Clinical Significance of hTERC Gene Amplification Detection by FISH in the Screening of Cervical Lesions

This study evaluated the clinical significance of hTERC gene amplification detection by fluorescence in sire hybridization （FISH） in the screening of cervical lesions. Cervical specimens of 50 high risk patients were detected by thin liquid-based cytology. The patients whose cytological resuits were classified as ASCUS or above were subjected to the subsequent colposcopic biopsies. Slides prepared from these 50 cervical specimens were analyzed for hTERC gene amplification using interphase FISH with the two-color hTERC probe. The results of the cytological analysis and those of subsequent biopsies, when available, were compared with the FISH-detected hTERC abnormalities. It was found that the positive rates of hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 28.57%, 57.14%, 100%, and 100%, respectively. The positive rates ofhTERC gene amplification in HSIL and SCC groups were significantly higher than those in NILM, ASCUS and LSIL groups （all P〈0.05）. The mean percentages of cells with hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 10.50%, 36.00%, 79.00%, and 96.50%, respectively. Patients with HSIL or SCC cytological diagnoses had significantly higher mean percent- ages of cells with hTERC gene amplification than did patients with NILM, ASCUS or LSIL cytological diagnoses （all P〈0.05）. It was concluded that two-color interphase FISH could detect hTERC gene amplification to accurately distinguish HS1L and ISIL of cervical cells. It may be an adjunct to cytology screening, especially high-risk patients.

INCREASED EXPRESSION OF PDGF AND C-MYC GENES IN LUNGS AND PULMONARY ARTERIES OF PULMONARY HYPERTENSIVE RATS INDUCED BY HYPOXIA

The role of growth factors and proto-oncogene in pulmonary vascular structural remodelling is not well known.The present study examined gene expression of platelet-derived growth factor(PDGF)-A and -B chain and proto-oncogene,c-myc,in lung tissue and pulmonary artery of rats exposed to hypoxia and compared to those levels of gene expression in normal rats.Normal lungs and pulmonary artery expressed PDGF-A chain transcript of 1.7 kb and PDGF-B chain transcript of 3.5 Kb.The c-myc transcript of 2.2 kb was expressed as well. After hypoxic exposure for 7 and 14 days mRNA levels of PDGF-B chain and cmyc were elevated significantly compared with those of control rats.PDGF-A chain mRNA increased after hypoxia for 7 days,and then declined.These results suggest that activation of autocrine and/or paracrine is important in proliferation mechanism of pulmonary artery smooth muscle cells in hypoxic pulmonary hypertensive rats.

INDUCTION OF C-MYC GENE AMPLIFICATION BY HYDROXYUREA AND ITS INHIBITION BY HOMOHARRINGTONINE

Induction of c-myc gene amplification in L1210 cells by hydroxyurea and its inhibition by homohar-ringtonine were investigated using the DNA-DNA molecular hybridization technique. When the cells were treated with hydroxyurea 1.0 mM for 16 hours, and incubated a further 16 hours in a drug-free medium, the c-myc gene amplified 23.5-fold. If homohar-ringtonine 50 μM was used at the same time as hydroxyurea, gene amplification did not occur. Cycloheximide, an inhibitor of protein biosynthesis, produced a similar effect. Our results indicated that a (or some) protein factor(s) might be involved in gene amplification. Detailed analysis showed that the synthesis of this protein factor(s) started 4 hours before the initiation of the S phase but did not continue in the S phase. It was also found that this protein factor(s) was very labile and began to degrade 2 hours after its appearance.

Gene therapy with antisense c-myc adenovirus for human gastric carcino-ma cell line in vitro and for implanted carcinoma in nude mice

Objective:To study the effects of recombinant antisense c-myc adenovirus (rAS-c-myc-Ad) on SGG 7901 human gastric carcinoma cell line in for and in nude mice. Methods:The effects of rAS-c-myc-Ad and LacZ-Ad on SGG 7901 gastric carcinoma cells were observed with X-galstaining, MTT, DNA gradient degradation test, TUNEL, flow cytometry, PCR and western blot. The therapeutic effects of rAS-c-myc-Ad on the implanted ax 7901 cells in nude mice were also ob served.Results: rAS-c-myc-Ad significantly inhibited the growth of SGG 7901 cells and induced their apoptosis. After the treatment of rAS-c-myc-Ad, the prolifetion rate of the cells was decreased by 44’ l% in de and SGC 7901 cells failed to form caxcinoma ther they were implanted into nude mice. Injection of rAS-c-myc-Ad into the carcinoma subcutaneously implanted to the nude mice significantly inhibited the growth of the implanted carcinoma with an inhibition rate of 68. 9%. Conclusion: rAS-c- myc- Ad significantly inhibits the growth of SGG 7901 human gastric carcinoma cells in vitro and in nude

THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

Deregulated c-myc expression in quiescent CHO cellsinduces target gene transcription and subsequent apoptotic phenotype

Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synthetic steroid, 4-hydroxy-tamoxifen (OHT),binds HBD. Activated c-MycER, likely c-Myc, can inducequiescent CHO cells reentry into S phase and subsequentcell death under serum-free condition. In addition, theexpression of some proposed c-myc target genes such asODC, MrDb, cad, rcc1 and rc1 were found to increase uponOHT induction before S, phase entry and apoptosis, indicating that these target genes are involved in cell cycleregulation and/or apoptosis control. However, the mutantD106-143c-MycER protein does not have above activities.

Cervical cancer screening: hTERC gene amplification detection by FISH in comparison with conventional methods

Aim: To assess the clinical significance of hTERC amplification for cervical cancer screening detected by fluorescence in situ hybridization (FISH) and compare it with that of current screening methods within the same group. Methods: A total of one hundred and nine women were recruited in this study. All of them had liquid-based thin-prep cytologic test (TCT), human papillomavirus (HPV) DNA testing and hTERC gene amplification analysis using interphase two-color FISH. In addition, colposcopically directed biopsy and/or cone biopsy were conducted for definite histopathologic diagnosis for each case. The optimal threashold of hTERC gene amplification by fluorescence in situ hybridization (FISH) were assecced by receiver operating characteristic (ROC) curve. The results of hTERC gene amplification analysis were compared with the cytological analysis, HPV DNA testing and those of subsequent biopsies. Results: Among the 109 patients, 18 were benign lesion, 17 were LSIL, 66 were HSIL and 8 were invasive carcinoma of cervix (ICC). Of them, hTERC-positive cases were found in 0.0% (0/18) of normal specimens, 11.8% (2/17) of LSIL, 72.7% (48/66) of HSIL and 100.0% (8/8) of ICC, respectively. The positive rate of hTERC gene amplification was significantly higher in HSIL and ICC compared with normal and LSIL (all P < 0.01).The optimal cut-off point of percentages of cells with hTERC amplification was determined as 5.5%. Using this threshold the hTERC test reached a much higher specificity(94.3%, 33/35) and a relatively lower sensitivity(77.0%, 57/74) to distinguish benign lesion and LSIL from HSIL and ICC in comparison with HR-HPV test (51.4%;91.9%) and TCT (74.3%;81.1%). Area Under the Curve revealed that hTERC amplification test performed more accurately (area under the curve = 0.857) compared to HPV test (area under the curve = 0.717) and cytology(area under the curve = 0.777) to discriminate HSIL or higher from LSIL or lower. This study also found a significant positive correlation between positive hTERC gain and HR-HPV infection, abnormal cytological or histopathologic lesions (all P < 0.01) in patients with cervical diseases. Conclusion: hTERC amplification testing may be a promising adjunct to screen women for cervical precancer or cancer with high specificity and accuracy.

Promoter hypermethylation of tissue specific tumor supressor genes and point mutation in K-ras, c-myc proto-oncogenes in urinary (transitional cell) bladder carcinoma

In a total of 83 UN specimens were investigated for proto-oncogene mutations, tumor supressor genes promoter methylation status and c-myc and Ki-67 expression. Point mutations in c-myc were detected in cases with high grade and proliferation index. Mutated K-ras proto-onco- gene profiles were detected in 17 (21%) tumoral spiecemens that examined. Tumor specimens were also showed hypermethylated promoter domain for the SFRP2, MGMT tumor supressor genes. These findings showed the combine effect of mutated c-myc and K-ras oncogene and epigenetic inactivation of tissue specific tumor supressor genes (TS) play a crucial role in tumor progression and recurrence in UN carcinogenesis.

EXPRESSION OF c－myc GENE AND BIOSYNTHESIS OF BIOLOGICAL MACROMOLECULES IN ANTISENSE TRANSFECTANT HL_（60）~R－9

The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL-9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of 3H-TdR, 3H-UR and 3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant Phenotypes of HL-9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.

