Disruption of gap junctional intercellular communication in human renal cancer cell lines

OBJECTIVES: Gap junctional intercellular communication (GJIC) is believed to play an important role in the maintenance of cell homeostasis, and its disruption may be associated with carcinogenesis. However, GJIC has not been detected in many human cancers. We therefore studied the regulation of GJIC in human renal cancer cell lines. METHODS: We examined the human renal cancer cell lines, ACHN and NT, as well as Madin-Darby canine kidney (MDCK) cells as a positive control, using GJIC assays, Northern blotting to detect connexin 43 mRNA, immunofluorescent staining, and Western blotting of connexin 43 protein. RESULTS: GJIC of ACHN and NT was completely blocked. In ACHN cells, connexin 43 mRNA was not altered. However, connexin 43 protein was aberrantly localized and phosphorylated connexin 43 protein had disappeared. Both connexin 43 protein and its mRNA were undetectable in NT cells. CONCLUSIONS: GJIC in human renal cancer cell lines is impaired and various pathways may inhibit this mechanism in renal cancer. We believe that connexin plays an important role in renal carcinogenesis.     

Role of gap junctions in lung neoplasia

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.     

Absorption and urinary excretion of the coffee diterpenes cafestol and kahweol in healthy ileostomy volunteers

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml(-1) HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml(-1) and 3 mg ml(-1). All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread.     

12-O-tetradecanoylphorbol-13-acetate-induced inhibition of gap junctional communication is differentially regulated in a transformation-sensitive Syrian hamster embryo cell line compared to early passage SHE cells

The transformation-sensitive cell-line BPNi was more susceptible to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibition of gap junctional intercellular communication (GJIC) than early passage Syrian hamster embryo (SHE) cells, while the potency of TPA to down-regulate EGF-binding was similar in the two cell types. The kinetics of TPA-induced inhibition of GJIC suggested that different mechanisms may operate at high and low TPA concentrations. The initial inhibition after exposure to high TPA concentrations was followed by a recovery of GJIC. The recovery was much more pronounced in SHE than in BPNi cells. This effect could not be explained by differences in down-regulation of protein kinase C. Removal of high TPA concentrations also resulted in a faster recovery of GJIC in SHE than in BPNi cells. In addition, although forskolin induced a similar protection against the inhibitory effect of TPA on GJIC, forskolin restored GJIC blocked by TPA much faster in SHE than in BPNi cells. Thus, BPNi cells are more sensitive to TPA induced inhibition of GJIC than SHE cells, and have reduced capability to recover from down-regulated GJIC as compared to SHE cells.     

Fatigue in cancer: a phenomenological perspective

Experiments were conducted by using scrape-loading and dye transfer (SLDT) method to study the gap junction intercellular communication (GJIC) of Chinese hamster lung cells (V79), mouse fibrous cells (Balb/c-3T3), rat liver cells (WB) and human embryonal lung cells (2BS). We also observed the inhibition of the GJIC by TPA and the antagonistic effect of Curcumin derivatives on TPA. The results indicated that V79, WB, 3T3 and 2BS normal cells showed medium level of GJIC, and TPA could inhibit the GJIC to some extents. Curcumin derivatives (91022, 91022-S) could counteract the inhibition of TPA-induced GJIC. It was also found that human lung adenocarcinoma cell (A549) and GLC lacked GJIC, and 91022 could improve the GJIC of A549 cell. It may be related to its anticancer activity.     

Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells

Mechanical stimulation was used to initiate Ca2+ waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca2+ to adjacent cells and to assess alternative Ca2+ signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca2+ wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca2+ wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18beta-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca2+ wave. In contrast, treatment with suramin, a P2-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca2+ wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca2+ wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.     

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.     

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.     

Long-term visual outcome of choroidal neovascularization in pathologic myopia: natural history and laser treatment

Tumour-endothelial cell adhesion forms a key role in the establishment of distant metastases. This study examined the effect of gamma linolenic acid (GLA), an anti-cancer polyunsaturated fatty acid (PUFA), on both the gap junction communication of human vascular endothelial cells and tumour cell-endothelial interactions. By using scrape loading of Lucifer yellow dye, we showed that GLA at non-toxic levels increased Lucifer yellow transfer, indicating improved gap junction communication. The fatty acid also corrected the communication that was reduced by the mitogenic and motogenic factor HGF/SF. GLA inhibited the tyrosine phosphorylation of connexin-43, a protein that formed gap junction in this cell. When human tumour cells were added to quiescent or HGF/SF-activated endothelial cells, the presence of GLA reduced adhesion of tumour cells to the endothelium. It is concluded that GLA reduces tumour-endothelium adhesion, partly by improved gap junction communications of the endothelium.     

