Relationship between telomere length and radiosensitivity in various human cancer cell lines

Objective： To investigate the relationship between telomere length and radiosensitivity in various human cancer cell lines with the expectation to find a valid and common predictor of radiosensitivity for different cancers. Methods： Eight human cancer cell lines were used, including five human breast cancer cell lines （ZR-75-30, MCF-7, MDA-MB-435S, T-47-D, F539-1590）, two human larynx squamous carcinoma cell lines （Hep-2 and Hep-2R） and a human malignant glioma cell line （U251）. Among them, the radioresistant cell line Hep-2R was isolated and established from a radiosensitive human larynx squamous carcinoma cell line Hep-2 by our center. The radiobiological characteristics of the eight lines were analyzed by the method of colony-forming assay and the radiosensitivity parameters were calculated. Telomere length was analyzed by TRF （mean Telomere Restriction Fragments） length assay. Results： The radioresistance of Hep-2R cell line proved to be stable in long-term passaged cultures as well as in frozen samples. Radiosensitivity parameters are different among those lines. The SF2 values of Hep-2 and U251 are 0.4148 and 0.7520, respectively; The SF2 values of breast cancer cell lines are between those of Hep-2 and U251. The TRF of Hep-2R is 11.12Kb, longer than three times that of its parental counterpart. There is a positive correlation both between SF2 and TRF （r=-0.786, P〈0.05）, and between Do and TRF （r=0.905, P〈0.01）. Conclusion： It is concluded that radiosensitivity and telomere length （TRF） are negatively correlated, TRF could be a valid predictor for radiosensitivity.     

Expression of Coxsackie and Adenovirums and its Significance in Human Lung Cancer

OBJECTIVE To study the relationship between the coxsackie and adenovirus receptor （CAR） and the development of human lung cancer. To optimize adenovirus vector-based gene therapy. METHODS The expression of CAR in 112 cases of lung cancer was examined using immunohistochemistry. At the same time, the relationship between CAR expression and clinicopathologic characteristics was analyzed, RESULTS ：lhere is a little expression of CAR in normal lung tissue. Compared with paraneoplastic epithelial tissue of the lung, the expression of CAR is generally up-regulated in tumor tissues showing a significant dif- ference （P〈0.01）. The positive rate of CAR expression in squamous cell carcinoma was 43.1%, and in adenocarcinoma 70.2%, with the difference between the two rates being statistically significant （P〈0.01）. Compared to the paraneoplastic tissues, the difference in CAR positive expression was 35.4% for squamous cell carcinoma and 38.3% for adenocarcinoma. But the difference in different stages of squamous cell carcinoma had no statistical significance （P〉0.05）. However, the expression of CAR was at a high level in the bronchioalveolar carcinomas as 80.4% were CAR positive. This research showed that there was a specially high expression of CAR in adenocarcinomas. CONCLUSION CAR is expressed in human lungs at a low level and up-regulated in the tumor tissues, suggesting that there is a relationship between adenocarcinoma and CAR. This research provides a basis for planning a regimen of gene therapy using an adenovirus vector,     

EXPRESSION OF FLIP IN HUMAN COLON CARCINOMAS： A NEW MECHANISM OF IMMUNE EVASION

Objective： It has been proposed that Fas ligand （FasL） may play an important role in immune escape of tumors and FLIP is an important mediator of Fas/FasL pathway. In this study, the expression of FLIP was determined in human colon carcinoma cell lines and tissue to investigate the new mechanism of immune evasion of human colon carcinomas. Methods： RT-PCR and immunohistochemistry （IHC） were performed to investigate the expression of FLIP in human colon carcinoma cell lines SW480, LS174 and twenty human primary colon carcinoma specimens. Results： It was shown that SW480 cells, LS174 cells and primary colon carcinoma specimen constitutively expressed FLIP at the mRNA and protein level. The expression of FLIP was not found in the epithelial cells of normal colon mucosa. Conclusion： FLIP was expressed in human primary colon carcinoma specimens but not in the normal counterpart. It suggested that the expression of FLIP may occur during the malignant transformation from normal colon epithelial cells to colon carcinoma cells. Tumor cells might obtain the ability to resist the Fas-mediated apoptosis by expressing FLIP. The expression of FLIP might contribute to the formation of colon carcinomas.     