EFFECT OF HYPOXIA ON DNA SYNTHESIS AND C－MYC GENE EXPRESSION OF PULMONARY ARTERY SMOOTH MUSCLE CELLS

The neonate is particularly susceptible to the development of hypoxic pulmonary hypertension. The present study was undertaken to observe the effect of hypoxia on DNA synthesis and c-myc gene expression between newborn calf and adult bovine PASMC in vilro. DNA synthesis measured by 3H- TdR incorporation was increased after hypoxic challenge for 24h. Hypoxia enhanced the increment in 3H-TdR incorporation induced by EGF. Northern blot analysis revealed that PASMC cultured in both normoxia and hypoxia expressed c- myc gene transcript of 2. 2Kb ,but there is a higher 2. 2Kb mRNA expression in hypoxic PASMC than that in normoxia. We speculate that newborn calf PASMC exhibited potential response to hypoxia than adult,which was augmented by EGF. Enhanced c-myc gene expression may lead to a great understanding of the mechanism of PASMC growth in the development of pulmonary hypertension.

Detection of Tumor Suppressor Gene and Oncogene in SO-Rb_(50) Human Retinoblastoma Cell Line

Retinoblastoma (Rb) is the most common malignant'cancer of eye.So-Rb_(50) is the first Rb cell line established in China in 1988.It has passed to the 387th passage now.We collected cells of the 327th passage of SO-Rb_(50),purified its genomic DNA and detected it with Rb and c-myc cDNA probes respectively(normal human white blood cells DNA was the control).We found the Rb gene was deleted while c-myc gene was amplified three times.This provides a basis for further study of the regulation of tumor development and tumor reversal with this cell line in vitro.Eye Science 1993;9:34-37.

应用荧光原位杂交法检测宫颈脱落细胞中hTERC基因的扩增

目的:研究荧光原位杂交技术(FISH)在检测宫颈脱落细胞hTERC基因扩增中的应用,探讨其用于辅助诊断宫颈上皮内瘤变(CIN)的可行性。方法:分别对80例CIN妇女和20例正常妇女的宫颈脱落细胞标本,应用FISH技术(TERC/CSP3DNA探针)检测人类端粒酶(TERC)基因,分析CIN中hTERC基因的扩增状况;并将FISH检测结果与人乳头瘤病毒(HPV)16/18型DNA PCR检测结果比较,观察其检测效果。结果:CIN1级组、CIN2级组、CIN3级组和正常对照组发生hTERC基因扩增的细胞所占百分比分别为(7.41±1.96)%、(10.46±1.58)%、(16.85±2.09)%和(4.10±1.71)%,差别有统计学意义(P<0.05);FISH技术和HPV16/18型DNA PCR检测CIN的敏感性、特异性、约登指数及粗一致性分别为66.3%、100%、0.663、73%和50.0%、85%、0.350、57%。结论:FISH技术检测有助于探索hTERC基因扩增,CIN组中,hTERC基因的拷贝数明显增加,且hTERC基因扩增随细胞学病变的严重程度加重而增加;FISH技术可用于CIN的辅助诊断,且其检测效果优于HPV检测。

宫颈病变中hTERC基因扩增与hTERT蛋白表达的关系及意义

目的探讨宫颈病变中hTERC基因扩增与hTERT蛋白表达的关系及临床意义。方法通过FISH技术检测宫颈石蜡标本中hTERC基因扩增,免疫组化SuperVisionTM二步法检测hTERT蛋白表达,并进行统计学分析。结果 hTERC基因扩增阳性率随宫颈病变恶性程度增高而增加,呈正相关关系(r=0.814,P<0.05)。hTERT蛋白阳性率亦随宫颈病变恶性程度增高而升高,呈正相关关系(r=0.554,P<0.05)。hTERT蛋白表达与hTERC基因扩增呈正相关关系(r=0.578,P<0.05)。在CIN II/III与宫颈癌中,hTERC基因扩增阳性率高于hTERT蛋白,但在CIN I组中却较低。结论 hTERC基因扩增和hTERT蛋白表达与宫颈癌发生密切相关。FISH检测hTERC基因扩增结果相对客观,但对低级别宫颈癌前病变的敏感性不如免疫组化hTERT蛋白的检测。因此,对于难以判断CIN级别的病例若能FISH联合免疫组化技术,同时检测hTERC基因与hTERT蛋白有利于宫颈癌前病变的早期筛查,提示低级别癌前病变要加强随访。

