RESUMO
BACKGROUND: Serum cytokine concentrations may reflect inflammatory processes occurring during the development of colorectal neoplasms. Flavonols, bioactive compounds found in plant-based foods and beverages, may inhibit colorectal neoplasms partly by attenuating inflammation. METHODS: Using logistic regression, we estimated odds ratios (ORs) and 95% confidence intervals (CIs) to investigate the association between serum concentrations of interleukin (IL) ß, 2, 8, 10, 12p70, granulocyte macrophage colony stimulating factor, interferon-γ, and tumour necrosis factor-α, measured over time, flavonol intake, estimated from a flavonol database used in conjunction with a food frequency questionnaire, and adenoma recurrence in 872 participants from the intervention arm of the Polyp Prevention Trial. RESULTS: Decreased IL-2 concentration during the trial increased the risk of any adenoma recurrence (4th vs 1st quartile, OR=1.68, 95% CI=1.13-2.49), whereas decreased IL-1ß or IL-10 reduced the risk of advanced adenoma recurrence (OR=0.37, 95% CI=0.15-0.94; OR=0.39, 95% CI=0.15-0.98, respectively). Individuals with flavonol intake above the median (29.7 mg per day) and decreased cytokine concentrations had the lowest risk of advanced adenoma recurrence. CONCLUSION: Overall, no consistent associations were observed between serum cytokine profile and colorectal adenoma recurrence; however, decreased cytokine concentrations during high flavonol consumption may indicate prevention of colorectal neoplasms.
Assuntos
Adenoma/sangue , Adenoma/prevenção & controle , Neoplasias Colorretais/sangue , Neoplasias Colorretais/prevenção & controle , Citocinas/sangue , Flavonóis/administração & dosagem , Idoso , Ensaios Clínicos como Assunto , Dieta , Feminino , Humanos , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , RecidivaRESUMO
Tumor promoters may bring about events that lead to neoplastic transformation by inducing specific promotion-relevant effector genes. Functional activation of the transacting transcription factor AP-1 by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) may play an essential role in this process. Clonal genetic variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to promotion of transformation by TPA were transfected with 3XTRE-CAT, a construct that has AP-1 cis-enhancer sequences attached to a reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfected JB6 P+, but not P- variants, showed TPA-inducible CAT synthesis. Epidermal growth factor, another transformation promoter in JB6 cells, also caused P+ specific induction of CAT gene expression. These results demonstrate an association between induced AP-1 function and sensitivity to promotion of neoplastic transformation.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/farmacologia , Epiderme , Regulação da Expressão Gênica , Variação Genética , Cinética , Camundongos , Hibridização de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-jun , Simplexvirus/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (-402/-350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region -152/-135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.
Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Invasividade Neoplásica/genética , Proteínas de Ligação a RNA/farmacologia , Receptores de Superfície Celular/genética , Fatores de Transcrição Sp/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Fator de Transcrição Sp1/farmacologia , Fator de Transcrição Sp3/farmacologia , Proteínas Supressoras de Tumor/farmacologiaRESUMO
Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P(+)) and nonpromotable (P(-)) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P(+) lines, JB6 Cl 41 or JB6 Cl 22, was CaPO(4) precipitated and used to transfect the P(-) cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 x 10(-8) M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P(-) JB6 Cl 30 cells yielded 40 to 50 colonies per 10(5) cells. In contrast, transfection of Cl 30 cells with "P(+) DNA" derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10(5) cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P(+) DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P(+) lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P(+) DNA, no evidence for acquisition of promotability was obtained. P(-) JB6 Cl 25, like Cl 30, also permitted expression of transfected P(+) DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P(+) DNA and requires recipient cells of the appropriate phenotype for expression.
