细胞间隙连接通讯、连接蛋白与肿瘤的抑制

细胞间隙连接通讯(gap junction intercellular communication．GJIC)是多细胞生物体内普遍存在的一种通讯方式，在肿瘤的发生、发展和组织自身稳定的维持中具有重要作用。近年的研究显示，肿瘤和转化细胞中普遍存在GJIC的缺陷。本文通过对GJIC的生物学特性及GJIC、连接蛋白基因家族抑瘤作用的文献回顾．旨在探讨细胞间隙连接通讯、连接蛋白在肿瘤抑制中的重要作用。     

肿瘤细胞的间隙连接

间隙连接是细胞信号传导的重要途径，在细胞的协调生长步伐等方面的作用不容忽视。肿瘤细胞的形成和癌症的治疗都与其有关，特别是癌症基因治疗中的旁观者效应(bystander effect)与间隙连接胞间通讯(GJIC)密切相关。现综述近年来有关间隙连接在肿瘤细胞的形成和治疗等方面的研究进展。     

复方藤梨根制剂对荷人肺巨细胞癌裸鼠移植瘤生长及Cx43基因表达水平的影响

细胞间隙连接通讯（gap junction intercellular communication,GJIC）是细胞间的一种重要连接方式,在细胞分化、生长控制和维持体内环境平衡等方面起重要作用。近年来研究表明,肿瘤和转化细胞中普遍存在GJIC的缺陷,GJIC在肿瘤的发生及转移过程中有着极为重要的作用。间隙连接蛋白是由许多同源性基因编码的多基因家族,它们的表达和分布异常与肿瘤的形成关系密切。在最近的一些实验中,人们把野生型连接蛋白（connexin,Cx）基因转染到肿瘤细胞中,可见肿瘤的生长受到明显抑制,间隙连接通讯功能上调,细胞恢复正常生长,认为连接蛋白基因是一类肿瘤抑制基因家族。Cx43是一种主要的细胞间隙连接蛋白,在多种肿瘤细胞中都有Cx43表达的下降。为此,许多学者正在研究上调Cx43表达的药物,     

肿瘤细胞的间隙连接

间隙连接是细胞信号传导的重要途径,在细胞的协调生长步伐等方面的作用不容忽视.肿瘤细胞的形成和癌症的治疗都与其有关,特别是癌症基因治疗中的旁观者效应(bystander effect)与间隙连接胞间通讯(GJIC)密切相关.现综述近年来有关间隙连接在肿瘤细胞的形成和治疗等方面的研究进展.     

茶多酚对人膀胱癌BIU-87细胞间隙连接蛋白43表达和细胞间隙连接通讯功能的影响

背景与目的:研究茶多酚的主要植物化学物质成分(-)-epigallocatechin-3-gallate(EGCG)对人膀胱癌BIU-87细胞间隙连接蛋白43(Cx43)的表达和细胞问通讯功能的影响,探讨EGCG防治膀胱肿瘤的机制.材料与方法:分别采用四甲基偶氮唑蓝(MTT)法、膜联蛋白/碘化丙锭双标流式细胞术、RT-PCR、Western印迹和划痕标记荧光染料传输技术,体外观察不同浓度的EGCG(0、5、10、20 mg/L)对BIU-87细胞的生长抑制率、凋亡率、Cx43 mRNA及其蛋白的表达、细胞间间隙连接通讯功能(GJIC)的变化.结果:10 mg/L和20 mg/L的EGCG均能明显抑制BIU-87细胞的生长和诱导细胞凋亡,其抑制率分别为(15.67±1.15)%、(18.33±1.53)%,凋亡率分别为(42.00±4.34)%、(27.33±3.21)%.与对照组和5 mg/L EGCG相比,EGCG 10 mg/L和20 mg/L组能显著上调Cx43 mRNA及其蛋白的表达,并增强细胞间的GJIC功能(P＜0.05).EGCG在10～20 mg/L范围内对BIU-87细胞的生长的抑制作用、诱导细胞凋亡和上调Cx43表达的效应均随浓度的增加而增强,呈剂量依赖性(P＜0.05).结论:EGCG(10、20 mg/L)能通过有效上调BIU-87细胞Cx43转录水平的表达、增强细胞间隙连接所介导的GJIC功能,从而诱导膀胱肿瘤细胞凋亡,抑制其生长.     

