Disruption Of Cell Cycle Control Research Articles

Dysregulated Transcriptional Upregulation of Cell-Cycle Regulators By KLF1 Mutation E325K Induces Cell-Cycle Arrest in Erythroid Cells Differentiated from the Congenital Dyserythropoietic Anemia (CDA)-Patient Specific Induced Pluripotent Stem Cells

The congenital dyserythropoietic anemias (CDAs) are congenital red blood cell disorders representing ineffective erythropoiesis and dyserythropoietic changes in the bone marrow. We diagnosed a female patient with undiagnosed congenital anemia as type IV CDA caused by a heterozygous missense mutation of the erythroid-specific transcription factor, KLF1; c.973G>A, p. E325K. Although the mutation has been reported in a male patient characterized as hydrops fetalis, severe neonatal jaundice and transfusion-dependent anemia (Arnaud L et al., Am J Hum Genet, 2010), the proband showed relatively mild phenotype showing moderate dyserythropoietic anemia. In order to investigate the pathological significance of mutant KLF1 during erythropoiesis, we generated induced pluripotent stem cells (iPSCs) from peripheral blood of the CDA patient (CDA-iPSCs), and utilized these cells for better understanding of its molecular basis.Hematopoietic precursors were induced from CDA-iPSCs by embryoid bodies formation. CD34(+) precursor cells were then isolated and cultured in the presence of cytokine cocktail for additional 1-3 weeks (Figure 1). Morphological analysis revealed that many erythroblastic cells derived from CDA-iPSCs were multinucleated, in accordance with that of bone marrow erythroblasts from CDA patient. Flow cytometric analysis showed that CDA-iPSC-derived cells contained significantly lower percentage of CD235a(+)/CD71(+) erythroid lineage cells than the cells derived from control iPSCs. CD71 expression decreased in time during erythroid differentiation culture in control CD235a(+) cells, whereas it maintained in CDA-iPSC-derived cells, indicating inappropriate maturation of erythroblastic cells.These data promoted us to explore the possibility of cell death in CDA-iPSC-derived cells during erythroid differentiation culture by comprehensive microarray analysis of erythroblastic cells. In CDA-iPSC-derived cells, many positive and negative regulators of cell-cycle, such as E2F2 and CDKN2C, were differentially expressed, indicating the disruption of cell-cycle control during late stage of erythropoiesis from CDA-iPSCs. In consistent with this, significantly lower percentage of BrdU-uptaking cells were detected in CD235a(+) cells derived from CDA-iPSCs after 3 hour pulse of BrdU during erythroid liquid culture.Most of the KLF1 mutations with functional alterations currently reported are classified into hypomorphic variants or truncating loss-of-function variants. KLF1 E325K has been reported to actively interfere with transcription of KLF1 target genes (Arnaud et al., 2010). Our findings suggest that KLF1 E325K variant might act to disrupt the transcriptional control of various cell cycle regulators including known KLF1 target genes E2F2 and CDKN2C, which lead to the pathological cell-cycle arrest in type IV CDA patients. Our model provides insights on understanding the mechanisms of type IV CDA, and it would be a useful tool for drug screening and identification of novel biomarkers. [Display omitted] DisclosuresKohara:neo ALA CO.,LTD.: Research Funding. Sugawara:neo ALA CO.,LTD.: Research Funding. Yoshie:neo ALA CO.,LTD.: Research Funding. Tani:neo ALA CO.,LTD.: Research Funding; SHINNIHON PHARMACEUTICAL CO., LTD: Research Funding.

