Gap junction expression following UVC-induced neoplastic transformation in human hybrid cell lines

Decreased connexin gene expression and loss of the capacity for either homologous or heterologous intercellular communication has been associated with neoplastic transformation. We tested the hypothesis that loss of gap junctional intercellular communication (GJIC) correlates with tumorigenic potential in the HeLa x skin fibroblast human hybrid cell system. Connexin gene expression, gap junction function and tumorigenicity were determined for the non-tumorigenic somatic hybrid cell line (CGL1) and a series of UVC-induced tumorigenic cell lines derived from CGL1. CGL1 and the parental skin fibroblasts express connexin43 (alpha1 gap junction gene) mRNA and protein, form gap junctional plaques and have functional gap junctions. UVC- irradiation of CGL1 cells produced a cell line (UV12) with an aggressive tumorigenic phenotype, which lost connexin43 expression as well as both homologous and heterologous GJIC and was in this respect similar to HeLa cells. However, the phenotype of UV12 cells exhibited some instability and revertants to a less aggressive tumorigenic phenotype were isolated. These cells expressed connexin43 mRNA and protein, and demonstrated homologous GJIC. Furthermore, cells reconstituted from a tumor derived from this revertant cell line retained significant connexin43 expression and homologous GJIC, although they exhibited an aggressive tumorigenic phenotype. Thus, functional homologous GJIC cannot be dissociated from tumorigenicity in this system. However, heterologous GJIC between these same UVC-induced tumorigenic cell lines and normal human skin fibroblasts was reduced, whereas the non-tumorigenic hybrid cells showed extensive heterologous GJIC. In summary, re-acquisition of connexin43 expression and homologous GJIC does not restore the non-tumorigenic phenotype in UVC- induced tumorigenic HeLa skin fibroblast human hybrid cells. However, reduction of heterologous GJIC does correlate with tumorigenicity in this cell system.      

Altered gap junctional intercellular communication in neoplastic rat esophageal epithelial cells

Gap junctional intercellular communication (GJIC) is reduced in many neoplastic cells, but few data exist for esophageal neoplasms. GJIC was examined by fluorescent dye microinjection in two nontumorigenic and two highly tumorigenic rat esophageal epithelial cell lines. All lines expressed high levels of dye coupling in homologous cell culture. In cocultures of nontumorigenic and tumorigenic cells, however, only one of six cell combinations displayed significant heterologous GJIC. Northern, Western, and immunohistochemical analyses indicated that all four cell lines expressed comparable levels of connexin43 (Cx43), but not connexin32 or connexin26, and formed Cx43-containing gap junction plaques at cell-cell interfaces. Immunostaining of rat esophageal frozen sections demonstrated that esophageal epithelial cells expressed Cx43 in vivo. In normal epithelium, the highest expression was seen in the basal cells and little suprabasal staining was evident. In preneoplastic and neoplastic lesions of the esophageal epithelium which were induced by treating rats with N-nitrosomethylbenzylamine, Cx43 staining of the basal layer was also seen but appeared to be more diffuse compared to normal epithelium. In addition, suprabasal Cx43 staining was apparent in dysplastic and papillomatous lesions. These results indicate that Cx43 is expressed in normal and neoplastic rat esophageal cells and that the cells exhibit extensive homologous GJIC, but little heterologous GJIC. This lack of heterologous GJIC may be due to differences in cell adhesion proteins or other factors.      

Gap junctional intercellular communication and connexin43 expression in human ovarian surface epithelial cells and ovarian carcinomas in vivo and in vitro.

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.     

Effective asymmetry in gap junctional intercellular communication between populations of human normal lung fibroblasts and lung carcinoma cells

The dysfunction of homologous and/or heterologous gap junctional intercellular communication (GJIC) has been implicated in tumorigenesis of many kinds of cells. Here we have characterized GJIC and the expression of connexins in six human lung carcinoma cell lines and normal lung fibroblasts (HLF). Compared with HLF, all the carcinoma cells showed reduced or little homologous GJIC. They expressed remarkably reduced connexin(Cx)43 mRNA and variable levels of Cx45 mRNA, but neither Cx43 nor Cx45 protein could be detected. However, using a preloading assay, transfer of calcein was observed between donor HLF cells and first order neighboring recipient tumor cells (recipient cells in 1000-fold excess). Transfer from tumor to HLF cells under the same conditions was not seen, although increasing the ratio of donor tumor cells to recipient HLF cells and plating the cells at low density did reveal weak transfer from tumor cells to HLF. Transfection of Cx43 into giant cell carcinoma PG cells increased homologous communication and eliminated the rectifying behavior of heterologous communication. This indicates that the apparent rectification of dye transfer between normal and tumor cells was a product of low rates of heterologous transfer linked to (i) rapid dilution of the dye to below detectable limits through a very well coupled cell population (tumor to HLF) and (ii) concentration of dye in immediate neighbors in a poorly coupled cell population (HLF to tumor cells). These results suggest that the coupling levels may need to exceed a certain threshold to allow propagation of signals over a sufficient distance to affect behavior of a cell population. We propose that the relative rates of heterologous and homologous coupling of cell populations and the 'pool size' of shared metabolites in tumor cells and the surrounding normal tissue are likely to be very important in the regulation of their growth.     

