Effects of miR-144-3p on Proliferation, Cell Cycle and Apoptosis of K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of miR-144-3p on cell proliferation, cell cycle and apoptosis of blast phase chronic myelogenous leukemia (CML) K562 cells.@*METHODS@#K562 cells were cultured in vitro and mimics negative control, hsa-miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor were respectively transfected into K562 cells with transfection reagents. The cells were divided into five groups including blank control, mimics negative control, miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor. After transfection, the cell proliferation activity was detected by CCK-8 assay. The cell cycle distribution and apoptosis were detected by flow cytometry.@*RESULTS@#Compared with the blank control and mimics negative control groups, the proliferation rate of miR-144-3p mimics group was significantly decreased (P<0.05), the proportion of S phase cells was markedly increased (P<0.05), while the proportion of G1 phase cells was obviously decreased (P<0.05), and the apoptosis rate was significantly increased (P<0.05). Compared with the blank control and inhibitor negative control groups, the proliferation rate of miR-144-3p inhibitor group was obviously increased (P<0.05), the proportion of S phase cells was markedly decreased (P<0.05), while the proportion of G1 phase cells was obviously increased (P<0.05), and the apoptosis rate was significantly decreased (P<0.05).@*CONCLUSION@#miR-144-3p can inhibit the proliferation and promote apoptosis of K562 cells, affect the cell cycle, and block K562 cells in S phase, which indicates that miR-144-3p is involved in the cell cycle activity of CML during blastic phase.

Cell cycle related long non-coding RNAs as the critical regulators of breast cancer progression and metastasis

Cell cycle is one of the main cellular mechanisms involved in tumor progression. Almost all of the active molecular pathways in tumor cells directly or indirectly target the cell cycle progression. Therefore, it is necessary to assess the molecular mechanisms involved in cell cycle regulation in tumor cells. Since, early diagnosis has pivotal role in better cancer management and treatment, it is required to introduce the non-invasive diagnostic markers. Long non-coding RNAs (LncRNAs) have higher stability in body fluids in comparison with mRNAs. Therefore, they can be used as efficient non-invasive markers for the early detection of breast cancer (BCa). In the present review we have summarized all of the reported lncRNAs involved in cell cycle regulation in BCa. It has been reported that lncRNAs mainly affect the cell cycle in G1/S transition through the CCND1/CDK4-6 complex. Present review paves the way of introducing the cell cycle related lncRNAs as efficient markers for the early detection of BCa.

Dysregulation of KIF14 regulates the cell cycle and predicts poor prognosis in cervical cancer: a study based on integrated approaches

Cervical cancer (CC) is the most common malignant tumor in females. Although persistent high-risk human papillomavirus (HPV) infection is a leading factor that causes CC, few women with HPV infection develop CC. Therefore, many mechanisms remain to be explored, such as aberrant expression of oncogenes and tumor suppressor genes. To identify promising prognostic factors and interpret the relevant mechanisms of CC, the RNA sequencing profile of CC was downloaded from the Cancer Genome Atlas and the Gene Expression Omnibus databases. The GSE63514 dataset was analyzed, and differentially expressed genes (DEGs) were obtained by weighted coexpression network analysis and the edgeR package in R. Fifty-three shared genes were mainly enriched in nuclear chromosome segregation and DNA replication signaling pathways. Through a protein-protein interaction network and prognosis analysis, the kinesin family member 14 (KIF14) hub gene was extracted from the set of 53 shared genes, which was overexpressed and associated with poor overall survival (OS) and disease-free survival (DFS) of CC patients. Mechanistically, gene set enrichment analysis showed that KIF14 was mainly enriched in the glycolysis/gluconeogenesis signaling pathway and DNA replication signaling pathway, especially in the cell cycle signaling pathway. RT-PCR and the Human Protein Atlas database confirmed that these genes were significantly increased in CC samples. Therefore, our findings indicated the biological function of KIF14 in cervical cancer and provided new ideas for CC diagnosis and therapies.