Targeting Cell Cycle to Suppress Cancer Aggressiveness

Background: Failure of cell division control due to mutations leading to inactivation or over activation of regulatory proteins is the leading cause of cancer development. Several mitogens and growth factors have been found to regulate cancer cell cycle progression. However, all signalling pathways converge to the cell cycle machinery and thus disruption of cell cycle control offers an attractive therapeutic target for the treatment of cancer. Method: We undertook a comprehensive search of bibliographic databases through PubMed and selected the most relevant and appropriate peer-reviewed research articles. Results: There has been a breakthrough in identification of the cell cycle regulatory molecules and elucidation of their roles in subtle adjustment of the balance between proliferation and apoptosis of cancer cells. This review will shed light on the current understanding of the regulation of the cell cycle pathway links and the feasibility of targeting cell cycle for fighting metastatic cancer. Conclusion: There are cross-talks among the diverse neoplastic cell types acting together on cell cycle to ensure survival, growth and metastasis, by inhibiting apoptosis, promoting angiogenesis, and avoiding immune system. Approaches should be undertaken to synergistically block the activation of the interconnected pathways for effective cancer therapy. Keywords: Cancer, drug targets, cell cycle, metabolic reprogramming, oxidative stress, immunomodulatory microRNAs, extraribosomal functions, CDK, cyclins, genomic instability, check points, p53, therapeutics.

Abstract 4718: ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications

Abstract Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered Phase 1 clinical trials. It binds bromodomains of BRD2/4/T with similar affinities (Ki of 1-2.2 nM), but exhibits roughly 10-fold weaker potency towards BRD3 (Ki of 12.2 nM). ABBV-075 is highly selective for 18 bromodomain proteins tested (Kd > 1 μM; more than 600-fold selectivity vs. BRD4) and has moderate activity towards CREBBP (Kd = 87 μM; 54-fold selectivity vs. BRD4). ABBV-075 exhibited robust single agent activity in cell viability assays across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to ABBV-075 indicated that ABBV-075 manifested diverse mechanisms of action in different cancer settings. These include 1): disruption of cell cycle control leading to G1 arrest followed by senescence, 2) inhibition of oncogenesis drivers leading to apoptosis and 3) potentially targeting tumor microenvironment to provide additional therapeutic benefit. Consistent with its broad spectrum of activities in vitro, ABBV-075 has comparable or superior efficacies to standard of care agents in flank xenograft mouse models of non-small-cell and small cell lung cancers, pancreatic, breast, prostate, head & neck cancers, multiple myeloma, diffuse large B cell lymphoma and leukemia. These results support the development of ABBV-075 in diverse hematological malignancies and solid tumor indications. Citation Format: Aparna Sarthy, Leiming Li, Daniel H. Albert, Xiaoyu Lin, Warder Scott, Emily Faivre, Mai H. Bui, Xiaoli Huang, Denise M. Wilcox, Terry Magoc, Fritz G. Buchanan, Paul Tapang, George S. Sheppard, Le Wang, Steve D. Fidanze, John Pratt, Dachun Liu, Lisa Hasvold, Paul Hessler, Tamar Uziel, Lloyd Lam, Ganesh Rajaraman, Guowei Fang, Steven W. Elmore, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4718.

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.

Differential regulation of proliferation, cell cycle control and gene expression in cultured human aortic and pulmonary artery endothelial cells by resveratrol

Resveratrol is a grape polyphenol with cardioprotective attributes, supported in part by its demonstrated anti-mitogenic, apoptosis-inducing and gene modulatory activities in various cell types known to play an integral role in atherogenesis. To test whether resveratrol exerts similar effects on systemic and pulmonary vasculature, cells derived from different anatomical sites, cultured human aortic and pulmonary artery endothelial cells, respectively denoted HAECs and HPAECs, were exposed to resveratrol for assessment of effects on proliferation, cell cycle distribution, induction of apoptosis, and specific gene expression. Resveratrol inhibited cell proliferation in a time- and dose-dependent manner in HAECs and HPAECs, accompanied by disruption of cell cycle control and progression as assayed by flow cytometry. Analysis of gene changes in resveratrol-treated endothelial cells by RT-PCR showed suppression of nitric oxide synthase (eNOS) and preproendothelin-1 (ppET-1) expression in both cell types. To discover group gene alterations resulting from exposure to resveratrol, changes in mRNA levels were determined by human signal transduction pathway finder cDNA array analysis. The results showed that resveratrol up-regulated levels of cyclin-dependent kinase inhibitor p57, egr-1, forkhead box A2 and c-jun in HAECs, and elevated expression of cathepsin D, ICAM-1, c-jun and patched 1 in HPAECs. In addition, treatment by resveratrol also resulted in attenuated expression of bcl-xl, fibronectin-1, HIP, mdm2, PIG3 and WSB1/SWIP-1 in HAECs, and CDX1, engrailed homolog 1, FASN, fibronectin-1, forkhead box A2, Hoxa-1, hsp27, PIG3, ELAM-1/E-selectin and WSB1/SWIP-1 in HPAECs. These results suggest that resveratrol acts by distinct and overlapping signaling pathways and mechanisms in HAECs and HPAECs, further supporting the notion that the cardioactive activities and effects of this grape polyphenol are contingent upon or influenced by the vascular bed of origin.