Detection of K-ras Gene Point Mutation＇s Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective： To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods： Three kinds of special sequence primers （SSP） for polymerase chain reaction （PCR） with regard to the mutation styles （OAT, COT and GOT） at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results： The style of K-ras gene point mutation at codon 12 was OAT in human pancreatic cancer cell line. Conclusion： PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.     

Expression of CD44s mRNA in Gastric Carcinoma

Objective： To study the expression of CD44s mRNA in the occurrence, development and invasion of gastric carcinoma （GC）. Methods： The expressions of CD44s mRNA in 66 cases of GC, 25 cases of superficial gastritis and 25 cases of atypical hyperplasia were examined by in situ hybridization （ISH）. Results： There was no expression of CD44s mRNA in the group of superficial gastritis; the positive rate was 20%（5/25） in the group of atypical hyperplasia and 62.12%（41/66） in the group of gastric carcinoma. The positive rate in poor differentiation group was significantly higher than that in well differentiation group （P〈0.05）, and the positive rate of lymph node metastasis group was significantly higher than that in negative lymph node metastasis group（P〈0.05）. Conclusion： The expression of CD44s mRNA was related to cell differentiation degree and lymph node metastasis, the activation of CD44s gene was related to strong invasion of cancer cells and poor prognosis.     

High Expression of the RECK Gene in Breast Cancer Cells is Related to Low Invasive Capacity

OBJECTIVE To investigate the expression of the RECK gene in human breast (cancer) cell lines, and to determine the relationship between RECK gene expression and the invasive capacity of the breast cancer cell lines. METHODS The invasive capacity of breast (cancer) cell lines including HBL-100, MCF-7 and MDA-MB-435S were determined by the Tran-swell method. The protein expression levels of RECK, MMP-2 and MMP-9 genes in these three cell lines were measured by immunocytochemical methods. The expressions of the RECK gene and protein level were measured by RT-PCR and Western blots in the cell lines respectively. RESULTS The order of the invasive capacity of the breast (cancer) cell lines was MDA-MB-435S, being the highest, and HBL-100, being the lowest. The invasive capacity difference between any two groups among the three groups was significant (P<0.01). The protein expression level of the RECK gene in the HBL-100 cell line was highest, and no expression was detected in MDA-MB-435S cells. Moreover, the expression of the RECK gene was negatively correlated with the expression of the MMP-2 and MMP-9 genes. The mRNA level of the RECK gene in HBL-100 cells was the highest, but no expression was found in the MDA-MB-435S cells (P<0.001). CONCLUSION There was a significant negative correlation between the expression level of the RECK gene and invasive capacity in vitro, and the RECK gene expression showed an inverse proportion to that of the MMP-2, MMP-9 genes.     

P73、ER、PR在食管癌及癌前病变中的表达及临床意义

Objective： To test the expression of mutant p73, p53, ER and PR proteins in the esophageal normal mucosa, hyperplasia, dysplasia and squamous cell carcinoma, and research the clinically pathological significance and the correlation, for the early diagnosis, prognostic measure and therapy in clinic. Methods： With Immunohistochemistry, it was examined to show these tumor markers＇ expression in different epithelial lesions of 40 esophageal squamous cell carcinomas, 14 dysplasias, 14 hyperplasias and 14 normal mucosas. Results： The expression of p73 was 55%, 21%, 0% and 0% in the esophageal carcinoma, dysplasia, hyperplasia and normal mucosa, respectively. The significant difference in expression of p73 （P〈0.001） was observed between the esophageal normal mucosia, hyperplasia, dysplasia and esophageal squamous cell carcinoma with Fisher＇s exact test. Difference in expression of p73 （P〈0.05） was observed between the esophageal squamous cell carcinoma and dysplasia with X^2 test. The expression of p73 showed non-correlation with the patient＇s age, sex, tumor＇s grade, lymph-node metastasis and invasive depth （P〉0.05）; Similarly, the expression of mutant p53 was 67.5%, 35.7%, 7% and 0%, respectively; In like manner, the expression of ER was 55%, 21.4%, 14.2% and 0%, respectively; The expression of PR was 57.5%,14.28%, 0% and 0%, respectively. The significant difference in expression of PR （P〈0.001） was observed with Fisher＇s exact test. Difference in expression of PR （P〈0.05） was observed between the esophageal squamous cell carcinoma and dysplasia with x2 test. The expression of PR （P〈0.05） was correlated with lymph-node metastasis, and showed non-correlation with the patient＇s age, sex, tumor＇s grade, and invasive depth （P〉0.05）. Moreover, over-expression of mutant p53 and p73 showed significant correlation with ER and PR protein＇s positive expression （P〈0.05）. Conclusion： P73 protein may become a new tumor＇s marker to diagnose esophageal squarnous celt carcinoma. Because the expression of p73 protein was closely correlated with ER and PR, they could be simultaneously examined to help to early diagnose, prognosticate and cure esophageal carcinoma.     

