G1-S Checkpoint Research Articles

CDK4 inhibition restores G₁-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G₁-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19INK4D overexpression partly restored G₁-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19INK4D, but not p16INK4A, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G₁-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G₁ checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G₁-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.

MicroRNA-224 Negatively Regulates p21 Expression During Late Neoplastic Progression in Inflammatory Bowel Disease

The development of colon cancer represents a major complication in patients with inflammatory bowel disease (IBD). The importance of microRNAs (miRs) in carcinogenesis is becoming clearer because miRs have been implicated in the regulation of cancer-related cellular processes to include apoptosis, differentiation, cell cycle progression, and immune function. In the current study, we sought to identify miR dysregulation specific to progression along the normal-inflammation-cancer axis in colonic specimens from patients with IBD. MiR microarrays and quantitative reverse transcription PCR were used to detect and confirm dysregulated miRs. Receiver operating characteristic curve analysis was applied to evaluate the potential use of miR-224 as a neoplastic disease marker in IBD. For miR-224 target messenger RNA (mRNA) identification, mRNA microarrays were employed in combination with bioinformatic analyses, Western blotting, and luciferase activity measurements. We identified 30 miRs that were differentially expressed between chronically inflamed mucosae and cancers arising from IBD tissues. MiR-224 levels increased successively at each stage of IBD progression and accurately discriminated cancers from normal or chronically inflamed IBD tissues. Moreover, mRNA arrays combined with bioinformatic analyses suggested the participation of miR-224 in cell cycle regulation. Subsequently, cell cycle experiments indicated that miR-224 regulates the G1-S checkpoint. Finally, in silico prediction analyses, confirmed by Western blotting and luciferase assays, identified p21 as a specific direct mRNA target of miR-224. These findings reveal miR dysregulation specific to IBD-associated colorectal carcinoma. MiR-224 is overexpressed in IBD cancers and targets p21, a key cell cycle regulator. Moreover, these results establish the participation of miR-224 in IBD carcinogenesis.

Non-Parkinson movement disorders

SOLUTIONS TO THE MAJOR RIDDLES IN MOVEMENT DISORDERS ARE APPEARING AT A BREATHTAKING PACE: 1) loss-of-function mutations in PRRT2, which encodes a cell surface protein expressed in neurons, have been found in many patients with paroxysmal kinesigenic dyskinesias; 2) mutations in CIZ1, which encodes a protein involved in cell-cycle control at the G1-S checkpoint, have been identified in a small percentage of patients with cervical dystonia; and 3) finally, after many years of genetics and identification of more than 25 disease-associated genes, cellular studies related to the pathobiology of hereditary spastic paraplegia are converging on defects in modeling the endoplasmic reticulum and membrane trafficking. On the treatment front, the distinctive syndromes of faciobrachial dystonic seizures with anti-LRI1 antibodies and anti-N-methyl-d-aspartic acid encephalitis with orobuccolingual dyskinesias are becoming increasingly recognized by clinicians as imminently treatable conditions. Also on the treatment front, the first phase I trial of MRI-guided high-intensity focused ultrasound for essential tremor has been completed and intraoperative MRI is currently being used to place electrodes in the brains of patients with medically intractable dystonia. Definitive etiologies and efficacious treatments for non-Parkinson disease movement disorders are no longer wishful thinking.

CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11p110

Checkpoint kinase 2 (CHK2) kinase is a key mediator in many cellular responses to genotoxic stresses, including ionizing radiation (IR) and topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis and DNA repair by phosphorylating downstream target proteins, such as p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer susceptibility gene. In this study, we used a tandem affinity purification strategy to identify proteins that interact with CHK2 kinase. Cyclin-dependent kinase 11 (CDK11)(p110) kinase, implicated in pre-mRNA splicing and transcription, was identified as a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11(p110) on serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated CDK11(p110) in a DNA damage-independent manner. At a molecular level, CDK11(p110) phosphorylation was required for homodimerization without affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA splicing. Conversely, CHK2 depletion decreased endogenous splicing activity. Mutation of the phosphorylation site in CDK11(p110) to alanine abrogated its splicing-activating activity. These results provide the first evidence that CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Posttranslational Modifications of the Retinoblastoma Tumor Suppressor Protein as Determinants of Function

