Antitumor effect of non-steroid glucocorticoid receptor ligand CpdA on leukemia cell lines CEM and K562.

Glucocorticoids (GCs) are widely used in chemotherapy of hematological malignancies, particularly leukemia. Their effect is mediated by glucocorticoid receptor (GR), a well-known transcription factor. Besides their therapeutic impact, GCs may cause a number of side effects leading to various metabolic complications. The goal of immediate interest is testing glucocorticoid analogs capable of induction/enhancement of GR transrepression, but preventing GR dimerization and transactivation leading to side effects. In this work we have investigated effects of a promising new selective GR agonist, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride (CpdA), on CEM and K562 leukemia cells. Both cell lines express functional GR. CpdA compared with the glucocorticoid fluocinolone acetonide (FA) exerted more prominent cytostatic and apoptotic effects on the cells. Both cell lines exhibited sensitivity to CpdA, demonstrating a good correlation with the effects of FA on cell growth and viability. In contrast to FA, CpdA did not induce GR transactivation evaluated by no obvious increase in expression of GR target (and dependent) gene FKBP51. At the same time, luciferase assay showed that CpdA efficiently activated transrepression of NF-κB and AP-1 factors. We also evaluated the effect of combined action of CpdA and the proteasome inhibitor Bortezomib. The latter induced a caspase-dependent apoptosis in both T-cell leukemia cell lines. By treatment of CEM cells with different CpdA/GC and Bortezomib doses, we have designed a protocol where CpdA shows potentiating effect on Bortezomib cytotoxic activity. Generally, the present work characterizes a novel non-steroid GR ligand, CpdA, as a promising compound for possible application in leukemia chemotherapy.

[Immunohistochemical study of the prostate-specific antigen in prostatic cancer]. / Immunogistokhimicheskoe issledovanie prostaticheskogo spetsificheskogo antigena v rake predstatel'noi zhelezy.

Prostate-specific antigen (PSA) is secreted both by normal epithelial prostatic cells and cell of prostatic carcinoma (PC). No parallelism exists between the degree of PC differentiation and the type of PSA secretion. PSA concentration in the peripheral blood not always corresponds to the intensity of its immunoreactivity on the tissue level. Amount of PSA in the blood depends on the stroma vascularisation and number of cells contacting with the organ stroma. A decrease of PSA in the peripheral blood due to therapy may be combined with high intensity of its synthesis in tumor cells. PSA immunohistochemistry may be recommended as a method of PC clinical course monitoring and dynamics of its changes in the course of carcinoma therapy.

Increased expression of p50-NF-kappaB and constitutive activation of NF-kappaB transcription factors during mouse skin carcinogenesis.

To elucidate the possible role of NF-kappaB in mouse skin carcinogenesis we studied the expression of p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA) and IkappaB-alpha inhibitor as well as kappaB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IkappaB-alpha protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in kappaB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive kappaB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IkappaB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different kappaB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related kappaB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.

Resistance of transformed mouse keratinocytes to growth inhibition by glucocorticoids.

Glucocorticoid hormones are strong inhibitors of normal keratinocyte proliferation, but established mouse skin papillomas and carcinomas become resistant to these hormones. The biological effect of glucocorticoids is mediated through a highly specific glucocorticoid receptor (GR). To study the possible mechanisms of glucocorticoid resistance of transformed mouse keratinocytes, we evaluated GR expression and function in non-tumorigenic (3PC), papilloma-producing (MT1/2 and P1/17), and squamous cell carcinoma-producing (Ca3/7 and Ca8/29) keratinocyte cell lines and analyzed the DNA sequence of GR in glucocorticoid-sensitive and glucocorticoid-resistant keratinocytes. All transformed keratinocyte cell lines studied appeared to be completely resistant to the growth inhibition by the glucocorticoid fluocinolone acetonide (FA), whereas the untransformed cell line 3PC was very sensitive to FA. Despite the glucocorticoid resistance, all the tumorigenic keratinocyte cell lines expressed high levels of GR mRNA and protein. Southern blot analysis and direct sequencing of the DNA-binding domain of the GR gene revealed no significant changes in GR gene structure in transformed keratinocytes. To test the functional capability of GR, we compared the effect of FA on the expression of glucocorticoid-responsive genes. FA strongly induced metallothionein 1 expression in 3PC cells, slightly induced metallothionein 1 expression in P1/17 and Ca3/7 cells, and did not affect its expression in MT1/2 and Ca8/29 cells. These data suggest that resistance to the growth inhibition of glucocorticoids is an important feature of tumorigenic keratinocyte cell lines. It is likely that this hormone-resistant phenotype is a result of alteration of GR function but not of GR expression or gene structure.