K-ras基因在人喉鳞状细胞癌细胞株(Hep-2)中的表达及其意义

目的 K-ras基因的突变激活,最常见于胰腺癌、结肠癌和肺癌等肿瘤。国外有人报道人喉鳞状细胞癌的发生与K-ras基因的突变激活没有相关性。本文旨在探讨K-ras基因在喉鳞状癌细胞株Hep-2中的表达及其意义,为喉癌发生与K-ras点突变的相关性研究奠定坚实的基础。方法采用RT-PCR技术检测K-ras基因mRNA在喉癌细胞株Hep-2和胰腺癌细胞株MIAPaCa-2中的表达。结果人胰腺癌细胞株MIAPaCa-2和人喉鳞状癌细胞株Hep-2中K-ras mRNA表达均为强阳性。结论人喉鳞状细胞癌细胞株Hep-2中K-ras基因表达阳性,喉癌的发生可能与其点突变引起的激活有关。     

mTOR siRNA对喉鳞状细胞癌Hep-2细胞生物学行为的影响

目的观察mTOR siRNA对喉鳞状细胞癌Hep-2细胞增殖能力的影响。方法选取45例喉鳞状细胞癌组织和45例正常喉黏膜组织,采用免疫组织化学法检测mTOR蛋白的表达;将喉鳞状细胞癌Hep-2细胞分为空白对照组、空载体组和mTOR siRNA组,及正常对照组、溶剂对照组和雷帕霉素组,分别利用mTOR siRNA和雷帕霉素进行治疗,采用Western bloting法检测不同浓度的mTOR siRNA和雷帕霉素分别处理细胞后对mTOR蛋白表达的影响,并采用CCK-8试剂盒分析mTOR蛋白表达下调对喉鳞状细胞癌Hep-2细胞增殖能力的影响。结果 mTOR蛋白在喉鳞状细胞癌组织中表达的阳性率显著高于正常喉黏膜组织(P<0.05)。3种不同浓度mTOR siRNA分别转染Hep-2细胞24、48、72 h,各组与空白对照组、空载体组相比,mTOR蛋白表达均有所降低(P<0.05),且mTOR蛋白表达随mTOR siRNA浓度的增加、作用时间的延长而降低(P<0.05)。3种不同浓度雷帕霉素处理Hep-2细胞24、48、72 h,各组与溶剂对照组及正常对照组相比,mTOR蛋白表达量均有所降低(P<0.05);且mTOR蛋白表达随雷帕霉素浓度的增加、处理时间的延长而降低(P<0.05)。与空白对照组、空载体组相比,各mTOR siRNA组的Hep-2细胞增殖在转染后24、48、72 h均受到明显抑制(P<0.05)。结论 mTOR siRNA均能降低喉鳞状细胞癌Hep-2细胞中mTOR蛋白的表达,有效抑制细胞的生长,为喉鳞状细胞癌的分子治疗提供了理论依据。     

Gankyrin蛋白在喉鳞癌中表达的实验研究

目的研究Gankyrin蛋白在喉鳞癌组织及喉鳞癌Hep-2细胞系中的表达。方法免疫组织化学方法检测24例喉鳞癌组织、相应癌旁组织及10例正常组织,免疫细胞化学法检测Hep-2细胞系中Gankyrin蛋白的表达与分布,并计算阳性细胞率,进行统计分析。结果喉鳞癌组织及Hep-2细胞系中,Gankyrin蛋白主要表达于细胞核。喉鳞癌组织中Gankyrin蛋白表达阳性细胞率(58.5%)明显高于相应癌旁组织(11.4%)及正常组织(18.1%),且差异有统计学意义(均P0.01)。结论喉鳞癌组织及喉鳞癌Hep-2细胞系中均有Gankyrin蛋白的表达,可为Gankyrin在喉鳞癌中表达的后续相关研究提供理论依据。     

