Regulation Of Cell Cycle Control Research Articles

S Phase–Coupled E2f1 Destruction Ensures Homeostasis in Proliferating Tissues

Precise control of cell cycle regulators is critical for normal development and tissue homeostasis. E2F transcription factors are activated during G1 to drive the G1-S transition and are then inhibited during S phase by a variety of mechanisms. Here, we genetically manipulate the single Drosophila activator E2F (E2f1) to explore the developmental requirement for S phase–coupled E2F down-regulation. Expression of an E2f1 mutant that is not destroyed during S phase drives cell cycle progression and causes apoptosis. Interestingly, this apoptosis is not exclusively the result of inappropriate cell cycle progression, because a stable E2f1 mutant that cannot function as a transcription factor or drive cell cycle progression also triggers apoptosis. This observation suggests that the inappropriate presence of E2f1 protein during S phase can trigger apoptosis by mechanisms that are independent of E2F acting directly at target genes. The ability of S phase-stabilized E2f1 to trigger apoptosis requires an interaction between E2f1 and the Drosophila pRb homolog, Rbf1, and involves induction of the pro-apoptotic gene, hid. Simultaneously blocking E2f1 destruction during S phase and inhibiting the induction of apoptosis results in tissue overgrowth and lethality. We propose that inappropriate accumulation of E2f1 protein during S phase triggers the elimination of potentially hyperplastic cells via apoptosis in order to ensure normal development of rapidly proliferating tissues.

Overview: Special issue on Drosophila spermatogenesis.

Overview: Special issue on Drosophila spermatogenesis.

SWOG S0826: A phase II trial of SCH 727965 (NSC 747135) in patients with stage IV melanoma.

8521 Background: Cyclin-dependent kinases (cdks) function to regulate cell cycle control and agents that can target cdks in malignant progression remain viable therapeutic strategies. Selective inhibition of cdk2, in particular, may be of therapeutic value in a subset of patients with melanoma. Methods: 60 patients with metastatic melanoma of cutaneous or mucosal origin were planned to be recruited to a multicenter, single-arm phase II trial of the cdk inhibitor, SCH 727965 (NSC747135). Patients were potentially eligible if they had 0-1 previous treatments, PS of 0-1, and adequate organ function. Ocular melanoma patients and patients with a history of brain metastases were excluded. SCH 727965 50 mg IV every 3 weeks was given until progression with disease assessment occurring every 2 cycles. Co-primary endpoints were 1-year overall survival (OS) and 6-month progression free survival (PFS). Results: 72 patients were enrolled from July 1, 2009 to November 1, 2010 at 24 institutions. 68% of patients had M1c disease and 43% had LDH elevation. 19% had prior therapy for metastatic disease. 28 patients (39%) experienced Grade 4 adverse events, including 20 cases of neutropenia, one case each of cardiac ischemia/infarction, cardiac troponin I elevation, dehydration, abdominal pain, leukopenia, muscle weakness, headache, syncope, and anterior ischemic optic neuropathy. 65 patients are currently evaluable for response. The response rate was 0/65 (95% C.I. (0-6%)). Stable disease was observed in 22%. The estimated median PFS was 1.5 months (95% CI: 1.4 – 1.5); 6-month PFS was 11% (5-20%). Median OS was 8 months (95% CI: 5-11 months); 1-year OS was 36% (95% CI: 24-48%). The null hypothesis of 1-year overall survival=25% was rejected (p=0.04) but 6-month PFS=11% was not (p=0.8). Data Analysis will be updated when missing data are received. Correlative studies of Rb phosphorylation and cyclin expression will be pursued. Conclusions: SCH 727965 appears to be reasonably well tolerated although grade 4 events were relatively common, particularly near the time of infusion. There were no responses but few patients had prolonged disease stabilization that may have resulted in improvement in the 1-year OS rate.

