Met-Controlled Allosteric Module of Neural Generation as A New Therapeutic Target in Rodent Brain Ischemia / 中国结合医学杂志

OBJECTIVE@#To investigate a Met-controlled allosteric module (AM) of neural generation as a potential therapeutic target for brain ischemia.@*METHODS@#We selected Markov clustering algorithm (MCL) to mine functional modules in the related target networks. According to the topological similarity, one functional module was predicted in the modules of baicalin (BA), jasminoidin (JA), cholic acid (CA), compared with I/R model modules. This functional module included three genes: Inppl1, Met and Dapk3 (IMD). By gene ontology enrichment analysis, biological process related to this functional module was obtained. This functional module participated in generation of neurons. Western blotting was applied to present the compound-dependent regulation of IMD. Co-immunoprecipitation was used to reveal the relationship among the three members. We used IF to determine the number of newborn neurons between compound treatment group and ischemia/reperfusion group. The expressions of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were supposed to show the changing circumstances for neural generation under cerebral ischemia.@*RESULTS@#Significant reduction in infarction volume and pathological changes were shown in the compound treatment groups compared with the I/R model group (P<0.05). Three nodes in one novel module of IMD were found to exert diverse compound-dependent ischemic-specific excitatory regulatory activities. An anti-ischemic excitatory allosteric module (AM@*CONCLUSIONS@#AM

Mechanisms involved in PERK-dependent autophagy under arsenite exposure / 军事医学

Objective To explore whether PERK is involved in the regulation of arsenite-induced autophagy.Methods Human hepatoma cells HepG2 were cultured and treated with arsenite.The expression level of autophagic hallmarks and the activation status of PERK were detected by Western blotting.The transactivation of p53 and the induction of its downstream target genes expression were also detected by Western blotting after knockdown of PERK expression.Transactivity of p53 was detected by dual luciferase reporter assay after knockdown of PERK expression.Results An increase in the LC3BII:I ratio,the induction of Beclin-1 expression and the degradation of p62 were readily observed in arsenite-treated HepG2 cells,but the effects were abolished after knockdown of PERK expression.Furthermore,phosphorylation of p53 at Ser15 and Ser392,transactivation of p53 and the induction of its downstream target gene DAPK1 expression were effectively inhibited under the same PERK knockdown conditions.Conclusion PERK regulates arsenite-induced autophagy by activating p53-dependent DAPK1 upregulation.

Correlation between promoter methylation of hMLH1 and DAPK genes and drug resistance of lung cancer / 肿瘤研究与临床

Drug resistance is one of the major obstacles for chemotherapy and targeted therapy in lung cancer with complex mechanisms.The researches have demonstrated that epigenetic changes are closely related to drug resistance of tumor.DNA methylation is an important epigenetic modification.In this paper, the relationships between hMLH1 promoter aberrant methylation and platinum-resistance, and DAPK promoter aberrant methylation and EGFR-TKI resistance in lung cancer will be briefly reviewed.

Methylation of E-cadherin, p16, RASSF1A, DAPK and MGMT in salivary gland tissues / 军事医学

Objective To investigate the methylation status of E-cadherin(E-cad), p16, RASSF1A, DAPK and MGMT in histologically normal salivary gland tissues and provide reference for determination of the methylation status of salivary gland tumors.Methods Methylation of E-cad, p16, RASSF1A,DAPK and MGMT was analyzed using methylation-specific polymerase chain reaction ( MSP) .The results were compared with the methylation status of these genes in salivary adenoid cystic carcinoma ( ACC) tumor tissues in our previous studies and the association between promoter methylation of E-cad, p16, RASSF1A, DAPK, and MGMT on one hand and the patients′gender, age, smoking and types of gland on the other hand was also analyzed .Results Promoter methylation was detected in 8 of the 60 (13%) salivary glands, E-cad in 4(7%), p16 in 2(4%), RASSF1A in 2(4%), DAPK in 2 (4%), and MGMT in 1(2%).Compared with our previous results, there was a significantly lower methylation ratio in promoter methylation of E-cad(P<0.01), p16 (P<0.01), RASSF1A (P<0.01),and DAPK (P<0.01) in salivary gland tissues than in ACC tumor tissues.Conclusion Promoter methylation of E-cad, p16 and RASSF1A is a rare event in histologically normal salivary gland tissues .