Fluorescent Epigenetic Small Molecule Induces Expression of the Tumor Suppressor Ras-Association Domain Family 1A and Inhibits Human Prostate Xenograft

Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance. Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.

Effects of methylated chrysenes on AhR-dependent and -independent toxic events in rat liver epithelial cells

Methylated chrysenes (MeChry) are important cigarette smoke constituents and 5-MeChry has been listed as possibly carcinogenic to humans. Although a major attention has been in past paid especially to mutagenic, tumor-initiating effects of MeChry, little is known about toxic effects of MeChry related to tumor promotion. As the position of methyl group has been repeatedly observed to determine genotoxic effects of MeChry, we examined both genotoxic and nongenotoxic effects of MeChry, using rat liver cell lines as experimental models. All six MeChry were relatively efficient aryl hydrocarbon receptor (AhR) agonists, with 3- and 6-MeChry being the most potent inducers of the AhR-mediated reporter gene activity. All six compounds disrupted contact inhibition in rat liver epithelial WB-F344 cells, a process previously reported to be AhR-dependent, suggesting that MeChry may interfere with cell cycle control in an AhR-dependent manner. In contrast, only 5- and 6-MeChry were found to acutely inhibit gap junctional intercellular communication (GJIC), another parameter correlating with tumor promoting effects of xenobiotics. Both 5- and 6-MeChry were efficient inducers of mRNA expression of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons, including cytochromes P450 1A1/1B1 and aldo-keto reductase 1C9. However, only 5-MeChry, and not 6-MeChry, induced significant formation of DNA adducts in rat liver epithelial cells, which corresponded with its ability to induce high accumulation of cells in S-phase. On the other hand, 5-MeChry induced neither apoptosis related to DNA damage nor phosphorylation of p53 tumor suppressor. Taken together, our results suggest that methyl group position may affect both genotoxic and nongenotoxic effects of MeChry, such as formation of DNA adducts and inhibition of GJIC. All MeChry showed a potency to disrupt cell proliferation control, while 5-MeChry was a single compound inducing DNA damage, disruption of cell cycle control and inhibition of GJIC in rat liver cells.

Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1

We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well-understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.

Mechanisms Involved in Resveratrol‐Induced Apoptosis and Cell Cycle Arrest in Prostate Cancer—Derived Cell Lines