The retinoblastoma tumor suppressor protein (pRB) plays an integral role in G1-S checkpoint control and consequently is a frequent target for inactivation in cancer. The RB protein can function as an adaptor, nucleating components such as E2Fs and chromatin regulating enzymes into the same complex. For this reason, pRB's regulation by posttranslational modifications is thought to be critical. pRB is phosphorylated by a number of different kinases such as cyclin dependent kinases (Cdks), p38 MAP kinase, Chk1/2, Abl, and Aurora b. Although phosphorylation of pRB by Cdks has been extensively studied, activities regulated through phosphorylation by other kinases are just starting to be understood. As well as being phosphorylated, pRB is acetylated, methylated, ubiquitylated, and SUMOylated. Acetylation, methylation, and SUMOylation play roles in pRB mediated gene silencing. Ubiquitinylation of pRB promotes its degradation and may be used to regulate apoptosis. Recent proteomic data have revealed that pRB is posttranslationally modified to a much greater extent than previously thought. This new information suggests that many unknown pathways affect pRB regulation. This review focuses on posttranslational modifications of pRB and how they influence its function. The final part of the review summarizes new phosphorylation sites from accumulated proteomic data and discusses the possibilities that might arise from this data.

Differential mechanisms of action are involved in chlorpyrifos effects in estrogen-dependent or -independent breast cancer cells exposed to low or high concentrations of the pesticide

It has reported that many environmental compounds may display estrogenic actions and these findings led to researchers to associate breast cancer risk with the use of some pesticides. The aim of this work was to investigate the effect of chlorpyrifos (CPF) on cell proliferation and the ERα-dependence of this action employing MCF-7 and MDA-MB-231 breast cancer cell lines. We have also analyzed CPF action on the cell cycle distribution and the cyclins that are implicated in G1-S and intra-S checkpoints. Finally, the action on cell death and ROS production were studied. We demonstrated the ability of CPF 0.05μM to induce cell proliferation through ERα in hormone-dependent breast cancer cells. In contrast, CPF 50μM induces intra-S arrest modifying checkpoints proteins, through a mechanism that may involve changes in redox balance in MCF-7. In MDA-MB-231, we have found that CPF 50μM produces an arrest in G2/M phase which could be related to the capacity of the pesticide for binding to tubulin sites altering microtubules polymerization. Altogether, our results provide new evidences on the action of the pesticide CPF as an environmental breast cancer risk factor due to the effects that causes on the mechanisms that modulate breast cell proliferation.

Induction of a G 1 -S checkpoint in fission yeast

Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.

Role of p16(INK4A) in Replicative Senescence and DNA Damage-Induced Premature Senescence in p53-Deficient Human Cells.

The p16INK4A (hereafter p16) tumor suppressor is encoded by the INK4A/ARF locus which is among the most commonly dysregulated sequences in human cancer. By inhibiting cyclin-dependent kinases, p16 activates the G1-S checkpoint, and this response is often considered to be critical for establishing a senescence-like growth arrest. Not all studies support a universal role for p16 in senescence. Single-cell analysis of noncancerous human fibroblast cultures undergoing senescence as a function of culture age (replicative senescence) has revealed that p16 is not expressed in the majority (>90%) of cells that exhibit features of senescence (e.g., flattened and enlarged morphology coupled with senescence-associated β-galactosidase expression), ruling out a requirement for p16 in this process. In addition, ionizing radiation triggers premature senescence in human cancer cell lines that do not express p16. These observations are made with cells that express wild-type p53, a key mediator of the DNA damage response. In this paper, we examine the growing evidence suggesting a negative regulatory relationship between p16 and p53 and discuss recent reports that implicate a role for p16 in replicative senescence and ionizing radiation-induced premature senescence in human cells that lack wild-type p53 function.