Inactivation of DAPK1 gene by methylated oligonucleotides and its effect on the proliferation of leukemia cell line K562 / 白血病·淋巴瘤

Objective To inactivate Death-associated protein kinase 1 gene (DAPK1) by transfecting complementary methylated oligonucleotides and studies its effect on the proliferation of myelogenous leukemia cell line K562. Methods Methylated oligonucleotides complementary to DAPK1 gene promoter were transfected into K562 cells by Iipo2000. Methylation specific PCR (MSP) and Reverse transcription PCR (RT-PCR) were applied to detect DAPK1 gene promoter methylation status and its mRNA expressions respectively. MTT was used to detect the proliferation of K562 cells pre- and post- oligonucleotides transfection. Results DAPK1 gene promoter in non-treated and control groups were unmethylated with detectable mRNA expressions, but it became methylated with inhibited mRNA expressions after methylated oligonucleotide transfection. Proliferation in methylated oligonucleotide treatment group was significantly higher than that in non-treated and control groups. Conclusion Complementary methylated oligonucleotides could inactivate DAPK1 gene and inhibit its expression in K562 cells, which could promote its proliferation.

O papel do gene p16 e do gene Dapk no controle do ciclo celular e na via carcinogênese vulvar / The role of p16 and Dark genes in cell cycle control and in the pathways of vulvar carcinogenesis

O câncer vulvar é o quarto tipo de câncer mais comum nas mulheres e representa 4,8% dos cânceres do trato genital inferior. O carcinoma de células escamosas é responsável por 80 a 90% de todos os cânceres de vulva. O carcinoma escamoso vulvar e suas lesões pré-malignas parecem desenvolver-se por dois caminhos distintos, baseados em características etiológicas e histopatológicas, tendo assim uma etiologia heterogênea. Um dos caminhos está relacionado com a infecção pelo HPV, e o outro, com as desordens epiteliais, tais como líquen escleroso e hiperplasia epitelial. O HPV é um importante fator causal das neoplasias do trato genital inferior. Ele está presente em cerca de 90% dos cânceres do colo uterino e 30 a 40% dos cânceres de vulva. O tipo mais prevalente é o 16, seguido pelos tipos 18, 45, 31 e 33. O estudo das alterações genéticas e epigenéticas, por meio da análise de metilação e imunoexpressão gênica, tem demonstrado uma grande versatilidade para o monitoramento molecular de pacientes com câncer, o que impulsiona pesquisas de métodos diagnósticos e terapêuticos do câncer. Nesta atualização pretendeu-se demonstrar as funções dos genes p16 e DAPK e as recentes pesquisas sobre a expressão destes genes nas vias da carcinogênse vulvar.

Vulvar cancer is the fourth commonest kind of cancer in women and it represents 4.8% of cancers in the lower genital tract squamous cell carcinoma is responsible for 80-90% of all vulvar cancers. Squamous cell carcinoma and it's premalignant lesions seem to develop in two distinct pathways, based on etiological and histopathological characteristics, thus forming a heterogeneous etiology. Whereas one of the pathways is related to HPV infection, the other is related to epithelial disorders such as: lichen sclerousus and epithelial hyperplasia. HPV is an important contributing factor of neoplasia in the lower genital tract. It is found in 90% of cervical cancers and in 30-40 % of vulvar cancers. The most prevalent kind is 16, followed by 18, 45, 31, and 33. The study of genetic and epigenetic alterations by means of methylation and genic immunoexpression has demonstrated great versatility to the monitoring ofpatients with cancer, which boosts researches of diagnostic and therapeutic methods for cancer. This update intends to demonstrate the role of p16 and DAPK genes as well as the recent researches regarding the expression of these genes in the pathways of vulvar carcinogenesis.

Establishment of PC12 cell line with stable inhibition of DAPK expression / 基础医学与临床

Objective To screen a cell line which stably suppresses DAPK expression and to observe the growth characters.Methods Four pairs of shRNA were designed,synthesized and inserted into the pDsRed1-N1-U6 vector.The recombinant plasmids were purified and transfected into PC12 cell.Meanwhile,a pDsRed1-N1-U6 vector was transfected as control.The cell clones were screened by G418,and the stable PC12 cell line was established.DAPK expression was detected by Western blot.MTT method and Flow Cytometry(FCM) assay were used to assess the growth characters of the cell line.Results The shRNAs were transfected into PC12 cell and the cell clones were successfully screened out.Of the four recombinant plasmids,the F2 was the best interfering shRNA.Beyond our expectation,the F1 recombinant plasmid had an enhanced effect on DAPK expression.Conclusion A stable PC12 cell line with stable inhibition of DAPK expression by was established using siRNA expression vectors.