Atoh1, an Essential Transcription Factor in Neurogenesis and Intestinal and Inner Ear Development: Function, Regulation, and Context Dependency

Atoh1 (also known as Math1, Hath1, and Cath1 in mouse, human, and chicken, respectively) is a proneural basic helix-loop-helix (bHLH) transcription factor that is required in a variety of developmental contexts. Atoh1 is involved in differentiation of neurons, secretory cells in the gut, and mechanoreceptors including auditory hair cells. Together with the two closely related bHLH genes, Neurog1 and NeuroD1, Atoh1 regulates neurosensory development in the ear as well as neurogenesis in the cerebellum. Atoh1 activity in the cochlea is both necessary and sufficient to drive auditory hair cell differentiation, in keeping with its known role as a regulator of various genes that are markers of terminal differentiation. Atoh1 is known in other fields as an oncogene and a tumor suppressor involved in regulation of cell cycle control and apoptosis. Aberrant Atoh1 activity in adult tissue is implicated in cancer progression, specifically in medullablastoma and adenomatous polyposis carcinoma. We demonstrate through protein sequence comparison that Atoh1 contains conserved phosphorylation sites outside the bHLH domain, which may allow regulation through post-translational modification. With such diverse roles, tight regulation of Atoh1 at both the transcriptional and protein level is essential.

Unraveling interactions of cell cycle‐regulating proteins Sic1 and B‐type cyclins in living yeast cells: a FLIM‐FRET approach

Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.

Cellular and molecular mechanisms of various types of oocyte aging.

It is well established that age-related decline of a woman's fertility is related to the poor developmental potential of her gametes. The age-associated decline in female fertility is largely attributable to the oocyte aging caused by ovarian aging. Age-associated oocyte aging results in a decrease in oocyte quality. In contrast to ovarian aging, there is a concept of postovulatory oocyte aging. Postovulatory aging of oocytes, not being fertilized for a prolonged time after ovulation, is known to significantly affect the development of oocytes. Both categories of oocyte aging have similar phenotypes of reproductive failure. However, the mechanisms of the decline in oocyte quality are not necessarily equivalent. An age-dependent increase in aneuploidy is a key determinant of oocyte quality. The reduced expression of molecules regulating cell cycle control during meiosis might be involved in the age-dependent increase in aneuploidy. The mechanism of age-associated oocyte aging might be involved in mitochondrial dysfunction, whose etiologies are still unknown. Alternatively, the mechanism of postovulatory oocyte aging might be involved in reactive oxygen species-induced mitochondrial injury pathways followed by abnormal intracellular Ca2+ regulation of the endoplasmic reticulum. We suggest that future research into the mechanism of oocyte aging will be necessary to develop a method to rescue the poor developmental potential of aged oocytes.

Abstract 5546: Screening the RbBP6 gene for abnormal alternative splicing in colon cancer

Abstract Cancer arises after a series of mutations that result in uncontrolled cellular differentiation and growth. These result because the genes required for normal cell growth and apoptotic processes are abnormally regulated and thus the progression of cancer and survival is favoured. The RbBP6 gene is amongst the genes which are down-regulated in cancerous cells. RbBP6 is a gene that has been shown to regulate cell cycle control and apoptosis. RbBP6 isoform 3 has been shown to regulate G2/M cell cycle checkpoint while the alternatively spliced RbBP6 transcript 1 product has been implicated in apoptosis regulation and cancer. Expression and regulation of the gene in colon cancer was examined by amplifying cDNA of RNA extracted from three colon adenocarcinoma cell lines obtained from different stages of colon cancer, using RT-PCR with the RbBP6 exon 16 primers and confirmation of the 2 splice variants by NCBI BLAST. Two bands appeared on a 1% Agarose gel, the ratio of the intensity of the two bands was found to be 1:1 in the primary cell line compared to down-regulation in grade II cell line while these alternatively spliced products could hardly be amplified in grade III cell line. RbBP6 has two splice isoforms which are regulated by alternative splicing in colon cancer. The expression of these splice variants was found to be indirectly proportional to colon cancer progression. The primary colon cancer cell line showed more expression of these alternatively spliced products, which decreased significantly in grade II and III. This suggests that RbBP6 alternatively spliced products could be anti-carcinogenic but are de-regulated in favour of carcinogenesis and this further suggests that RbBP6 gene might be somehow involved in the development and progression of colon adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5546. doi:10.1158/1538-7445.AM2011-5546

The regulatory network of cell-cycle progression is fundamentally different in plants versus yeast or metazoans

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell-cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell-cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell-cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell-cycle regulators, e.g. CDKs, cyclins, or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.