Overexpression of CDC25C affects the cell cycle of ovarian granulosa cells from adult and young goats

Background: CDC25 is a dual-specificity phosphatase that was first identified in the yeast Schizosaccharomyces pombe as a cell cycle-defective mutant. Although CDC25 is involved in the cell cycle of ovarian granulosa cells, the CDC25 signaling pathway has not been clarified fully. To explore the role of CDC25C in the cell cycle of goat ovarian granulosa cells, a CDC25C-overexpressing vector, pCMV-HA-CDC25C, was constructed and transfected into granulosa cells from adult and young white goats from Jiangsu Nantong. RT-PCR was used to measure CDC25C, CDK1, and WEE1 gene expression levels, and flow cytometry was used to distinguish ovarian granulosa cells in different phases of the cell cycle. Progesterone and estradiol levels in transfected ovarian granulosa cells were also measured. Results: In adult goat follicular granulosa cells transfected with pCMV-HA-CDC25C, CDC25C expression increased significantly, which greatly increased the relative gene expression levels of both CDK1 and WEE1. Additionally, progesterone and estradiol levels were increased in goat follicular granulosa cells overexpressing CDC25C. And the cell cycle results showed that transfection of pCMV-HA-CDC25C leads to a higher proportion of cells in S phase compared to the no vector-transfected groups. Conclusions: The results of this study indicated that the overexpression of CDC25C may increase the gene expression levels of both WEE1 and CDK1 in S phase and accelerate the transition of cells from G1 phase to S phase.

Knockout of p16INK4a promotes aggregative growth of dermal papilla cells

Summary Objective: Dermal papilla cells (DPCs) are located in the hair follicles and play an important role in hair growth. These cells have the ability to induce hair follicle formation when they display aggregative behavior. DPCs derived from the androgenetic alopecia (AGA) area undergo premature senescence in vitro, associated with p16INK4a expression. The aim of the current study was to investigate the expression of p16INK4a in aggregative and non-aggregative DPCs and the effect of p16INK4a down-regulation in these cells by adenovirus-mediated RNA interference (RNAi). Method: DPCs were isolated and cultured from healthy human scalp. p16INK4a gene and protein were detected in aggregative and non-aggregative cells. Expression of p16INK4a in DPCs was silenced by infection with rAd5-CDKN1A-1p2shRNA. Cell fate was monitored after infection. The growth of cells was measured by MTT assay. Cell cycle was evaluated by flow cytometry (FCM). Results: DPCs were isolated by digestion and showed aggregative behavior for six passages. The expression of p16INK4a showed a clear upward trend in non-aggregative cells when compared with aggregative group. p16INK4a expression was silenced by rAd5-CDKN1A-1p2shRNA (p<0.05). The p16INK4a-silenced cells grew more rapidly and exhibited a trend towards aggregative growth. There was an increase in the proportion of cells in G1 phase, while those in S phase were reduced after p16INK4a gene silencing (p<0.05). Conclusion: Our results suggest that p16INK4a plays an important role in the premature senescence and aggregative behavior of DPCs. These observations can lead to novel therapeutic strategies for treatment of AGA.

Candidate genes and pathogenesis investigation for sepsis-related acute respiratory distress syndrome based on gene expression profile

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS. RESULTS: A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein-protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes. CONCLUSIONS: Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

BACKGROUND: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. RESULTS: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. CONCLUSIONS: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Dynamics of Kirsten Ras and isoforms of protein kinasis in the progression of oral potentially malignant to malignant lesions