Changes in protein expression during multistage mouse skin carcinogenesis.

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene-initiated, 12-O-tetradecanoylphorbol-13-acetate-promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-beta1 (TGFbeta1) were strongly expressed in the suprabasal layers, whereas integrin alpha6beta4 was expressed only in basal cells and only moderate staining for transforming growth factor-alpha (TGFalpha) was seen. In hyperplastic skin, TGFalpha expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of alpha6beta4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20-30 wk), focal areas of gamma-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFbeta1 expression began to decrease. Cx26 and TGFalpha staining became patchier in some late-stage papillomas (30-40 wk), whereas suprabasal alpha6beta4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFalpha, HB-EGF, TGFbeta1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for alpha6beta4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity.

Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis.

Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

The mouse skin carcinogenesis model.

Skin carcinogenesis can be divided into at least three major stages: initiation, promotion, and progression. In the mouse skin model, the first stage is thought to involve the interaction of a tumor initiator with the genetic material of stem cells, leading to an irreversible alteration in growth control or differentiation, probably by activation of the Ha-ras oncogene. The major effect of all skin-tumor promoters seems to be the specific expansion of the initiated stem cells. The correlation between the abilities of tumor promoters to induce sustained hyperplasia and their tumor-promoting activities is very good. We found that the appearance of alpha-glutamyl transpeptidase (GGT) and keratin 13 and the lack of expression of keratins 1 and 10 are good markers for skin tumor progression. These alterations occur when papillomas change from a diploid to an aneuploid state, mainly as a result of developing trisomies 6 and 7. To evaluate the role of GGT in skin-tumor progression, we transfected a functional GGT cDNA into two cell lines that normally produce papillomas when grafted into the skin of nude mice. When injected subcutaneously, all of the GGT-transfected clones formed malignant tumors, whereas only 24% of vector-transfected cells did. When GGT-transfected clones were placed into grafts, the grafts had an average mass almost three times that of grafts of vector-transfected cells. Our recent studies also suggest that the ribonucleoprotein telomerase and the gap-junctional proteins connexins (Cxs) are also important in skin-tumor progression. A progressive increase in telomerase activity was associated with the increased level of genomic instability during tumor progression. In addition, the level and expression of Cx26, Cx43, and Cx31.1 were significantly altered during skin tumor promotion and progression. The differences of various mouse stocks and strains in susceptibility to multistage skin carcinogenesis seem to be related more to alterations in tumor promotion than to tumor initiation; however, the critical events have not been determined. Results with an inbred strain of SENCAR mice, which are very sensitive to papilloma formation by the two-stage protocol, also suggest that susceptibility is related to promotion. Despite the high incidence of papillomas in these inbred SENCAR mice, the number of malignant tumors was extremely low, suggesting that sensitivity to promotion and progression are independent in these mice.

Connexin expression in epidermal cell lines from SENCAR mouse skin tumors.