间隙连接细胞通讯与肿瘤的抑制

间隙连接细胞通讯(gap junctional intercellular communication,GJIC)是多细胞生物体内普遍存在的一种通讯方式,在胚胎发育、生长控制、自身稳定的维持等方面发挥着重要作用.近来的研究显示,肿瘤组织中常伴有GJIC的异常,主要表现为肿瘤细胞间的同型GJIC减少,肿瘤细胞与周围正常细胞间的异型GJIC选择性丧失,从而使肿瘤细胞脱离机体的调控,导致肿瘤细胞无限增殖.而另一方面,共同培养、药物作用或连接蛋白(connexin,Cx)基因转染引起肿瘤细胞GJIC的恢复,又可作为抑瘤措施而发挥作用,逆转恶性表型,这将是一种温和的新型疗法,可能在今后的肿瘤治疗中发挥重要的辅助作用.     

藤梨根提取物对人食管癌Eca-109细胞生长和凋亡的调节

[目的]探讨藤梨根提取物对人食管癌Eca-109细胞生长和凋亡的调节作用。[方法]MTT比色法检测藤梨根正丁醇提取物及乙酸乙酯提取物在不同浓度及不同时间对Eca-109细胞生长的抑制作用：用TUNEL法检测藤梨根不同提取物对诱导肿瘤细胞凋亡效应。[结果]藤梨根正丁醇提取物及乙酸乙酯提取物对人食管癌Eca-109细胞的生长具有明显的抑制作用．并具有浓度依赖性和时间依赖性：其中正丁醇提取物对肿瘤细胞的抑制作用较乙酸乙酯提取物更为明显。两种提取物均能明显地诱导肿瘤细胞的凋亡。[结论]藤梨根提取物有较好的抗肿瘤作用．抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡可能是藤梨根抗肿瘤作用的机制之一。     

全反式维甲酸与单纯疱疹病毒胸苷激酶基因对胶质瘤的协同杀伤作用

背景与目的：细胞间缝隙连接通讯（gap junctional intercellular communication,GJIC)是介导单纯疱疹病毒胸苷激酶（herpes simplx virus thymidine kinase,HSV-tk)基因治疗中旁观者效应的重要机制。全反式维甲酸(all-trans-retinotic acid,ATRA)或能通过上调胶质细胞GJIC和抑制肿瘤细胞生长的双重作用,促进HSV-tk基因治疗的疗效。本研究的目的在于证实HSV-tk和ATRA联合应用对胶质瘤细胞的协同杀伤作用。方法：分别以1μmol/L、10μmol/L、100μmol/L3种浓度的ARTA作用于培养的大鼠C6胶质瘤细胞,并研究ATRA对C6胶质瘤细胞分化、增殖、GJIC及connexin43(Cx43)基因转录等方面的影响。将C6细胞与稳定表达HSV-tk基因的C6tk细胞按不同比例混合,分别在含GCV和不同浓度ATRA或仅含GCV的培养基中培养,并于药物作用7天后用MTT法检测旁观者效应。结果：经3种浓度的ARTA作用后,C6胶质瘤细胞均显示出细胞分化的形态学特征。C6细胞的增殖也明显受到ATRA的抑制,绝大多数的活细胞都静止于G1期,100μmol/L ATRA对C6细胞增殖的抑制尤其明显,并诱导细胞凋亡。经3种浓度的ATRA分别作用后,C6细胞的GJIC也明显提高,而Cx43基因的转录未受明显影响。旁观者效应实验的结果显示,3种浓度的ATRA均可明显增强旁观者效应。结论：联合应用ATRA与HSV-TK基因治疗可以产生显著的杀伤胶质瘤细胞的作用。     