FISH检测宫颈脱落细胞中hTERC基因的意义

目的探讨在宫颈脱落细胞中检测不同级别宫颈病变人端粒酶基因(hTERC)的扩增情况及其临床意义。方法用荧光原位杂交技术(FISH)检测22例正常宫颈脱落细胞、38例低度鳞状上皮内病变(LSIL)、43例高度鳞状上皮内病变(HSIL)及36例宫颈鳞状细胞癌脱落细胞中hTERC的表达情况。结果 hTERC基因在正常宫颈脱落细胞、LSIL、HSIL及宫颈癌中的阳性表达率分别为4.55%、23.68%、86.05%和91.67%。各组hTERC基因阳性率与正常对照组相比均差异显著(P<0.05);高度鳞状上皮内病变及宫颈癌组与低度鳞状上皮内病变组相比差异极显著(P<0.01)。结论 hTERC基因高表达在宫颈癌发生、发展中可能起重要作用,经细胞学检测hTERC基因有望作为宫颈癌筛查及宫颈病变预后预测的辅助指标。

FISH技术检测宫颈hTERC基因的表达及其临床意义

目的探讨荧光原位杂交技术检测宫颈上皮细胞hTERC基因的表达及其临床意义。方法应用荧光原位杂交技术(FISH)检测138例宫颈脱落细胞中hTERC基因的表达情况。结果118例各级宫颈病变中,CIN176例,CIN214例,CIN3/原位癌16例,宫颈浸润癌12例,用FISH检测hTERC基因的表达其阳性率分别是13%、64%、81%、100%,子宫颈癌hTERC基因的表达较宫颈上皮内瘤变各组差异有统计学意义(P<0.05)。结论hTERC基因在各级宫颈病变均有一定的表达。hTERC基因的阳性表达率随着宫颈病变分级呈逐渐上升趋势。且hTERC基因在高度病变中的阳性表达率明显高于低度病变。

人乳头瘤病毒感染的宫颈上皮内瘤变中的hTERC基因分析

目的探讨人乳头瘤病毒感染的宫颈上皮内瘤变细胞中3号染色体hTERC基因扩增情况。方法对57例病理诊断为CIN(CIN18例,CIN215例,CIN334例;其中包括HPV感染46例与无HPV感染11例)患者的液基细胞学检测剩余样本应用荧光原位杂交技术检测hTERC基因,同时以20例无HPV感染正常宫颈鳞状上皮作为对照,将结果与病理诊断进行比较。结果在正常宫颈鳞状上皮中,3号染色体hTERC基因主要表现为2个杂交荧光信号,宫颈病变与HPV感染的宫颈鳞状上皮中,出现了hTERC基因拷贝数增多,在CIN1～CIN3中,HPV感染率分别为12.5%、86.7%、94.1%,hTERC基因扩增的阳性率分别为25.0%、60.0%、82.3%,46例HPV感染的CIN中,hTERC基因扩增的阳性率为80.4%,而11例HPV阴性的CIN患者中,其扩增阳性率为18.2%,两组间比较,差异具有统计学意义(P<0.05)。结论3号染色体hTERC基因的扩增与HPV感染及宫颈病变程度相关,hTERC基因的扩增可能是HPV感染致端粒酶活性增加的早期事件。

荧光原位杂交技术检测宫颈癌及癌前病变组织中hTERC基因的扩增及临床意义

目的检测子宫颈病变组织中人端粒酶RNA基因(hTERC)的异常扩增情况,探讨其临床病理学意义。方法收集不同宫颈病变组织195例,包括慢性宫颈炎33例,CINⅠ级34例,CINⅡ-Ⅲ级37例,宫颈鳞癌30例,宫颈腺癌61例。应用双色荧光原位杂交(FISH)技术检测宫颈病变组织中hTERC基因的异常扩增情况,并分析其与临床病理学参数的关系。结果 hTERC基因扩增的阳性率分别为:慢性宫颈炎3.03%,CINⅠ级29.41%,CINⅡ-Ⅲ级72.97%,宫颈鳞癌100%,宫颈腺癌91.8%。宫颈鳞癌及腺癌hTERC基因扩增率明显高于CINⅠ级、CINⅡ-Ⅲ级(P<0.05),CINⅡ-Ⅲ级hTERC扩增率显著高于CINⅠ级(P<0.05),但宫颈鳞癌与腺癌hTERC基因扩增率比较差异无统计学意义(P>0.05)。结论应用FISH技术检测hTERC基因的异常扩增可作为组织学诊断困难病变的确诊、病变预测及治疗后风险评估手段。

