c-myc对肿瘤细胞生长抑制基因的转录调控

c-myc基因在许多肿瘤的发生、发展过程中起着重要作用,它主要通过对靶基因的转录调控发挥作用。对c-myc如何调节肿瘤细胞周期生长抑制基因转录机制进行综述。c-myc对细胞周期的调控主要通过对生长抑制基因转录抑制实现,主要有2个不同机制一个通过myc/Max二聚体中c-myc蛋白的羧基端(C末端)与Mil-1形成复合物与生长抑制基因p15等启动子Inr区域结合,拮抗其他转录激活因子如Mil-1与Inr结构结合。另一机制c-myc及可能存在的抑制物与转录激活因子Sp1及SMad(smaandmadhomologue)等形成复合物干扰Sp1等对生长抑制基因的激活。造成细胞恶性转化及增殖。

肿瘤生长抑制基因ING1在基底细胞上皮瘤中的表达及突变分析

目的研究肿瘤生长抑制基因ING1在基底细胞上皮瘤中是否存在过度表达及基因突变。方法搜集了54份基底细胞上皮瘤标本,运用免疫组织化学染色,DNA单链构象多态性分析(SSCP)及DNA序列测定的方法研究ING1基因在基底细胞上皮瘤中的表达及突变情况。结果免疫组化显示ING1基因在25%(6/24)基底细胞上皮瘤标本中过度表达。SSCP及DNA序列测定结果显示54例基底细胞上皮瘤标本中仅1例标本(1.8%)发生实质突变。突变发生在ING1基因编码区exon2,因而可能影响所有ING1异构体结构,并影响PHD锌指基序的功能。结论ING1基因在基底细胞上皮瘤中无明显过度表达及突变。

Downregulation of survivin by RNAi inhibits growth of human gastric carcinoma cells

AIM: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901. METHODS: To knockdown survivin expression, a small interfering RNA targeting against survivin was synthesized and transfected into SGC-7901 cells with lipofectamineTM2000. The downregulation of survivin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell proliferation inhibition rates were determined by methyl thiazolyl tetrazolium (MTT) assay. The effect of survivin siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RESULTS: RNA interference could efficiently suppress the survivin expression in SGC-7901 cells. At 48 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. Downregulation of survivin resulted in significant inhibition of tumor cell growth in vitro. The cell proliferation inhibition rates at 24, 48 and 72 h after survivin siRNA and non-siliencing siRNA transfection, were 34.06%, 47.61% and 40.36%, respectively. The apoptosis rate was 3.56% and the number of cells was increased in G0/G1 phase from 38.2% to 88.6%, and decreased in S and G2/M phase at 48 h after transfection. CONCLUSION: Downregulation of survivin results in significant inhibition of tumor growth in vitro. The inhibition of survivin expression can induce apoptosis of SGC-7901 cells. The use of survivin siRNA deserves further investigation as a novel approach to cancer therapy.

Suppression of gastric cancer growth by baculovirus vector-mediated transfer of normal epithelial cell specific-1 gene

AIM: To study the inhibitory effect of baculovirus- mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry. RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.

Effect of breast-cancer metastasis suppressor 1 (BRMS1) on growth and metastasis of human gastric cancer cells in vivo

Objective： The aim of the study was to investigate inhibitory effects of breast-cancer metastasis suppressor 1 protein （BRMS1） on primary tumor growth and metastasis of human gastric cancer （GC） cells in nude mice. Methods： We compared the expression of BRMS1 in the primary gastric tumor and metastatic gastric tumor by immunohistochemistry. Expression of BRMS1 also was detected in the GC cells by RT-PCR and Western blot. Three groups of cultured human GC cell line SGC-7901, were maintained： transfected cells with pcDNA3.1（-）B/myc-BRMS1; negative control cells with pcD- NA3.1/myc-his（-）B; and blank control ceils without any transfection. Histologically intact samples of the cells, maintained by passage in the subcutis of nude mice, were transplanted orthotopically into stomach walls of nude mice to establish a nude mouse model of human gastric carcinoma. Their primary tumor growth and metastasis were then observed. Results： The expression of BRMS1 was markedly stronger in the primary gastric tumor compared with metastatic gastric tumor. We also detected BRMS1 gene and protein in the gastric cancer cell tines. Numbers of metastasis tumors significantly differed among mice infected with transfected cells, with negative controls and with blank controls （4.38 ± 0.60, 7.75 ± 0.59, and 7.63± 0.65, respectively; P 〈 0.05）. However, there were no significant differences in the size of orthotopic tumors among mice infected with transfected, negative control and blank control cells [（12.02 ± 0.70）, （12.71 ± 0.63） and （12.89 ± 0.71） mm, respectively; P 〉 0.05]. Conclusion： BRMS1 suppresses metastasis of GC cells, but does not inhibit growth of gastric tumors.

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer. Its mRNA expression was suppressed in most hepatoma samples. In order to study the biological effect of TTR gene on the growth of hepatoma cells, a recombinant vector containing TTR cDNA was constructed by pCMV, then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50%in strength compared with that of the control. This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid. Hepatoma cells of cell lines PLC/PRF/5, SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization. The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721cells transfected with pCMV alone. However, a remarkable TTR mRNA expression was observed in hepatoma SMMC-7721 cells transfected with pCMV-TTR. It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.

Effect of tumor suppressor in lung cancer-1 on growth inhibition of MG63 cell line

Objective： The aim of this study was to establish the osteosarcoma cell sublines which stably expressing tumor suppressor in lung cancer-1 （TSLC1） gene and evaluate its effect on growth inhibition of human osteosarcoma cell line MG63. Methods： The recombinant plasmid pCI-TSLC1 was stably transfected into MG63 cells with Lipofectamine 2000. The posi- tive clones were developed by selection by G418. Biological characteristics of one of the 6 cell lines which highly expressing TSLC1, namely, the M8T were studied. Cell growth was analyzed with MTT assay. 2 x 10^7cells suspended in 0.2 mL phosphate buffered saline （PBS） were injected into the two flanks of 5-6-week-old female BALB/C nu/nu athymic nude mice. The volumes of subcutaneous of tumor growth were evaluated and calculated by the formula V= Length x Width x Height x 0.5 once a week. Results： The MST cell subline which stably expressing TSLC1 was characterized by Western blot. The genetic stability and purity of M8T cells were stable. TSLC1 significantly suppressed the growth of M8T cells in vitro. Moreover, the tumorigenicity of MST cells was suppressed in vivo. Conclusion： The osteosarcoma cell sublines MST which stably expressing TSLC1 had been successfully established. The ability of growth and metastasis of MST was significantly suppressed both in vitro and in vivo.