Assuntos
Transformação Celular Neoplásica , DNA/genética , Animais , Adesão Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Camundongos , Fenótipo , Pele , Acetato de Tetradecanoilforbol/toxicidade , TransfecçãoRESUMO
Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity. In view of this difference and the differences in both recipient cells and 12-O-tetradecanoyl-phorbol-13-acetate dependence of expression, it appears that the transforming activity and the promotion-sensitive activity are specified by different genes. The JB6 promotion-sensitive cell lines may be useful for detecting and cloning transforming genes that escape detection in the NIH 3T3 cell focus assay.
Assuntos
Linhagem Celular , Transformação Celular Neoplásica , Animais , Adesão Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Inibição de Contato , DNA/genética , Enzimas de Restrição do DNA , Células Epidérmicas , Camundongos , Fenótipo , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Collagen synthesis was inhibited in JB-6 mouse epidermal cells after exposure to 12-O-tetradecanoylphorbol-13-acetate under conditions leading to irreversible neoplastic transformation. In vitro translation and hybridization studies demonstrated a dramatic decrease in collagen mRNA in 12-O-tetradecanoylphorbol-13-acetate-treated cells, suggesting that the inhibition of collagen synthesis in response to 12-O-tetradecanoylphorbol-13-acetate is due to regulation at a pretranslational level.
Assuntos
Forbóis/farmacologia , Pró-Colágeno/genética , Acetato de Tetradecanoilforbol/farmacologia , Animais , Células Cultivadas , Epiderme , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Pró-Colágeno/biossíntese , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Programmed cell death 4 (Pdcd4), a tumor invasion suppressor, is frequently downregulated in colorectal cancer and other cancers. In this study, we find that loss of Pdcd4 increases the activity of mammalian target of rapamycin complex 2 (mTORC2) and thereby upregulates Snail expression. Examining the components of mTORC2 showed that Pdcd4 knockdown increased the protein but not mRNA level of stress-activated-protein kinase interacting protein 1 (Sin1), which resulted from enhanced Sin1 translation. To understand how Pdcd4 regulates Sin1 translation, the SIN1 5' untranslated region (5'UTR) was fused with luciferase reporter and named as 5'Sin1-Luc. Pdcd4 knockdown/knockout significantly increased the translation of 5'Sin1-Luc but not the control luciferase without the SIN1 5'UTR, suggesting that Sin1 5'UTR is necessary for Pdcd4 to inhibit Sin1 translation. Ectopic expression of wild-type Pdcd4 and Pdcd4(157-469), a deletion mutant that binds to translation initiation factor 4A (eIF4A), sufficiently inhibited Sin1 translation, and thus suppressed mTORC2 kinase activity and invasion in colon tumor cells. By contrast, Pdcd4(157-469)(D253A,D418A), a mutant that does not bind to eIF4A, failed to inhibit Sin1 translation, and consequently failed to repress mTORC2 activity and invasion. In addition, directly inhibiting eIF4A with silvestrol significantly suppressed Sin1 translation and attenuated invasion. These results indicate that Pdcd4-inhibited Sin1 translation is through suppressing eIF4A, and functionally important for suppression of mTORC2 activity and invasion. Moreover, in colorectal cancer tissues, the Sin1 protein but not mRNA was significantly upregulated while Pdcd4 protein was downregulated, suggesting that loss of Pdcd4 might correlate with Sin1 protein level but not mRNA level in colorectal cancer patients. Taken together, our work reveals a novel mechanism by which Pdcd4 inhibits Sin1 translation to attenuatemTORC2 activity and thereby suppresses invasion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/genética , Neoplasias do Colo/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fator de Iniciação 4A em Eucariotos/metabolismo , Genes Supressores de Tumor , Células HCT116 , Células HT29 , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Invasividade Neoplásica , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , TransfecçãoRESUMO
Since cell surface gangliosides have been implicated in the regulation of cell growth and differentiation and since tumor-promoting phorbol esters produce a number of membrane changes, the possibility was investigated that ganglioside changes may play a role in promotion of transformation in JB6 mouse epidermal cells. The studies showed that tumor-promoting but not non-promoting phorbol esters produced decreased precursor incorporation into disialoganglioside GD1b and an unknown ganglioside, Gx. These ganglioside responses to promoters were antagonized by the antipromoter retinoic acid, thus suggesting that alterations in ganglioside levels may be involved in promotion of transformation.
Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Epiderme/metabolismo , Gangliosídeos/metabolismo , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Camundongos , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Fatores de TempoRESUMO
The JB6 mouse epidermal cell line has been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or other tumor promoters resulted in the irreversible acquisition by JB6 cells of tumorigenicity in nude mice and anchorage-independent growth in soft agar. As previously reported, one of the biochemical responses that occurs during TPA treatment is a greater than 75% reduction in a mannose-labeled glycoprotein with an apparent molecular weight of 180,000. This TPA-sensitive glycoprotein has now been identified on the basis of collagenase and pepsin sensitivity as procollagen pro-alpha 1(I) chain. [3H]proline labeling also demonstrated a parallel decrease in the 150,000 procollagen pro-alpha 2(I) component. Two nonphorbol promoters, mezerein and epidermal growth factor, were also active in decreasing procollagen synthesis. Promotion-sensitive and promotion-resistant clonal derivatives of JB6 showed similar basal levels of collagen synthesis as well as similar degrees of TPA-dependent procollagen loss indicating that the collagen decrease may be necessary but is not sufficient to produce the promotion response. Comparison of chemically transformed mouse epidermal cell lines with paried nontransformants suggests the reduced procollagen synthesis is a stably acquired phenotypic change and may therefore be involved in maintenance of the transformed phenotype as well as in its induction.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Forbóis/farmacologia , Pró-Colágeno/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Animais , Linhagem Celular , Epiderme , Camundongos , Ésteres de Forbol/farmacologia , Tretinoína/farmacologiaRESUMO
A specific decrease in the net de novo synthesis ([1-14C]-glucosamine incorporation) of cell surface trisialoganglioside (GT) occurs in preneoplastic mouse JB6 epidermal cells in response to tumor-promoting phorbol esters, mezerein, or epidermal growth factor, all of which promote neoplastic transformation in JB6 cells, but not in response to the bladder promoter sodium cyclamate, a nonpromoter in JB6 cells. The ganglioside showing elevated synthesis after mezerein or epidermal growth factor exposure is monosialoganglioside 1, whereas disialoganglioside 1b synthesis is elevated after phorbol ester exposure. Primary mouse epidermal cells and putatively initiated epidermal cell lines selected for their resistance to induction of terminal differentiation by high calcium are resistant to promotion of anchorage-independent transformation by 2-week exposure to 12-O-tetradecanoylphorbol-13-acetate. In both cell types, little or no decrease in GT synthesis occurs in response to short-term 12-O-tetradecanoylphorbol-13-acetate exposure, thus extending further our previous observation that this GT response is restricted to promotable cells. A decreased synthesis of GT also occurs consistently in cell lines transformed by 12-O-tetradecanoylphorbol-13-acetate or N-methyl-N-nitro-nitrosoguanidine as compared with their nontransformed counterparts but not in cell lines transformed by a cloned integrated murine sarcoma provirus containing the oncogenic sequence v-mos. Thus, reduced cell surface GT synthesis may be important both in the induction and in the maintenance of the chemically transformed but not viral oncogene mos-transformed phenotype in mouse epidermal cells.