Alteration of gap-junctional intercellular communication (GJIC) has long been proposed to be involved in carcinogenesis. Previously, we reported that the level of gap junctional intercellular communication in mouse skin carcinoma cell lines is significantly lower than in papilloma cell lines and normal mouse keratinocytes Klann et al., Cancer Res 49:699-705, 1989). Here, we present data on expression of the gap-junctional protein connexins (Cx) 26, Cx31.1, and Cx43 in a comprehensive panel of keratinocyte cell lines representing different stages of mouse skin carcinogenesis and the effect of different conditions of propagation on Cx phenotype. Northern and western blot analyses and immunostaining showed that all cell lines studied in vitro expressed Cx43 but most did not express Cx31.1 or Cx26. The abundance of Cx43 expression on plasma membranes correlated well with the level of GJIC. In vivo expression of Cx43 and Cx26 was strongly increased. Whereas none of tumorigenic cell lines expressed Cx26 gap junctions in culture, those growing as tumors in nude mice began to express Cx26 protein. The comparison of Cx expression on the keratinocyte membranes in three different groups of tumors (papillomas and squamous cell and spindle cell carcinomas) clearly revealed that the abundance of Cx43 and Cx26 expression directly correlated with the level of tumor differentiation. All studied tumors were Cx31.1 negative. These results suggest that both Cx expression and gap-junction permeability are gradually reduced during the tumor progression stage of mouse skin carcinogenesis.

Effect of diverse tumor promoters on the expression of gap-junctional proteins connexin (Cx)26, Cx31.1, and Cx43 in SENCAR mouse epidermis.

The inhibition of gap-junctional intercellular communication (GJIC) between initiated and surrounding normal cells by tumor promoters is believed to be important in the promotion stage of carcinogenesis. Therefore, we examined the effect of skin-tumor promoters on the expression of the gap-junctional proteins connexin (Cx) 26, Cx43, and Cx31.1 in SENCAR mouse skin. Animals were treated with 12-0-tetradecanoylphorbol-13-acetate (TPA) (8.3 nmol), okadaic acid (OA) (2.5 nmol), chrysarobin (220 nmol), or benzoyl peroxide (BzPo) (83 micromol). Northern blot and immunofluorescence analyses revealed that keratinocytes in adult mouse skin expressed Cx31.1 and Cx43 but not Cx26. All four of the skin-tumor promoters switched on the Cx26 gene, transiently increased expression of Cx43, and significantly inhibited the expression of Cx31.1. The time courses for changes in Cx26, Cx3l. 1, and Cx43 mRNA levels coincided in most cases and in general corresponded well to the time-response curves for hyperplastic changes in mouse skin. The peaks of Cx26 and Cx43 expression and Cx31.1 inhibition appeared 12 h after TPA application and 24 h after OA and chrysarobin application. BzPo elevated the levels of Cx26 and Cx43 transcripts later (peak at 2-4 d). In tumor promoter-treated skin, Cx26 and Cx43 plaques were on the plasma membrane of most keratinocytes. Cx31.1 staining was much weaker than in untreated epidermis. Thus, tumor promoters induce a large change in the expression of several Cxs, which in turn may affect both the level of GJIC and the sensitivity of GJlC to regulatory factors.

The expression of gap junctional proteins during different stages of mouse skin carcinogenesis.

The loss or alteration of gap junctional intercellular communication (GJIC) has long been proposed to play an important role in the process of carcinogenesis. In this study we examined the expression of three gap junction proteins, connexins (Cx)26, 43 and 31.1, in mouse hyperplastic skin, papillomas and squamous cell carcinomas (SCC). Tumors were induced in SENCAR mice by either of two initiation/promotion protocols: 7,12-dimethylbenz-[a] anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA) or DMBA/benzoyl-peroxide (BzPo). Keratinocytes in adult mouse skin expressed Cx31.1 and Cx43 but did not express Cx26. Skin hyperplasia induced by one topical application of TPA was accompanied by hyperexpression of both Cx26 and Cx43. In addition, TPA significantly inhibited the expression of Cx31.1. After repetitive application. Connexin 26 and Cx43 were hyperexpressed in most of the papillomas studied (20-40 weeks after initiation). However, in some late papillomas, immunostaining revealed a focal loss of Cx26. Immunostaining of mouse skin SCC revealed decreased Cx43 and Cx26 levels in 65% and 85% of cases respectively. The high levels of Cx26 and Cx43 mRNA in most of the SCC did not correlate with the decreased abundance or disappearance of Cx26 and Cx43 immunoreactive spots from tumor plasma membranes. Thus, the expression of these two connexins in SCC was impaired at the post-translation level. Cx31.1 expression was strongly inhibited during all stages of carcinogenesis. Taken together, our results suggest that three different connexin genes are differentially regulated during mouse skin carcinogenesis and the decrease of connexin expression may be an important marker of skin tumor expression.

