Onset Of DNA Synthesis Research Articles

Non-Canonical Replication Initiation: You're Fired!

The division of prokaryotic and eukaryotic cells produces two cells that inherit a perfect copy of the genetic material originally derived from the mother cell. The initiation of canonical DNA replication must be coordinated to the cell cycle to ensure the accuracy of genome duplication. Controlled replication initiation depends on a complex interplay of cis-acting DNA sequences, the so-called origins of replication (ori), with trans-acting factors involved in the onset of DNA synthesis. The interplay of cis-acting elements and trans-acting factors ensures that cells initiate replication at sequence-specific sites only once, and in a timely order, to avoid chromosomal endoreplication. However, chromosome breakage and excessive RNA:DNA hybrid formation can cause break-induced (BIR) or transcription-initiated replication (TIR), respectively. These non-canonical replication events are expected to affect eukaryotic genome function and maintenance, and could be important for genome evolution and disease development. In this review, we describe the difference between canonical and non-canonical DNA replication, and focus on mechanistic differences and common features between BIR and TIR. Finally, we discuss open issues on the factors and molecular mechanisms involved in TIR.

Indirect semiquantitative determination of p34cdc2 levels in G1 and G2 cells of the carbohydrate-starved root meristems in Vicia faba var. minor

In eukaryotes, the 34kDa kinase (p34) encoded by the <em>cdc2</em> gene is a key regulator of both the onset of DNA synthesis (G<sub>1</sub> to S phase transition) and the onset of mitosis (G<sub>1</sub> to M phase transition). Using mouse anti-human PSTAIRE and FITClabelled goat antibodies, indirect semiquantitative determination of p34<sup><em>cdc2</em></sup> levels was performed in meristematic cells from the control (intact) and excised, carbohydrate-starved main roots of Vicia faba var. minor. No evident differences in the intensity of fluorescence was found either between the G<sub>1</sub> and G<sub>2</sub> cells or between the control cells and the cells arrested at both Principal Control Points by carbohydrate starvation. It seems thus, that the cell cycle block induced in meristematic cells of <em>V. faba</em> var. <em>minor</em> is not correlated with the absolute level of the key cell cycle enzyme responsible for phosphory-lution of cellular proteins, but primarily with the altered activity of p34<sup><em>cdc2</em></sup>.

36 EFFECT OF ACTIVATION METHODS ON DNA SYNTHESIS AND DEVELOPMENT OF CANINE PARTHENOGENETIC EMBRYOS

Artificial activation is an important step for successful somatic cell nuclear transfer. In mammals, different methods of parthenogenesis have been studied to increase the developmental efficiency of cloned embryos. In an attempt to improve the techniques of nuclear transfer in canine species, this study investigated the timing of DNA synthesis and in vivo development of canine parthenotes produced by different activation treatments. For canine parthenotes, in vivo matured oocytes were obtained by flushing (~72 h after ovulation) the oviducts of mixed breed bitches. Denuded oocytes were cultured for 4 min in 10 μM calcium ionophore, and then they were divided into 2 groups: (1) 2DMAP group was cultured for 2 h in 6-DMAP; (2) 4DMAP group was cultured for 4 h in 6-DMAP. The first experiment determined DNA synthesis of parthenotes by 1 h treatment with incorporation and immunofluorescent detection of thymidine analogue 5-bromo-2′-deoxyuridine-5′-triphosphate (BrdU). The primary antibody was mouse anti-BrdU (Sigma, St. Louis, MO, USA), and the secondary antibody was fluorescein (FITC)-conjugated affinity purified goat anti-mouse IgG (Jackson). In order to examine the pronuclear formation and the onset of DNA synthesis in experimental groups, parthenotes derived from 2DMAP and 4DMAP groups were observed by BrdU incorporation at 2, 4, and 12 h post-activation (hpa). Data were analysed using Graph Prism software (GraphPad, San Diego, CA, USa). The next experiment observed in vivo development as follows: parthenotes were surgically transferred to synchronized recipient female dogs. The implantation rate of parthenogenetic fetuses was observed in uterus of recipients on Day 26 of pregnancy. All parthenotes of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP parthenotes showed 96% BrdU incorporation at 2 hpa. Incorporation of BrdU was detected in all parthenotes of both experimental groups after 4 hpa. A total of 98 parthenotes were transferred to 9 surrogate mothers (53 parthenotes into 5 recipients in 2DMAP group and 45 parthenotes into 4 recipients in 4DMAP group). There was no significant difference in pregnancy rate between the 2 groups (2DMAP: 60% v. 4DMAP: 50%), whereas the implantation rates were significantly higher in 2DMAP (24.5%) compared with 4DMAP (4.4%; P < 0.001). The recovered parthenotes were able to develop to the stages of limb-bud formation, but few parthenotes showed the small and degenerating formation. Regardless of treatment group, the implantation site of the fetuses indicated either one side or both of the uterus. In conclusion, the present results demonstrated that the protocols using combined treatment with 10 μM of calcium ionophore (4 min) followed by a 2-h culture with 1.9 mM of DMAP resulted in completing pronuclear formation and enhancing fetal formation. This result could be useful method for improving canine cloned embryos production. This study was supported by IPET (#311062–04–2-SB010), RNL Bio (#550–20130013), RDA (PJ008975022013), and Research Institute for Veterinary Science and Natural Balance.

Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes

Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.

Visualization of the MCM DNA helicase at replication factories before the onset of DNA synthesis

In mammalian cells, DNA synthesis takes place at defined nuclear structures termed "replication foci" (RF) that follow the same order of activation in each cell cycle. Intriguingly, immunofluorescence studies have failed to visualize the DNA helicase minichromosome maintenance (MCM) at RF, raising doubts about its physical presence at the sites of DNA synthesis. We have revisited this paradox by pulse-labeling RF during the S phase and analyzing the localization of MCM at labeled DNA in the following cell cycle. Using high-throughput confocal microscopy, we provide direct evidence that MCM proteins concentrate in G1 at the chromosome structures bound to become RF in the S phase. Upon initiation of DNA synthesis, an active "MCM eviction" mechanism contributes to reduce the excess of DNA helicases at RF. Most MCM complexes are released from chromatin, except for a small but detectable fraction that remains at the forks during the S phase, as expected for a replicative helicase.

Abstract 3918: A structural role of androgen receptor in progression of prostate cancer cells from G1 to S phase

Abstract The androgen receptor (AR) plays a critical role in proliferation of prostate cancer cells. However, the mechanism of its action in proliferation remains unknown. We have shown previously that an AR-antagonist, Bicalutamide (Casodex), inhibits progression of synchronized LNCaP cells from G1 to S phase suggesting AR requirement in G1 phase for the cells to enter S phase (J Cell Physiol 195:337-345). In the present study we observed that a 4-hour pulse treatment with Casodex during mid- to late-G1 phase was sufficient to decrease the ability of cells to enter S phase. This brief pulse treatment with Casodex had no noticeable affect on either AR transcriptional activity or DNA synthesis in cells that had entered S phase. These observations suggested AR involvement in regulatory events, such as the assembly of pre-replication complex (pre-RC), that occur during mid- to late-G1 and are necessary for the onset of DNA synthesis and the progression of cells from G1 to S phase. Consistent with this possibility, we observed that AR co-localized and co-immunoprecipitated with Cdc6, an essential component of pre-RC, as determined by confocal microscopy and immunoprecipitation studies, respectively. Co-localization and co-precipitation of AR and Cdc6 were seen in synchronized cells that entered S phase, but not in those that were either in G0/G1 phase or prevented from entering into S phase by Casodex treatment indicating AR role in pre-RC. AR imunoprecipitate (AR-IP) also contained cyclin E and cyclin A that play a critical role in pre-RC assembly. In addition, AR also co-localized with DNA polymerase and AR-IP prepared from nuclear extracts of exponentially growing LNCaP cells contained DNA polymerase, PCNA, and ribonucleotide reductase. AR interaction with pre-RC components and the enzymes of DNA synthesis was highly specific since several other proteins such as cyclin B (a mitotic cyclin), caspase-3 (an apoptotic enzyme), PSA (AR-target gene), or GAPDH and actin (housekeeping proteins) were not associated with AR-IP. Thus AR interaction is limited to those proteins and enzymes that are involved in the onset of DNA synthesis and, therefore, the progression of prostate cancer cells from G1 to S phase. Furthermore, sucrose-density gradient analysis revealed AR co-sedimentation with cell cycle regulatory proteins, viz., cyclins E and A, and the enzymes of DNA synthesis, viz., DNA polymerase, PCNA and ribonucleotide reductase, in nuclear extracts that were prepared from S, but not G0/G1, phase cells. Thus AR exhibits a cell cycle-dependent interaction with proteins and enzymes involved in the initiation of DNA synthesis. Together, these observations point to a novel role of AR as a structural component of DNA replication machinery to exert control over proliferation of prostate cancer cells through a mechanism that is independent of its role as a transcription factor (Supported by NIH grant DK57864 and DoD grant W81XWH-05-1-0071). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3918. doi:1538-7445.AM2012-3918