Oral premalignant lesions and oral squamous cell carcinoma are significant health problems influenced by different predisposing factors and genetic alterations .The clinical and histological features alone cannot accurately predict whether potentially premalignant lesions of the oral mucosa will remain stable, regress or progress to malignancy. Methodology included extraction of RNA from paraffin embedded tissue samples, RT-PCR followed by PCR in 53 biopsy specimens of premalignant and malignant lesions. Results of this study indicate that Akt levels show a continuous rise from pre-malignant lesions to grade II. [Akt1: Premalignant lesions 0.06 + 0.01 and in moderately differentiated Grade II OSCC 0.12 + 0.03; Akt2: Premalignant lesions 0.03 +0.00 and moderately differentiated Grade II OSCC 0.13 + 0.03 and Akt3: Premalignant lesions 0.02 +0.00 and moderately differentiated Grade II OSCC 0.06 + 0.01. KRas expression also increases as the grade of the cancer increases [premalignant lesions 0.02 +0.02 and Grade II OSCC 0.17 +0.01]. Blocking these genes at the level of premalignant lesions may help to decrease the chances of progression into oral squamous cell carcinoma

Role of protein kinases and Kirsten Ras pathways in oral squamous cell carcinoma and poorly differentiated oral squamous cell carcinoma in Punjab [Pakistan] population

Oral Squamous Cell Carcinoma [OSCC] develops by accumulation of multiple genetic alterations, influenced by the patient's genetic predisposition as well as by environmental influences, that includes pan, chaalyia, tobacco, alcohol, chronic inflammation, and viral infection. This study was carried out to understand the molecular alterations which contribute to the development of OSCC in Pakistani population. The study was conducted on a sample of 53 patients collected from different hospitals of Lahore. Results of this study indicate that Akt levels shows higher expression with increase in grades. The value of Akt1 in well differentiated grade 1 tissue is of the order of 0.09+0.00, Akt2 is 0.04+0.00 and Akt3 is 0.02+0.00 while in poorly differentiated tissues the values of Akt 1, Akt and Akt3 are 0.22+0.09, 0.13+0.03 and 0.06+0.01 respectively. kRas is an oncogene which is highly elevated in both grades [well differentiated: 0.09+0.02 and poorly differentiated: 0.13+0.02] in all samples of OSCC. In conclusion, our data demonstrated that Akt isoforms and kRas significantly control the cancer transition pathway. It is seen that Akt 1expression rises from 2.5- fold in well differentiated tissues to 3.5-fold in poorly differentiated tissues. The Akt-2 on the other hand shows only 0.5-fold increases from normal tissue in grade 1 tissues, but rises to 4-fold in grade-3 tissue. On the other hand there was no change in Akt-3 as compared to normal in grade I tissues yet, 3-fold increase has been recorded in grade III tissue. The oncogene K-RaS shows consistent increase of the order of 2.5-fold in grade I and 3-fold in grade III. This information combined with histopathological reports can further improve our understanding of the prognosis of oral squamous cell carcinoma

Meta-analysis of the cell cycle related C12orf48

The cell cycle is a conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division. The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during the cycle. Although the genes involved in interphase are well studied, most of the genes which are involved in mitotic events still remain unidentified. Since, the discovery of mitosis related genes is still incomplete, we performed a co-expression and gene ontology analysis for revealing novel mitosis regulated genes. In this study, we showed that C12orf48 is co-expressed with well-known mitotic genes. Moreover, it is also co-expressed with the genes that have roles in interphase such as DNA replication. Furthermore, our results showed that C12orf48 is also differentially expressed in various cancers. Therefore, the results presented in this study suggest that C12orf48 may be an important molecule for both interphase and mitosis. Since, the molecules involved in these mechanisms are crucial for proliferation as well as in carcinogenesis, C12orf48 should be considered as a novel cell cycle and carcinogenesis related gene.

Mecanismos da toxidez de FGF2 em células malignas dependentes de Ras: bloqueio de divisão celular e estresse proteotóxico / Mechanisms of FGF2 toxicity in Ras-driven malignant cells: cell division blockage and proteotoxic stress