Skin carcinogenesis: characteristics, mechanisms, and prevention.

Skin carcinogenesis can be operationally and mechanistically divided into at least three major stages - initiation, promotion and progression. Variations among stocks and strains of mice to susceptibility to multistage skin carcinogenesis appear to be more related to alterations in tumor promotion than tumor initiation; however, the critical events have not been determined. In the mouse skin model the first stage is thought to involve the interaction of a tumor initiator with the genetic material of stem cells leading to an irreversible alteration in some aspect of growth control and/or differentiation, probably activating the Ha-ras oncogene. Some skin tumor promoters such as the phorbol esters, indole alkaloids, and polyacetates, appear to act through protein kinase C leading to specific phosphorylation of cellular proteins whereas others such as okadaic acid class of compounds appear to act through phosphatases also leading to an increase in phosphorylation. In addition, other types of tumor promoters such as peroxides, benzo(e)pyrene, and chrysarobin may act through a free radical mechanism. Regardless of the type, the major effect of the skin tumor promoters appears to be the specific expansion of the initiated stem cells in the skin. There is a very good correlation between the abilities of tumor promoters to induce a sustained hyperplasia and their tumor promoting activities. This appears to occur by both direct and indirect mechanisms involving the loss of glucocorticoid receptors, differentiation alterations, a direct growth stimulation of the initiated cells and/or selective cytotoxicity. A number of growth factors have recently been found to be increased during tumor promotion and may be responsible for the increase in cell proliferation. An inhibition of cell-cell communication and stimulation of differentiation of non-initiated cells appear to be important indirect mechanisms of further expanding the initiated cell population. The appearance of GGT and keratin 13 (K13) and the lack of expression of K1 and K10 were found to be good markers for skin tumor progression. These alterations occur at the time papillomas change from a diploid to aneuploid state which is mainly due to trisomies of chromosome 6 and 7. In order to evaluate a casual role for GGT in skin tumor progression, a functional GGT cDNA was transfected into two of our cell lines which normally produce papillomas when grafted into the skin of nude mice. The GGT positive cells and the vector transfected cells (controls) from one of the cell lines were cloned and injected into nude mice and placed into transplantation chambers.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell culture assays for chemicals with tumor-promoting or tumor-inhibiting activity based on the modulation of intercellular communication.

The ability of chemicals with tumor-promoting or tumor-inhibiting activity to modulate gap junctional intercellular communication is reviewed. The two most extensively used types of assays for screening tests are (1) metabolic cooperation assays involving exchange between cells of precursors of nucleic acid synthesis and (2) dye-transfer assays that measure exchange of fluorescent dye from loaded cells to adjacent cells. About 300 substances of different biological activities have been studied using various assays. For tumor promoters/epigenetic carcinogens, metabolic cooperation assays have a sensitivity of 62% and dye-transfer assays 60%. Thirty percent of DNA-reactive carcinogens also possess the ability to uncouple cells. The complete estimation of the predictive power of these assays could not be made because the majority of the substances studied for intercellular communication effects in vitro have not yet been studied for promoting activity in vivo. Both metabolic cooperation assays and dye transfer assays respond well to the following classes of substances: phorbol esters, organochlorine pesticides, polybrominated biphenyls, promoters for urinary bladder, some biological toxins, peroxisome proliferators, and some complex mixtures. Results of in vitro assays for such tumor promoters/nongenotoxic carcinogens, such as some bile acids, some peroxides, alkanes, some hormones, mineral dusts, ascorbic acid, okadaic acid, and benz(e)pyrene, do not correlate with the data of in vivo two-stage or complete carcinogenesis. Enhancement of intercellular communication was found for 18 chemicals. Among these, cAMP, retinoids, and carotenoids have demonstrated inhibition of carcinogenesis. We examine a number of factors that are important for routine screening, including the requirement for biotransformation for some agents to exert effects on gap junctions. We also discuss the mechanisms of tumor promoter and tumor inhibitor effects on gap junctional permeability, including influences of protein kinase activation, changes in proton and Ca2+ intracellular concentrations, and effects of oxy radical production.