Proteomic and Genomic Profile of High-Risk MDS After Treatment with 5-Azacytidine,

Abstract 3818 BackgroundConventional cytogenetics is the major prognostic factor in predicting outcome of myelodysplasia (MDS), but at least 50% of patients have a normal karyotype. 5-azacytidine (5-AZA) is a DNA methyltransferase inhibitor used in treatment of high-risk MDS, which is able to delay the progression to AML and improve clinical outcomes, but targets of the methylation status are poor known. AimsTo evaluate molecular changes, at genomic or proteomic level, which can be identified as additional targets of 5-azacytidine in MDS patients with normal conventional cytogenetics. MethodsBy reverse-phase protein microarray (RPMA), we analyzed bone marrow mononuclear cells from 19 patients affected of high risk MDS, treated with 5-azacytidine (median age 71 years, M/F=12/5). Treatment consisted of 4 cycles of 100mg flat dose for 7 days+21 days of wash-out. In 7 cases the sample after 4 cycles of therapy was matched to the sample collected before starting treatment. RPMA was used to quantitatively map 45 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. For the first 4 patients we used also a genome-wide approach based on SNP (single nucleotide polymorphism) array (6.0 Affymetrix platform) to detect copy number and allelotype data in order to identify new genetic markers in terms of SNP, CNV and LOH. ResultsAll patients were evaluable for response one month after the 4th cycle. Three patients were refractory and progressed to AML and 1 was a late responder (after 7 cycles). All other patients experienced hematologic improvement. After 4 cycles of 5-AZA, at proteomic level we found three main signaling pathways were increased and did not correlate with the response: 1) pro-survival signaling: PLC-y-1-Tyr783 (p=0.0017), and its upstream regulators Src-Tyr416, c-Abl-Tyr735 (p=0.002) and downstream target STAT5Tyr694 (p=0.0017) were increased, without affecting proliferative pathways, such as AKT activation status on Ser473 and Thr308 or mTORSer2448. 2) Autophagy: ATG5, Beclin 1 and LC3B were significantly elevated after treatment (p values respectively <0.0001, 0.0056 and 0.0124). Activation of the autophagy pathway occurred downstream of mTOR, since mTORSer2448, AktSer473, AktThr308, ERKThr202Tyr204 (and in general proliferation markers) were not affected. 3) Restore of cell cycle G1-S checkpoint: c-Abl-Tyr735, Rb-Ser608, Rb-Ser780Chk1-Ser345, p53, p53Ser15, and in particular HDAC1, HDAC3 were significantly elevated (p<0.001).At genomic level, we identified 46 losses and 25 gains. In three cases we found a deletion on chromosome 20q13.12 (from 46125 kb to 46178 kb) at level of NCOA3 gene, a transcriptional coactivator protein that contains several nuclear receptor interacting domains and an intrinsic histone acetyltransferase activity. In 2 cases we found a loss in the region 22q13 (from 40871 kb to 40910 kb), at level of MKL1 gene, encoding a nuclear protein involved in transduction signals from the cytoskeleton to the nucleus. MKL1 is involved in a specific translocation event associated with acute megakaryocytic leukemia. Moreover in one case we found a deletion on chromosome 4q24 (from 105800 kb to 107200 kb) in a region involving TET2 gene. Loss of heterozygosity 4q24 and TET2 mutations have been implicated in the pathogenesis of myelodysplastic syndromes. Another patient showed autozigosity regions with an extensive CN-LOH on chromosomes 7 and 11, involving the centromers and recently their neoplastic potential in colon cancer have been reported and never, before, observed in MDS. ConclusionMore informative findings can be detected using a proteomic approach in combination with the genomic one. Autophagy activation can be considered an escape pathway promoting survival in neoplastic cells and the observation that 5-AZA does not affect proliferative pathways suggests the possibility to combine it with anti-proliferative agents. The increase of HDAC1 and HDAC3 can provide the molecular rationale for the development of a combination of 5-azacytidine with HDAC inhibitors. Disclosures:No relevant conflicts of interest to declare.