Resveratrol is a polyphenol found at high concentrations in grapes and red wine with reported anticarcinogenic effects. We studied the molecular mechanism of resveratrol-induced apoptosis and proliferation arrest in prostate derived cells PZ-HPV-7 (nontumorigenic line), LNCaP (androgen-sensitive cancer line), and PC-3 (androgen-insensitive cancer line). Apoptosis and cell cycle distribution were evaluated by flow cytometry and proliferation by MTT assay and direct cell counting. Caspases, bax, bcl-2, cyclins, Cdks, p53, p21, and p27 were measured by Western blot and kinase activities of cyclin/Cdk complexes by immunoprecipitation followed by kinase assays with appropriate substrates. Resveratrol induced a decrease in proliferation rates and an increase in apoptosis in cancer cell lines in a dose- and time-dependent manner. These effects were coincident with cell accumulation at the G0/G1 phase. In LNCaP and PC-3, the apoptosis induced by resveratrol was mediated by activation of caspases 9 and 3 and a change in the ratio of bax/bcl-2. Expressions of cyclin D1, E, and Cdk4 as well as cyclin D1/Cdk4 kinase activity were reduced by resveratrol only in LNCaP cells. In contrast, cyclin B and Cdk1 expression and cyclin B/Cdk1 kinase activity were decreased in both cell lines in the presence of resveratrol. However, modulator proteins p53, p21, and p27 were increased by resveratrol only in LNCaP cells. These effects probably result in the observed proliferation arrest and disruption of cell cycle control. In addition, the specific differences found between LNCaP and PC-3 suggest that resveratrol acts through different mechanisms upon the androgen or estrogen receptor cell status.

Dibenzanthracenes and benzochrysenes elicit both genotoxic and nongenotoxic events in rat liver ‘stem-like’ cells

Polycyclic aromatic hydrocarbons (PAHs) with molecular weight 278 are a group of PAHs that are mostly not covered by the current monitoring programs, despite their relative abundance in environmental samples and possible carcinogenicity. Although benzo[ g]chrysene (BgChry) and dibenz[ a,h]anthracene (DBahA) have been for a long time studied as genotoxic, tumour-initiating compounds, little is known about the potential tumour-promoting effects of this group of PAHs. In the present study, we investigated their impact on activation of the aryl hydrocarbon receptor (AhR), induction of enzymes involved in metabolic activation of PAHs, disruption of cell cycle control in confluent cell population and inhibition of gap junctional intercellular communication (GJIC), using the rat liver epithelial cell line WB-F344 as a model of liver progenitor cells. We found that BgChry was the weakest inducer of the AhR-mediated activity, while relative potencies of benzo[ b]chrysene (BbChry) and benzo[ c]chrysene (BcChry) were comparable to the previously reported values for dibenzanthracenes. All compounds increased expression of cytochromes P450 1A1 and 1B1, and aldo-keto reductase 1C9. BgChry was found to induce high amounts of DNA adducts, which corresponded with induction of p53 phosphorylation at Ser15, apoptosis and accumulation of cells in S-phase of cell cycle, leading to a decrease in cell numbers. All other compounds were found to stimulate cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. We found that only BgChry, and to a lesser extent also BcChry, inhibited GJIC at high concentrations. Taken together, dibenzanthracenes and benzochrysenes, with exception of BgChry, seem to act primarily through deregulation of cell proliferation in liver epithelial cells, which is related to their relatively high AhR-mediated activity. The disruption of cell cycle control might contribute to their carcinogenic effects, as well as to carcinogenicity of complex environmental mixtures containing high levels of PAHs with molecular weight 278.

Inhibitors of Leishmania mexicana CRK3 cyclin-dependent kinase: chemical library screen and antileishmanial activity.

The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED50) of less than 10 microM. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED50s for these inhibitors were 5.8 and 7.6 microM, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development.

