间隙连接基因Cx43表达对肺癌细胞体内成瘤生长的抑制

目的探讨间隙连接基因表达和细胞通讯功能对肿瘤生长的抑制作用。方法以高转移性人肺癌ＰＧ细胞为材料,该细胞的间隙连接基因Ｃｘ４３表达抑制,细胞通讯功能缺陷。用Ｃｘ４３ｃＤＮＡ转染ＰＧ细胞,分离转染子克隆,与只转染空载体ｃＤＮＡ的对照组ＰＧ进行比较。用Ｎｏｒｔｈｅｒｎ分子杂交和染料传输方法检查间隙连接表达情况,并观察细胞在体外和裸鼠体内生长。结果空载体对照组与未转染组ＰＧ相似,Ｃｘ４３ｍＲＮＡ无表达,通讯功能缺陷,细胞生长快,在软琼脂内集落形成率高（１１．６％）,植入裸鼠体内２８天,平均瘤重３．４７ｇ。转染组细胞Ｃｘ４３ｍＲＮＡ表达升高,通讯功能增强,细胞生长慢,在软琼脂内集落形成率和在裸鼠体内生长速度明显低于对照组,抑制率分别为９０％和７５％。结论间隙连接基因Ｃｘ４３表达对肺癌细胞有抑瘤作用。     

Suppression of tumorigenicity of a human lung carcinoma line by nontumorigenic bronchial epithelial cells in somatic cell hybrids

Hybrid cell lines between HuT292-DM, a human lung carcinoma line resistant to 6-thioguanine and ouabain, and either normal human bronchial epithelial cells (NHBE) or an SV40 "immortalized" but nontumorigenic derivative thereof (BEAS-2B), have been isolated by double selection. Hybrids of NHBE and HuT292-DM cells senesced after 40-43 population doublings in culture. In contrast, hybrids of BEAS-2B and HuT292-DM showed no sign of a culture "crisis" and have an indefinite life span. HuT292-DM cells produced tumors in 100% of athymic nude mice with a mean latency of 27 days, whereas tumorigenicity was totally suppressed in 76% of the BEAS-2B x HuT292-DM hybrids, with a 2- to 3-fold increased tumor latency in the remaining 24% of these hybrids. While the hybrids are hypotriploid to hypotetraploid, the parental lines are hypodiploid. The growth of HuT292-DM cells is stimulated, whereas NHBE and BEAS-2B cells are inhibited by serum. The growth response of the BEAS-2B x HuT292-DM hybrids to serum is similar to that of HuT292-DM cells. Thus, tumorigenicity and culture longevity are dominantly controlled by the nontumorigenic parent (NHBE or BEAS-2B). On the other hand, serum responsiveness is more similar to that of the tumorigenic parent (HuT292-DM).     

Silencing of connexin 43 suppresses invasion, migration and lung metastasis of rat hepatocellular carcinoma cells

To reduce cancer mortality, understanding of mechanisms of cancer metastasis is crucial. We have established six rat hepatocellular carcinoma (HCC) cell lines, which exhibit differing metastatic potential to the lung after inoculation into the tail veins of nude mice. In the present experiment, we investigated the process of cell attachment to metastatic sites and possible regulating factors. One hour after inoculation, two of two HCC cell lines with high metastatic potential and one of two HCC cell lines with low metastatic potential exhibited many attached cells in the lung. One day after inoculation, lung metastatic foci were observed only with highly-metastatic cells with elevated connexin 43 (Cx43) expression as assessed by cDNA array analysis. Furthermore, 24 or 48 h after transfection of an siRNA targeting Cx43, in vitro invasion and migration were suppressed by 68% (P < 0.001) and 36% (P < 0.05) compared with control-siRNA transfected cells, despite no differences in cellular morphology, cell proliferation or apoptotic activity. Moreover, the number of metastatic nodules per lung area in nude mice was significantly (P < 0.01) reduced. In conclusion, suppression of Cx43 expression in tumor cells reduced in vitro migration and invasion capacity and in vivo metastatic ability so that Cx43 has potential as a molecular target for prevention of cancer metastasis with Cx43 overexpressing tumors.     