Emergence of undifferentiated rat tracheal cell carcinomas, but not squamous cell carcinomas, is associated with a loss of expression of E- cadherin and of gap junction communication

A series of cells representing normal, non-tumorigenic cell lines, as well as differentiating neoplastic and undifferentiated neoplastic rat tracheal epithelial cell populations were evaluated for their ability to establish homologous and/or heterologous cell-cell gap junction communication in culture. Gap junction communication was evaluated by flow cytometric quantitation of the transfer of the fluorescent dye calcein from a donor to a recipient cell population via gap junctions. The data indicate that normal primary cultures of rat tracheal epithelial cells, as well as non-tumorigenic cell lines and squamous cell carcinomas cell populations, retain the ability to establish both homologous and heterologous gap junction communication. In all cases an average of >48% of recipient cells had acquired calcein label during a 5-h interval of co-culture of donor and recipient cells at confluent densities. Cells harvested directly from squamous cell carcinoma tumors exhibited similar levels of cell-cell communication. In contrast, cells giving rise to undifferentiated carcinomas, as well as cells harvested from undifferentiated carcinomas, exhibited very low levels or no homologous or heterologous cell-cell communication. Cell populations exhibiting distinctly different communication phenotypes were evaluated by Northern blot analysis for expression of connexins (Cx 26, 32 and 43) and E-cadherin. Neither communicating nor non-communicating cells expressed connexin 32. Those cell populations, which established functional gap junctions, expressed E-cadherin as well as connexin 26 and/or 43. In contrast, those cell populations that lacked the ability to communicate universally lacked expression of E-cadherin, and a quarter also lacked expression of detectable levels of connexin.      

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.     

Reduced levels of connexin43 in cervical dysplasia: inducible expression in a cervical carcinoma cell line decreases neoplastic potential with implications for tumor progression

Loss of gap junctional intercellular communication (GJIC) has been linked to aberrant proliferation and an enhanced neoplastic phenotype. Many human tumors, including the cervical carcinoma line HeLa, have been reported to be deficient in expression of the gap junction protein connexin43 (Cx43) and GJIC. To determine if this is an early event in carcinogenesis, we utilized immunohistochemistry to screen a series of cervical biopsy samples and demonstrated a major reduction in Cx43 expression in dysplastic regions compared to normal epithelia. To determine whether this loss influences the neoplastic behavior of cervical carcinoma cells, we have constructed HeLa cell lines in which Cx43 expression can be induced in response to doxycycline. This approach allows for the discrimination of Cx43-mediated effects from those due to pre-existing clonal heterogeneity. Cx43 induction in these cells led to assembly of functional junctions but did not alter growth control in vitro as measured by logarithmic growth, saturation density or focus formation when in co-culture with growth-controlled fibroblasts. However, Cx43 induction decreased two indices of neoplasia: it reduced anchorage-independent growth and attenuated the growth rate of tumor xenografts. These results indicate that established HeLa cell lines are unresponsive to Cx43-mediated signals which are thought to mediate growth control of non-transformed cells, however, Cx43 expression can still reduce aspects of the neoplastic phenotype of these cells, indicating that loss of connexin signaling in dysplastic cells may contribute to their neoplastic progression.     

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.      

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.     