Effects of seawater acidification on cell cycle control mechanisms in Strongylocentrotus purpuratus embryos.

Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered. Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos. To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1) the onset of DNA synthesis, (2) production of a mitotic regulator, cyclin B, and (3) formation of the mitotic spindle. We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ∼7.0, 7.5, and 8.0 by CO2 infusion. Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints. We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G1/S, G2/M and metaphase/anaphase transitions at relatively similar rates. These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics.

Hypoxia. Cross talk between oxygen sensing and the cell cycle machinery

A fundamental physiological property of mammalian cells is the regulation of proliferation according to O(2) availability. Progression through the cell cycle is inhibited under hypoxic conditions in many, but not all, cell types, and this G1 arrest is dependent on hypoxia-inducible factor (HIF) 1α. Components of the hexameric MCM helicase, which binds to replication origins before the onset of DNA synthesis, are present in large excess in mammalian cells relative to origins, suggesting that they may have additional functions. Screens for HIF-1α interacting proteins revealed that MCM7 binds to the amino-terminal PER-SIM-ARNT (PAS) domain of HIF-1α and stimulates prolyl hydroxylation-dependent ubiquitination and degradation of HIF-1α, whereas MCM3 binds to the carboxyl terminus of HIF-1α and enhances asparaginyl hydroxylation-dependent inhibition of HIF-1α transactivation domain function. Thus MCM proteins inhibit HIF activity via two distinct O(2)-dependent mechanisms. Under prolonged hypoxic conditions, MCM mRNA expression is inhibited in a HIF-1α-dependent manner. Thus HIF and MCM proteins act in a mutually antagonistic manner, providing a novel molecular mechanism for homeostatic regulation of cell proliferation based on the relative levels of these proteins.

Effects of Activation Methods on DNA Synthesis and Development of Parthenogenetic Porcine Embryos

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.

Reduced expression of GINS complex members induces hallmarks of pre-malignancy in primary untransformed human cells

In cancer cells ablation of the GINS complex member Psf2 elicits chromosome mis-segregation yet the precise role of Psf2 in mitosis is unknown. We investigated the putative mitotic role of the GINS complex using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Metaphase spreads from Psf1/Psf2-depleted HDF were normal and mitotic exit of Psf1/Psf2-depleted cells was only slightly delayed, suggesting no direct role for the GINS complex in mitosis of untransformed cells. Because the GINS complex is required for initiation and elongation events during DNA replication we hypothesized that the mitotic delay of Psf1/Psf2-deficient cells resulted indirectly from defective DNA synthesis during a prior S-phase. Therefore, we investigated the effects of Psf1/Psf2-depletion on DNA replication. Recruitment of Mcm7 to chromatin during G1 was unaffected by Psf1/Psf2-ablation, indicating that replication licensing does not require GINS. However, chromatin-binding of Cdc45 and PCNA, onset of DNA synthesis and accumulation of G2/M markers were delayed in Psf1/Psf2-ablated cells. The cell cycle delay of Psf1/Psf2-depleted HDF was associated with several hallmarks of pre-malignancy including γH2AX, Thr 68-phosphorylated Chk2, and increased numbers of aberrant fragmented nuclei. Ectopic expression of catalytically-inactive Chk2 promoted S-phase and G2/M progression in Psf1/Psf2-depleted cells, as evidenced by modestly-increased rates of DNA synthesis and increased dephosphorylation of Cdc2. Therefore, S-phase progression of untransformed cells containing sub-optimal levels of Psf1/2 is associated with replication stress and acquisition of DNA damage. The ensuing Chk2-mediated DNA damage signalling likely contributes to maintenance of chromosomal integrity.