Survivin基因在喉癌中的表达及意义

目的研究Survivin基因在喉癌中的表达及其与各临床病理因素的关系.方法应用RT-PCR法检测2株喉癌细胞株,40例喉癌组织标本及相应的癌旁组织中Survivin基因的表达;应用免疫组织化学链霉菌抗生物素蛋白-过氧化酶连接SP法检测Survivin基因在120例喉癌组织中的表达情况.结果 RT-PCR显示喉癌细胞株和67.5%(27例)的喉癌组织表达Survivin mRNA,而癌旁组织内无1例阳性表达.SP法显示在Survivin基因在喉癌中阳性表达率为66.7%,其阳性表达率在有无淋巴结转移、复发、临床分期、5年生存率等方面差异有显著性(P＜0.05).结论 Survivin基因与喉癌的自然的发展过程有关,同时可作为判断肿瘤恶性程度,判断预后的有效指标.Survivin基因可能成为喉癌新的诊断标志及基因治疗的靶点.     

S100A4与MMP-9在喉鳞癌中的表达和相关性研究

分析正常喉组织、喉鳞癌组织中S100A4和MMP-9的表达及临床意义,探讨其在喉鳞癌发生发展中的作用及相关性。方法：采用免疫组织化学SP法检测S100A4和MMP-9蛋白的表达并分析二者的相关性及其与临床病理因素的关系;采用Western blot法检测S100A4和MMP-9蛋白的表达。结果：免疫组化结果表明S100A4和MMP-9蛋白在癌旁喉组织和喉鳞癌中的阳性表达率均呈升高趋势,分别为8.6%和14.3%,63.8%和71.3%,并且二者表达均与喉鳞癌淋巴结转移相关。统计学结果显示80例喉鳞癌组织中,S100A4和MMP-9蛋白表达呈正相关（P<0.01,r=0.440）。Western blot结果表明S100A4和MMP-9蛋白在喉鳞癌组织中表达高于癌旁喉组织。在喉癌细胞系Hep-2中干扰S100A4蛋白的表达后,MMP-9的蛋白水平下调。结论：S100A4和MMP-9在喉鳞癌发生发展中均存在异常,并且S100A4基因抑制后能够显著下调MMP-9的表达。     

凋亡抑制蛋白FLIP在喉部病变组织中的表达及意义

目的：检测凋亡抑制蛋白FLIP在人喉部常见病变组织中的表达并揭示其在喉癌等病变发生发展中的意义。方法：采用免疫组织化学EliVision法检测38例喉鳞状细胞癌标本,30例声带息肉组织标本,26例喉乳头状瘤标本,15例正常喉黏膜组织标本,喉癌Hep-2细胞系10个细胞爬片样本中FLIP蛋白的表达并进行统计学分析比较。结果：凋亡抑制蛋白FLIP在38例喉癌标本中有32例阳性表达（84.2%）,在喉癌Hep-2细胞系10个细胞爬片样本中全部阳性表达（100%）,26例喉乳头状瘤中5例阳性表达（19.2%）,30例声带息肉标本中仅2例阳性表达（6.7%）,而正常喉黏膜组织中均为阴性。凋亡抑制蛋白FLIP在喉癌组织中表达水平与喉部良性肿瘤和良性病变组织中的表达水平有显著差异（P〈0.01）。结论：凋亡抑制蛋白FLIP的表达水平与喉部病变组织的良恶性程度密切相关,提示其可能与喉癌的发生相关。     

Growth inhibition of human laryngeal cancer cell with the adenovirus-mediated p53 gene

Mostmalignanciesarediseasegeneratedwithaprocessofgeneticalteration.Basedonthistheory,newapproachestocancertherapyarebeingdevel0ped.Oneoftheseisgenetherapy.Amongthegeneshavingtherapeuticpotentialforcancertreatment,thep53tumorsuppressorgenehasbeenmostextensivelystudied.[l]Wild-typep53hasbeenshownt0beinvolvedintranscirptionalregulation.ItarrestscellsattheGllScheckpoint,blocksDNAreplicati0nandinducesap0ptosis."'Recentstudieshavesh0wnthatwild-typep53genecansuppressthegrowthofsomehumancancercelll…     