Small-interfering RNA-mediated silencing of the MAPK p42 gene induces dual effects in HeLa cells

The genesis and progression of cervical cancer involve the mutation or deviant expression of numerous genes, including the activation of oncogenes (Ha-ras, C-myc, C-erbB2 and Bcl-2) and inactivation of tumor-suppressor genes (p53 and Rb). Previous studies showed that small-interfering RNAs (siRNAs) targeting the MAPK p42 gene partly inhibit proliferation and increase apoptosis in human cervical carcinoma HeLa cells. Results of a microarray analysis showed that MAPK p42 siRNA inhibited cell growth through the regulation of cell cycle control and apoptosis and induced interferon-like response in HeLa cells. In order to confirm the dual effects of MAPK p42 siRNA, we compared the roles of siRNA and U0126, an inhibitor of MAPK p42, in HeLa cells. Short 21-mer double-stranded/siRNAs were synthesized to target MAPK p42 mRNA in HeLa cells. The siRNAs were transfected into HeLa cells using Lipofectamine. The cells were treated with siRNA or U0126 at different concentrations for a period of 48 h. The biological effect of siRNA and U0126 on HeLa cells was measured by MTT and flow cytometry. MAPK1, NUP188, P38, STAT1, STAT2, PML and OAS1 were analyzed by real-time quantitative PCR. HeLa cell growth was inhibited by siRNA or U0126, and the effect of siRNA inhibition was greater than that of U0126. Cell cycle phases were different for siRNA or U0126, but HeLa cell growth was arrested at the S phase by siRNA and at G1 phase by U0126. A down-regulation in MAPK p42 expression by siRNA and up-regulation by U0126 were noted. The results of real-time quantitative PCR showed that P38 was up-regulated and NUP188 was down-regulated by siRNA in comparison with the control groups, and the results were consistent with those of U0126. Expression levels of STAT1, STAT2, PML and OAS1 induced by siRNA differed from those induced by U0126. siRNA-mediated silencing and deactivation induced by U0126 in MAPK p42 led to growth inhibition in the HeLa cells. The effects of siRNA on HeLa cell growth were different from those of U0126. Dual effects of MAPK p42 siRNA-2 on HeLa cell growth were noted: one consisted of a specific effect induced by siRNA-mediated p42 MAPK silencing and the other exhibited a non-specific interferon-like response.

Abstract 924: Possible joint effects between single nucleotide polymorphisms in inflammation genes and serum androgen levels on risk of prostate cancer

Abstract Both inflammation and androgens are likely to be involved in the etiology of prostate cancer. There is evidence for cross-talk between androgen and inflammation pathways, yet little is known about the joint contribution of inflammation and androgens to prostate cancer risk. Thus, we evaluated the joint effects of 9,932 tag single nucleotide polymorphisms (SNPs) in 774 inflammation-related genes and four serum androgen measures (total testosterone [T], bioavailable T, 3α-androstanediol glucuronide [3α diol G], and androstenedione) on risk of prostate cancer in 516 incident cancer cases and 560 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. SNPs included in this study were genotyped as part of the National Cancer Institute Cancer Genetic Markers of Susceptibility genome-wide association study. Likelihood ratio tests (LRT) were used to compare logistic models of prostate cancer regressed on age, center, the androgen (in quartiles), the SNP (additive genetic coding) and the SNP x hormone interaction term to logistic models of prostate cancer regressed on age, center, and the androgen. False discovery rate (FDR) control was used to adjust for multiple testing. Statistically significant LRT P-values were noted for T and three intronic SNPs in linkage disequilibrium (r2>0.7) within the HIPK2 gene on 7q34 (rs10256326, rs7788362, and rs12539357; FDR-adjusted P=0.04 for all SNPs). For each SNP, the minor allele was associated with a reduced prostate cancer risk in men with serum T levels in the lower three quartiles, but was associated with an increased risk among men in the highest quartile of serum T. LRT P-values of borderline statistical significance were also seen for joint effects of these three SNPs and bioavailable T on risk of prostate cancer (FDR-adjusted P=0.07 for all SNPs). There were no significant joint effects for any of the SNPs with 3αdiol G or androstenedione after adjustment for multiple tests. Our preliminary results suggest that T levels could interact with HIPK2 gene variants to influence prostate cancer risk. HIPK2 is an attractive candidate gene for further evaluation because it codes for a serine/threonine kinase involved in transcriptional regulation, particularly involved in p53-dependent and independent regulation of cell cycle control and apoptosis. Future studies are needed to confirm our preliminary findings and to further understand the possible interaction between T and genetic variants in HIPK2, which could contribute to prostate cancer risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 924.