Connexin 43 transfection in basaloid squamous cell carcinoma cells

To investigate the relationship between the expression of connexin in basaloid squamous cell carcinomas (BSCC) and their rapid proliferation and invasive potential, we examined the effect of overexpression of connexin 43 (Cx43) in a BSCC-derived cell line (BSC-OF). BSC-OF was transfected with Cx43 to obtain 15 clones with a stable expression of Cx43. In these cells, although Cx43 was distributed throughout the cytoplasm, it did not form connexon plaque. In almost all of the clones, cell proliferation was clearly suppressed. Furthermore, we investigated cell migration and invasion in three clones that showed a remarkable down-regulation in cell growth, and found that Cx43 transfection showed no significant effect on either. These results suggest that Cx43 plays a role as a tumor suppressor in the cytoplasm of Cx43-transfected BSC-OF cells. However, no definite correlation was found between Cx43 and cell migration and invasion.     

间隙连接蛋白Cx43、Cx45在鼻咽癌组织中的表达

背景与目的：间隙连接在细胞间的营养物质,离子和细胞调节因子的交换中起重要作用。间隙连接异常,细胞间的物质交换障碍,往往导致细胞分裂失控,在有些癌组织和癌细胞中存在间隙连接的功能异常。恢复这些癌细胞的间隙连接功能,它们表现出正常细胞的生物学表型,因此,探讨细胞间隙连接蛋白在鼻咽癌中的表达,将有可能为临床诊断提供方法。为鼻咽癌的发病机理提供新思路。方法;采用免疫组化技术检测间障连接蛋白Cx43,Cx45在鼻咽组织中的表达。结果：（1）Cx43,Cx45在鼻咽慢性炎症组织和鼻咽癌组织中有表达差异,在鼻咽癌中,Cx43,Cx45阳性率为44．8％,46．6％,与鼻咽慢性炎症柱状上皮细胞阳性率为85％和100％比较,显著性下降（P＜0．01）,（2）鼻咽慢性炎症组织比鼻咽癌组织含鳞状细胞的百分率低（P＜0．01）,分别为29．7％和56．9％,（3）Cx43,Cx45在鼻咽癌旁柱状上皮细胞中的表达低于癌旁鳞状上皮细胞（P＜0．001）,高于癌细胞（P＜0．01）,结论：Cx43,Cx45在鼻咽组织中的表达异常,可能与鼻咽组织鳞化和癌变有关。     

Influence of transfection with connexin 26 gene on malignant potential of human hepatoma cells

We investigated the effect of transfection with connexin (Cx) 26 gene on the malignant potential of PLC/PRF/5 hepatoma cells, observing changes in their morphological features, alpha-fetoprotein (AFP) expression, cell proliferation and apoptosis in vitro, and their tumor growth in vivo. Fluorescence-activated cell sorting (FACS) analysis showed that 10.6% of PLC/PRF/5 hepatoma cells transfected with Cx26 cDNA expressed excessive Cx26, and the spread of lucifer yellow was wider in the colony of stable transfectants (PLC/Cx26) after its microinjection than in control. Nucleo-cytoplasmic (N/C) ratio was significantly lower in PLC/Cx26 (P < 0.0001). Cell proliferation assay showed significantly lower numbers in PLC/Cx26 on day 10 after seeding than in control (P = 0.0039), and AFP level /10(5) cells was significantly lower in medium of PLC/Cx26 (P = 0.0039). The number of proliferating cell nuclear antigen (PCNA)-positive cells was less in PLC/Cx26 in vitro than in control (P = 0.0039), and single-stranded DNA (ssDNA)-positive cells were more abundant in the colony of PLC/Cx26 (P = 0.029). Tumor volume in SCID mice was significantly smaller in the group of PLC/Cx26 than in the control (P < 0.01) throughout the observation period, and tumor weight of PLC/Cx26 was significantly lower (P = 0.0019) week 9 after inoculation. Transfection with Cx26 cDNA inhibited dedifferentiation, suppressed cell proliferation, and apoptosis was induced. Tumor growth of PLC/Cx26 was retarded. These findings suggest that transfection with Cx26 gene into human hepatoma cells reduces their malignant potential.     

