The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis.

Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules <1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.

Metal salts as promoters of in vitro morphological transformation of hamster embryo cells initiated by benzo(a)pyrene.

The hamster embryo cell bioassay has been used to study the effect of metal salts on morphological transformation. A synergistic enhancement of the transformation frequency was found for the combined treatment with organic carcinogens [benzo(a)pyrene, N-hydroxy-2-acetylaminofluorene, and 4-nitroquinoline 1-oxide] and nickel sulfate, cadmium acetate, or potassium chromate. Chromic chloride and zinc chloride did not induce transformation themselves, and they had no effect on the transformation frequency when tested in combination with benzo(a)pyrene. The synergistic effect between benzo(a)pyrene and nickel sulfate or cadmium acetate was also apparent when the cells were treated sequentially with the chemicals. When the cells were first exposed to benzo(a)pyrene, both nickel sulfate and cadmium acetate showed a promotion-like effect similar to that obtained with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Moreover, when 12-O-tetradecanoylphorbol-13-acetate or benzo(a)pyrene were used as promoting agents, both nickel sulfate and cadmium acetate were able to initiate morphological transformation. The data suggest that the metal salts are more potent as promoters than they are as initiators. The present findings may be of importance in relation to carcinogenicity of metal compounds to humans.

Contrasting duration of inhibition of cell-cell communication in primary mouse epidermal cells by phorbol 12,13-dibutyrate and by bryostatin 1.

The bryostatins, macrocyclic lactones isolated on the basis of their antineoplastic activity, activate protein kinase C in vitro and inhibit phorbol ester binding to the enzyme. In intact cells, the bryostatins induce some phorbol ester responses, such as neutrophil activation, but paradoxically they not only fail to induce other responses, e.g., differentiation in HL-60 promyelocytic leukemia cells, but actually block response to the phorbol esters. We compare here bryostatin I and phorbol 12,13-dibutyrate as inhibitors of cell-cell communication in cultured primary mouse epidermal cells. Like phorbol 12,13-dibutyrate, bryostatin I at nanomolar concentrations markedly inhibited cell coupling. It differed from the phorbol esters, however, in that its action was more transient. By 4 h of incubation bryostatin 1 caused little inhibition of coupling. Moreover, coincubation of bryostatin 1 and phorbol 12,13-dibutyrate gave no greater response at this time than that found for bryostatin 1 alone. Time-dependent inhibition of the protein kinase C pathway could account for many of the observed differences between the actions of the phorbol esters and bryostatin 1.

Role of ions in the regulation of porcine lactate dehydrogenase.

Different ions affect the H4 and M4 isoenzymes of porcine lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) in the same way, inhibiting the enzyme at low pyruvate concentrations, whereas at high pyruvate concentrations, the activities were enhanced. The inhibition was competitive with regard to pyruvate and NADH. The enhancement of the enzyme activity at high pyruvate concentration is due to the increase in the Km value for pyruvate, implying that higher substrate concentrations are needed to obtain substrate inhibition. Sulphate behaved differently from the other ions. It inhibited in a noncompetitive manner with regard to pyruvate and did not activate the enzyme at high pryvuate concentration. The effect of ions increased with the size of the anion. The ionic strength was of less importance.

Role of plasma fibronectin in the morphological transformation of hamster embryo cells.

Replacement of fetal bovine serum (FBS) by newborn serum in the hamster embryo cell transformation assay blocks the induction of morphologically transformed colonies by chemical carcinogens. Moreover, the addition of newborn serum strongly inhibits the formation of morphologically transformed colonies in the standard assay using 20% FBS. The present experiments show that the inhibitory factor is heat labile and not dialyzable, but is removed when the newborn serum is submitted to gelatin affinity chromatography. The inhibitory factor is recovered in the eluted gelatin bound material which gives essentially one band in SDS electrophoresis corresponding to that of plasma fibronectin. In separate experiments fibronectin from bovine plasma is shown to inhibit cell transformation in a similar way. The experiments suggest that fibronectin plays a central role in the mechanism of morphological cell transformation.

Reversal of the promotional effect of 12-O-tetradecanoylphorbol-13-acetate on morphological transformation of hamster embryo cells by glucocorticoids.