Role of protein kinase C in the regulation of gap junctional communication.

We have examined the effect of protein kinase (PKC) depletion in SV40-transformed Djungarian hamster fibroblasts (DM15 cells) on the level of gap junction permeability. Cx43 electrophoretic mobility, and cell sensitivity to different uncoupling stimuli. After 24 hr exposure to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the total PKC activity in DM15 cells was reduced to 20-25% in comparison with intact cells. In PKC-depleted cells the level of dye coupling was 30-40% higher than in the same untreated cultures. Western blot analysis revealed multiple forms of the gap junction protein connexin 43, which correspond to known phosphorylated and dephosphorylated forms of this protein. No decrease in the level of connexin 43 phosphorylation after PKC depletion was observed. TPA (10(-7) g/ml), mezerein (10(-7) g/ml), teleocidin (10(-8) g/ml), Ca-ionophore A23187 (10(-6) g/ml), insecticide 1,1,1-trichloro-2,2-bis-(p-chlorphenyl)-ethane (DDT) (10(-4) g/ml), and nigericin (10(-5) M in hydrolysate lactalbumin solution, pH 6.3) induced a four-to six-fold decrease in the number of recipient cells in the dye-coupling assay. PKC-depleted cells became almost completely resistant to the uncoupling effect of mezerein, teleocidin, and A23187, as well as to new exposure to TPA, and became partially resistant to the effect of DDT. Nigericin inhibited intercellular communication between PKC-depleted cells to the same extent as between control cells. Thus, in the cell system studied, PKC plays a certain role in maintaining the basal level of gap junction permeability and has an important significance as a mediator of the uncoupling effects of such substances as TPA, mezerein, teleocidin, and Ca2+.

The carcinogen benzo(e)pyrene is metabolized by DM15 cells without an uncoupling effect on their gap junctions.

Benzo(e)pyrene (B(e)P) promotes carcinogenesis in the skin. Unlike some other promoters however, B(e)P does not produce an uncoupling effect on gap junction permeability in DM15 transformed fibroblasts. This study demonstrates that DM15 cells exhibit a relatively high level of B(e)P metabolism. Moreover, although pretreatment of DM15 cells with benz(a)anthracene results in an 8-fold increase of arylhydrocarbon hydroxylase activity and a 2-fold increase in the rate of B(e)P metabolism, it did not enable B(e)P to affect Lucifer Yellow transfer between DM15 cells. We conclude that neither B(e)P nor its metabolites are capable of uncoupling gap junction permeability in DM15 cells.

Effect of tumor promoting stimuli on gap junction permeability and connexin43 expression in ARL18 rat liver cell line.

The ARL18 rat liver cell line has previously been used for screening tumor promoters in the metabolic cooperation assay (Williams 1980; Williams et al. 1981; Telang et al. 1982). These cells display high levels of gap junctional communication, as assessed functionally and immunologically. Intracellularly injected Lucifer Yellow diffused extensively and there was rapid fluorescent recovery after photobleaching. Moreover, expression of connexin43 (Cx43) was high as evaluated by immunocytochemistry of cell monolayers and Western blot analysis of total cell homogenates. Western blot analysis revealed multiple forms of Cx43, which presumably correspond to known dephosphorylated and phosphorylated states of this protein. Gap junction permeability and Cx43 expression in ARL18 cells were studied after exposure to the tumor promoters 12-0-tetradecanoyl-phorbol-13-acetate (TPA), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT), and after wounding the cell monolayer. TPA and DDT strongly inhibited gap junction permeability; whereas monolayer wounding did not affect the degree of fluorescent recovery after injury, either in the cells on the edge of the wound or in distal regions. No changes in the cellular distribution of Cx43 were observed after any of these treatments, although Western blots revealed a decrease in total Cx43 after 24-h exposure to DDT (10 micrograms/ml) and a slight increase after TPA treatment (30 min, 0.1 microgram/ml). Relative abundance of different phosphorylated Cx43 forms was increased after 1 h exposure to DDT (10 micrograms) and 30 min exposure to TPA (0.1 microgram/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of K+/H+ antiporter nigericin on gap junction permeability.