Expression and function of connexin in normal and transformed human keratinocytes in culture

We have studied the gap junctional intercellular communication(GJIC) of immortalized and tumourigenic human keratinocyte celllines and of a spontaneously immortalized non-tumourigenic anda highly differentiating keratinocyte cell line (HaCaT) as thecontrol. In homologous cultures, the GJIC capacity of five squamouscell carcinoma-derived cell lines was 1–27% that of theHaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenicpotential and an SV40 DNA-immortalized cell line had markedlyreduced GJIC capacities. Northern analysis and immunohisto-chemistryshowed that connexin (Cx) 43 is the major gap junction proteinexpressed in the communicating cells. They do not express Cx26 or 32. The low or absent communication observed in certaincell lines was due in some to a lack of Cx 43 gene expression,but in others to aberrant localization of the gap junction protein.GJIC of these cell lines, as well as that of primary normalhuman epidermal keratinocytes, was susceptible to 12-O-tetra-decanoylphorbol-13-acetate-mediatedinhibition. Moreover, GJIC of HaCaT cells and their tumourigenicderivatives is Ca²⁺-dependent. These results, when comparedwith those previously obtained for mouse keratinocyte cell lines,reveal that GJIC of human keratinocytes was correlated to thedegree of differentiation and is controlled in a similar wayto that of murine keratinocytes. Aberrant GJIC seems to be acommon feature of human and murine skin carcinogenesis.     

Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells

Loss of gap junctional intercellular communication (GJIC) is a characteristic of cancer cells. Since a coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis, the present study was designed to test the hypothesis that skin-derived tumor cells may modulate homologous and heterologous GJIC. While homologous GJIC of human dermal fibroblasts as well as epidermal keratinocytes was detected, no communication was measured between SCL-1 cells derived from squamous cell carcinoma of human skin. Interestingly, co-cultures of dermal fibroblasts and SCL-1 tumor cells in serum-containing medium resulted in a 52-70% lowering of the number of communicating fibroblasts. Furthermore, incubation of confluent fibroblast cultures with serum-free supernatant fractions (20-30 kDa) from tumor cells, termed the 20/30 fraction, lowered the homologous gap junction communication of fibroblasts by >90%. This novel aspect of down-regulated homologous GJIC of dermal fibroblasts, which is reversible, was neither mediated by alteration of the expression of connexin43, the major gap junctional protein of dermal fibroblasts, nor by aberrant localization of connexin43 in the plasma membrane. Furthermore, post-translational modifications of connexins, such as phosphorylation, was not measured by mobility shift studies. Tumor cell-mediated GJIC down-regulation between fibroblasts was suppressed using EGTA-containing serum-free tumor cell-derived supernatants suggesting that calcium ions (Ca2+) might mediate the transduction of this effect. The involvement of Ca2+ in down-regulation of homologous GJIC of fibroblasts was supported by an increase in fluorescence intensity of the intracellular calcium-sensitive indicator Fura-2 upon treatment of fibroblasts with the active 20/30 fraction. In conclusion, these data establish homologous GJIC of (stromal) fibroblasts as a parameter modulated by a paracrine acting factor(s) of epithelial tumor cells during tumor-stroma interaction of skin cells.     

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。     

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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Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells

We examined gap junctional intercellular communication (GJIC)of primary human mesothelial cells and cell lines of asbestos-associatedhuman pleural mesotheliomas, and the effect of asbestos andother mineral fibres on these cells. In homologous cultures,the GJIC capacity of six out of seven tumour cell lines wasmarkedly less than for primary mesothelial cells. This defectin GJIC appeared not to be at the expression level of mRNA andprotein of the gene encoding the 43 kDa gap junction protein.In heterologous cocultures of tumour cells and primary mesothelialcells, however, 80–90% of the tumour cell/normal cellcontacts were functional. Exposure of primary mesothelial cellsto TPA, a phorbol ester tumour promoter, resulted in markedinhibition of GJIC, being an action common to numerous tumourpromoters. Such an effect though was not observed with the carcinogenicmesothelioma-inducing mineral fibres chrysotile and amosite,neither with glass wool. These results suggest that a permanentdefect in GJIC capacity is a common feature of human mesotheliomacells, but how mineral fibres are involved in the process ofmesotheliomagenesis is still unclear.     

Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT-1080 human fibrosarcoma cells in vitro and in vivo

Numerous studies have demonstrated a correlation between dysregulation/loss of connexin expression or gap junction intercellular communication (GJIC) function and decreased growth control both in human tumors and tumor cell lines. Likewise, restoration of constitutive connexin expression/function is correlated with increased growth control/decreased tumorigenicity. Here, we show for the first time that inducible restoration of connexin43 (Cx43) expression and GJIC function in a human tumor line of mesenchymal origin (HT-1080, fibrosarcoma) resulted in a lowered neoplastic potential. Specifically, HT-1080 cells induced to express Cx43 demonstrated diminished foci formation when in co-culture with normal fibroblasts, decreased colony formation under anchorage-independent conditions, and reduced tumor growth when injected into immunodeficient mice. These results, obtained utilizing an inducible system that helps address issues of clonal heterogeneity, strongly implicate Cx43 as a tumor suppressor in human tissue of mesenchymal origin and GJIC as a regulatory mechanism for cellular growth control both in vitro and in vivo. This study also further supports the hypothesis that loss of Cx43/GJIC in human tumors may play an important role in the dysregulation of normal growth control.     