FGF2 (Fibroblast Growth Factor 2) é o membro fundador de uma grande família de fatores de crescimento protéicos. Sua atividade se dá através da ligação e ativação de receptores específicos de membrana (FGFRs) com atividade de tirosina quinase. No organismo adulto, a sinalização de FGF2 está envolvida na indução de processos de sobrevivência, proliferação e diferenciação celular; além de cicatrização e angiogênese. Por atuar como um clássico fator de crescimento, a atividade de FGF2 está freqüentemente implicada em mecanismos pró-tumorais. Entretanto, alguns grupos, incluindo o nosso, têm reportado que FGF2 também pode apresentar efeitos antiproliferativos a até citotóxicos seletivamente em células malignas. Em 2008, publicamos um compreensivo relato mostrando que FGF2 bloqueia irreversivelmente a proliferação de linhagens murinas malignas dependentes de Ras. Alterações que levem a atividade aumentada de proteínas Ras estão presentes em diversos cânceres humanos e, freqüentemente, resultando em problemas no tratamento e prognóstico ruim. No presente trabalho, utilizamos principalmente a linhagem murina maligna dependente de Ras Y1 D1G, que apresenta um controle estrito de quiescência/proliferação em função da presença de soro; e é por isso mesmo um bom modelo para a análise dos efeitos de FGF2 sobre o ciclo celular. Análises por citometria de fluxo mostraram que, nessas células, apesar de disparar a transição G0→G1→S, FGF2 provoca um atraso na fase S seguido de um bloqueio do ciclo em G2. Embora bloqueie a progressão no ciclo (proliferação), FGF2 induz em Y1 D1G o crescimento celular em termos de massa e volume. Assim, nessas células FGF2 "desconecta" crescimento celular de proliferação. Esse desarranjo do ciclo celular provocado por FGF2 nas células Y1 D1G tem como resultado a instabilidade genotípica e morte celular; evidenciada pela perda da integridade de membrana plasmática e altas taxas de fragmentação de DNA observadas após o estímulo por esse fator. Esse efeito tóxico de FGF2 depende da atividade da proteína Src; porque a inibição química dessa proteína apresentou proteção total frente aos efeitos tóxicos de FGF2. Análises por espectrometria de massas mostraram que FGF2 induz aumento dos níveis de proteínas relacionadas à síntese protéica, e também de proteínas relacionadas ao estresse proteotóxico. Sabe-se que células malignas lidam com níveis basais altos de diferentes tipos de estresse; incluindo o estresse proteotóxico. Esse quadro mostra que o efeito tóxico disparado por FGF2 em Y1 D1G está relacionado a um acumulo de proteínas/célula, perda da homeostase de proteínas e estresse proteotóxico. Corrobora essas proposições o fato de que a inibição química de Src, que protege totalmente as células do efeito tóxico de FGF2, impede completamente o acúmulo de proteínas/célula. Além disso, em células Y1 D1G resistentes ao efeito tóxico de FGF2, e que inclusive dependem deste para proliferar em cultura, a atividade de FGF2 tem efeito oposto; ou seja, provoca diminuição dos níveis estacionários de proteínas/célula. Juntos, esses resultados demonstram que FGF2 é capaz de atacar uma vulnerabilidade de células malignas dependentes de Ras; e no caso estudado, essa vulnerabilidade decorre do desequilíbrio na homeostase de proteínas