Neuroprotective and thrombolytic agents: advances in stroke treatment

Malignant mesothelioma (MM) is resistant to all conventional forms of therapy though there is considerable evidence from clinical trials and animal models of the disease that an immune response can be elicited to the tumour. In order to define those target antigens expressed by MM cells which might provide a focus for an effective immune response we tested patients' sera for the presence of MM autoantibodies by Western blot analysis. Eight of 29 (28%) patients with MM had serum antibodies of the IgG class in high titre and each antiserum recognised different protein antigens. In those individuals where sequential samples were available, the antibody titre increased with the progression of the disease though the number of target antigens remained constant. Sera from the eight patients were studied further: six of the antigen complexes were expressed at least partially in the nucleus; two showed some specificity for the tumour in that they discriminated antigens that were highly expressed in all human MM cell lines, but were not expressed in a human SV40 transformed mesothelial line; four of the antisera recognised a homologue in mouse tissue and each of these had a different pattern of expression. Collectively, these antisera define a subset of nuclear autoantigens that are over-expressed in dividing cells.     

BAX frameshift mutations in cell lines derived from human haemopoietic malignancies are associated with resistance to apoptosis and microsatellite instability

Bax suppresses tumorigenesis in a mouse model system and Bax-deficient mice exhibit lymphoid hyperplasia suggesting that BAX functions as a tumour suppressor in human haemopoietic cells. We examined BAX expression in 20 cell lines derived from human haemopoietic malignancies and consistent with a potential tumour suppressor function, identified two cell lines, DG75 (a Burkitt lymphoma cell line) and Jurkat (a T-cell leukaemia line), which lacked detectable BAX expression. Apoptosis of DG75 cells induced by low serum or ionomycin was significantly delayed relative to similar Burkitt lymphoma cell lines with normal BAX levels. Although DG75 and Jurkat cells expressed several BAX RNA species including the prototypical BAX alpha RNA, the absence of BAX protein was due to single base deletions and additions in a polyguanine tract within the BAX open reading frame. These frameshift mutations result in premature termination of translation and have recently also been identified in some colon cancers with microsatellite instability. Although mismatch repair defects are not considered a common feature of haemopoietic malignancies, DG75 and Jurkat cells had widespread microsatellite instability and did not express detectable levels of MSH2. In Jurkat cells, lack of MSH2 expression was due to a point mutation in exon 13 of MSH2 resulting in premature termination of translation. Our results suggest that a pathway linking mismatch repair defects, BAX tumour suppressor frameshift mutations and resistance to apoptosis may be a key feature of some lymphomas and leukaemias.     

Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy

Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) 'prodrug' system. Since retinoids have been reported to increase GJIC by induction of connexin expression, we hypothesized that these compounds could be used to augment the HSVtk/GCV bystander effect. Addition of all-trans retinoic acid increased GJIC in tumor cell lines, augmented expression of connexin 43, and was associated with more efficient GCV-induced in vitro bystander killing in cells transduced with HSVtk via either retrovirus or adenovirus vectors. This augmentation of bystander effect could also be seen in vivo. HSVtk-transduced tumors in mice treated with the combination of GCV and retinoids were significantly smaller than those treated with GCV or retinoids alone. These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effects and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk/GCV system to treat tumors or vascular restenosis.     

Selective astrocytic transgene expression in vitro and in vivo from the GFAP promoter in a HSV RL1 null mutant vector--potential glioblastoma targeting

Due to the lack of any effective therapy, novel approaches are currently being explored for the treatment of primary brain tumours. It has previously been demonstrated that variants of HSV-1 which are deleted in the RL1 gene and fail to produce the virulence factor ICP34.5 are potential candidates for tumour therapy. The RL1 variant 1716 replicates selectively within tumour cells and has the potential to deliver a therapeutic or tumour killing gene directly to the site of tumour growth. As many intracerebral tumours are glial and predominantly astrocytic in origin, we have evaluated the ability of 1716 to deliver a reporter gene specifically to astrocytes in vivo and in vitro using a 2.2 kb fragment which controls expression of the glial fibrillary acidic protein (GFAP), an astrocyte specific protein. Two 1716 variants, 1774 and 1775, were constructed which contain the GFAP-promoter element linked to the E. coli beta-galactosidase gene, inserted into the HSV-1 UL43 and US5 loci, respectively. In primary cultures, human primary tumour cell lines and established tumour cell lines in vitro, 1774 and 1775 gave high levels of expression of beta-galactosidase specifically in astrocytes. In vivo following intracerebral inoculation, both viruses demonstrated high levels of beta-galactosidase expression predominantly in astrocytes. These results indicate that the GFAP promoter element could be used for efficient and selective transgene delivery to human gliomas.     