hTERC基因表达在宫颈癌筛查中的意义

目的:探讨宫颈病变脱落细胞中人类染色体端粒酶基因(hTERC)的表达及hTERC基因检测在宫颈癌筛查中的临床意义。方法:收集宫颈病变患者的宫颈脱落细胞标本129例,依据组织病理学结果分为3组:宫颈上皮内瘤变低度病变组(CINⅠ)、高度病变组(CINⅡ/Ⅲ)、宫颈癌组(SCC);同时选择健康妇女的宫颈脱落细胞标本20例作为对照组建立实验室阈值;应用荧光原位杂交技术(FISH)检测两组标本hTERC基因的扩增情况,最终以病理学诊断为金标准,比较FISH检测结果。结果:研究各组中CINⅠ,CINⅡ/Ⅲ,SCC的hTERC基因阳性表达率分别为7.31%,56.25%,75.00%,hTERC基因扩增比例随病变进展而增加;研究组与对照组hTERC基因阳性表达率差异有统计学意义(P<0.05);研究各组两两比较,hTERC基因阳性表达率低度病变组与高度病变组、宫颈癌组差异有统计学意义(P<0.05),而高度病变组与宫颈癌组的差异无统计学意义(P>0.05)。结论:FISH方法检测hTERC基因可用于筛查宫颈高度病变及宫颈癌,其阳性扩增信号多倍体型可能是宫颈病变进展的预测性指标。

hTERC基因在不同级别宫颈病变中的表达与意义

目的探讨hTERC基因扩增在不同级别宫颈病变中的表达及其临床意义。方法应用荧光原位杂交技术(FISH)检测150例宫颈脱落细胞中hTERC基因的表达情况,其中106例为高危型HPV阳性,44例为阴性。结果 hTERC基因扩增在组织学正常或炎症、CINⅠ、CINⅡ、CINⅢ及浸润性宫颈癌中的表达率分别为5.0%、22.2%、57.1%、81.3%、96.3%,各组间hTERC基因异常扩增率差异均有统计学意义(P<0.05);HPV阳性患者中hTERC基因扩增率为72.6%(77/106),阴性患者中hTERC基因扩增率为9.1%(4/44);hTERC基因扩增的患者中,HPV阳性所占的比例为95.1%(77/81),阴性所占比例仅为4.9%(4/81),两者比较差异有统计学意义(P<0.05)。结论高危型HPV感染可能是hTERC基因异常扩增的早期事件。

hTERC基因检测对筛查高危子宫颈病变预防宫颈癌的临床意义

目的探讨应用荧光原位杂交(FISH)技术检测人染色体端粒酶RNA组分(hTERC基因)异常扩增在子宫颈病变中的临床意义。方法收集经组织病理学确诊的子宫颈病变组织245例,包括单纯性子宫颈炎75例、CINⅠ64例、CINⅡ63例和CINⅢ43例,另取20例健康宫颈组织作对照。用FISH方法检测265例宫颈活检组织的hTERC基因表达,用表面等离子体谐振核酸检测法对其中196例进行HPV-DNA检测。结果正常宫颈组织hTERC基因的异常扩增检出率为0,宫颈炎、CINⅠ、CINⅡ和CINⅢ组织分别为2.7%(2/75)、9.4%(6/64)、61.9%(39/63)和62.8%(27/43)。正常宫颈组织HPV阳性率为10.0%(1/10),宫颈炎、CINⅠ、CINⅡ组织分别为10.8%(7/65)、25.0%(11/44)和29.8%(14/47),而CINⅢ组织则高达63.3%(19/30),差异有统计学意义(P<0.05)。同时有hTERC基因异常扩增和HPV阳性的检出率在CINⅢ组织中为50.0%,显著高于CINⅡ的27.7%和CINⅠ的2.3%(P<0.05),未见于正常宫颈和宫颈炎;hTERC基因异常扩增的检出率在HPV阳性人群为55.8%(29/52),显著高于HPV阴性人群的18.1%(26/144),差异有统计学意义(P<0.05)。对宫颈病变高危性的诊断,hTERC基因异常扩增检测的敏感性为63.6%,优于HPV检测的39.0%(P<0.05)。71例CINⅡ、CINⅢ高危患者行宫颈锥切治疗,取切缘正常组织检测,hTERC基因呈阴性,术后经4个月～2年随访追踪,病灶修复部位未见有hTERC基因转为阳性。结论 hTERC基因检测有助于筛查宫颈高危病变和选择治疗方案,对预防宫颈癌有益;同时进行hTERC基因联合HPV检测将更有意义。