identifying lysines 873 and 874 as being acetylated. Having done this, the authors assessed whether acetylation might influence pRb phosphorylation, by using constructs where the lysines at residues 873 and 874 were replaced by arginines or glutamines (873/874RR and 873/874QQ, respectively). A change to glutamine mimics the acetylation status, whereas a change to arginine prevents acetylation. Experiments using these constructs showed that wild-type pRb and 873/874 RR were phosphorylated to a similar extent, but the 873/874 QQ construct showed a significant reduction in phosphorylation. The p300-dependent acetylation of pRb is increased by adenovirus E1A, through recruitment of p300 and pRb into the same complex. This process is dependent on E1A concentrations: at high E1A concentrations the acetylation of pRb is lost. To assess the function of acetylation, the authors looked at proteins that interact with the C-terminus of the pRb protein. For example, the MDM2 oncoprotein bound more efficiently to pRb in its acytelated form, both in vitro and in vivo. Similar experiments with E2F failed to detect any differences.These recent advances suggest acetylation as a new control mechanism in regulating pRb activity and a new mechanism through which viral oncoproteins can affect tumour-suppressor activity.

Conserved promoter motif is required for cell cycle timing of dnaX transcription in Caulobacter.

Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including dnaX, dnaA, dnaN, gyrB, and dnaK, are induced at the G(1)/S-phase transition. By analyzing mutations in the dnaX promoter, we identified a motif between the -10 and -35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

High cyclin E/low p27Kip1 expression is associated with poor prognosis in astrocytomas

Cyclin E and p27Kip1 are co-regulators of the G1- to S-phase transition and closely related to tumor behavior. The purpose of this study was to examine expression of cyclin E and p27Kip1 in astrocytomas and to evaluate the relationships between expression of these cell-cycle regulators and prognosis of patients with astrocytoma. Tissue samples from 130 astrocytomas (WHO grade 1 n = 5, grade 2 n = 23, grade 3 n = 64, grade 4 n = 38) were examined immunohistochemically for cyclin E and p27Kip1 expression. Patient charts were reviewed for clinical presentation, and survival was followed. The cyclin E labeling index (LI) tended to increase with tumor grade (Kruskal-Wallis, P = 0.0104). For patients with primary astrocytomas, the 50% survival times for the low cyclin E LI (< 5%) group and the high cyclin E LI (> or = 5%) group were 53.7 months and 19.8 months. In combined analysis of cyclin E and p27Kip1 expression, the low cyclin E/high p27Kip1 LI (> or = 50%) group had the best survival (50% survival time: 103.2 months), the low cyclin E/low p27Kip1 LI (> or = 50%) and the high cyclin E/high p27Kip1 LI groups moderate survival (24.1 and 27.5 months), and the high cyclin E/low p27Kip1 LI group the worst survival (13.1 months). Multivariate analysis identified the combined factor, high cyclin E/low p27Kip1, as a novel independent prognostic factor for survival time (P = 0.0037, relative risk = 2.4). This study suggested that combined analysis of cyclin E and p27Kip1 expression was considered to be potentially useful in predicting the prognosis of patients with astrocytoma.

A mathematical model of in vitro cancer cell growth and treatment with the antimitotic agent curacin A

A mathematical model of cancer cell growth and response to treatment with the experimental antimitotic agent curacin A is presented. Rate parameters for the untreated growth of MCF-7/LY2 breast cancer and A2780 ovarian cell lines are determined from in vitro growth studies. Subsequent growth studies following treatments with 2.5, 25 and 50 nanomolar (nM), concentrations of curacin A are used to determine effects on the cell cycle and cell viability. The model's system of ordinary differential equations yields an approximate analytical solution which predicts the minimum concentration necessary to prevent growth. The model shows that cell growth is arrested when the apoptotic rate is greater than the mitotic rate and that the S-phase transition rate acts to amplify this effect. Analysis of the data suggests that curacin A is rapidly absorbed into both cell lines causing an increase in the S-phase transition and a decrease in the M-phase transition. The model also indicates that the rate of apoptosis remains virtually constant for MCF-7/LY2 while that of A2780 increases 38% at 2.5 nM and 59% at 50 nM as compared to the untreated apoptotic rate.