REDD1 (regulated in development and DNA damage 1) modulates the glucocorticoid receptor function in keratinocytes.

Glucocorticoids (GCs) are widely used for the treatment of inflammatory skin diseases despite significant adverse effects including skin atrophy. Effects of GCs are mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Previously, we discovered that one of the GR target genes, REDD1, is causatively involved in skin atrophy. Here, we investigated its role in GR function using HaCaT REDD1 knockout (KO) keratinocytes. We found large differences in transcriptome of REDD1 KO and control Cas9 cells in response to glucocorticoid fluocinolone acetonide (FA): both the scope and amplitude of response were significantly decreased in REDD1 KO. The status of REDD1 did not affect GR stability/degradation during self-desensitization, and major steps in GR activation-its nuclear import and phosphorylation at activating Ser211. However, the amount of GR phosphorylated at Ser226 that may play negative role in GR signalling, was increased in the nuclei of REDD1 KO cells. GR nuclear import and transcriptional activity also depend on the composition of GR chaperone complex: exchange of chaperone FKBP51 (FK506-binding protein 5) for FKBP52 (FK506-binding protein 4) being a necessary step in GR activation. We found the increased expression and abnormal nuclear translocation of FKBP51 in both untreated and FA-treated REDD1 KO cells. Overall, our results suggest the existence of a feed-forward loop in GR signalling mediated by its target gene REDD1, which has translational potential for the development of safer GR-targeted therapies.

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.

The tumor suppressor effect of the glucocorticoid receptor in skin is mediated via its effect on follicular epithelial stem cells.

Glucocorticoids are potent inhibitors of mouse skin tumorigenesis. The glucocorticoid control of cellular functions is mediated via the glucocorticoid receptor (GR), a well-known transcription factor. Recently, we generated transgenic mice overexpressing GR under control of the keratin5 (K5) promoter, and showed that K5.GR animals are resistant to skin carcinogenesis. Follicular epithelial stem cells (SCs), located in the bulge region of the hair follicle, are believed to be one of the target cells for skin carcinogenesis. We found that the number of putative hair follicle SC detected as label-retaining cells was significantly less in the K5.GR transgenics compared to wild type (w.t.) littermates. We also showed that GR overexpression led to a reduction in the clonogenicity of the follicular epithelial SCs. We evaluated the global effect of GR on gene expression in a population of follicular SC-enriched bulge keratinocytes isolated by fluorescence activated cell sorting. We found that GR affected the expression of numerous bulge SC 'signature' genes, genes involved in the maintenance of SC and progenitor cells of non-epidermal origin and proapoptotic genes. Our findings underscore the important role of GR signaling in the homeostasis of follicular epithelial SCs, and suggest that the reduction in their number may underlie the tumor suppressor effect of GR in the skin.

Tumor suppressor activity of glucocorticoid receptor in the prostate.

Glucocorticoids are extensively used in combination chemotherapy of advanced prostate cancer (PC). Little is known, however, about the status of the glucocorticoid receptor (GR) in PC. We evaluated over 200 prostate samples and determined that GR expression was strongly decreased or absent in 70-85% of PC. Similar to PC tumors, some PC cell lines, including LNCaP, also lack GR. To understand the role of GR, we reconstituted its expression in LNCaP cells using lentiviral approach. Treatment of LNCaP-GR cells with the glucocorticoids strongly inhibited proliferation in the monolayer cultures and blocked anchorage-independent growth. This was accompanied by upregulation of p21 and p27, down-regulation of cyclin D1 expression and c-Myc phosphorylation. Importantly, the activation of GR resulted in normalized expression of PC markers hepsin, AMACR, and maspin. On the signaling level, GR decreased expression and inhibited activity of the MAP-kinases (MAPKs) including p38, JNK/SAPK, Mek1/2 and Erk1/2. We also found that activation of GR inhibited activity of numerous transcription factors (TF) including AP-1, SRF, NF-kappaB, p53, ATF-2, CEBPalpha, Ets-1, Elk-1, STAT1 and others, many of which are regulated via MAPK cascade. The structural analysis of hepsin and AMACR promoters provided the mechanistic rationale for PC marker downregulation by glucocorticoids via inhibition of specific TFs. Our data suggest that GR functions as a tumor suppressor in prostate, and inhibits multiple signaling pathways and transcriptional factors involved in proliferation and transformation.