Abstract 3884: Beta-2 spectrin opposes the CDK4-mediated suppression of TGF-beta signaling by rescuing Smad3 nuclear localization

Abstract Background: Transforming growth factor beta (TGF-β) signaling inhibits cell proliferation by inducing a G1 cell cycle arrest. By contrast, cyclin-dependent kinase 4 (CDK4) inactivates retinoblastoma function early in G1, thus promoting cell cycle progression. However, CDK4 can also inhibit TGF-β signaling by phosphorylating the linker region of Smad3, a key mediator of TGF-β signaling. We have previously demonstrated that Smad3 nuclear translocation and TGF-β signaling are dependent upon the function of the adaptor protein, beta-2 spectrin (β2SP or ELF). Furthermore, loss of β2SP in a knockout model results in the spontaneous development of hepatocellular cancer (HCC). HCCs from these elf+/− mice overexpress CDK4, and haploinsufficiency of CDK4 in elf+/−/CDK4neo/+ mice suppresses hepatocarcinogenesis. These findings point towards competing activities of β2SP and CDK4 on TGF-β signaling, which may be determined by their modulation of Smad3. Our goal was to determine the mechanisms for these competing effects. Methods: We have overexpressed CDK4, β2SP, or both in human HCC cell lines. Using these stable cell lines, we examined Smad3 phosphorylation by western blot, Smad3 nuclear localization by immunocytochemistry, and TGF-β-regulated gene expression by quantitative RT-PCR (qPCR). Results: We sought to determine whether β2SP can rescue the CDK4-mediated suppression of TGF-β signaling. Over-expression of CDK4 blocked TGF-β-induced Smad3 nuclear translocation, whereas a CDK4/6-specific inhibitor, PD-0332991 (Pfizer, Inc) rescued Smad3 nuclear translocation. Similarly, over-expression of β2SP rescued Smad3 nuclear translocation in these CDK4 overexpressing cells. Furthermore, both PD-0332991 and β2SP overexpression enhanced the expression of TGF-β-regulated genes, as determined by qPCR. We hypothesize that the opposing effects of CDK4 and β2SP are mediated by the balance between the CDK4-mediated phosphorylation of the Smad3 linker region, and the β2SP-mediated phosphorylation of the Smad3 C-terminal domain. Thus, we also examined the domain-specific phosphorylation of Smad3 in CDK4-overexpressing cells. PD-0332991 specifically reduces phosphorylation of the linker region of Smad3 (T179 and S208), but has no effect on c-terminal phosphorylation (S423/S425), while overexpression of β2SP increases c-terminal phosphorylation. Assessment of Smad3 nuclear localization and the effects on TGF-β signaling in the presence of phospho-specific Smad3 mutants is ongoing. Conclusions: Our results have begun to clarify the competing roles of CDK4 and β2SP on Smad3 nuclear localization and thus TGF-β signaling. Based on our results, targeted-inhibition of the CDK4-Smad3 interaction may be critical in rescuing TGF-β-mediated regulation of cell cycle control, but β2SP expression may be required, and may serve as a critical marker predictive of such activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3884.

Yeast Cell Cycle Analysis: Combining DNA Staining with Cell and Nuclear Morphology

In studies of eukaryotic cell cycle regulation, the budding yeast Saccharoymyces cerevisiae offers many advantages as a model system. Due to its simple growth requirements and genetic tractability, this organism is a powerful tool for investigating the molecular regulation of cell cycle control. One earlier disadvantage to performing cell cycle analyses in yeast was that existing methods were restricted to either visual analysis or flow cytometry, both of which present limitations in the scope and accuracy of the data obtained. This unit demonstrates the combined use of DNA content measurements and bright-field image analysis using multispectral imaging flow cytometry (MIFC) to provide a more precise quantitation of yeast cell cycle distribution. The advantage of this method is the ability to analyze large numbers of cells using multiple cell cycle indicators in a relatively short amount of time.