From Chemical Embryology to Nucleosome Patterning - an interview with Roumen G. Tsanev

Roumen Tsanev was a prominent Bulgarian scientist whose pioneering ideas about the role for chromatin in cell differentiation and development led him to propose the first hypothesis for epigenetic information based on a histone code. To test experimentally his ideas, Dr.Tsanev explored nucleosome structure and heterogeneity and generated seminal data on nucleosome segregation in replicating chromatin. Roumen Tsanev made significant contributions to the understanding of chromatin changes that underlie zygotic gene activation. He identified sperm specific chromatin components tightly bound to DNA and demonstrated that the histone complement of the male pronucleus appears before the onset of DNA synthesis in the mouse zygote. In this interview, Roumen Tsanev talks about his passion for science and literature, reminisces about surmounting the harsh realities in post-war communist Bulgaria through creativity and determination, and explains what led him to propose that histones were carriers of epigenetic information. Dr. Tsanev discusses mathematical models of gene regulation and recalls computer simulations that reveal the non-linearity of genetic networks. He explains how this non-linearity could affect cell proliferation, differentiation, development and evolution.

Calcium/calmodulin-dependent protein kinase II-δ isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

26 NUCLEAR REPROGRAMMING AND EMBRYONIC AND FETAL DEVELOPMENT OF CLONED OVINE EMBRYOS PRODUCED USING CAFFEINE-TREATED OOCYTES AS CYTOPLAST RECIPIENTS

We have previously reported that treatment of ovine oocytes with 10 mM caffeine prior to their use as cytoplast recipients for somatic cell nuclear transfer (SCNT) increased the activities of both MPF and MAPK kinases, increased the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC), increased total cell numbers at the blastocyst stage, and altered the expression profiles of a number of developmentally regulated genes including Oct-4 and Oct-4-regulated genes and the stress response genes (HSP27 and HSP70). In addition, an increase in nuclear translocation of HSP27 was observed (Lee and Campbell 2006 Biol. Reprod. 74, 691–698; Choi et al. 2006 Reprod. Fertil. Dev. 18, 122; Choi et al. 2007 Reprod. Fertil. Dev. 19, 134). The objectives of the present study were to determine the potential mechanisms by which caffeine increased cell numbers and improved nuclear reprogramming. We previously hypothesized that caffeine treatment increased the degree of PCC and removed chromatin-bound somatic factors, allowing access of oocyte factors involved in reprogramming. With the use of a TUNEL assay, the accessibility of DNaseI (determined by fluorescence intensity) was increased in caffeine-treated embryos (P &lt; 0.05), supporting the hypothesis. The timing of pronuclear formation, onset of DNA synthesis, and first cleavage were also studied. At 6 h post-activation (hpa), no difference in frequency of pronuclear formation was observed between groups but higher numbers of Brdu-labelled nuclei were detected in caffeine-treated embryos (67.6% v. 43.5%; P &lt; 0.01). In caffeine-treated SCNT embryos, 40.5%, 54.3%, and 65% were cleaved at 20, 22, and 24 hpa as compared to 27.3%, 38.9%, and 48.4% in non-treated controls. On Day 7 there were no significant differences in the frequency of development to the blastocyst stage between treatment and control groups (26.06% v. 22.88%); however, treated embryos had increased total cell numbers (98.5 v. 76.6; P &lt; 0.05) and lower percentages of apoptotic nuclei (11.27% v. 20.3%; P &lt; 0.05). A total of 106 treated and 120 control blastocysts were transferred to the uteri of 43 and 49 recipient ewes, respectively. Five and six ewes established pregnancy, respectively. In the treatment group, three ewes maintained pregnancy for over 100 days and one live lamb was born; in contrast, all controls aborted within 100 days. Taken together, these results suggest that the increased cell numbers and improved reprogramming in SCNT embryos produced using caffeine-treated oocytes as cytoplast recipients may be mediated by increased NEBD and PCC facilitating chromatin access. In addition, increased MAP kinase activity may result in translocation of HSP27 to the nucleus, resulting in reduced apoptosis. Furthermore, caffeine-treated embryos maintained pregnancies for a longer period, supporting our previous observations (Lee et al. 2006 Reprod. Fertil. Dev. 18, 135–136) and suggesting that in sheep the use of caffeine to increase oocyte kinase activities results in embryos with a greater developmental competence.

This Month in Gastroenterology

This Month in Gastroenterology

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (> 40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin beta1 (HRG-beta1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.