幽门螺杆菌感染与胃上皮细胞间隙连接功能的体外研究

目的　探讨幽门螺杆菌(H.pylon,Hp)对胃上皮细胞间隙连接通讯(GJIC)功能的影响及其发病机制。方法　应用Hp(包括Cag A 和Cag A-株)全菌和超声粉碎上清分别作用胃上皮细胞株SGC- 790 1。采用激光扫描共焦显微镜(L SCM,L eica TCS SP)以荧光光漂白后再恢复(FRAP)技术测定SGC- 790 1细胞GJIC功能(以荧光传递速率K表示,×10 - 3/ s)。结果　Cag A 和Cag A- Hp株均下调SGC- 790 1细胞GJIC功能(Cag A :F=5 7.98;Ca-g A- :F=2 9.5 9,P<0 .0 1) ;且Cag A 与Cag A- Hp相比前者更显著下调胃上皮细胞GJIC功能(全菌:t=13.86 ,P<0 .0 1;超声粉碎上清:t=11.87,P<0 .0 1)。结论　Cag A 比Cag A- Hp显著抑制胃上皮细胞的GJIC功能,提示其在胃癌发生中起重要作用。     

洛伐他汀对 MCF-7细胞增殖分化功能及间隙连接细胞通讯的影响

背景与目的:洛伐他汀( lovastatin)是细胞内源性胆固醇合成的抑制剂,临床已普遍用于治疗高脂血症.现有研究报道,洛伐他汀具有抗肿瘤作用,但分子机制尚不清楚.本文探讨洛伐他汀对人乳腺癌细胞 MCF-7增殖功能以及间隙连接细胞通讯( gap junctional intercellular communication,GJIC)的影响.方法:分别以 4、 8、 16 μ mol/L洛伐他汀处理 MCF-7细胞 24～ 72 h后,流式细胞仪检测细胞周期的时相分布及细胞凋亡率,硝基蓝四氮唑( NBT)还原实验鉴定细胞分化,并采用划痕标记染料示踪技术观察洛伐他汀对 MCF-7细胞 GJIC功能的影响.结果:不同浓度洛伐他汀处理细胞不同时间后, MCF-7细胞的增殖明显受抑,抑制率最高可达 75.8%,且同一处理时相点各组间比较差异均有显著性 (P< 0.05);细胞周期分析显示,各处理组内 G0/G1期细胞明显增多,处理 72 h后可高达 80%左右;同时洛伐他汀能明显促进 MCF-7细胞分化、各处理组间比较差异均有显著性 (P< 0.01),但洛伐他汀诱导该细胞凋亡的作用不明显.另外,洛伐他汀有上调或恢复 MCF-7细胞 GJIC的作用; 16 μ mol/L洛伐他汀处理细胞 72 h后,荧光染料传输的范围可达 4～ 5层细胞.以上作用均有浓度-效应和时间依赖关系.结论:洛伐他汀可抑制 MCF-7细胞增殖,使细胞生长阻滞于 G0/G1期,并能促进细胞分化,该作用可能与洛伐他汀上调或恢复 MCF-7细胞的 GJIC功能有关.     

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。     

Homologous and heterologous gap-junctional intercellular communication in v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial cell lines.