Monitoring of Long‐Term Effects of Resveratrol on Cell Cycle Progression of Human HeLa Cells after Administration of a Single Dose

Expression of the human papillomavirus-encoded oncoproteins E6 and E7 in human HeLa cervical carcinoma cells results in their escape from the proper control of the cell cycle progression. Therefore, their susceptibility to agents modulating cell cycle differs from that in cells in which the control of cell cycle regulation is intact. Recently, a number of experimental studies revealed that polyphenols, especially resveratrol, could exert a strong antiproliferative effect. Polyphenols (e.g., resveratrol or epicatechins), potent antioxidant agents, are abundant components of our diet and, therefore, may not only affect the proliferation of healthy cells in the organism but also modulate the action of distinct anticancer drugs. Indeed, it has been shown that resveratrol enhances the antimitotic effect exerted by roscovitine (ROSC), a potent cyclin-dependent kinase inhibitor, on human MCF-7 breast cancer cells. In the present contribution the action of resveratrol alone and in combination with ROSC on human HeLa cells was determined. Resveratrol inhibited proliferation of exponentially growing HeLa cells. Exposure of HeLa cells to 50 micromol/L resveratrol blocked cells in the S phase in a time-dependent manner. After 12 h the population of G(2)/M-phase cells completely disappeared, and during a further 12 h the frequency of S-phase cells markedly increased and reached approximately 90%. Thus, resveratrol synchronized HeLa cells in the S phase. After removal of resveratrol, synchronized HeLa cells rapidly progressed through the cell cycle. Four hours after medium change, more than 70% of cells moved into the G(2)/M phase. Moreover, resveratrol combined with ROSC enhanced the antiproliferative action of resveratrol.

EXPRESSION OF PTEN AND SHP1, INVESTIGATED FROM TISSUE MICROARRAYS IN PEDIATRIC ACUTE LYMPHOBLASTIC, LEUKEMIA

PTEN and SHP1 are tumor suppressor genes involved in the regulation of cell cycle control and apoptosis. The authors investigated the protein expression of PTEN and SHP1, by immunohistochemistry in tissue microarrays from bone marrow samples in children, diagnosed with acute lymphoblastic leukaemia and nonmalignant controls. PTEN was overexpressed in diagnostic ALL samples, while SHP1 showed a low expression. Both proteins showed a significant difference in expression compared to nonmalignant controls. The roles of PTEN and SHP1 are not well investigated in pediatric leukemia and could in the future play a role as prognostic factors.

Cdc25B Phosphatase Inhibitors in Cancer Therapy: Latest Developments, Trends and Medicinal Chemistry Perspective

The Cdc25 phosphatases (Cdc25A, Cdc25B, and Cdc25C in humans), which are responsible for dephosphorylating specific tyrosine/threonine residues on cyclin dependent kinases (CDKs), function as essential regulators of cell cycle control during normal eukaryotic cell division and as mediators of the checkpoint response in cells with DNA damage. Because overexpression of Cdc25A and Cdc25B has been linked to numerous cancers and often correlates with a poor clinical outcome, both academia and industry have devoted substantial research effort in establishing the basic underlying molecular mechanisms and in identifying novel, specific and potentially useful inhibitors of Cdc25 as potential anticancer drugs. Over the past year, dozens of research papers and patent applications describing new Cdc25 inhibitors belonging to different structural classes have been disclosed. In this review, we give an overview on the current status in the field of medicinal chemistry of Cdc25B inhibitors. In addition, molecular modeling studies aimed to clarify the molecular mechanism of inhibition as well as the pharmacophoric features critical for design of new and selective Cdc25B inhibitors are also discussed.