FGF2 is the first member of a large family of peptide growth factors. It binds and activates specific membrane receptors (FGFRs) belonging to a family of tyrosine kinase receptors (RTK). In adult organisms, FGF2 signaling is involved in the induction of cell surveillance, proliferation and differentiation; and also wound healing and angiogenesis. FGF2 is a bona fide growth factor and, as such, it is often implicated in pro-tumor mechanisms. However, several groups, including ours, have reported that FGF2 can also display antiproliferative and even cytotoxic effects selectively in malignant cells. In 2008, we fully reported that FGF2 irreversibly blocks the proliferation of Ras-driven mouse malignant lineages. Alterations leading to Ras proteins overactivity are present in many human cancers frequently with bad prognosis. In the present work, we used mainly the Ras-driven mouse malignant lineage Y1 D1G that shows a strict control of quiescence/proliferation by serum factors, making it a great model to analyze the FGF2 effects upon cell cycle control. Flow cytometry analyses showed that in these cells, in spite of triggering G0→G1→S transition, FGF2 causes a delay on S phase followed by cell cycle arrest in G2. Despite blocking cell division, FGF2 induces cell growth in terms of mass and volume. Therefore, in these cells FGF2 "disconnects" cell growth from proliferation. This malfunction of cell cycle control caused by FGF2 on Y1 D1G cells leads to genotypic instability and cell death, highlighted by loss of plasma membrane integrity and high rates of DNA fragmentation. This FGF2 toxic effect depends on the activity of Src protein, because Src chemical inhibition completely protects cells from the FGF2 toxic effects. Mass spec analyses showed that FGF2 increases the levels of proteins involved in the protein synthesis machinery, and also of proteins active in proteostasis, indicating proteotoxic stress. It is known that malignant cells deal with high basal levels of different stresses, including the proteotoxic stress. This picture shows that the toxic effects triggered by FGF2 in Y1 D1G involve accumulation of proteins/cell, loss of protein homeostasis and proteotoxic stress. Corroborating these propositions, chemical inhibition of Src, which completely protects the cells from FGF2 toxic effects, totally abrogates the accumulation of proteins/cell. Moreover, in FGF2-resistant Y1 D1G cells, which depend on this factor for proliferation, FGF2 shows the opposite effect, causing decrease in steady state levels of protein/cell. Altogether, these results show that FGF2 causes a severe proteostasis imbalance in these Ras-driven mouse malignant cells

Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose.

Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.

Biologia molecular: aspectos básicos da genética: parte I / Molecular biology: genetic basic aspects: part I

A presente revisão pretende analisar os mais recentes métodos existentes na Biologia molecular-genética médica, com direcionamento quanto à detecção e ao acompanhamento das diferentes neoplasias ginecológicas. Inicialmente, neste estudo serão discutidos os aspectos básicos e as características celulares importantes, as diversas fases celulares e os métodos de análise existentes para avaliação dos cromossomos e dos genes. São revistos os mais importantes fatores oncogênicos responsáveis pela carcinogênese, bem como a ação dos oncogenes e dos genes supressores dos tumores.

This review intends to analyze the latest existing methods in Molecular Biology-Medical genetics, focusing on the detection and tracking of different gynecological neoplasias. This study will initially discusses basic aspects and important cellular characteristics, as well as the different cellular phases and existing analytical methods for the study of chromosomes and genes. It also reviews the most important oncogenic factors which are responsible for carcinogenesis, as well as the action of oncogenes and tumor-supressing factors.

Avaliação do ciclo celular de células tronco/progenitoras hemopoéticas da medula óssea de camundongos submetidos à desnutrição protéica / Hematopoietic stem/progenitor cell cycle evaluation from bone marrow of malnourished mice

A hemopoese é um processo dinâmico regulado pelo microambiente no qual se situa. O principal tecido hemopoético após o nascimento, a medula óssea, é constituído basicamente por substâncias solúveis, como fatores de crescimento, por uma matriz extracelular (MEC) e por células estromais, além das células hemopoéticas. Esse microambiente indutor íntegro é capaz de regular os processos de sobrevivência, proliferação e diferenciação celular, induzindo a célula a sair de um estado quiescente e entrar em ciclo celular. Contudo, na desnutrição protéica (DP) observa-se redução significativa da celularidade das células hemopoéticas, tanto no compartimento periférico quanto no central, a medula óssea. O comprometimento estrutural do microambinte medular decorrente da desnutrição pode prejudicar a sinalização de indução do ciclo celular, fato este que justificaria o quadro de pancitopenia. Portanto, no presente estudo nos propusemos avaliar o ciclo celular de células tronco/progenitoras hemopoéticas (CTPH) da medula óssea de camundongos desnutridos. Para tanto, utilizamos um modelo murino, sendo a desnutrição induzida a partir de uma ração hipoprotéica. As CTPH foram obtidas por método de depleção imunomagnética e utilizadas para a avaliação do ciclo celular a partir da incorporação de Iodeto de Propídeo (PI) e Laranja de Acridina (AO). Também, foram quantificadas proteínas regulatórias do ciclo celular por western blot e avaliada a expressão de receptores para fibronectina, VLA4 e VLA5. Paralelamente, em modelo ex vivo, avaliou-se a influência de fatores de crescimento e de uma matriz de fibronectina sobre a proliferação das CTPH. Considerando a importância das células estromais na sinalização celular, realizamos o ensaio de CFU-F para a quantificação de células estromais e dos fatores de crescimento secretados. Por fim, avaliamos a eficácia da recuperação nutricional frente às alterações no ciclo celular observadas no modelo de desnutrição...