Downregulation of connexin 43 in nasopharyngeal carcinoma cells is related to promoter methylation

Down-regulation of Cx43 expression had been shown to occur in nasopharyngeal carcinoma cells. The present study was undertaken to estimate if methylation of the promoter region in Cx43 gene was responsible for the repression of Cx43 expression in the CNE-1 nasopharyngeal carcinoma cells. Calcein transfer and lucifer yellow transfer were detected to evaluate gap junction intercellular communication (GJIC) in CNE-1 cells. It was found that the control CNE-1 cells showed no fluorescent dye transfer. After treatment with DNA methyltransferase inhibitor 5-aza-CdR, fluorescent dye transfer between cells became obvious. RT-PCR and Western blot were performed to determine the expression of Cx43 gene. The control CNE-1 cells showed a low expression level of Cx43, whereas 5-aza-CdR-treated CNE-1 cells showed an enhanced level of Cx43 expression. Methylation-sensitive restriction enzyme and PCR analysis showed that the methylation of the Cx43 gene promoter region occurred in CNE-1 cells. In addition, treatment with 5-aza-CdR inhibited the growth (including anchorage-independent growth) of CNE-1 cells, and resulted in an accumulation of cells in G0/G1 phase. These results indicate the promoter methylation as an important role in inactivation of Cx43 in CNE-1 cells.     

Effect of v-rasH oncogene transfection on estrogen-independent tumorigenicity of estrogen-dependent human breast cancer cells

Spontaneous or therapeutically induced progression of hormone-dependent human breast cancer to a form not amenable to endocrine treatment has been frequently recorded in clinical settings. In an experimental model system, we have changed the estrogen-dependent tumorigenicity of a human breast cancer cell line, MCF-7, to an independent state by stably introducing a model oncogene, v-rasH, into this cell line by means of DNA transfection. We now show that the oncogene-transfected hormone-independent MCF-7 cells may secrete diffusible tumorigenic factors that not only support their own tumor growth in vivo, but are also humorally active in partially triggering the tumor growth of wild type previously nontumorigenic MCF-7 cells, even when the wild type cells are implanted at a distant anatomical site in the same animal. Estrogen-independent tumor formation by MCF-7 cells was also induced in 50% of animals given injection by continuous administration of conditioned media from MCF-7-ras cells. However, the wild type tumors had limited tumor growth. Tumors were verified as adenocarcinomas and by Southern blotting were shown to be derived from the cells injected. In an in vitro coculture assay, a 5- to 7-fold enhancement in anchorage-independent growth of MCF-7 cells was observed in the presence of MCF-7-ras feeder cell layer. These data suggest that v-rasH-induced estrogen-independent tumorigenicity of human breast cancer cells occurs by secretion of mitogens which may function in an endocrine manner.     

Fusion of human umbilical cord mesenchymal stem cells with esophageal carcinoma cells inhibits the tumorigenicity of esophageal carcinoma cells

Prior studies on the biology and therapeutic application of human stem cells in human malignancies have reported mixed results. Some evidence shows the use of stem cell transplantation is an important tool in the treatment of several hematologic and non-hematologic malignancies while some others suggest both human stem cells and mature stromal cells can contribute to the development and growth of human malignancies. Aiming to provide more evidence on this controversial issue, we investigated the effect of cell fusion of mesenchymal stem cells with esophageal carcinoma cells on tumorigenesis. Results suggest that artificial fusion of human umbilical cord mesenchymal stem cells with esophageal carcinoma cells resulted in hybrids with declined cell growth, increased apoptosis and suppressed tumorigenicity. The comparison of gene expression profiles of human mesenchymal stem cells, esophageal carcinoma cells and hybrids indicated that fusion induced activation of apoptosis. Furthermore, the expression of DUSP6/MKP3 in MAPK pathway increased strikingly and the exogenous overexpression confirmed the growth suppression. Our results demonstrate fusion of human mesenchymal stem cells with esophageal carcinoma cells induced apoptosis and benign transdifferentiation rather than reprogramming to cancer stem cells.     

Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell-mediated chromosome transfer

To map tumor suppressor genes for lung adenocarcinomas, we introduced normal human chromosomes 3, 7, and 11 into the A549 tumor cell line by microcell-mediated chromosome transfer to test which chromosomes had the ability to suppress tumorigenicity. These human chromosomes, which contain the neomycin gene as a selectable marker, were transferred into A549 lung adenocarcinoma cells at frequencies of 0.3–1.8 × 10^?6. Two microcell hybrid clones with an introduced chromosome 3, two with an introduced chromosome 7, and six with an introduced chromosome 11 were isolated and examined for their growth properties and tumorigenicity in nude mice. Whereas parental A549 cells formed tumors with an average latency of 68 d, both microcell hybrids with an introduced chromosome 3 failed to form tumors for over 360 d. Similar tumorigenicity results were obtained when the clones were implanted into denuded tracheas, a more orthotopic transplantation site. The two clones with an introduced chromosome 7 were still tumorigenic; they formed tumors within 100–123 d after injection and grew progressively, although the tumors grew slightly slower than the parental cells did. Among the six clones with an introduced chromosome 11, one clone was still highly tumorigenic but did not contain an extra copy of an intact introduced chromosome 11. Three clones with a single intact copy of introduced chromosome 11 formed tumors with latency periods significantly longer than those of the parental cells. Two clones had two copies of the introduced chromosome 11, and both failed to form tumors within 1 yr of injection. These results indicate that chromosomes 3 and 11 can suppress the tumorigenicity of A549 lung adenocarcinoma cells.     

Restoration of TGF-beta signalling reduces tumorigenicity in human lung cancer cells

Members of the transforming growth factor-beta (TGF-beta) family regulate a wide range of biological processes including cell proliferation, migration, differentiation, apoptosis, and extracellular matrix deposition. Resistance to TGF-beta-mediated tumour suppressor function in human lung cancer may occur through the loss of type II receptor (TbetaRII) expression. In this study, we investigated the expression pattern of TbetaRII in human lung cancer tissues by RT-PCR and Western blot analyses. We observed downregulation of TbetaRII in 30 out of 46 NSCLC samples (65%) by semiquantitative RT-PCR. Western blot analyses with tumour lysates showed reduced expression of TbetaRII in 77% cases. We also determined the effect of TbetaRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-beta due to lack of TbetaRII expression. Stable expression of TbetaRII in these cells restored TGF-beta-mediated effects including Smad2/3 and Smad4 complex formation, TGF-beta-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis. Clones expressing TbetaRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice. Therefore, these results suggest that reestablishment of TGF-beta signalling in TbetaRII null cells by stable expression of TbetaRII can reverse malignant behaviour of cells and loss of TbetaRII expression may be involved in lung tumour progression.     

Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43).

Connexin 43 (Cx43), a structural component of gap junctions, is believed to function as a tumor suppressor gene. Previously, we showed that expression of Cx43 suppresses cell proliferation and tumorigenicity of human glioblastoma cells [Huang et al., Cancer Res 58:5089-5096, 1998] and enhances apoptosis in response to chemotherapeutic agents [Huang et al., Int J Cancer 92:130-138, 2001]. In the present study, we demonstrated that expression of Cx43 in human glioblastoma cells potentiated an apoptotic program under low-serum conditions. The Cx43-mediated effect was coupled with a decreased expression of the specific apoptosis-inhibitor bcl-2. Overexpression of bcl-2 in Cx43-transfected cells conferred resistance to apoptosis induced under low-serum conditions, suggesting that the Cx43-mediated apoptosis under low-serum conditions is regulated, in part, through the downregulation of bcl-2 expression. Furthermore, application of the phosphatidylinositol-3'-OH kinase inhibitor LY294002 specifically induced apoptosis in Cx43-transfected cells. Our results demonstrate a new role of Cx43 in the mediation of apoptosis under low serum conditions.     

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.     

Selective enhancement of metastatic capacity in mouse bladder carcinoma cells after transfection with DNA from liver metastases of human colon carcinoma

To identify sequences associated with a metastatic phenotype, DNA fragments isolated from 2 separate human colon carcinoma metastases were transfected into a mouse bladder carcinoma cell line together with the neoR gene as selectable marker. It was found that bulk populations of neomycin resistant cells carrying these human sequences caused more metastases in syngeneic mice than did control cells transfected with calf thymus DNA. Cells isolated from metastases retained the highly metastatic phenotype when transferred to secondary hosts.     