Androgen receptor regulates Cdc6 in synchronized LNCaP cells progressing from G1 to S phase

We have shown previously that androgen receptor (AR) activity is required for the progression of cells from G(1) to S phase. In an attempt to elucidate the mechanism of androgen- and androgen-receptor-mediated proliferation of prostate cancer cells, we studied the effect of anti-androgen bicalutamide (Casodex) on the expression of cell-cycle regulatory genes in synchronized LNCaP cells progressing from G(1) to S phase. LNCaP cells were synchronized by isoleucine-deprivation. Expression of cell-cycle regulatory genes in S phase control cells versus Casodex-treated cells that fail to enter S phase was studied using a microarray containing cDNA probes for 111 cell-cycle specific genes. RT-PCR and Western-blots were used to validate microarray data. Casodex blocked synchronized LNCaP cells from entering S phase. Microarrays revealed downregulation of eight genes in cells prevented from entering into S phase by Casodex. Of these eight genes, only Cdc6, cyclin A, and cyclin B were downregulated at both the mRNA and protein level in Casodex treated cells as compared to control cells. The mRNA and protein levels of Cdc6 increased as synchronized LNCaP cells progressed from G(1) to S phase, and were attenuated in Casodex-treated cells failed to enter S phase. Cyclins A and B were detected when cells entered S phase, but not when they were in G(1) phase. Like Cdc6, the levels of both cyclins A and B were attenuated in Casodex-treated cells. AR may play an important role in the onset of DNA synthesis in prostate cancer cells by regulating the expression and stability of Cdc6, which is critically required for the assembly of the pre-replication complex(pre-RC).

The role of intranuclear lipids

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCß1 having a role in the onset of DNA synthesis and PC-PLCγ1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.

Proliferation of Epidermal Growth Factor-stimulated Hepatocytes in a Hormonally Defined Serum-free Medium

The present study shows that adult rat hepatocytes in primary culture, which normally exhibit a restricted capacity to proliferate, can proceed through the cell cycle when cultured in a mixture of minimal essential medium (MEM) and Medium 199 (MEM-M199; 3:1, v/v), containing epidermal growth factor (EGF; 50 ng/ml), low glucose (0.75 g/l) and low levels of inorganic salts, amino acids and vitamins. Under these conditions, hepatocytes flatten and cell extensions appear. In contrast, Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5g/l) levels enriched with inorganic salts, amino acids and vitamins favours maintenance of differentiated functional hepatocyte capacities (albumin secretion), but does not allow proliferation or cell spreading. Cultivation of hepatocytes in MEM-M199 (3:1, v/v) results in the onset of DNA synthesis at 48 hours of culture and a concomitant induction of cyclin D1 protein. Under these conditions, cells successively progress through the mitogen-dependent restriction point in mid-late G1 phase, G1/S transition and S phase, as evidenced by Western blot analysis of the markers cyclins E and A and cyclin dependent kinase (CDK)2 and CDK1, respectively. Progression through the cell cycle is accompanied by a decrease in albumin secretion, indicating a decline in differentiated capacities. This study demonstrates that hepatocytes cultured in a mixture of MEM-M199 (3:1) provide a useful in vitro model for studying the regulation of hepatocyte proliferation.

Mutations of the CK2 phosphorylation site of Sic1 affect cell size and S-Cdk kinase activity in Saccharomyces cerevisiae.

By sequence analysis we found an amino acid stretch centred on Serine201 matching a stringent CK2 consensus site within the C-terminal, inhibitory domain of Sic1. Here we show by direct mass spectrometry analysis that Sic1, but not a mutant protein whose CK2 phospho-acceptor site has been mutated to alanine, Sic1S201A, is actually phosphorylated in vitro by CK2 on Serine 201. Mutation of Serine 201 alters the coordination between growth and cell cycle progression. A significant increase of average protein content and of the average protein content at the onset of DNA synthesis is observed for exponentially growing cells harbouring the Sic1S201A protein. A strong reduction of the same parameters is observed in cells harbouring Sic1S201E. The deregulated coordination between cell size and cell cycle is also apparent at the level of S-Cdk activity.

Role of androgen receptor in prostate cancer cell cycle progression from G 1 into S phase and in the onset of DNA synthesis

Role of androgen receptor in prostate cancer cell cycle progression from G 1 into S phase and in the onset of DNA synthesis