Methylation of the PTEN Promoter Leads to PTEN Inactivation in Multiple Myeloma

Aberrant methylation of CpG island of the promoter regions of genes is an important epigenetic mechanism, alternative to deletions or mutations, that cause the silencing of genes involved in carcinogenesis. We therefore investigated epigenetic events involved in multiple myeloma by screening 6 genes of interest for evidence of promoter hypermethylation.PATIENTS AND METHODS: The methylation patterns of the tumor suppressor genes p16INK4a(p16), E-cadherin (ECAD), FHIT and PTEN (negative regulator of AKT/PKB signalling pathway), Wnt signalling pathway antagonist genes WIF-1 and DKK-3 were determined in the bone marrow aspirates of 50 patients with Multiple Myeloma (MM) (37 at diagnosis; male 22; female 28; 68.8 median age. ISS stage: 47% stage I, 8% stage II and 45% stage III.), using the methylation-specific polymerase chain reaction after DNA bisulphite modification. Ten patients with monoclonal gammopathy of undetermined significance (MGUS) and 4 peripheral blood from controls were also evaluated. For statistical analysis Student t and Chi squared or Fisher exact tests were used.RESULTS: We found at least one hypermethylated gene in all MM patients. Gene methylation frequencies varied from 6% to 74%. ECAD, FHIT and p16 genes demonstrated a relatively high frequency of aberrant methylation with frequencies of 74%, 69% and 42%, respectively. WIF-1, PTEN, DKK-3 genes showed a low frequency of methylation with values of 28%, 14%, 6%, respectively. The methylation frequencies detected in MGUS were 91.6% for ECAD and FHIT, 41.6% for WIF-1, 16,6% for p16 and DKK3. The difference in methylation profile between MM and MGUS was statistically significant (X2= 37, df=5; p=0.0000). PTEN was found hypermethylated in 7/50 (14%) MM but none of MGUS or control samples were methylated. Of these 6 patients had IgG K isotype, 4 had abnormal cariotype with 13q deletion, one hyperdiploidia, one IgH rearrangements. Two patients were simultaneously hypermethylated in p16 gene promoter. No statistically significant correlation between methylation data and clinical parameters: gender, age, isotype of M component, type of light chain, haemoglobin, serum albumin level, calcium, β2 microglobulin, serum creatinine level, LDH, lytic bone lesions were found for any of examined genes. When correlation with Durie-Salmon Stage disease was tested hypermethylation of p16 and WIF-1 resulted more frequently associated with stage IIIA/B (p values 0.006 and 0.000, respectively). When gene methylation status and cytogenetics abnormalities were compared, we found that aberrant methylation of p16 and ECAD were significantly more frequent in MM patients with chromosome 13 abnormalities as sole entity or with 13q deletion associated to IgH translocations. By contrast, promoter hypermethylation of Wnt signalling antagonist genes was associated with 13q deletion co-occurring with a hyperdiploide cariotype. (X2=17; df=8 p value: 0.000). According to the number of methylated genes observed in each sample 48% MM had 3–4 hypermethylated genes. When differences between methylator phenotype and cytogenetics abnormalities were analyzed we found that 3–4 hypermethylated genes have the tendency to be associated with chromosome 13 abnormalities more often than with hyperdiploide cariotype (p value 0.05).CONCLUSION: Our results indicate that the accumulation of epigenetic events affecting genes regulating cell cycle control, cell adhesion and apoptosis is a common phenomenon in plasma cell disorders and may further contribute to the phenotype of MM cells. This is the first demonstration of PTEN inactivation as a result of promoter hypermethylation in MM patients. Since talidomide seems to have a role in the PTEN/PI3K/AKT pathway, the PTEN function is worthy to be further investigated in Multiple Myeloma.

Connections between polar growth and cell cycle arrest during the induction of the virulence program in the phytopathogenic fungus Ustilago maydis

Elegant work by others has highlighted the importance of connections between polar growth and cell cycle regulation in budding and fission yeast. However, it is striking that little attention has been paid to the study of these connections in phytopathogenic fungi. In these crop pests, germination of spores, the main infective agent, requires a strict control of cell cycle regulation as well as polarity growth. Our finding that a cyclin-cdk pair controls both processes in the corn smut fungus Ustilago maydis supports the importance of such a regulation during the pathogenic development of fungi.

Expression of p21waf1/cip1, p27kip1, p63 and Androgen Receptor in Low and High Gleason Score Prostate Cancer

The aim of this study was to investigate the expression of p21(waf1/cip1), p27(kip1), p63 and androgen receptor proteins in relation to serum prostate specific antigen levels in low and high Gleason score prostate cancers. Biopsies of patients suffering from prostate adenocarcinoma of low (3 + 3 to 3 + 4) and high (5 + 4 to 5 + 5) Gleason scores (13 cases each group) were immunostained for positive regulators of cell cycle control (p21(waf1/cip1) and p27(kip1)), and essential markers of normal prostate gland ontogeny (p63) and growth (androgen receptor) to find differentially expressed markers of malignant progression. Serum prostate specific antigen levels were also monitored at the time of biopsy and following anti-androgen therapy. All cases except one in each group were androgen receptor positive. P63 and p21(waf1/cip1) proteins detected in normal basal cell nuclei were lost in all but one studied tumors respectively. P27(kip1) protein, however, was detected in all low Gleason score prostate cancers, but it was found in only 7/13 high score cases. Prostate specific antigen levels, either pre- or post-treatment, did not show strict correlation with the p27(kip1) results. The low to high grade dedifferentiation of prostate adenocarcinoma is accompanied with the down-regulation of p27(kip1) protein, which may be an important molecular sign of the lost cell cycle control.