We examined gap-junctional intercellular communication (GJIC) in a series of normal and v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial (RLE) cell lines using the scrape loading-dye transfer and fluorescence-recovery-after-photobleaching (FRAP) assays. Whereas the normal RLE cell line, the control helper virus-transduced cell line, and the v-myc-transduced cell line all showed excellent GJIC, the v-raf-transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v-raf/v-myc-transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. Heterologous GJIC of these oncogene-transduced cell lines was also compared with that in the normal RLE cells. A modified FRAP assay, using fluorescent-microbead labelling to identify the oncogene-transduced cell from surrounding normal cells, was used to quantify the heterologous GJIC. The v-raf/v-myc-transformed RLE cells had no heterologous communication with the normal RLE cells, whereas v-raf- and v-myc-transduced cell lines maintained heterologous GJIC. Northern analysis showed that connexin 43 was the only gap-junction protein message expressed in these cell lines; connexin 32 and connexin 26 were not expressed. The levels of connexin 43 mRNA expression were relatively unchanged in all cell lines, suggesting that the reduction in GJIC was primarily at the posttranslational level. These findings suggest that reduction of homologous GJIC in v-raf- and v-raf/v-myc-transformed RLE cells is linked to their tumorigenic potential. Furthermore, the loss of heterologous GJIC, which we observed only in the v-raf/v-myc-transformed cells, might release such cells from the growth-regulating effects of surrounding normal cells, possibly contributing to their enhanced tumorigenic potential.     

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.      

Changes in gap junctional intercellular communication in mouse skin carcinogenesis

Gap junction intercellular communication (GJIC) has been measured in cell lines that represent different stages of chemically induced mouse skin carcinogenesis. No significant difference in GJIC, as measured by dye spread, was found in cultures of normal keratinocyte, papilloma or squamous carcinoma cell lines. There was no correlation, in this system, between the presence of a mutant Ha-ras gene and down- regulation of communication. There was, however, a marked decrease in GJIC (80-90%) on progression from squamous to spindle carcinoma cells. Measurement of GJIC in somatic cell hybrids shows that the genetic defect responsible for this down-regulation is recessive and is common to two independently isolated spindle cell lines. No abnormalities were found in the spindle cells in expression of connexin 43, a cell component involved in gap junction formation and permeability. However, expression of E-cadherin, a cell-cell adhesion molecule implicated in the process of gap junction formation, was missing in the spindle carcinoma cells. Introduction of an E-cadherin cDNA into the spindle cells partially restored junctional communication without causing any noticeable alterations in cell morphology. During the study a non- tumourigenic keratinocyte line, a sub-clone of a normal keratinocyte line, was also found to have a low level of GJIC. However, the defect in this line was shown, by genetic complementation in somatic cell hybrids, to be different from that in the spindle carcinoma cell lines. Consistent with these data, analysis by immunofluorescence shows an abnormal distribution of connexin 43 in these cells.      

Growth control of 3T3 fibroblast cell lines established from connexin 43–deficient mice

Connexins are considered to be involved in cell growth control, on the basis of studies mainly with tumorigenic cells. To study the role of connexin genes in normal cell growth control, we established fibroblast cell lines from connexin 43 (Cx43)–deficient mice and characterized their growth. Embryonic fibroblasts from wild-type mice (Cx43^+/+) and those with heterozygous (Cx43^+/–) and homozygous (Cx43^–/–) deficiencies of the Cx43 gene were cultured and passaged by a 3T3 protocol (every 3 d, 3 × 10⁵ cells/60-mm dish). All cell lines showed a growth crisis during passages 6–15 and then started to grow well. All cell lines grew at similar rates under the 3T3 protocol, but Cx43-deficient (Cx43^–/–) cell lines tended to grow faster when they were plated at 10⁵ cells per dish. Cx43^–/– cells did not express Cx43 and showed little gap-junctional intercellular communication (GJIC), confirming that Cx43 is the major connexin responsible for GJIC of these fibroblasts. While all Cx43^+/+ and Cx43^+/– cell lines expressed Cx43 protein, some of them showed very little GJIC. Those cell lines with high GJIC showed higher levels of the P2 form of Cx43 protein, and more Cx43 was localized in the plasma membrane than in cell lines with lower GJIC levels. We investigated effects of serum concentration on cell growth in these cell lines. Although different cell lines responded differentially to these agents, there was no clear relationship between Cx43 expression and cell growth stimulation by them. This suggests that Cx43 expression alone is not a strong regulator of mouse fibroblast growth. Mol. Carcinog. 23:121–128, 1998. © 1998 Wiley-Liss, Inc.     