Suppression of anchorage-independent growth after gene transfection.

A novel procedure for isolating anchorage-dependent cells has been developed. It involves negative selection of cells growing in suspension followed by clonal replica screening for anchorage-dependent growth. Cells which have regained anchorage-dependent growth have been isolated from a library of the Chinese hamster ovary cell line, CHO-K1, transfected with pSV2neo and human genomic DNA. One anchorage-dependent clone, 1042AC, has been studied in detail. Anchorage-dependent growth of 1042AC is stable when cultured as adherent monolayers, but revertants appear rapidly when cultured in suspension. Suppression is unlikely to be due to loss or mutation of hamster genes conferring anchorage-independent growth as hybrids between 1042AC and CHO-K1 have the suppressed phenotype of 1042AC. Furthermore, a population of cells obtained from the hybrid by selecting for revertants to anchorage-independent growth showed selective loss of the transgenome derived from 1042AC. The growth suppression was not due to transfection of the human Krev-1 gene, which has previously been shown to restore anchorage-dependent growth, nor was there any evidence of alteration in the endogenous hamster Krev-1 gene. However, evidence for a human gene being responsible for the suppressed phenotype has not been obtained yet.     

HLCDG1基因转染对肺癌细胞生长的影响

背景与目的:HLCDGI基因是我们实验室最近克隆的新基因,它位于5q33,介于D5S436～D5S470之间,在肺癌组织中明显表达下调或缺失。本研究旨在观察HLCDGI基因是否具有抑制肺癌细胞生长的能力。方法:构建HLCDGI基因的重组质粒pcDNA3.1(+)/HLCDGI,通过脂质体转染,将HLCDGI基因cDNA导入肺癌细胞系A549中,经G418筛选,获得稳定表达的细胞克隆。RT-PCR检测HLCDGI基因表达,并采用细胞生长抑制实验、软琼脂集落形成实验及裸鼠致瘤性实验,分析HLCDGI基因转染细胞恶性表型。结果:RT-PCR结果表明转染HLCDGI 基因的细胞HLCDGI mRNA明显表达。转染HLCDGI基因组、转染空载体组和未转染组,3组细胞生长倍增时间分别为70.0 h、43.3 h和39.5 h,转染HLCDGI基因组与两对照组之间的差异有显著性(P<0.05)。计算软琼脂中细胞克隆形成率,结果分别为8.5％、29.0％和35.0％,转染HLCDGI基因的细胞克隆形成率明显降低。将转染HLCDGI基因的细胞、转染空载体的细胞和未转染的细胞分别注射入无胸腺的裸鼠体内,43天后处死动物,剥离出肿瘤组织并称重,各组瘤块平均重量分别为0.120g、0.612g和0.924g,转染HLCDGI基因的细胞成瘤能力与两对照组细胞比较,差异有显著性(P<0.05)。结论:HLCDGI在肺癌A549细胞中表达,具有抑制细胞增殖和抑制裸鼠移植瘤形     

Suppression of tumorigenicity in cervical carcinoma HeLa cells by an episomal form of adeno-associated virus.

To compare the oncosuppressive activity of integrated and episomal adeno-associated virus (AAV), two Epstein-Barr virus (EBV) derived episomal form vectors, p205 and p220, were used to generate plasmids containing episomal AAV. HeLa cells transfected with p205, p220, and the plasmids with forward (pA205-1, pA220-1) and reverse (pA205-1, pA220-2) orientation of inserted AAV DNA were designated H205, H220, HA205-1, HA220-1, HA205-1, and Ha220-2, respectively. The respective average copy numbers of pA205 and pA220 per cell are 4.9-6.9 and 4.3. The AAV-transfected HeLa cells displayed growth inhibition when compared to parental and the vector-transfected HeLa cells. Nude mice assay showed that the tumor size from HA205-1 and HA205-2 cells were 11.3 and 22.6%, and those from HA220-1 and HA220-2 cells were 54.8 and 57.3%, respectively, when compared to parental HeLa cells. HA205-1 cells containing forward AAV insert exerted more oncosuppressive effect than HA205-2 cells containing reverse AAV insert. Our data indicate that the episomal AAV can exert oncosuppressive activity as compared to the integrated AAV in HeLa cells.