Advances of the regulatory mechanism of cyclin, cyclin- dependent kinases and related kinase inhibitors in cell cycle progression / 生物工程学报

Cell cycle plays a crucial role in cell development. Cell cycle progression is mainly regulated by cyclin dependent kinase (CDK), cyclin and endogenous CDK inhibitor (CKI). Among these, CDK is the main cell cycle regulator, binding to cyclin to form the cyclin-CDK complex, which phosphorylates hundreds of substrates and regulates interphase and mitotic progression. Abnormal activity of various cell cycle proteins can cause uncontrolled proliferation of cancer cells, which leads to cancer development. Therefore, understanding the changes in CDK activity, cyclin-CDK assembly and the role of CDK inhibitors will help to understand the underlying regulatory processes in cell cycle progression, as well as provide a basis for the treatment of cancer and disease and the development of CDK inhibitor-based therapeutic agents. This review focuses on the key events of CDK activation or inactivation, and summarizes the regulatory processes of cyclin-CDK at specific times and locations, as well as the progress of research on relevant CDK inhibitor therapeutics in cancer and disease. The review concludes with a brief description of the current challenges of the cell cycle process, with the aim to provide scientific references and new ideas for further research on cell cycle process.

Sheep gastrointestinal helminthiasis in the Sertão region of Paraíba State, Northeastern Brazil: prevalence and risk factors / Helmintoses gastrintestinais de ovinos no Sertão do Estado da Paraíba, Nordeste do Brasil: prevalência e fatores de riscos

In this study, we aimed to establish the prevalence and risk factors relating to gastrointestinal helminthiasis, and to characterize the sanitary management practiced among sheep herds in the Sertão region of the state of Paraíba, northeastern Brazil, based on factors that condition the ways of controlling these parasites in these herds. The research was carried out between April and July 2012. We visited 54 farms, where fecal and blood samples were individually collected from 465 animals. On each farm, a questionnaire was applied to gather information on variables relating to potential risk factors. The prevalence of sheep gastrointestinal helminthiasis in the region was 75.9%. At least one animal tested positive for this helminthiasis on 53 (98.1%) of the 54 farms evaluated. The eggs per gram of feces (EPG) analysis showed the following infection burdens: 51.8% with mild infection, 27.1% moderate infection, 9.9% heavy infection and 11.2% fatal infection. Among the sheep farms visited, anthelmintics were used on 81.5% (p <0.05). The most relevant risk factor in this study was the farm area, because it defines the area available for grazing animals. Properties with many animals and little pasture area, which are the most abundant type in the Sertão region of Paraíba, tend to have high prevalence of gastrointestinal helminthiasis, because the animals are more prone to reinfection. The Sertão region of Paraíba presents high prevalence of gastrointestinal helminthiasis among sheep, and the farm area is the most relevant risk factor for the development of these parasites.

Objetivou-se determinar a prevalência e os fatores de risco para as helmintoses gastrintestinais, caracterizando o manejo sanitário sob fatores condicionantes das formas de controle dessas parasitoses em rebanhos de ovinos da região do Sertão da Paraíba. A pesquisa foi desenvolvida no período de abril a julho de 2012. Foram visitadas propriedades, utilizando-se 465 animais, sendo coletadas individualmente amostras de fezes e sangue durante as visitas. Em cada propriedade, foi aplicado questionário para a coleta de informações acerca de variáveis que atuariam como possíveis fatores de risco. Observou-se que a prevalência das helmintoses gastrintestinais de ovinos na região do Sertão da Paraíba foi de 75,9%. Pelo menos um animal foi positivo para essas helmintoses, em 53 (98,1%) das 54 propriedades avaliadas. A análise de OPG (Ovos Por Gramas de Fezes) demonstrou que 51,8% dos animais apresentaram infecção leve, 27,1% infecção moderada, 9,9% infecção pesada e 11,2% infecção fatal. A utilização de anti-helmínticos ocorreu em 81,5% das propriedades (p <0,05). O fator de risco mais relevante neste estudo foi a área da propriedade, porque delimita a área de pastejo do animal. Propriedades com muitos animais e pouca área de pastejo, que são as mais abundantes no Sertão da Paraíba, tendem a apresentar alta prevalência de helmintoses gastrintestinais, pois os animais estão mais propensos à reinfecção. A região do Sertão da Paraíba apresenta uma elevada prevalência de helmintoses gastrintestinais em ovinos, e a área das propriedades é o fator de risco mais relevante para o desenvolvimento dessas parasitoses.

Maltrato institucional hacia el adulto mayor: percepciones del prestador de servicios de salud y de los ancianos / Institutional abuse toward the elderly: Perceptions of health care providers and older adults

Objetivo. Analizar la percepción que el prestador de servicios de salud y el adulto mayor (AM) tienen sobre el maltrato al AM en los servicios públicos de salud, en ciudades seleccionadas de México. Material y métodos. De 2009 a 2012 se realizó un estudio con diseño cualitativo y estrategia de triangulación de fuentes de datos; se efectuaron entrevistas semiestructuradas a 13 prestadores y a 12 ancianos para recuperar su experiencia en el tema. El análisis utilizó procedimientos de la Teoría Fundamentada. Resultados. El maltrato contra el AM es una práctica naturalizada por el personal y por el anciano, la cual se manifiesta de formas diversas. Conclusiones. La institucionalización, profesionalización histórica y falta de conciencia sobre las necesidades de los AM demandan cambios de planeación, organización y supervisión del Sistema de Salud. El personal requiere intervenciones de formación, capacitación y cambio de actitudes/comportamiento, para otorgar atención integral, digna, humana y de respeto a los Derechos Humanos de los AM.