The effects of glucocorticoids and cholesterol on morphological transformation have been studied using hamster embryo cells. The cells were exposed sequentially to benzo[a]pyrene (BP) (3 days) and the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) (4 days) before scoring for morphologically transformed colonies. Dexamethasone and hydrocortisone strongly inhibited the formation of morphologically transformed colonies when applied to the cells in the second period of exposure together with TPA, but had no effect when present with BP during the first period. Corticosterone had only a weak effect, while cortisone had no effect on the transformation frequency. Cholesterol gave rise to an enhanced number of transformed colonies. Dexamethasone, being the most potent compound tested, inhibited morphological transformation by more than 50% when present in a concentration of 0.25 nM. The finding that dexamethasone reduced the frequency of transformation even when added only 6 h prior to staining, indicates that the glucocorticoids may also reduce the transformation frequency by reversing morphologically transformed cell colonies to a normal appearance.

Synergistic effect on morphological transformation of hamster embryo cells by nickel sulphate and benz[a]pyrene.

Morphological transformation and induction of somatic mutation in the hamster embryo cell bioassay have been used to study whether the carcinogenicity of nickel is affected by polycyclic hydrocarbons. The transformation frequency was found to increase with increasing concentration of nickel sulphate, benz[a]pyrene (BP) and methylcholanthrene. In experiments with combinations of nickel sulphate and BP, the transformation frequencies used for all concentrations were higher than for compounds tested separately. The greatest enhancement was found using 5 micrograms/ml NiSO4 . 6H2O and 0.78 microgram/ml BP. The transformation frequency obtained with this combination was 10.7%, compared to 0.5% and 0.6% for the individual substances. No synergistic effect could be detected between nickel sulphate and methylcholanthrene (MC). In experiments measuring somatic mutation by selection for ouabain resistance, the mutation frequency was likewise found to be significantly higher than expected in mixtures of nickel sulphate and BP. The present demonstration of the synergistic effect between nickel sulphate and BP is of interest with the potentiating effect of cigarette smoking on development of lung cancer among nickel refinery workers.

Potentiating effect of cigarette smoke extract on morphological transformation of hamster embryo cells by benzo[alpha]pyrene.

Morphological transformation in the hamster embryo cell bioassay has been used to study the possibility that carcinogenicity of benzo[alpha]pyrene (BP) is affected by cigarette smoke extract and whether smoke extract will promote transformations initiated by BP. The transformation frequency increases wth increasing concentrations of BP and smoke extract. In experiments with a combined treatment of BP and smoke extract, the transformation rates were higher than expected for all concentrations from experiments with the compounds tested separately. The greatest potentiating effect was found using 0.01 micrograms/ml BP and 1 microgram/ml smoke extract. The transformation frequency obtained with this combination was 4.3% compared to 1.4% and zero respectively for the individual substances. In experiments where cells were treated sequentially with BP (0.05 micrograms/ml) for 4 days followed by smoke extract (1 or 5 micrograms/ml) for the next 4 days, the transformation frequency was significantly higher than expected on the basis of the compounds tested separately. The demonstration of a synergistic effect between BP and cigarette smoke and the promotion-like effect of smoke extract on BP-initiated transformations of hamster embryo cells are of interest in relation to the higher frequency of lung cancer found in areas with high air pollution compared to rural areas.

Promotional effect of different phorbol esters on morphological transformation of hamster embryo cells.

Hamster embryo cells were sequentially exposed to benzo[a]pyrene (BaP) (3 days) and different phorbol esters (4 days). Compounds which have been shown to act as tumor promoters in mouse skin carcinogenesis, showed a promotion-like effect on the formation of morphologically transformed colonies, while non-promoting analogs did not enhance the transformation frequency. In experiments where the exposure sequence was reversed, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) in the first exposure period and BaP in the second period, no significant enhancement of the transformation frequency was observed. The exposure to a low concentration of BaP in the second exposure period following a higher concentration in the first period, resulted in a strong enhancement of the transformation frequency suggesting that BaP also acts as a promoter.

Caffeine and other phosphodiesterase inhibitors are potent inhibitors of the promotional effect of TPA on morphological transformation of hamster embryo cells.

The phosphodiesterase inhibitors caffeine, theophylline, aminophylline and isobutyl-methylxanthine (IBMX) were found to inhibit induction of morphologically transformed hamster embryo cell colonies by sequential exposure to benzo[a]pyrene (BaP) and the tumor promoter TPA. Almost complete inhibition of cell transformation was observed when 50 micrograms/ml theophylline, aminophylline, IBMX, or 200 micrograms/ml caffeine was present together with the tumor promoter. The compounds had no effect on the transformation frequency when present together with the initiator, BaP, in the first exposure period. Substances that stimulate the adenylate cyclase and the addition of exogenous dibutyryl-cAMP had similar inhibitory effects.