Bystander effect in suicide gene therapy is directly proportional to the degree of gap junctional intercellular communication in esophageal cancer

Gap junctional intercellular communication (GJIC) has been shown to be involved in the bystander effect through herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy. In this study, we examined the expression of connexins, the components of gap junction, and the degree of GJIC in esophageal cancer cell lines and compared the bystander effect in cells with different capacities of GJIC. We found loss in connexin 26 expression and reduced connexin 43 in esophageal cancer. GJIC capacity varied among cell lines and was dependent on the connexin 43 expression in the cell-cell contact areas. In mixing assay, the extent of the bystander effect was tightly correlated with the degree of GJIC capacity. The effects of retinoic acid and cAMP on the bystander effect were also investigated. Treatment with retinoic acid, but not with cAMP, was associated with augmented bystander killing by increase in GJIC in some esophageal cancer cell lines. Our results indicated that the degree of GJIC was predictive to identify a tumor as suitable for gene therapy with the HSV-tk/GCV system. Also GJIC chemically-enhanced with retinoic acid might be useful to improve response in suicide gene therapy.     

In-vitro effects of antineoplastic prostaglandins on human leukemic cell growth and normal myelopoiesis

The effects of prostaglandin (PG)E1, PGD2 and 9-deoxy-delta 9-PGD2 (PGJ2) on the clonogenic growth of six kinds of human leukemic cell lines (K562, KG1, HL60, U937, THP1 and Molt4) and normal human myeloid progenitor cells (CFU-GM) were studied using semisolid agar cultures. While the degree of suppression of leukemic growth by PGE1 varied from cell line to cell line, PGD2 and PGJ2 equally suppressed the growth of all leukemic cell lines. The potency of growth inhibition was as follows: PGJ2 greater than PGD2 greater than PGE1. The increase of cellular cAMP level induced by prostaglandin treatment did not parallel their cytotoxic potency. Normal myeloid colony formation was also suppressed by PGE1, PGD2 or PGJ2. In contrast to the preferential inhibition of macrophage colony formation by PGE1, such lineage-selective suppression was not observed for PGD2 or PGJ2. These findings suggest that PGD2 and PGJ2 potently inhibit the leukemic growth by a different mechanism from that of PGE1 and by a cAMP-independent mechanism. These prostaglandins seem to be promising chemotherapeutic agents for acute leukemia.     

EFFECTS OF LIMONENE,SALVIA MILTIORRHIZA AND TURMERIC DERIVATIVES ON H-RAS ONCOGENE EXPRESSION AND GAP JUNCTION INTERCELLULAR COMMUNICATION IN HUMAN SOLID TUMOR CELL LINES

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The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication

The effects of five non-mutagenic carcinogens—Aroclor1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate(TPA) and 1, 1'-(2, 2, 2-trichloroethylidene)bis[4-chlorobenzene](DDT)—on gap junctional intercellular communication (GJIC)were tested in a cell line consisting of initiated cells (3PC).Four agents suspected of tumor promotion activity—O-anisidine,clofibrate, L-ethionone and d-limonene—were also testedfor their effects on GJIC. Finally sodium fluoride (NaF), whosecarcinogenic property is still unclear, was tested for its effectson GJIC in the 3PC cell line. Four of the five selected tumorpromoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC betweenthese initiated epidermal cells. The four non-mutagenic carcinogenswith tumor-promoting activity in vivo (o-anisidine, clofibrate,L-ethionine and d-limonene) all inhibited GJIC, whereas NaFhad no effect. Seven compounds (o-anisidine, Aroclor 1260, BP,DDT, L-ethionine, d-limonene and TPA) had a dose-dependent aswell as time-dependent inhibitory effect on GJIC. Under theexperimental conditions used, clofibrate showed only a dose-relatedinhibition of GJIC. PB showed no inhibitory effect on GJIC inthe 3PC cell line. In order to determine the role of biotransformationin the tumor-promoting activity of PB, its effect on GJIC wasalso examined in the presence of an Aroclor 1254-induced ratliver homogenate (S9 mix) and in the hepatoma cell line HepG2.In the presence of rat liver homogenate PB decreased GJIC inthe 3PC cell line, whereas in the HepG2 cells PB showed a time-anddose-dependent inhibitory effect. To study the potential differencesin susceptibility of cells representing different stages inthe process of tumor formation, the effect of the selected tumorpromoters on GJIC was also investigated in primary mouse keratinocytesand in a mouse skin carcinoma-derived cell line (CA3/7). Primarykeratinocytes were sometimes more (BP and clofibrate) and sometimesless sensitive (ethionineand limonene) for inhibitory effectson GJIC compared to the effects in the cell line 3PC. Exceptfor TPA and anisidin, GJIC between the CA3/7 cells was lessaffected by the selected agents compared to the 3PC cell line.These results show that, during the process of tumor formationthe susceptibility of cells to inhibition of GJIC by tumor promotersis variable. Overall the CA3/7 cells are less sensitive comparedto 3PC cells. The susceptibility of primary keratinocytes isvariable compared to 3PC cells, depending on the agent used.These results also show that GJIC is a valid parameter for testingthe tumor-promoting activity of compounds. Finally, this studydemonstrates that mouse keratinocyte cell lines could serveas an in vitro model for the detection of non-mutagenic carcinogenswith diverse target organs in vivo. For this use the cell lineconsisting of initiated cells (3PC) is more sensitive than thecarcinoma-derived cell line CA3/7.     

SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. II. Changes in gap junctional intercellular communication

Poor gap junctional intercellular communication (GJIC) has been associated with uncontrolled cell growth and neoplasia. We have successfully propagated normal first trimester invasive extravillous trophoblast (EVT) cells, and have produced premalignant EVT lines after SV40 Tagtransformation: RSVT-2 is an uncloned line that is long-lived; RSVT2/C is a clonal line that is immortal. Both are hyperproliferative, hyperinvasive and variably refractory to the anti-proliferative and anti-invasive effects of transforming growth factor β (TGFβ). Possible changes in gap junctions during the transition of normal invasive EVT cells to the premalignant stage were examined by comparing expression of connexin proteins (by immunolabeling for Cx26, Cx32, Cx40, Cx43), and mRNA (by Northern blot with cDNA probes for Cx26, Cx32, Cx43), and functional GJIC (by dye transfer using the preloading method) in normal parental EVT cells and their SV40 Tag transformants. Results from immunofluorescence and Northern blot analysis revealed that, of the panel of connexins examined, only Cx43 was variably expressed in these cell lines in vitro. Expression of Cx43 protein and mRNA was abundant in normal EVT cell line HTR8, reduced in long-lived RSVT-2 cells and undetectable in immortalized RSVT2/C cells. GJIC, as measured by dye transfer between donor and recipient cells, was also similarly reduced in recipient RSVT-2 cells, and drastically reduced in RSVT2/C cells, irrespective of whether the dye donor was of the same cell type (homocellular coupling) or HTR8 cells (heterocellular coupling). Treatment with TGFβ reduced Cx43 mRNA expression as well as GJIC in normal EVT cells, but not in the SV40 Tag transformants. Our findings suggest that downregulation of connexins with the resultant impairment in GJIC is an early event in tumor progression, as observed in the premalignant SV40 Tag transformants. Int. J. Cancer77:440–448, 1998. © 1998 Wiley-Liss, Inc.