Objective. To analyze the health care providers (HCP) and elderly patients' perceptions about abuse of the elderly by health personnel of public health services, in selected cities in Mexico. Materials and methods. A qualitative study and a strategy of data triangulation were performed during 2009 and 2012; 13 HCPs and 12 elders were interviewed, in order to obtain their experience regarding elder abuse. Grounded Theory proceedings were used for the analysis. Results. Elder abuse is a naturalized practice, from HCP and elderly people's point of view; these perceptions are showed in different ways. Conclusion. Institutionalization, historical professionalization and lack of consciousness about needs of the elderly (sociocultural and economic), require changes in planning, organization and monitoring process in the Health System; training and educational interventions on staff and exchange attitudes and behavior are necessary in order to offer a health care that is comprehensive, decent, human and with respect for the human rights.

Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture

Human embryonic stem cells (hESCs) are considered to be able to stably maintain their characteristics in vitro for prolonged periods, but we had previously encountered changes in proliferative ability and differentiation potential during extended culture of hESCs. Therefore, we investigated the proliferative ability and differentiation potential of hESCs during long-term culture. The hESCs, SNUhES3, were used to analyze population-doubling time, proliferation rate and differentiation potential. We classified hESCs into three groups according to culture period. Ten colonies of hESCs for each group were daily measured colony area and population-doubling time was assessed by the changes of colony area. Proliferation rate of hESCs was measured by 5-bromo-2'-deoxyuridine (BrdU) assay and telomerase activity. To evaluate differentiation potentials for hESCs, expression levels of undifferentiated and/or differentiated hESCs markers were examined by FACS, RT-PCR and immunostaining. Population-doubling time of early passage hESCs was longer than those of middle or late passage. Proliferative ability of hESCs was accelerated depending on culture periods. Cellular morphologies and the expression level of each three germ layer markers were obviously different from each passage of reattached embryoid bodies (EBs) after spontaneous differentiation. Differentiated cells of late passage expressed higher levels of undifferentiated markers such as Oct4 and SSEA4 than those of early and middle passage. But differentiated cells of early and middle passage expressed higher level of differentiated state markers, Nestin (ectoderm), Brachyury (mesoderm), HNF3beta (endoderm). From these results, it can be inferred that hESCs show higher proliferative abilities and reduced differentiation potentials as the passage number increased. Therefore, we conclude that early passage hESCs could be more suitable than middle and late passage hESCs in differentiation studies.

Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100æM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3 percent) and initial embryonic development (35.2 percent) than the single ionomycin treatment (69.4 percent for activation and 21.9 percent for development); and also lead to a more uniform activation (nearly 90 percent single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.

Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100æM) e diferentes tempos de exposição (2, 4 ou 6 horas) à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV) pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos MIV foram expostos à ionomicina seguido ou não pelo tratamento com inibidores específicos das quinases dependentes de ciclina (CDKI), e avaliados quanto à configuração nuclear, taxa de ativação e desenvolvimento até blastocisto. Os tratamentos combinados (ionomicina+CDKI) apresentaram melhor taxa de ativação (77,3 por cento) e desenvolvimento embrionário inicial (35,2 por cento) do que a ionomicina sozinha (69,4 por cento e 21,9 por cento, respectivamente), e também promoveram ativação mais uniforme (aproximadamente 90 por cento de formação de um pronúcleo). Estes resultados demonstram que os CDKIs potencializam o efeito da ionomicina na ativação e desenvolvimento embrionário inicial e podem auxiliar na obtenção de protocolos de ativação mais eficientes, aumentando a capacidade de desenvolvimento de embriões produzidos por meio de biotécnicas reprodutivas.

Effect of metanil yellow and malachite green on DNA synthesis in N-nitrosodiethylamine induced preneoplastic rat livers.

Metanil yellow (MY) and malachite green (MG) are textile dyes, which, despite the ban occurs unsrupulously as food colouring agents. Accordingly they constitute a serious public health hazard and are of sufficient environmental concern. We have earlier reported that both MY and MG have tumor enhancing effects on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine in rats. In order to understand the possible mechanisms by which MY and MG enhance tumor development, in this study we have tested the effects of MY and MG on DNA synthesis and PCNA expression in preneoplastic hepatic lesions during N-nitrosodiethylamine (DEN) induced hepatocarcinogenesis in male Wistar (WR) rats. Rats were administered 200 ppm DEN through drinking water for a period of one month. Administration of DEN for a period of one month showed an upregulation of cell cycle regulatory proteins namely cyclin D1, CDK4, cyclin E and CDK2. Accordingly, in other experiments, the animals were further administered MY and MG for a period of one month following one month DEN treatment. The effects of MY and MG were monitored on the basis of cell proliferation markers--DNA synthesis and PCNA expression both by immunohistochemical and immunoblotting. Following DEN administration, MY, MG and PB showed stimulation of DNA synthesis and increased PCNA expression when compared with either the corresponding controls or only DEN treated animals. In the present study, enhancing effect of MY, MG and PB on the cell proliferation markers during DEN-induced hepatic preneoplasia in rats was observed.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Expression of cyclins in ductal hyperplasia, atypical ductal hyperplasia and ductal carcinoma in situ of the breast