Regulation of the activation of the Fanconi anemia pathway by the p21 cyclin-dependent kinase inhibitor

Fanconi anemia (FA) is a rare disease characterized by congenital defects, progressive bone marrow failure and heightened cancer susceptibility. The FA proteins, BRCA1 and FANCD1/BRCA2 function cooperatively in the FA-BRCA pathway to repair damaged DNA. Activation of the FA-BRCA pathway occurs via the monoubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete nuclear foci where they function in DNA repair. The cellular regulation of FANCD2/I monoubiquitination, however, remains poorly understood. In this study, we have examined the roles of the p53 tumor suppressor protein, as well as its downstream target, the p21(Cip1/Waf1) cyclin-dependent kinase inhibitor, in the regulation of the activation of the FA-BRCA pathway. We demonstrate that, in contrast to p53, p21 has a major role in the regulation of the activation of the FA-BRCA pathway: p21 promotes S-phase and DNA damage-inducible FANCD2/I monoubiquitination and nuclear foci formation. Several lines of evidence establish that this effect is not a consequence of a defective G1-S checkpoint or altered cell-cycle progression in the absence of p21. Instead, we demonstrate that p21 is required for the transcriptional repression of the USP1 deubiquitinating enzyme upon exposure to DNA-damaging agents. In the absence of p21, persistent USP1 expression precludes the DNA damage-inducible accumulation of monoubiquitinated FANCD2 and FANCI. Consequently, p21(-/-) cells exhibit increased levels of mitomycin C-inducible complex chromosomal aberrations and elevated γH2AX nuclear foci formation. Our results demonstrate that p21 has a critical role in the regulation of the activation of the FA-BRCA pathway and suggest a broader role for p21 in the orchestration of DNA repair processes following exposure to DNA crosslinking agents.

DNA Damage-Sensing Kinases Mediate the Mouse 2-Cell Embryo's Response to Genotoxic Stress

A critical function of cells is the maintenance of their genomic integrity. A family of phosphoinositide-3-kinase-related protein kinases, which includes ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) kinases, play key roles in sensing DNA damage. ATM and ATR were demonstrated in the cleavage stages of mouse embryo development. Genotoxic stress was imposed by exposure to ultraviolet (UV) radiation (causes DNA strand breaks) or cisplatin (causes strand cross-links). UV irradiation or cisplatin treatment of 2-cell embryos in the G(2) phase of the cell cycle caused DNA damage as defined by increased phosphorylation of the H2A histone family, member X (H2AFX; previously H2AX) variant. UV irradiation caused a stable G(2)-M arrest, and cisplatin treatment allowed progression through mitosis followed by activation of a G(1)-S checkpoint. Both checkpoints were transformation-related protein 53-independent. Caffeine (inhibits both ATM and ATR), but not KU55933 (ATM-selective inhibitor), reversed the G(2)-M block induced by UV, inferring a primary role for ATR in sensing this form of DNA damage. Caffeine and KU55933 were equally effective in reversing the cisplatin-induced G(1)-S block, implicating ATM as the primary sensing enzyme. Breaching of either checkpoint by treatment with caffeine or KU55933 allowed embryos to progress through several further cell cycles, yet none developed to blastocysts. The results show, to our knowledge for the first time, that the G(2)-M and G(1)-S cell-cycle checkpoints in the early embryo are differentially regulated by ATM and ATR in response to genotoxic stress and that they act as an initial point for containment of genomic damage. Under conditions of extensive or persistent DNA damage, the demise of the embryo is the ultimate method of protecting genomic integrity.

Abstract 2888: SOCS3-deficient cells are defective in the DNA damage response

Abstract The SOCS family of proteins are negative feedback inhibitors of cytokine and growth factor signal transduction, targeting primarily the STAT family of transcription factors. Several SOCS genes, including SOCS1 and SOCS3 are hypermethylated and silenced in solid tumors, which is believed to impart a growth advantage via hyperactivation of the STAT pathway. Despite the growing role of SOCS in the pathogenesis of cancer, very little is known of their role in modulating tumor cell responses to therapy. We recently identified an unexpected function for SOCS3 in the context of the DNA damage response. We found that cells lacking SOCS3 are sensitive to ionizing radiation (IR) in a clonogenic survival assay due to impaired p21 (Cip1/Waf-1) upregulation, which results in failure of the radiation-induced G1-S checkpoint. Normal cell cycle arrest and p21 upregulation could be restored in SOCS3-/- MEFs by overexpression of a phosphorylation-deficient, dominant-negative (DN) mutant of STAT3. Two STAT3-dependent genes known to suppress p21 expression, c-Myc and BCL6 are overrepresented in SOCS3-deficient MEFs. Likewise, human tumor cells that repress SOCS3 expression by epigenetic silencing are also more radiosensitive than their SOCS3+ counterparts. In agreemwnt with our data in MEFs, c-Myc and BCL6 are overexpressed in human tumor cells that silence SOCS3 expression by promoter hypermethulation, suggesting that tumors lacking SOCS3 may be more vulnerable to IR-based therapies due to impaired cell cycle arrest and DNA repair. Our data suggest that therapeutic targeting of SOCS3 in tumor cells could sensitize cells to IR and improve the effectiveness of radiotherapy. 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 2888. doi:10.1158/1538-7445.AM2011-2888