Effect of glucocorticoids on TPA-induced inhibition of gap-junctional communication and morphological transformation in Syrian hamster embryo cells.

The effect of glucocorticoids on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibition of gap-junctional intercellular communication (GJIC) and morphological transformation in Syrian hamster embryo (SHE) cells was examined. Fluocinolone acetonide (FA) and dexamethasone (DEX) almost completely suppressed the effect of TPA on induction of transformed morphology. On the other hand, up to 1000 times higher FA and DEX concentrations did not influence the inhibitory effect of TPA on GJIC. Neither treatment with these glucocorticoids for 4, 24 or 48 h before TPA exposure nor 24 h co-exposure with TPA altered the effect of TPA on GJIC. Thus the potent effect of glucocorticoids as inhibitors of the promotional effect of TPA on morphological transformation in SHE cells does not result in alterations of TPA-induced inhibition of GJIC.

Nickel-induced alterations in human renal epithelial cells.

Cellular progression to malignancy appears to require a number of distinct steps in which genetic damage in key regulatory genes accumulates. Immortalization, or escape from senescence, is considered to be one of the first phenotypic changes. Ni2+ treatment of normal human kidney epithelial (NHKE) cells in vitro resulted in immortalization of the cells IHKE cells). The combined action of Ni2+ and v-Ha-ras oncogene fully transformed the cells to tumorigenicity in athymic nude mice. Sequence analysis of DNA from IHKE cells revealed point mutation in the p53 gene at codon 238 with T-->C transition. These findings suggest that Ni-induced mutation in the p53 gene can be involved in the immortalization of the NHKE cells. The results also show that changes in the responses to EGF and TGF beta and in the expression of their receptors occur during malignant progression in vitro.

Gap junctional intercellular communication is not a major mediator in the bystander effect in photodynamic treatment of MDCK II cells.

Photodynamic treatment (PDT) of confluent MDCK II cells resulted in a noticeable clustering of dead cells, consistent with a significant bystander effect. Likewise, PDT of cells in microcolonies resulted in an overabundance of microcolonies that had responded to the treatment as a single unit, that is, in which either all or no cells were dead. Confluent MDCK II cells appeared to communicate via gap junction channels, while cells in microcolonies did not. Monte Carlo simulation models were fitted to the distributions of dead cells in confluent monolayers and in microcolonies. The simulations showed that the degree of the bystander effect was higher in microcolonies than in confluent cells, suggesting that gap junction communication may be involved in the bystander effect. However, when the gap junction hypothesis was tested by treatment of microcolonies with 30 microM dieldrin, an inhibitor of gap junctional intercellular communication, there was no reduction of the bystander effect, indicating that this effect was not mediated by gap junctional intercellular communication. PDT influenced phosphorylation of tyrosine residues in several proteins in the cells. Protein phosphorylation is important in cellular signaling pathways and may be involved in the bystander effect, for example by influencing the mode of cell death.

Effect of 1,1'-(2,2,2-trichloroethylidene)-bis(4-chlorobenzene) (DDT) on gap junctional intercellular communication and morphological transformation of Syrian hamster embryo cells.

The organochlorine insecticide 1,1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene) (DDT) did not induce or promote induction of morphological transformation in Syrian hamster embryo (SHE) cells, but it was a potent inhibitor of gap junctional intercellular communication (GJIC). The kinase inhibitor staurosporine did not affect DDT induced inhibition of GJIC, although it has been shown to decrease the inhibitory effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on GJIC. In addition, pretreatment with TPA made the cells refractory to further TPA induced inhibition of GJIC, while they remained sensitive to DDT. Thus, DDT and TPA inhibit GJIC through different mechanisms. Elevation of cellular cyclic adenosine monophosphate (cAMP) level by exposure to forskolin counteracted the inhibitory effect of DDT similar to that observed for TPA. Continuous exposure to DDT at concentrations near the effective concentration (50%) value (EC50 value) resulted in a slight recovery of GJIC following the initial inhibition. This recovery was not accompanied by the cells becoming refractory to further DDT induced inhibition of GJIC. The recovery of GJIC after removal of the DDT containing medium seemed to be related to a reduction in the amount of cell-associated DDT.

Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project.

While the accumulation of genetic changes in a somatic cell is considered essential for the genesis of a cancer, it has become clear that not all carcinogens are genotoxic, suggesting that some carcinogens indirectly participate in the generation of genetic changes during carcinogenesis. A European project funded by the European Community was thus conceived to study mechanisms of nongenotoxic aspects of carcinogenesis. Two main strategical approaches were adapted: (i) to study whether and how Syrian hamster embryo (SHE), Syrian hamster dermal (SHD) and BALB/c 3T3 cell transformation systems simulate in vivo carcinogenesis, and to examine whether they can detect nongenotoxic carcinogens; (ii) to study, refine and validate mechanisms-based end-points for detection of nongenotoxic carcinogens. For mechanisms-based research, the proposed end-points included gap junctional intercellular communication (GJIC) inhibition, altered expression of critical genes, immortalization and aberrant cell proliferation. We also selected model compounds commonly usable for various endpoints. Our major results can be summarized as follows: (1) SHE and BALB/c 3T3 transformation systems reflect both genotoxic and nongenotoxic carcinogenic events; they detect not only genotoxic but also many although not all, nongenotoxic carcinogens. This is further supported by the fact that both genotoxic and nongenotoxic carcinogens were able to immortalize SHD cells. (2) Many nongenotoxic carcinogens, although not all, inhibit GJIC in vitro as well as in vivo. Mechanistic studies suggest an important role of blocked GJIC in carcinogenesis and that different mechanisms are involved in inhibition of the communication by different agents used. However, inhibition of GJIC is not a prerequisite for the enhancement (or induction) of transformation of SHE or BALB/c 3T3 cells. (3) Among compounds examined, there was a good correlation between induction of micronuclei and cell transformation in SHE cells while no such correlation was found between the induction of cell transformation and ornithine decarboxylase activity. (4) Two transgenic mouse mutation assays (lacI and lacZ) were established and validated. The genotoxin dimethylnitrosamine was shown to be mutagenic to the liver in both assays. Ortho-anisidine, a bladder-specific carcinogen that was inactive in standard rodent genetic toxicity assays was uniquely mutagenic to the bladder of the transgenic mice. The peroxisome proliferator methyl clofenipate was established as nonmutagenic to the liver of both transgenic mice. That eliminated DNA damage as a cause of the liver tumours produced by this chemical and weakened the idea that induced cell division leads to mutation induction. (5) With an in vitro DNA replication model, it was found that DNA damage induced by genotoxic agents can be responsible for inhibition of DNA replication, while certain nongenotoxic agents such as phorbol esters increase DNA replication. (6) An attempt to use structure-activity relationship for subfamilies of nongenotoxic carcinogens, e.g., receptor-mediated carcinogens, has been initiated with some promising results. Our results support the idea that there are multiple nongenotoxic mechanisms in carcinogenesis, and that working hypothesis-oriented approaches are encouraged rather than simple screening of chemicals in developing test systems for the detection of nongenotoxic carcinogens.

Role of serum in the morphological transformation of Syrian hamster embryo cells: Characterization and partial purification of protein factors in foetal bovine serum.

The expression of transformed colony morphology in Syrian hamster embryo (SHE) cells, and thus the results obtained in the SHE cell transformation assay, is dependent on the source of the foetal bovine serum (FBS) used. The purpose of this study was to characterize the factors in FBS that are necessary for the expression of transformed morphology. The factors were of protein nature (precipitated by ammonium sulfate and non-dialysable), sensitive to heating and thiol reagents, but resistant to acid and solvent treatment. The active factor(s) were found to bind to a number of protein purification media for ion exchange or affinity purification, and it was initially difficult to reconstitute the biological activity from eluted fractions. This loss of activity was not caused by the separation of more than one necessary factor, but by the factors being highly hydrophobic and negatively charged, and therefore strongly bound to the column material. The active factors could be eluted from Affigel Blue in 50% ethylene glycol, but not in 4m NaCl. The bioactive protein fraction could be further fractionated by gel permeation chromatography on Biogel P-60 in 1 m acetic acid, and cation exchange chromatography on MonoS with 20% acetonitrile added to the buffers. Isoelectric focusing on a Rotofor cell indicated two peaks of transforming activity, one with isoelectric point at about pH 8.5, and one at pH 9.5. The finding of two peaks of biological activity is supported by reversed phase chromatography studies. Bioactivity of two fractions from isoelectric focusing with pI around 8.5 and 9.5 were eluted at propanol concentrations of 20 and 27%, respectively. In the present studies, we were unable to identify the factors with transformation supporting activity, probably because of the high content of protein/peptides with similar biochemical properties in FBS. In further studies we will seek to demonstrate whether previously isolated growth factors, or signalling substances, with similar biochemical properties support the expression of the morphologically transformed phenotype in SHE cells.