Cyclin/cdc complexes are known to function in cell-cycle regulation. Cyclin D1/cdk4 and -6 complexes, which functions as a G1-S checkpoint and cyclin B1/cdc2 complexes, a G2-M checkpoint are essential for DNA synthesis and mitosis, respectively. Thus, dysregulated overexpression of cyclins appears to be involved in uncontrollable cell proliferation and early tumor development. We investigated the expression and proliferative index of cyclin D1 (PIcyclin D1), cyclin B1 (PIcyclin B1) and Ki-67 (PIKi-67) using immunohistochemical staining on 15 cases of ductal hyperplasia (DH), 26 cases of atypical ductal hyperplasia (ADH) and 43 cases of ductal carcinoma in situ (DCIS) of the breast in order to evaluate whether these cyclins are associated with abnormal cell proliferation and play a role in tumor development from ADH to carcinoma. Furthermore, we investigated whether the expression and proliferative index of the cyclins and Ki-67 are correlated with the histologic grade according to the Van Nuys classification and with the histologic subtype according to traditional classification. Finally, we estimated the correlation coefficient among PIcyclin D1, PIcyclin B1, PIKi-67 and estrogen receptor in ADH and DCIS. The expression of cyclin D1 was detected in 39.5% of DCIS and 7.7% of ADH cases. In the DH cases, expression of cyclin D1 was not found. Expression of cyclin B1 was also detected in 69.7% of DCIS, 50.0% of ADH and 93.3% of the DH cases. The PIcyclin D1 was significantly different among these three groups. Moreover, the PIcyclin D1 and PIKi-67 were differed significantly between the low grade DCIS and ADH cases. However, PIcyclin B1 only appeared to be significantly different between the total DCIS and ADH. Results of the correlation coefficient among PIcyclin D1, PIcyclin B1 and PIKi-67 were positively correlated with each other. No significant correlation was found between the expression of ER and cyclin D1 in ADH and DCIS. In summary, our results support the hypothesis that a cyclin D1 and cyclin B1 protein aberration, along with Ki-67, may act as a relatively early event in the tumor development from ADH to carcinoma.

Changes in expression of cell cycle regulators and their hepatic lobular distribution in partial hepatectomy-induced regenerating rat liver

Partial hepatectomy (PH) endorses quiescent hepatocytes to reenter the cell cycle. The regenerating liver returns to its preresection weight after 7 days, following one or two cell division and maintains nearly its original volume after then. We focused on the inhibition of further hepatocyte proliferation, hypothesizing possible involvement of cell cycle upregulators and inhibitors. We studied protein levels in expression of cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs), and their in situ hepatic lobular distributions in partial hepatectomized rat liver. Cyclin E was expressed in the same levels in normal liver and after PH. Expression of cyclin A, not detected in normal liver, increased in following times after PH and reached a maximum at 7 day. CDK2 and 4 showed increased expression toward terminal period. Contradictory findings of cyclin A and these CDKs might play an important role in the inhibition of further cell division, although still unclear. Constitutively expressed CDK6 decreased after 1 day. p18 showed peak expression within 1 day, and p16, p21, p27 and p57 were stronger at terminal periods. During the expected period of their activity, intranuclear translocations were observed in cyclin E, p18 and p16. There was no evidence of regional distribution in hepatic lobular architecture, instead, diffuse in situ expression, corroborating synchronous event, was found.

The effects of transferring tumor suppressor gene p16INK4A to p16INK4A-deleted cancer cells

OBJECTIVES: p16 is known to be an important tumor suppressor gene and is also called MTS1 (multiple tumor suppressive gene 1). Especially in the case of non-small cell lung cancer, it was not expressed in more than 70% of cell lines examined. To determine changes in cell-cycle related proteins and the tumorigenic effect, we, therefore, transfected p16INK4A gene into lung cancer cell lines. METHODS: We transfected p16INK4A gene into lung cancer cell lines which do not express p16 protein. We evaluated the effect by clonogenic assay and observed the changes of cell-cycle related proteins. RESULTS: The newly-expressed p16 formed a complex with cdk4, and phosphorylated pRB was decreased, although cyclin D1 and pRB:cyclin D1 complex were unchanged. Clonogenic assay after selection with G418 showed that, in the cell lines transfected with p16, tumorigenicity was significantly less than in the control. CONCLUSION: These results suggest that the p16INK4A gene can be a candidate for gene therapy in cases of NSCLC in which p16INK4A gene is inactivated.