Abstract 228: A comprehensive study to functionally classify the BRCA2 missense mutations found in Fanconi anemia patients

Abstract Biallelic mutations in breast cancer susceptibility gene 2 (BRCA2) cause a severe Fanconi Anemia (FA) phenotype that is a rare recessive disorder and reflects the cellular and phenotypic consequences of genetic instability. A number of mutations found in FA patients are missense mutations in BRCA2 and classified as variants of unknown clinical significance. This study is aimed towards the evaluation of functional significance of a few BRCA2 unclassified variants. In this study, six of the human mutations (L2510P, R2336H, I2490T, W2626C, K2729N and IVS7+2T&amp;gt;G) were generated in human BRCA2 gene cloned in a bacterial artificial chromosome vector. The effect of these variants on BRCA2 function was examined by using a mouse embryonic stem (ES) cell-based assay. We have shown the ability of wild-type human BRCA2 and neutral BRCA2 variants to rescue the lethality of Brca2-null mouse ES cells and functionally complement the loss of endogenous mouse Brca2. W2626C and IVS 7+2T&amp;gt;G mutations failed to restore the viability of ES cell colonies after deletion of the endogenous mouse Brca2. The K2729N BRCA2 rescued the ES cell lethality and appeared similar to wild-type BRCA2 in various functional assays. In contrast, L2510P and R2336H BRCA2 exhibited reduced ES cell viability. In addition, ES cells expressing L2510P BRCA2 are sensitive to DNA damaging agents. Furthermore, L2510P BRCA2 mutant ES cells are slow growing, have increased genomic instability, defective in G1-S checkpoint control and show upregulation of p53 and p21. These mutant cells are defective in homologous recombination mediated DNA repair. IVS7+2T&amp;gt;G and R2336H mutations result in exon-skipping leading to premature protein truncation. Alterations in L2510P, I2490T, W2626C and K2729N map to a highly conserved region of BRCA2 and the homology based molecular modeling of the mutant proteins supports our ES-cell results. L2510P and W2626C mutations disrupt the BRCA2 structure and the interaction of BRCA2 with a highly conserved protein DSS1, whereas I2490T and K2729N mutations do not affect the BRCA2 structure and are neutral in nature. It is possible that the patients with the variant alleles that are neutral in this assay have other undetected mutation in that allele or have mutations in some other FA genes. In conclusion, this study provides functional classification of a few variants of unknown clinical significance that should help in counseling individuals carrying these alleles. . 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 228. doi:10.1158/1538-7445.AM2011-228

The coupling of epidermal growth factor receptor down regulation by 1alpha,25-dihydroxyvitamin D3 to the hormone-induced cell cycle arrest at the G1-S checkpoint in ovarian cancer cells

1alpha,25-dihydroxyvitamin D3, 1,25(OH) 2D 3, regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH) 2D 3 in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH) 2D 3 under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH) 2D 3 suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH) 2D 3-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH) 2D 3-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH) 2D 3 suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone.

Prognostic Value of Cell Cycle Proteins in Squamous Cell Carcinomas of the Oral Cavity