Aberrant Methylation of p16 Tumor Suppressor Gene and Death-Associated Protein Kinase in Non-Small Cell Lung Carcinoma / 결핵

BACKGROUND: The p16INK4a (p16) tumor suppressor gene is frequently inactivated in human non-small cell lung cancers (NSCLCs), predominantly through homozygous deletion or in association with aberrant promotor hypermethylation. Death-associated protein kinase (DAPK) gene influences interferon γ-induced apoptotic cell death and has important role in metastasis of lung cancer in animal model. Hypermethylation of promoter region of DAP kinase gene may suppress the expression of this gene. METHODS: This study was performed to investigate the aberrant methylation of p16 or DAP kinase in 35 resected primary NSCLCs by methylation-specific PCR (MSP), and demonstrated frequency, diagnostic value and clinical implication of aberrant methylation of two genes. RESULTS: Thirty-two cases were male patients, and 3 cases were female patients with an average age was 57.8±10.5 years. The histologic types of lung cancer were 22 of squamous cell carcinoma, 12 of adenocarcinoma, 1 of large cell carcinoma. Pathologic stages were 11 cases of stage I(1 IA,10 IB), 13 cases of stage II (1 IIA, 12 IIB), and 11 cases of stage III(9 IIIA, 2 IIIB). Regarding for the cancer tissue, p16 aberrant methylation was noted in 13 case of 33 cases (39.4%), DAP kinase in 21 cases of 35 cases (60%). Age over 55 year was associated with p16 aberrant methylation significantly (p<0.05). Methylation status of two genes was not different by smoking history, histologic type, size of tumor, lymph node metastasis and disease progression of lung cancer. There was no correlation between p16 and DAP kinase hypermethylation. CONCLUSION: This investigation demonstrates that aberrant methylation of p16 tumor suppressor gene or DAP kinase showed relatively high frequency (74.3%) in NSCLCs, and that these genes could be a biologic marker for early detection of lung cancer.

Study on the relevancy between OPCML and DAPK gene methylation and CasKi cell apoptosis / 中国药理学通报

Aim To study tumor suppressor gene OPCML,DAPK methylation changes during the process of CasKi cell apoptosis induced by trichosanthin, and to explore the correlation and the role between cervical cancer cell apoptosis and tumor suppressor gene methylation so as to find new demethylation drugs.Methods ① MTT was applied to assay the inhibition of TCS to CasKi cell proliferation and flow cytometry was used to analyze cervical CasKi cell apoptosis induced by trichosanthin; ② Methylation-specific PCR(MSP) technology was applied to detect cervical cancer and during cell apoptosis process OPCML and DAPK gene promoter methylation status of CpG islands.Results In CasKi cervical cancer cells,OPCML and DAPK gene promoter region showed a high degree of CpG island methylation status, by trichosanthin treatment,the growth of CasKi markedly was inhibited, and flow cytometry analysed the characteristic sub-G1 peak,OPCML and DAPK gene promoter region showed no CpG island methylation of performance.Conclusions During the process of CasKi cell apoptosis induced by trichosanthin,OPCML and DAPK gene demethylated significantly,accordingly, trichosanthin might be a new methylation inhibitor,and there might be some correlation between cell apoptosis and tumor suppressor gene methylation.And OPCML and DAPK gene methylation tests might become new clinical indicators for the early detection of cervical cancer.

Effect of 5-azacytidine on the methylation of DAPK1 in cervical carcinoma cell line / 西安交通大学学报(医学版)

Objective To investigate the correlation between methylation status and death-associated protein kinase 1 (DAPK1) in SiHa and Hela cell line of cervical carcinoma and intervention of DNA methyltransferase inhibitor 5-azacytidine on the expression of DAPK1 and the proliferation of the cells. Methods DAPK1 methylation status was analyzed using methylation-specific PCR methods. The expression of mRNA and protein of DAPK1 were analyzed by RT-PCR and SABC methods after the treatment with 5-azacytidine. MTT assay was used to observe the changes of proliferation activity of the cells after 5-azacytidine treatment. Results DAPK1 genes were methylated and did not express in SiHa cells in the cervical carcinoma. Its expression could be restored by 5-azacytidine. MTT assay showed 5-azacytidine could weaken the proliferation of cancerous cells. Conclusion DAPK1 methylation plays an important role in the carcinogenesis of cervical cells and can reexpress after the treatment with 5-azacytidine which also restored its inhibitory function on carcinoma.