Hypermethylation of the p16 Gene and Lack of p16 Expression in Hepatoblastoma

Hepatoblastoma is the most frequent pediatric liver tumor that develops mostly in young children. Abnormal regulation of cell cycle regulatory genes including p16 has been described, displaying no p16 mRNA and p16 protein in hepatoblastomas. The inactivation of p16, leading to the disruption of cell cycle control is involved in many types of human malignancies. However, the mechanism of the p16 inactivation in hepatoblastomas has not yet been elucidated. In this present study, we examined the methylation status of the p16 gene promoter by using methylation-specific PCR in 24 cases of hepatoblastomas and in 20 cases of corresponding non-neoplastic liver tissue. Aberrant methylation of 5′ CpG islands of p16 was present in 12 of 24 (50.0%) cases of hepatoblastoma. Clinicopathologic parameters were not associated with the methylation status of p16. To correlate the methylation status of p16 with the expression of p16, immunohistochemical staining was done in tumors and non-neoplastic liver tissue. All non-neoplastic liver tissues displayed moderate, but heterogeneous immunoreactivity for p16. Eight of 12 (66.6%) methylation-positive hepatoblastomas showed a complete lack of immunoreactivity for p16. The other 4 methylation-positive hepatoblastomas had heterogeneous immunoreactivity. Nine of 12 (75.0%) unmethylated cases of hepatoblastoma displayed diffuse immunoreactivity, whereas 3 cases of unmethylated hepatoblastoma were not immunostained for p16. Our data indicate that the hypermethylation of p16 is a major mechanism of the transcriptional repression of p16 in hepatoblastomas, and we suggest that the inactivation of p16, leading to the lack of p16, may play an important role in the tumorigenesis of hepatoblastomas.

Regulation of the Cell Cycle in Normal and Pathological Glia

Precise regulation of the glial cell cycle is essential during nervous system development and in response to injury, whereas disruption of cell cycle control is associated with malignant glial tumors and other nervous system diseases. The Ras signaling pathway plays a central role in regulating the mammalian cell cycle, and uncontrolled Ras signaling has been implicated in a wide range of human cancers, including malignant gliomas. Recent studies in glia have demonstrated that activation of Ras can either induce or inhibit proliferation through complex interactions among downstream signaling pathways impinging on cell cycle regulatory proteins. Studies in Schwann cells have begun to delineate the pathways by which Ras regulates the cell cycle in normal and pathological glia, and have identified promising targets for therapeutic intervention in the treatment of PNS and CNS malignant glial tumors.

Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett's mucosa.

Minichromosome maintenance (Mcm) proteins are essential for eukaryotic DNA replication, and their expression implies potential for cell proliferation. Expression is dysregulated in dysplastic states but data for oesophageal squamous mucosa and Barrett's mucosa have not been published. To test the hypothesis that Mcm proteins are downregulated together with the proliferation marker Ki-67 in differentiating epithelial compartments of non-dysplastic squamous and Barrett's epithelium, and that this process does not occur in dysplastic mucosae. METHODS AND CASES: Forty five patients with Barrett's oesophagus included 20 with glandular dysplasia (10 low grade, eight high grade, two both, and four with invasive adenocarcinoma). Twenty five other patients included 12 with oesophageal squamous dysplasia (three low grade, six high grade, three both, and four with invasive squamous carcinoma). Formalin fixed paraffin embedded tissue sections from biopsy series and resections were immunostained using antibodies to Mcm2, Mcm5, and Ki-67. Percentage of nuclei positive for Mcm2, Mcm5, and Ki-67 was estimated and scored from 0 to 6 as: 0, none +; 1, <10%+; 2, 10-30%+; 3, 30-70%+; 4, 70-90%+; 5, >90%+; 6, all+. Four separate epithelial strata were scored: in squamous epithelium the basal layer and thirds to the surface, in Barrett's mucosa the luminal surface, upper and lower crypt, and deep glands. In non-dysplastic squamous epithelium and Barrett's mucosa, high level expression of Mcm2, Mcm5, and Ki-67 proteins was largely confined to the proliferative compartments and downregulated in differentiated compartments. Expression persisted up to the mucosal surface in dysplastic squamous epithelium and Barrett's mucosa. Persistent expression of Mcm2, Mcm5, and Ki-67 proteins in luminal compartments of dysplastic oesophageal squamous epithelium and dysplastic Barrett's mucosa may be diagnostic markers and imply disruption of cell cycle control and differentiation in these dysplastic epithelia.

Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

Purpose : Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes, Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4A overexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizing radiation. Methods and Materials : The normal mammary epithelial cell line MCF10A was stably transfected with full-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selected and treated with either 8 Gy of ionizing radiation or 7 J/M 2 of UV radiation. The profile of cell cycle progression and the accumulation of several cell cycle proteins were analyzed. Results : We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint in response to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizing radiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in the mock-transfected MCF10A cells, but not in the Cul-4A transfectants. Conclusion : Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly through disruption of cell cycle control.

Disruption of cell cycle control by human papillomaviruses with special reference to cervical carcinoma.

Human papillomaviruses (HPVs) play a major role in neoplastic transformation of squamous epithelial cells. The viral genome is small in size and only encodes a limited number of proteins, so one of the major functions of the viral proteins is to modulate the function of key cellular proteins involved in cell cycle control and DNA replication. During this process important host cell cycle checkpoints are lost which may lead to the accumulation of genetic abnormalities and eventual malignant transformation. This review briefly describes the normal cell cycle and also the mechanisms by which HPVs interfere with cell cycle control both as part of their productive life cycle and in the process of neoplastic transformation.

Genetic steps in the development of squamous cell carcinoma of the esophagus.

Esophageal squamous cell carcinoma (ESCC) is a frequent form of cancer that shows striking variations in geographic distribution, reflecting exposure to specific environmental factors that are still poorly defined. ESCC develops as the result of a sequence of histopathological changes that typically involves esophagitis, atrophy, mild to severe dysplasia, carcinoma in situ and finally, invasive cancer. Genetic changes associated with the development of ESCC include mutation of the p53 gene, disruption of cell-cycle control in G1 by several mechanisms (inactivation of p16MTS1, amplification of Cyclin D1, alterations of RB), activation of oncogenes (e.g., EGFR, c-MYC) and inactivation of several tumor suppressor genes. Loss of heterozygosity on chromosome 17q25 has been linked with tylosis, a rare autosomal dominant syndrome associated with high predisposition to ESCC. Whether this locus is also involved in sporadic ESCC remains to be elucidated. Chronic esophagitis is a frequent occurrence in populations at high risk of ESCC. These lesions often show focal accumulation of p53 protein and in some instances, patches of positive cells in esophagitis area at the margins of tumors were found to contain a mutation in the p53 gene. This observation is consistent with field cancerization in the esophagus and suggests that esophagitis may represent an interesting target for early detection of ESCC as well as for intervention strategies.

The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest.

Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.

Disruption of cell-cycle control by viral oncoproteins

28. Zhang, W.-W. and Yaneva, M. (1 992) Biochem. Biophys. Res. Commun. 186,574-579 Yaneva, M., Ochs, R., McRorie, D. K., Zweig, S. and Busch, H. (1985) Biochim. Biophys. Acta 841,22-29 Francoeur, A.-M.. Peebles, C. L., Gompper, P. T. and Tan, E. M. (1986) J. Immunol. 136,1648-1653 Reeves, W. H. and Sthoeger, Z. M. (1989) J. Biol. Chem. 264,5047-5052 Yaneva, M. and Busch, H. (1986) Biochemistry 25, 5057-5063 Porges, A. J., Ng, T. and Reeves, W. H. (1990) J. Immunol. 145,4222-4228 Stuiver, M. H., Coenjaerts, F. E. J. and van der Vliet, 1’. C. (1990) J. Exp. Med. 172, 1049-1054 May, G., Sutton, C. and Could, H. (1991) J. Biol. Chem. 266,3052-3059 Chan, J. Y. C., Lerman, M. I., Prabhakar, €3. S., Isozaki, O., Santisteban, P., Kuppers, R. C., Oates, E. I,., Notkins, A. I,. and Kohn, L. D. (1989) J. Biol. Chem. 264,365 1-3654 Yaneva, M., Wen, J., Ayala, A. and Cook, R. (1989) J. Biol. Chem. 264, 13407134 1 1 Mimori, T., Ohosone, Y., Hama, N., Suwa, A., Akizuki, M., Homma, J., Griffith, A. J. and Hardin, J. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 57,1777-1781 29.