DNA甲基化导致肝细胞癌SYK基因失表达

目的：探讨SYK（Spleen tyrosine kinase，脾酪氨酸激酶）在肝细胞癌中的表达和不表达的机制。方法：分别用逆转录-聚合酶链反应（RT—PCR）方法和甲基化特异性聚合酶链反应（Methylation—specific PCR，MSP）检测SYK基因在肝癌细胞系（HepG2和Hep3B）和34例肝细胞癌组织、癌旁非瘤组织中的表达和甲基化情况。结果：肝癌细胞系Hep3B表达SYKmRNA，而HepG2不表达SYK mRNA、DNA甲基化转移酶抑制剂5-aza-2’-deoxyeytidine处理HepG2后，SYK重新表达．Hep3B细胞SYK甲基化阴性，HepG2细胞SYK甲基化阳性；34例肝细胞癌组织标本中，5例SYKmRNA表达阴性，SYK基因甲基化均阳性；29例SYK mRNA表达阳性，其中3例SYK甲基化阳性．其余26例SYK甲基化阴性。肿瘤组织SYK基因的甲基化率为23．5％（8／34），而瘤旁肝组织中为8．8％（3／34）。结论：SYK基因启动子甲基化导致肝细胞癌SYK mRNA失表达、可能是肝癌发病的机制之一。     

Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers

A variety of tumor suppressor genes are down-regulated by hypermethylation during carcinogenesis. Using methylated CpG amplification-representation difference analysis, we identified a DNA fragment corresponding to the Tazarotene-induced gene 1 (TIG1) promoter-associated CpG island as one of the genes hypermethylated in the leukemia cell line K562. Because TIG1 has been proposed to act as a tumor suppressor, we tested the hypothesis that cytosine methylation of the TIG1 promoter suppresses its expression and causes a loss of responsiveness to retinoic acid in some neoplastic cells. We examined TIG1 methylation and expression status in 53 human cancer cell lines and 74 primary tumors, including leukemia and head and neck, breast, colon, skin, brain, lung, and prostate cancer. Loss of TIG1 expression was strongly associated with TIG1 promoter hypermethylation (P < 0.001). There was no correlation between TIG1 promoter methylation and that of retinoid acid receptor beta2 (RARbeta2), another retinoic-induced putative tumor suppressor gene (P = 0.78). Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 5 days restored TIG1 expression in all eight silenced cell lines tested. TIG1 expression was also inducible by treatment with 1 micro M all-trans-retinoic acid for 3 days except in densely methylated cell lines. Treatment of the K562 leukemia cells with demethylating agent combined with all-trans-retinoic acid induced apoptosis. These findings indicate that silencing of TIG1 promoter by hypermethylation is common in human cancers and may contribute to the loss of retinoic acid responsiveness in some neoplastic cells.     

Epigenetic silencing of maspin gene expression in human breast cancers

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.     

Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.     

Delivery of gene silencing agents for breast cancer therapy

The discovery of RNA interference has opened the door for the development of a new class of cancer therapeutics. Small inhibitory RNA oligos are being designed to specifically suppress expression of proteins that are traditionally considered nondruggable, and microRNAs are being evaluated to exert broad control of gene expression for inhibition of tumor growth. Since most naked molecules are not optimized for in vivo applications, the gene silencing agents need to be packaged into delivery vehicles in order to reach the target tissues as their destinations. Thus, the selection of the right delivery vehicles serves as a crucial step in the development of cancer therapeutics. The current review summarizes the status of gene silencing agents in breast cancer and recent development of candidate cancer drugs in clinical trials. Nanotechnology-based delivery vectors for the formulation and packaging of gene silencing agents are also described.     

Hypermethylation of ribosomal DNA in human breast carcinoma

We examined the methylation status of the transcribed domain of ribosomal DNA (rDNA) in 58 patients with breast cancer. The mean percent of methylation was significantly higher in breast tumours than that of normal control samples (P < 0.0001). This increased rDNA methylation was associated with oestrogen receptor non-expression (P < 0.0273) and with moderately or poorly differentiated tumours as compared to well differentiated tumours (P < 0.0475). Our results suggest that rDNA can be a useful marker for monitoring aberrant methylation during breast tumour progression.     

Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer

Introduction  

BRCA1 or BRCA2 germline mutations increase the risk of developing breast cancer. Tumour cells from germline mutation carriers have frequently lost the wild-type allele. This is predicted to result in genomic instability where cell survival depends upon dysfunctional checkpoint mechanisms. Tumorigenic potential could then be acquired through further genomic alterations. Surprisingly, somatic BRCA mutations are not found in sporadic breast tumours. BRCA1 methylation has been shown to occur in sporadic breast tumours and to be associated with reduced gene expression. We examined the frequency of BRCA1 methylation in 143 primary sporadic breast tumours along with BRCA1 copy number alterations and tumour phenotype.     