The cell cycle is under strict regulation. Cyclin-dependent kinases (CDKs) at the G1-S and G2-M checkpoints are positively regulated by cyclins and negatively regulated by CDK inhibitors. Oral squamous cell carcinoma (OSCC) is a common malignancy that is characterized by a high degree of local aggression and lymph node metastasis. We examined positive regulators of the cell cycle (cyclin A, cyclin B1, and cyclin D1), cell cycle inhibitors (p16, p21, p27, p53, and Rb), and the proliferation markers Ki-67 and topoisomerase IIA by immunohistochemistry in a tissue microarray, comprising 136 cases of OSCC. Expression of cyclin A, cyclin B1, cyclin D1, topoisomerase IIA, p53, and Rb was positively associated with Ki-67. Overexpression of cyclin D1 correlated with the presence of lymph node metastasis. Overexpression of cyclin A, cyclin D1, topoisomerase IIA, and Rb and downregulation of p21 and p27 were associated with reduced overall survival in OSCC patients. Cyclin A, p21 and p27, were implicated an independent prognostic factor, by multivariate analysis, in OSCC patients. In conclusion, cyclin A, cyclin D1, p21 and p27 expression can be a valuable marker of poor prognosis and tumor aggressiveness in OSCC.

Characterization of a Candidate Tumor Suppressor Gene Uroplakin 1A in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is increasing in incidence, but the knowledge of the genetic underpinnings of this disease remains limited. In this study, we identified the tetraspanin cell surface receptor uroplakin 1A (UPK1A) as a candidate tumor suppressor gene (TSG), and we investigated its function and mechanism in ESCC cells. UPK1A downregulation occurred in 68% of primary ESCCs examined, where it was correlated significantly with promoter hypermethylation (P < 0.05). Ectopic expression of UPK1A in ESCC cells inhibited cell proliferation, clonogenicity, cell motility, and tumor formation in nude mice. Mechanistic investigations suggested that these effects may be mediated by inhibiting nuclear translocation of β-catenin and inactivation of its downstream targets, including cyclin-D1, c-jun, c-myc, and matrix metalloproteinase 7 (MMP7). Cell cycle arrest elicited by UPK1A at the G(1)-S checkpoint was associated with downregulation of cyclin D1 and cyclin-dependent kinase 4, whereas metastasis suppression was associated with reduction of MMP7. These findings were consistent with evidence derived from clinical samples, where UPK1A downregulation was correlated with lymph node metastasis (P = 0.009), stage (P = 0.015), and overall survival (P < 0.0001). Indeed, multivariate cyclooxygenase regression analysis showed that UPK1A was an independent prognostic factor for overall survival. Taken together, our findings define a function for UPK1A as an important TSG in ESCC development.

Prognostic Significance of the Alterations of the G1-S Checkpoint in Localized Leiomyosarcoma of the Peripheral Soft Tissue

The aim of this study was to investigate the expression of cell cycle regulators p53, p16, cyclin-D1, and retinoblastoma (Rb) gene protein in leiomyosarcoma of the peripheral soft in order to identify expression profiles potentially useful for clinical prognostic purposes. A tissue microarray representing 70 localized leiomyosarcomas of the limbs and limb girdles was created with 3 representative cores from each tumor. Immunohistochemical staining was performed for p53, p16, cyclin-D1, and Rb using standard techniques. Staining was scored as either absent-low (<20% of neoplastic cells) or moderate-diffuse (≥20%). Outcome analysis was performed for local recurrence-free survival (LFS), metastatic disease-free survival (MDFS), and disease-specific survival (DSS). Kaplan-Meier analysis of survival revealed that no single alteration of the factors examined was associated with outcome, but tumors showing concomitant alteration of p16 and p53 were characterized by reduced MDFS and DSS (P=0.01 and P<0.001, respectively). In addition, patients who received adjuvant therapy consisting of radiotherapy alone or radiotherapy and chemotherapy had a better DSS than those receiving surgery alone or surgery and chemotherapy (P=0.05). In multivariate analysis, altered p16/p53 remained the only parameter predictive of MDFS and DSS (P=0.048, hazard ratio [HR]=2.488, 95% confidence interval [95% CI] 1.006-5.116; P=0.043, HR=2.498, 95% CI 1.029-5.909, respectively). Accumulation of cell cycle alterations represents a prognostic indicator in localized soft tissue leiomyosarcoma, and in particular altered p16/p53 expression is associated with an unfavorable prognosis. This may help the clinical management of patients with leiomyosarcomas.

MicroRNA Gene Regulatory Pathways in the Establishment and Maintenance of ESC Identity

Understanding the molecular foundations of embryonic stem cell (ESC) self-renewal and pluripotency will facilitate therapeutic exploitation of these remarkable cells. Here we discuss the emerging roles of microRNAs in the establishment and maintenance of ESC identity and summarize our current understanding of the mechanisms controlling microRNA expression and function in ESCs.