Effects of IKK inhibitor PS1145 on NF-kappaB function, proliferation, apoptosis and invasion activity in prostate carcinoma cells.

A key antiapoptotic transcription factor, nuclear factor kappa-B (NF-kappaB), is known to be critically important for tumor cell growth, angiogenesis and development of metastatic lesions. We and others showed previously that NF-kappaB transcription factor was constitutively activated in androgen-independent prostate carcinoma (PC) cell lines due to the upregulated activity of inhibitor of NF-kappaB kinases (IKK). In this work, using luciferase assay, electrophoretic mobility shift assay and Northern blot analysis of expression of endogenous kappaB-responsive genes, we demonstrate that a novel highly specific small-molecule IKK inhibitor, PS1145, efficiently inhibited both basal and induced NF-kappaB activity in PC cells. We found that PS1145 induced caspase 3/7-dependent apoptosis in PC cells and significantly sensitized PC cells to apoptosis induced by tumor necrosis factor alpha. We also showed that PS1145 inhibited PC cell proliferation. Effects of PS1145 on proliferation and apoptosis correlated with inhibition of interleukin (IL)-6, cyclin D1, D2, inhibitor of apoptosis (IAP)-1 and IAP-2 gene expression and decreased IL-6 protein level. In addition, we found that incubation with PS1145 inhibited the invasion activity of highly invasive PC3-S cells in invasion chamber assay in a dose-dependent manner. Overall, this study provides the framework for development of a novel therapeutic approach targeting NF-kappaB transcription factor to treat advanced PC.

Increased telomerase activity in mouse skin premalignant progression.

It has been postulated that the expression of the ribonucleoprotein telomerase is necessary to overcome cellular senescence and that malignant tumors must express telomerase to maintain their immortality. In most human adult tissues, telomerase activity is not detected. In contrast, several murine tissues express various levels of telomerase. Mouse skin however, does not show telomerase activity. Using the mouse skin chemical carcinogenesis system, a well-characterized model for studying premalignant and malignant progression, we assayed telomerase activity at various stages of premalignant papilloma progression by means of the recently developed telomeric repeat amplification protocol. We observed that at 10 weeks of promotion, only one mouse skin papilloma of 11 analyzed showed high levels of telomerase activity. The number of papillomas showing higher levels of telomerase activity increased at 20 weeks, and at 30 weeks of promotion, 100% of papillomas expressed significantly higher levels of telomerase. We learned from previous studies that early papillomas are diploid, well-differentiated lesions, whereas late papillomas are aneuploid and very dysplastic. It appears that the progressive increase in telomerase activity is associated with the increased level of genomic instability and the phenotypic progression of these premalignant tumors. It is also possible, however, that the increase in telomerase activity could be in part a consequence of an increase in the proportion of proliferating cells. Nevertheless, the mouse skin system may be a very useful in vivo model for the study and development of anti-telomerase therapeutic strategies.

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.

An upward shift of intracellular pH rather than the final absolute pH value is critical for controlling gap junction permeability in tumor promoter-treated cells.

The tumor promoter TPA is found to inhibit gap junction permeability in monolayer cultures of hamster fibroblasts. This effect is associated with an increase in intracellular pH. Here we show that neither an increase in pHi alone nor TPA treatment under conditions preventing pHi-shift affect gap junction permeability. It is not the level of pHi reached, but rather the pHi-shift itself that is essential for the inhibition of gap junction permeability in the presence of TPA.

Inhibition of the glucocorticoid receptor expression by diverse tumor promoters in SENCAR mouse epidermis.