Notch基因在人乳腺癌中的作用

目的检测Notch1和JAG1在乳腺癌中的表达,及其与乳腺癌相关临床指标的关系,分析Notch基因在人类乳腺癌中的作用和意义。方法应用逆转录聚合酶链反应（RT—PCR）检测60例乳腺癌组织和25例癌旁正常乳腺组织Notch1和JAG1的表达,对乳腺癌组织与癌旁组织进行表达率和表达强度标准化系数的统计学比较,并在不同的腋窝淋巴结转移情况间、不同TNM分期和病理学分级间进行Notch1表达强度标准化系数的统计学比较。结果乳腺癌组织Notch1的表达率和标准化系数分别为93．3％（56／60）和0．83,均明显高于癌旁组织,乳腺癌组织中JAG1的表达率为10％（6／60）,癌旁组织中无JAG1表达,伴腋窝淋巴结转移的病例Notch1标准化系数高于无腋窝淋巴结转移的病例;乳腺癌Ⅰ期病例Notch1标准化系数（0．57）低于Ⅱ期（1．05）Ⅱ期高于Ⅲ期（0．59）,Ⅰ期与Ⅲ期差异无统计学意义;乳腺癌Ⅰ级病例Notch1标准化系数（0．55）低于Ⅱ级（0．83）和Ⅱ级低于Ⅲ级（1．05）,Notch1可能在分化较好的人乳腺癌中的表达是低的,在分化较差的人乳腺癌中的表达是增高的。结论人类乳腺癌中存在Notch1和JAG1的异常高表达,提示Notch1的异常表达与活化可能与人类乳腺癌的形成有关,在人类乳腺癌不同发展阶段的作用可能不同。     

Hypermethylation of the PTEN gene in ovarian cancer cell lines

This study was performed to investigate the hypermethylation status of the PTEN gene in ovarian cancer. To this end, we incubated eight ovarian cancer cell lines with the demethylating agent 5-aza-2' deoxycytidine in three different concentrations for 5 days. Subsequently, the PTEN expression was quantified by both real time RT-PCR and quantitative western analyses. PTEN mRNA varied considerably in response to demethylation whereas PTEN protein concentrations remained constant in all cell lines except OAW42 cells (12.5%). The data suggest that PTEN is highly regulated at translational level. However, methylation of the PTEN gene plays a subordinate role in ovarian cancer.     

Cytokeratin 8 silencing in human nasopharyngeal carcinoma cells leads to cisplatin sensitization

By comparing protein profiles of nasopharyngeal carcinoma HONE1 cells to transformed nasopharyngeal epithelial NP 69 cells, several clusters of differentially expressed proteins were identified. The increased expression of cytokeratin 8 (CK8) and pyruvate kinase M2 was a common feature in four NPC cell lines compared to the two transformed epithelial cell lines. Suppression of CK8 was associated with the sensitivity to cisplatin in HONE1 cells; while overexpression of CK8 provided resistance to cisplatin-mediated apoptosis; and this protection occurred through an enhanced phosphorylation of c-Jun NH(2)-terminal kinase (JNK). Our findings implicate an underlying molecular mechanism in which CK8 is required for cisplatin resistance.     

siRNA沉默hTERT基因表达对人乳腺癌细胞增殖和凋亡的影响

目的应用小干扰RNA（small interferingRNA，siRNA）抑制乳腺癌MCF-7细胞hTERT的表达，探讨其对乳腺癌细胞增殖和凋亡的效应。方法体外化学合成针对hTERT基因的siRNA序列，在脂质体介导下转染MCF-7细胞，实时定量PCR检测hTERTmRNA表达水平，Westernblot检测hTERT蛋白表达水平，流式细胞仪检测细胞凋亡状况，MTT法检测细胞增殖活性，平板克隆形成实验检测克隆形成率。结果所设计的3对针对不同靶点的siRNA与对照组相比均可有效抑制hTERT的表达，hTERTsiRNA转染MCF-7细胞48h后，siRNAl-siRNA3组hTERTmRNA表达分别为（35．3±4．2）％、（30．7±2．8）％、（31．3±3．6）％，与阴性对照组（96．4±2．8％）相比，差异有统计学意义。MTT结果显示MCF-7细胞增殖能力显著降低，转染48h后，siRNAl～siRNA4细胞抑制率分别为（57．6±3．6）％、（61．3±4．3）％、（65．6±6．3）％和（3．1±4．5）％，细胞克隆形成能力下降，凋亡率明显增加。结论体外化学合成的hTERTsiRNA可以有效地抑制MCF-7细胞hTERT的表达，从而抑制细胞增殖，促进细胞凋亡。     

TIPIN depletion leads to apoptosis in breast cancer cells

Triple‐negative breast cancer (TNBC) is the breast cancer subgroup with the most aggressive clinical behavior. Alternatives to conventional chemotherapy are required to improve the survival of TNBC patients. Gene‐expression analyses for different breast cancer subtypes revealed significant overexpression of the Timeless‐interacting protein (TIPIN), which is involved in the stability of DNA replication forks, in the highly proliferative associated TNBC samples. Immunohistochemistry analysis showed higher expression of TIPIN in the most proliferative and aggressive breast cancer subtypes including TNBC, and no TIPIN expression in healthy breast tissues. The depletion of TIPIN by RNA interference impairs the proliferation of both human breast cancer and non‐tumorigenic cell lines. However, this effect may be specifically associated with apoptosis in breast cancer cells. TIPIN silencing results in higher levels of single‐stranded DNA (ssDNA), indicative of replicative stress (RS), in TNBC compared to non‐tumorigenic cells. Upon TIPIN depletion, the speed of DNA replication fork was significantly decreased in all BC cells. However, TIPIN‐depleted TNBC cells are unable to fire additional replication origins in response to RS and therefore undergo apoptosis. TIPIN knockdown in TNBC cells decreases tumorigenicity in vitro and delays tumor growth in vivo. Our findings suggest that TIPIN is important for the maintenance of DNA replication and represents a potential treatment target for the worst prognosis associated breast cancers, such as TNBC.