Negative Regulation of Cell Cycle Kinetics of Human Hematopoietic Cells by the Transcription Factor Dmtf1.

Hematopoietic stem cells (HSCs) are predominantly quiescent with only a small number entering active phases of cell cycle at any time point. With such tightly regulated cell cycle kinetics, HSCs ensure preservation of the stem cell pool. Many cell cycle-related proteins, mainly tumor suppressor genes, are involved in maintenance of HSCs quiescence. Dmtf1 (Cyclin D-binding Myb-like Protein1) is a transcription factor that negatively regulates cell cycle by inducing Arf expression, and its deletion has been reported in some leukemias (Bodner SM et al, 1999). As there is no information regarding the role of Dmtf1 in hematopoiesis, we examined the impact of Dmtf1 on regulating cell cycle kinetics of hematopoietic progenitor cells. Dmtf1 mRNA was expressed in human granulocytes, lymphocytes, CD34+ cells, and in murine Sca1+lin-CD117+ (KSL) cells. Using retroviral vectors (MIEG3/IRES-GFP), we first investigated cell cycle progression in 293 cells transduced with four different constructs; GFP only control vector (−), wild type Dmtf1 (WT), and two dominant negative mutants expressing a point mutation (K319E) or a deleted myb-like repeat box (dMHR). A total of 36.0% of sorted and cultured GFP+ control (−) cells were in S/G2+M 24hr after initiation of culture. Whereas 30.8% of cells expressing (WT) were in S/G2+M at the same time point, expression of the two dominant negative mutants K319E and dMHR induced 46.6% and 45.5% of the cells into S/G2+M, respectively suggesting that loss of Dmtf1 activity results in rapid cell proliferation. Interestingly, a 2.5-fold increase in the ecxpression of Arf and p21 mesured by qPCR was detected in cells transduced with WT only whereas other transduced cells did not show any change in expression of both Arf and p21. The impact of these constructs was then evaluated in cord blood cells using CFU assays. Cord blood CD34+ cells were transduced with the four vectors mentioned above and GFP+ cells were subsequently sorted and cultured. Both K319E and dMHR induced a 25% increase in the number of clonogenic progenitors relative to (−) while a modest decrease of 10% in colony numbers was detected in the WT group. Cells cycle analysis of these cells is currently under investigation. These results demonstrate that Dmtf1 acts as a negative regulator of cell cycle control in hematopoietic cells and suggests that it may play a role in the maintenance of HSC quiescence.

The pattern of p53 gene mutations on oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the result of accumulation genetic lesion and caused by specific mutation in specific key regulation genes. p53 gene is key target specific regulatory genes which function as negative regulators in cell cycle control. The highest mutation rates found in human cancers and the etiology in high risk populations and the pattern of molecular pathogenesis mechanism involved in the OSCC remain unclear. The purpose of this study was to determine the presence of alteration or mutation of p53 gene and to associate these mutations histopathological status of the patients such as well differentiated and poorly differentiated in OSCC in order to elucidate the molecular pathogenesis mechanism of OSCC based on the pattern of p53 gene. Using 40 untreated well and poorly differentiated OSCC biopsy samples and 16 normal patients were analyzed for the presence of mutation in the conserved region of the p53 gene exons 5 and or 7 by PCR-SSCP mutational analysis for p53 gene showed 70% of total samples : exon 5: 27.5% with heterozygous mutation 81.8%, exon 7: 55% with heterozygous mutation 100%. The incidence of p53 mutation was not significantly associated with well and poorly differentiated OSCC with the exception in exon 5 of p53 gene (p = 0.013) using contingency coefficient. This study concludes that mutation of p53 gene especially in exon 7 may not indirectly play in the progressivity of OSCC with the exception of mutation in exon 5 of p53 which indicates the essential role in the progressivity of OSCC.