Glucocorticoid hormones are very potent inhibitors of keratinocyte proliferation. Their function is mediated by the glucocorticoid receptor (GR) which is highly expressed in mouse epidermis. In the study reported here we compared the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and non-phorbol ester tumor promoters such as okadaic acid, chrysarobin, and benzoyl peroxide on the levels of GR protein and mRNA in SENCAR mouse epidermis. Glucocorticoid binding assay and Northern blot analysis revealed that all four tumor promoters decreased both GR protein and mRNA levels in keratinocytes in vivo. We also found that TPA and okadaic acid inhibited GR expression in keratinocyte cell line. These results suggest that GR inhibition may play an important role in mouse skin hyperplasia and promotion of skin carcinogenesis.

[Reparative DNA synthesis in human and murine embryonic liver cell cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine]. / Reparativnyi sintez DNK v kul'ture kletok émbrional'noi pecheni cheloveka i myshi, obrabotannykh N-metil-N'-nitro-N-nitrosoguanidinom.

The carcinogenic agents--UV-light and N-methyl-N'-nitro-N-nitroso-guanidine (NMNG) were shown to induce DNA repair synthesis in human and mouse liver cell cultures. The DNA repair synthesis, measured autoradiographically, was less active in liver cells than in fibroblasts from the same embryo. The hepatocytes exhibited a higher resistance to the toxic effect of both UV-light and NMNG. This phenomenon is suggested to be due to the active non-excision DNA repair in liver cells.

[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.

[Species and tissue differences of reparative DNA synthesis in embryonic cell cultures treated with carcinogens]. / Vidovye i tkanevye razlichiia reparativnogo sinteza DNK v kul'turakh émbrional'nykh kletok, obrabotannykh kantserogenami.

DNA repair synthesis (RS) was studied in embryonic cell cultures exposed to different carcinogenic factors: UV-light, N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, aflatoxin BI and 7,12-dimethylbenz(a)anthracene. DNA RS level was shown to be higher in human liver cells than in murine ones. Tissue-dependent differences in DNA RS of cells damaged by carcinogens were found, too. RS-activity was higher in human, mouse and rat fibroblast cultures than in liver cultures of the same species. RS level in human kidney cultures was similar to that in human fibroblasts. The said differences should be taken into account in the evaluation of the results of testing of chemical agents for carcinogenicity, using their ability to cause DNA repair synthesis.

[DNA reparative synthesis in liver cell cultures from mice with various susceptibilities to spontaneous hepatic carcinogenesis]. / Reparativnyi sintez DNK v kul'turakh kletok pecheni myshei s razlicnoi predraspolozhennost'iu k spontannomu gepatokantserogenezu.

Ultra-violet radiation-induced unscheduled DNA synthesis in liver cell cultures from CBA, C57Bl/6j and AKR adult male mice was studied autoradiographically. Ultra-violet radiation (254 nm; 1.5-36 J/m2) triggered on an active unscheduled DNA synthesis in all cell cultures studied. The levels of unscheduled DNA synthesis in the liver cell nuclei of the spontaneous hepatocarcinogenesis-susceptible strain (CBA) and relatively, resistant ones (C57Bl/6j and AKR) were similar. It is suggested that the susceptibility to hepatocarcinogenesis in different murine strains is determined by such parameters as DNA damage and repair (stage of initiation) as well as factors peculiar to the stage of promotion.

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.

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.

[Effect of benz(a)pyrene on a monolayer culture of normal and malignant mouse liver cells]. / Vliianie benz(a)pirena na monosloinuiu kul'turu normal'nykh i zlokachestvennykh kletok pecheni myshi.

Both the cells of monolayer culture of mouse embryonic liver and that of highly malignant hepatoma 22A transplanted for 20 years actively metabolized the carcinogenic hydrocarbon benz(a)pyrene and were highly sensitive to iits toxic action. Since hepatic tissue was resistant in vivo to carcinogenic carbohydrates it is suggested that the resistance depended on factors acting at the organ or the organism but not as the cellular level. The mechanism of retention of hepatoma 22A sensitivity to the toxic action of benz(a)pyrene is also 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.

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.

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)

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+.