Expression of cell adhesion molecules and connexins in gap junctional intercellular communication deficient human mesothelioma tumour cell lines and communication competent primary mesothelial cells

Gap junctional intercellular communication (GJIC) has been reportedto be markedly reduced in human mesothelloma tumour cell linescompared with primary mesothelial cells. Iminunofluorescencestainings have shown that the gap junction protein connexin43(Cx43) is expressed In both malignant and normal mesothelialcells. In this study the mRNA expression of Cx43 and three differentconnexlns—Cx37, Cx40 and Cx45, which are highly expressedin lung tissue—was investigated in eight human mesotheliomacell lines, and in human primary mesothellal cells from severaldonors. The expression of the intercellular adhesion moleculesA-CAM (N-cadherln) and L-CAM (E-cadherin) was studied at theprotein level. No mRNA expression of Cx37, Cx40 or Cx45 in eithermesothelioma tumour cells or the primary mesothelial cells wasdetected. Cx43 was expressed at both the mRNA and the proteinlevel, in seven out of eight mesothelloma cell lines, as wellas in all the primary mesothellal cell cultures. The intercellularadhesion molecule A-CAM was expressed at the cell—cellborders In six out of seven mesothelioma cell lines, as wellas in normal mesothellal cells. No expression of L-CAM was observedin these cells. The results suggest that Cx43 and A-CAM arethe major proteins in gap and adherens Junctions respectivelyin human mesothellal cells. Most mesothelioma tumour cell lineswith markedly reduced GJIC still express both Cx43 and A-CAM.Only one of our mesothelloma tumour cell lines severely deficientin GJIC lacks both the gap junction protein Cx43 and the celladhesion molecule A-CAM.     

Enhancement of liver cell gap junction protein expression by glucocorticoids

Gap junctional intercellular communication (GJIC) and the expressionof gap junction proteins (connexins) may be involved in growthregulation and neoplastic transformation. The mechanisms ofconnexin gene regulation in normal and neoplastic tissues arepoorly understood. In this study, the glucocorticoids, dexamethasoneand hydrocortisone, enhanced fluorescent dye-coupling in primarycultured rat hepatocytes and MH₁C₁ rat hepatoma cells. Othertypes of steroids (ß-estradiol, testosterone, aldosteroneand progesterone) had no effect. Northern blot, Western blot,nuclear run-on and immunohistochemical analyses showed thatglucocorticoids enhanced the expression of connexin32 in thesecells in a dose- and time-dependent fashion. Connexin26 expressionwas also enhanced slightly by dexamethasone in hepatocytes,but not MH₁C₁ cells. Connexin43 expression in these cells wasnot affected by steroids. In WB-F344 rat liver epithelial cells,which were highly coupled and expressed high levels of connexin43and no detectable connexin32 or connexin26, dexamethasone hadno effect on coupling or connexin expression. These resultsindicate that dye-coupling and the expression of connexin32and connexin26, but not connexin43, were upregulated by glucocorticoidsin a cell-specific manner. These effects on GJIC and connexinexpression may be involved in the induction of hepatic differentiationand inhibition of growth.     

Gap junctional communication in cultured human lung carcinoma cells.

Animal tumor models have demonstrated a close correlation between gap junctional, intercellular communication (GJIC) and tumor metastasis. To examine GJIC levels in human lung carcinoma cells, a novel technique was developed: cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse which opens transient pores on the plasma membrane was applied in the presence of the fluorescent dye, Lucifer yellow, causing the dye's penetration into the cells growing on the conductive part of the slide. The migration of the dye through gap junctions to the non-electroporated cells growing on the non-conductive area was then observed microscopically under fluorescence illumination. The results show that this is a rapid, precise and highly reproducible assay for GJIC assessment in lines established from lung carcinomas or freshly explanted lung tumor cells. Out of 17 established lines only two had extensive junctional communication, while out of 16 fresh tumor specimens none displayed GJIC. On the other hand, fibroblasts isolated from the same tumors had extensive junctional permeability. The examination of GJIC in a large number of samples could establish a correlation between GJIC and metastasis which might have prognostic value.