TPA inhibition of gap junctional intercellular communication in Syrian hamster embryo cells through two different pathways.

Exposure of early passage Syrian hamster embryo cells to high TPA concentrations (8 or 16 mum) decreased gap junctional intercellular communication (GJIC) to about 20% of the control during the first hour. Subsequently, the GJIC capacity recovered to 70-80% after 4-10 hr. Exposure to lower TPA concentrations also reduced GJIC, but did not result in the subsequent rapid enhancement of communication. Treatment of the cells to different TPA concentrations down-regulated protein kinase C (PKC), but this down-regulation did not seem to explain fully the induced recurrence of GJIC following high TPA concentrations. The maximal down-regulation of PKC took place at TPA concentrations above 0.16 mum. Addition of 8 mum TPA to cells pretreated with 0.016 and 8 mum for 10 hr reduced GJIC from 80 to 40%, whereas pretreatment with 0.16 or 1.6 mum TPA made the cells refractory to further TPA-induced inhibition. The PKC inhibitor staurosporine suppressed the effect of TPA on GJIC in cells exposed to 0.016 mum TPA, whereas no suppression was observed in cells depleted of PKC. The results indicate that TPA is capable of inhibiting GJIC through two different mechanisms, a staurosporine-sensitive mechanism at low TPA concentrations (EC(50) = 0.18 mum) and a staurosporine-insensitive mechanism at higher concentrations (EC(50) = 7 mum).

Effect of protein kinase C inhibitors on TPA-induced inhibition of gap junctional intercellular communication and induction of transformed morphology in Syrian hamster embryo cells.

Staurosporine and H-7, but not polymyxin B, were found partly to suppress the effect of TPA on both inhibition of gap junctional intercellular communication (GJIC), down-regulation of EGF-binding and induction of transformed morphology in Syrian hamster embryo (SHE) cells. The parallel effect of these kinase inhibitors suggests that TPA induces the effects through activation of protein kinase C (PKC). This is consistent with the view that PKC is involved in both the induction of transformed morphology and the inhibition of GJIC. All the inhibitors studied reduced PKC activity in SHE cell extracts. IC(50) values were determined to be 0.008 mum for staurosporine, 55 mum for H-7 and 16 mum for polymyxin B. The values for staurosporine and H-7 corresponded to those concentrations that suppressed TPA-induced cellular responses. The lack of effect of polymyxin B on whole SHE cells indicates that this compound does not enter the cells.

Morphological transformation and effect on gap junction intercellular communication in Syrian hamster embryo cells as screening tests for carcinogens devoid of mutagenic activity.

A large fraction of chemicals observed to cause cancer in experimental animals is devoid of mutagenic activity. It is therefore of importance to develop methods that can be used to detect and study environmental carcinogenic agents that do not interact directly with DNA. Previous studies have indicated that induction of in vitro cell transformation and inhibition of gap junction intercellular communication are endpoints that could be useful for the detection of non-genotoxic carcinogens. In the present work, 13 compounds [chlordane, Arochlor 1260, di(2-ethylhexyl)phthalate, 1,1,1-trichloro-2, 2-bis(4-chlorophenyl)ethane, limonene, sodium fluoride, ethionine, o-anisidine, benzoyl peroxide, o-vanadate, phenobarbital, 12-O-tetradecanoylphorbol 13-acetate and clofibrate] have been tested for their ability to induce morphological transformation and affect intercellular communication in Syrian hamster embryo cells. The substances were selected on the basis of being proven or suspected non-genotoxic carcinogens, and thus difficult to detect in short-term tests. The data show that nine of the 13 compounds induced morphological transformation, and seven of the 13 inhibited intercellular communication in hamster embryo cells. Taken together, 12 of the 13 substances either induced transformation or caused inhibition of communication. The data suggest that the combined use of morphological transformation and gap junction intercellular communication in Syrian hamster embryo cells may be beneficial when screening for non-genotoxic carcinogens.