Hematopoiesis is a dynamic process governed by the microenvironment in witch it is located. Basically, the main hematopoietic tissue, the bone marrow, is composed by soluble factors such growth factors, extracellular matrix (ECM) and stromal cells, besides the hematopoietic cells. This intact inducible microenvironment is capable to control cell survival, proliferation and differentiation, inducing cell to exit a quiescent state and enter the cell cycle. However, in protein malnutrition (PM) is observed a significant reduction of hematopoietic cells, both in peripheral and central compartments. The bone marrow structural impairment due to malnutrition could harm the cell cycle signaling, a fact that could justify the establishment of pancitopenia. Therefore, in this study we set out to assess the cell cycle of hematopoietic stem/progenitors cells (HSPC) from bone marrow of malnourished mice. We used a murine model, and malnutrition induced from a low protein diet. The HPSC were obtained by immunomagnetic depletion method and used for the evaluation of cell cycle from the incorporation of propidium iodide (PI) and Acridine Orange (AO). Also, we quantified the cell cycle regulatory proteins by western blot and evaluated the expression of receptors for fibronectin, VLA4 and VLA5. Meanwhile, in ex vivo model, we evaluated the influence of growth factors and a matrix of fibronectin on the proliferation of HSPC. Considering the importance of stromal cells in cell signaling, we performed the CFU-F assay for the quantification of stromal cells and growth factors secreted. Finally, we evaluated the efficacy of nutritional recovery in the face of cell cycle alterations observed in the model of malnutrition. Briefly, we observed an impairment in the cell cycle of HSPC of undernourished mice, with an increase in this cell population in G0/G1 phase. The proteins that induce cell cycle showed a reduced expression whereas the inhibitory proteins showed increased...

The Pneumocystis life cycle

First recognised as "schizonts" of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.

The chromatin structure of the lysozyme GAS41 origin of DNA replication changes during the cell cycle

We used a rapid and simple protocol using lysolecithin for mapping HS sites in vivo. The protocol is based on partial digestion with DNase I of exponentially growing cells following permeabilization by short treatment with lysolecithin. Using this protocol, we analyzed the chromatin structure of the region surrounding two overlapping elements, an origin of bidirectional DNA replication and the GAS41 promoter, in chicken myelomonocytic HD11 cells arrested in G0, G0 and S phases as well as at the G0/S border. The results show that the chromatin of this region became more nuclease sensitive when cells were arrested in G0 phase and that this change in chromatin structure was reversible after the cells began to enter S phase.

Visualization of the ribosomal DNA (45S rDNA) of Indica rice with FISH on some phases of cell cycle and extended DNA fibers

The ribosomal DNA (45S rDNA) behaviors during the cell cycle were analyzed on interphase nuclei, prophases, metaphases, pachytene chromosomes and extended DNA fibers in rice (Oryza,sativa ssp.indica cv.Guangluai No.4) by using high-resolution fluorescent in situ hybridization (FISH). The results show that 45S rDNA is located at the ends of short arms of chromosomes 9 and 10. But the signals are much more intense on chromosome 9 than on chromosome 10 in metaphase. Pachytene chromosome has rDNA signal arrays on chromosome 9. Different phases are described and discussed. These results indicate that the activity of rDNA at individual loci may also vary through the cell cycle in rice. on extended DNA fibers, 45S rDNA signals appear as strings of numerous red spots, but some signals are missed in some regions, probably result from weak signals or intergenic spacers