The K+/H+ antiporter nigericin inhibits the intercellular exchange of the fluorescent dye Lucifer Yellow between DM15-transformed fibroblasts derived from the Djungarian hamster. The efficacy of nigericin action was related to its concentration and time of incubation. The nigericin-induced uncoupling effect on gap junctions was reversible and was shown to be based on its ability to cause cystolic acidification. The effect of nigericin on dye-coupling in intact and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-pretreated cells did not differ, indicating that the uncoupling effect of H+ on gap junctions in DM15 cells was not mediated by the TPA-dependent isoform of protein kinase C.

The effect of complete carcinogens on intercellular transfer of lucifer yellow in fibroblast culture.

The effect on permeability of gap junctions of complete powerful carcinogens, 3-methylcholanthrene (MC), 7,12-dimethylbenz(a)anthracene (DMBA), ethyl methanesulfonate (EMS), and weak carcinogens, benz(a)anthracene (BA), benzo(e)pyrene (B(e)P) as well as the aryl-hydroxylase inhibitor 7,8-benzoflavone (7,8-BF) has been studied with the use of a dye-coupling technique and transformed Djungarian hamster DM15 fibroblasts. MC, EMS and 7,8-BF were found to exert a strong inhibitory effect on cell-to-cell dye transfer. BA and DMBA had the uncoupling activity only in 2 out of 4 experiments. B(e)P was not shown to affect LY transfer between DM15 cells. The uncoupling effect of MC, 7,8-BF and EMS (only when EMS used at the concentration of 600 micrograms/ml but not 1000 micrograms/ml) appeared reversible. The causes of failure to detect DMBA and B(e)P effects on gap junctions are discussed.

Identification of tumor promoters by their inhibitory effect on intercellular transfer of lucifer yellow.

The effect of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein, teleocidin, anthralin, the Ca2+-ionophore A23187, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT) and phenobarbital (PB) on lucifer yellow transfer in cultures of SV-40-transformed Djungarian hamster fibroblasts was studied. TPA, mezerein, teleocidin, A23187, DDT and BHT exerted a strong inhibitory effect on cell-to-cell dye transfer. Anthralin uncoupled cells in 3 experiments out of 6. PB appeared to enhance lucifer yellow transfer. Sodium nitrite, a substance with unknown promoting activity, effectively uncoupled cells. All the promoters investigated had a reversible effect on the dye transfer. The value of the dye transfer method for promoter screening is discussed.

[Possibility of identifying tumor promoters by their inhibitory action on the intercellular exchange of lucifer yellow]. / Vozmozhnost' vyiavleniia opukholevykh promotorov po ikh ingibiruiushchemu deistviiu na mezhkletochnyi obmen liutsiferovym zheltym.

The effects of tumor promoter--12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein, anthralin, Ca2+-ionophore A23187, butylated hydroxytoluene (BHT), DDT and phenobarbital--on cell-to-cell exchange of Lucifer Yellow were studied in cultures of SV40-transformed Djungarian hamster fibroblasts. TPA, mezerein, A23187, DDT and BHT strongly inhibited cell-to-cell exchange of Lucifer Yellow. Anthralin uncoupled cells in 3 out of 6 experiments. Phenobarbital, in contrast to other promoters, enhanced dye transfer. The effects of all the promoters tested were fully reversible. The potential use of Lucifer Yellow exchange inhibition as a test for the screening of tumor promoters is discussed.