Suppression of luteal steroidogenesis by an LHRH antagonist (Nal-Lys antagonist: antide) in vitro during early pregnancy in the rat

Gap-junctional intercellular communication (GJIC) in 20 primary human liver tumors with different degrees of malignancy has been studied at the functional and molecular levels. When GJIC capacity was determined by dye-transfer assay performed directly with freshly removed tumor tissue, significant reduction was found in all samples, regardless of their morphology. In addition, a selective lack of GJIC between tumor and surrounding non-tumorous cells was observed in some cases, probably due to the physical separation between them resulting from encapsulation of tumors. There was, however, no essential change in the level of expression of the major liver gap-junction protein, connexin (cx) 32, in liver tumors as measured by Northern and Western blot analyses. Immunohistochemical study revealed aberrant localization of cx 32 in the majority of malignant liver tumors. Instead of cytoplasmic membrane localization at intercellular contacts, cx 32 was detected mainly either intracytoplasmically or in plasma membrane free from contact with other cells. We did not detect any mutation in the coding sequence of the cx 32 gene from any of the human liver tumors we tested. Thus it is likely that the aberrant localization of cx 32 in tumor cells is due to disruption of the mechanisms for establishment of this protein into gap-junction plaques, rather than to structural abnormality of the cx 32 protein itself. Another member of the connexin family, cx 43, not detectable in non-tumorigenic hepatocytes, was expressed in several tumors, especially in invasive areas, but was detected in only a few tumor cells and was localized intracytoplasmically, suggesting that cx 43 protein is not involved in GJIC in the tumors.     

Influence of catheter materials and tissue composition on low dose rate iridium-192 seed implant dosimetry

Drug sensitivity was studied for the tubulin inhibitors taxol, taxotere, rhizoxin and for doxorubucin and cisplatin, in human lung and breast cancer cell lines, including drug-selected cell lines, overexpressing the membrane transporter P-glycoprotein (Pgp) or the multidrug resistance protein (MRP). All tubulin-inhibiting agents were more potent than doxorubicin and cisplatin in all cell lines. In the drug resistance-selected cell lines (doxorubicin or mitoxantrone resistant) there was cross-resistance between the tubulin inhibitors and the selecting agent; however, MRP overexpressing cells were relatively less resistant to taxanes than the Pgp overexpressing cells. Polymerization of microtubules after exposure to taxol was observed in drug sensitive cell lines, but not in resistant cell lines, even at high taxol concentrations and after long exposure times. In the Pgp overexpressing cell lines, steady accumulation of 14C-taxol was defective and could be reverted by verapamil. MRP overexpressing cells did not have a significant accumulation defect of taxol, compared to the parental cell lines, and verapamil did not have any effect. These data confirm that the Pgp overexpression is an important mechanism of resistance to taxanes and rhizoxin in human lung and breast tumor cells. However, the presence of mechanisms other than transport defects may play an important role in non-Pgp expressing cells, and these may include an altered function of tubulins.     

Studies on mechanism about ATP inhibited the proliferation of MGC-803 cells

This experiment was to study the mechanism of ATP anticancer effects. By using flow cytometry, Scrape-Loading and dye transfer (SLDT), dot hybridization methods, changes of cell cycle phase distribution, gap junctional intercellular communication (GJIC), and oncogene expression were observed in human stomach mucous glandular carcinoma (MGC-803) cells treated with ATP (0.23 mg/ml). It was found that ATP inhibited the proliferation and arrested cell cycle in S phase. The ATP-treated MGC-803 cells increased in GJIC, and decreased in expression of c-Ha-ras oncogene. These results indicated that the inhibition of proliferation and increased GJIC was closely correlated with the reduction of c-Ha-ras oncogene expression.     

Growth inhibitory response to activin A and B by human prostate tumour cell lines, LNCaP and DU145

Activins are growth and differentiation factors which have been shown to have proliferative and antiproliferative actions in many tissues. In addition, they have been implicated in tumourigenesis in reproductive tissues. Although activin and inhibin are present in rat ventral prostate, inhibin beta, but not alpha, subunit proteins have been detected in the human prostate epithelial tumour cell lines LNCaP, DU145 and PC3. With this absence of capacity to produce inhibins, the aims of this study were to determine the effect of activin A and B and follistatin on DNA synthesis by these human prostate tumour cell lines. The results demonstrate a differential response to exogenously added activin A and B on DNA synthesis in vitro by the tumour cell lines. The inhibitory effects were observed on LNCaP cells in the absence or presence of stimulation with 1 nM 5 alpha-dihydrotestosterone and on the androgen-independent DU145 cells, but not the PC3 cells. Activin A caused a dose-dependent inhibition of DNA synthesis and proliferation by LNCaP and androgen-independent DU145 cells which was maximal at 8 ng/ml. The effect of exogenously added activin A was completely reversed by follistatin, but not by inhibin A. The addition of human recombinant FS 288 alone (400 ng/ml) did not have any effect on DNA synthesis, whereas inhibin A alone (400 ng/ml) caused a significant inhibition of DNA synthesis. The capacity of all three cell lines to produce activins and follistatins was demonstrated by the expression of the mRNAs and confirmed by the localisation of immunoreactivity for these ligands to the cytoplasm of the tumour cells. The growth inhibitory response to activins A and B by LNCaP and DU145 cells, and the ability of follistatin to block these effects, suggest that the autocrine interactions between activins and follistatins have a role in the regulation of LNCaP and DU145 prostate tumour cell growth.