The Limitations of the G1-S Checkpoint

It has been proposed that the G(1)-S checkpoint is the critical regulator of genomic stability, preventing the cell cycle progression of cells with a single DNA double-strand break. Using fluorescence-activated cell sorting analysis of asynchronous cells and microscopic analysis of asynchronous and synchronized cells, we show that full blockage of S-phase entry is only observed >4 hours after irradiation. The process is ataxia-telangiectasia mutated (ATM) dependent and Chk1/2 independent and can be activated throughout G(1) phase. By monitoring S-phase entry of irradiated synchronized cells, we show that the duration of arrest is dose dependent, with S-phase entry recommencing after arrest with kinetics similar to that observed in unirradiated cells. Thus, G(1)-S checkpoint arrest is not always permanent. Following exposure to higher doses (> or =2 Gy), G(1)-S arrest is inefficiently maintained, allowing progression of G(1)-phase cells into G(2) with elevated gammaH2AX foci and chromosome breaks. At early times after irradiation (< or =4 h), G(1)-S checkpoint arrest is not established but cells enter S phase at a reduced rate. This early slowing in S-phase entry is ATM and Chk2 dependent and detectable after 100 mGy, showing a novel and sensitive damage response. However, the time needed to establish G(1)-S checkpoint arrest provides a window when cells can progress to G(2) and form chromosome breaks. Our findings detail the efficacy of the G(1)-S checkpoint and define two significant limitations: At early times after IR, the activated checkpoint fails to efficiently prevent S-phase entry, and at later times, the checkpoint is inefficiently maintained.

Abstract 2029: Identification of novel tumor suppressor microRNAs implicated in epithelial ovarian cancer from the 19q13.41 non-coding RNA cluster

Abstract Background: Epithelial ovarian cancer is the 5th leading cause of cancer death in women. Our objective was to identify key genetic events important for the pathogenesis of this lethal disease. Methods: Levels of microRNA (miRNA) expression were examined in specimens of primary papillary serous carcinomas, normal ovary and distal fallopian tube using Next Generation Sequencing and a custom expression array. Chromosomal gains and losses were also examined by CGH. SYBR Green reagents were used to measure relative expression of target gene expression by quantitative real-time PCR. Functional impact of altered miRNA expression was tested using standard MTT and Caspase 3/7 assays to measure proliferation and apoptosis (Promega). Key outcome demographics were coded and correlated with miRNA and gene expression by Kaplan-Meier analysis. Results: A total of 140 miRNAs were differentially expressed when papillary serous ovarian cancers were compared to either fimbrae of normal fallopian or normal ovary. Of these, 36 miRNAs were found to correlate with either overall survival, disease free interval (DFI) or platinum sensitivity. Nineteen (19) of these clinically significant miRNAs mapped to single primate-specific genomic locus located at 19q13.41. This locus spanned 125 Kb of non-coding DNA and encoded a total of 44 miRNAs, most all of which showed significant copy number variation in papillary serous ovarian cancers (n = 178) and showed copy losses in the majority of tumors. Using established algorithms for target prediction, we found that this miRNA cluster collectively targeted more than 2800 distinct genes. Key loci included gene products implicated in the epithelial-to-mesenchymal transition (Snail, Slug) as well as both the G1-S and G2-M cell cycle checkpoints (MYCN and Wee1). Transfection of established ovarian cancer cell lines with individual 19q13.41 miRNAs significantly reduced expression of Snail, Slug, Wee1, resulting in altered proliferation and apoptosis. Conclusions: Altered expression of 19q13.41 cluster miRNAs correlate with significant clinical outcomes for women with papillary serous ovarian cancers. These miRNAs appear to play a key role in regulating the expression of gene products critical for the ongoing proliferation and metastasis of ovarian cancer. Future work will focus on dissecting the role of individual 19q13.41 miRNAs in ovarian and other cancers as well as validating the novel nanoparticle-based strategies we have developed for therapeutic miRNA delivery. Supported by NIH TCGA and the Ovarian Cancer Research Foundation (OCRF) 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 2029.