尼美舒利对人喉鳞癌Hep-2细胞祼鼠移植瘤CD44和MMP-7表达的影响

目的探讨尼美舒利对喉鳞状细胞癌裸鼠皮下移植模型的抑瘤作用及机制。方法建立人喉鳞癌Hep-2细胞株裸鼠移植瘤模型,应用尼美舒利处理裸鼠,观察肿瘤生长情况并绘制生长曲线,免疫组织化学技术检测移植瘤组织中COX-2、CD44和MMP-7蛋白表达,RT-PCR技术检测移植瘤组织中COX-2、CD44 和MMP-7mRNA表达情况。结果与对照组相比,治疗组肿瘤体积增长较对照组缓慢,体积抑瘤率为63.36%,重量抑瘤率达51.81%,肿瘤体积和重量均明显低于对照组（P均＜0.05）。实验组和对照组治疗前后裸鼠体重增长值差异无统计学意义（P＞0.05）。实验组COX-2、CD44和MMP-7蛋白及mRNA表达明显低于对照组,差异有统计学意义（P均＜0.05）。结论尼美舒利可有效抑制人喉鳞癌细胞系Hep-2裸鼠移植瘤的生长,其机制可能与抑制COX-2、CD44及MMP-7表达有关。     

RNA干扰hTERT基因治疗喉鳞状细胞癌的实验研究

目的:探讨RNA干扰hTERT(人端粒酶逆转录酶)基因对人喉鳞状细胞癌的治疗作用.方法:根据hTERT cDNA序列构建表达hTERT mRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1、pshRNA2.将shRNA质粒分别转染人喉癌Hep-2细胞株及荷瘤裸鼠瘤体内.以激光共聚焦显微镜观察质粒在Hep-2细胞及瘤体内的表达;以MTT法观察质粒对Hep-2细胞增殖的抑制作用;以蛋白印迹法(Western blot法)检测Hep-2细胞中hTERT蛋白的表达;以免疫组化SP法检测裸鼠瘤体内hTERT蛋白的表达.结果:pshRNA1、pshRNA2转染Hep-2细胞及pshRNA1转染瘤体后,共聚焦显微镜下见大量的癌细胞表达绿色荧光,hTERT蛋白表达明显下降.细胞受pshRNA1、pshRNA2转染后,其生长活性受到明显抑制.体内抑瘤实验表明,与空质粒载体组(pshRNA4)和生理盐水组相比,pshRNA1组移植瘤生长明显受到抑制.结论:表达shRNA的、hTERT基因特异的真核表达质粒能有效转染体内、外喉鳞癌细胞,并可有效抑制人喉鳞癌细胞的生长,为今后应用RNA干扰基因治疗喉癌提供重要的实验参考.     

The involvement of CHD5 hypermethylation in laryngeal squamous cell carcinoma

Chromodomain helicase DNA-binding protein 5 (CHD5) has been found to be a candidate tumor suppressor gene (TSG) in malignant neural tumors. In mice heterozygous for chd5 deficiency, the first tumor observed was pathological squamous cell carcinoma. More than 95% of primary laryngeal cancer is squamous cell carcinoma. Thus, we explored the expression of CHD5 in 65 patients with laryngeal squamous cell carcinoma (LSCC) using real-time PCR, immunohistochemistry and Western blotting. DNA methylation was detected using bisulfate-specific sequencing. The potential function of CHD5 was determined using MTT, apoptosis and transwell migration assays in CHD5-transfected Hep-2 cells. Our results revealed that the mRNA and protein expression levels of CHD5 in LSCC tissues were significantly lower than those in clear surgical margin tissues (p<0.05), and there is a significant correlation between the mRNA and protein expression levels of CHD5 (p<0.01). In addition, there were significant differences in CHD5 mRNA and protein levels with respect to the patient's clinical stage (p<0.05). Aberrant methylation of the CHD5 promoter was frequently found in the Hep-2 cell line and LSCC tumor tissues, especially tumor tissues from advanced TNM (p<0.05) or older patients (p<0.05). Finally, ectopic expression of CHD5 in laryngeal cancer cells led to significant inhibition of growth and invasiveness. Our data suggest that CHD5 is a tumor suppressor gene that is epigenetically downregulated in LSCC.