Assuntos
Carcinógenos/farmacologia , Transformação Celular Neoplásica , Diterpenos , Fator de Crescimento Epidérmico/farmacologia , Gangliosídeos/biossíntese , Ésteres de Forbol/farmacologia , Forbóis/farmacologia , Pele/metabolismo , Terpenos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Radioisótopos de Carbono , Linhagem Celular , Glucosamina/metabolismo , Camundongos , Pele/efeitos dos fármacosRESUMO
The JB6 mouse epidermal cell lines have been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 1 to 100 ng/ml results in the irreversible acquisition of tumorigenicity in nude mice and anchorage-independent growth. Among the biochemical responses which occur during TPA treatment is a decrease in procollagen synthesis. During a study of the possible role of H2O2 in the process of promotion, it was observed that catalase purchased commercially would inhibit TPA promotion as well as the reduction of collagen synthesis in a dose-dependent manner. A highly purified catalase preparation failed to demonstrate the TPA blocking activity, suggesting that this activity was due to a contaminating factor. We have separated the TPA blocking factor from the catalase itself using concanavalin A affinity chromatography. The factor is a Mr 60,000 glycoprotein showing TPA hydrolase activity. The enzyme, which is similar to a murine liver-derived TPA hydrolase, produces a single phorbol product from TPA that has been identified as phorbol-13-acetate. TPA hydrolase was used to terminate TPA action in soft agar. This made it possible to establish that approximately 4 days of exposure to phorbol esters are required for promotion of transformation in the JB6 model system.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Pele/citologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Catalase/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Cromatografia de Afinidade , Colágeno/biossíntese , Epiderme/efeitos dos fármacos , Hidrolases/metabolismo , Camundongos , Peso Molecular , Fatores de TempoRESUMO
Extracellular calcium is required in the induction of neoplastic transformation of preneoplastic mouse JB6 epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Depleting extra-cellular calcium by chelation or by use of commercial calcium-depleted medium inhibited TPA-promoted transformation of promotion-sensitive JB6 cells with a half-maximal inhibition at 1.2 mM calcium. Inhibition was reversible by the addition of calcium. The calcium channel blockers lanthanum and nifedipine inhibited promotion of anchorage-independent transformation by TPA maximally at 10.0 microM and 1.0 nM, respectively, suggesting that calcium entry occurs partially via cell channels. None of the above treatments altered expression of transformation in anchorage-independent tumorigenic JB6 cell lines, indicating that the extracellular calcium requirement was at the level of induction, not expression of transformation. The calcium requirement was not merely a requirement for proliferation; calcium concentrations from 0.2 to 1.8 mM had no effect on JB6 cell monolayer growth. Extracellular calcium was required for 7 days for maximal colony induction. The calcium ionophore A23187 was not a promoter and moderately inhibited TPA-promoted transformation, indicating that increases in free cytosolic calcium concentrations are not sufficient for promotion of transformation and may even activate calcium-dependent antipromoting events. The results suggest that a cellular calcium pool supplied by extracellular calcium, but not distinguishable by ionophoretic increases in free cytosolic calcium, is essential in TPA-promoted neoplastic transformation.
Assuntos
Cálcio/fisiologia , Transformação Celular Neoplásica/efeitos dos fármacos , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Calcimicina/farmacologia , Linhagem Celular , Ácido Egtázico/farmacologia , Espaço Extracelular/fisiologia , Lantânio/farmacologia , Camundongos , Nifedipino/farmacologiaRESUMO
TPA (12-O-tetradecanoylphorbol-13-acetate), a potent tumor promoter, has been shown to stimulate or inhibit cell growth depending on the cell type investigated. We recently found that RT101 cells, a transformed mouse JB6 epidermal cell line, acquired a greater growth inhibition response to TPA during conventional subcultivation. The growth of low-passage RT101 cells was slightly inhibited by TPA in monolayer culture but stimulated in soft agar. In contrast, the growth of high-passage cells was greatly inhibited by TPA in both monolayer culture and in soft agar. Inhibition was dose dependent, directly correlated with protein kinase C-activating activities of tumor promoters, and was found to be reversible. TPA-treated high-passage cells were greatly reduced in volume, showed extensive abnormal mitoses, and were more susceptible to detachment. High-passage cells were also found to be less tumorigenic as indicated by in vivo tumorigenicity assay in nude mice. TPA treatment rendered cells still less tumorigenic in the case of both cell lines. The mechanism for acquisition of increased sensitivity to TPA of RT101 cells during subculture was investigated; it involved nonrandom DNA damage and detachment of nonviable cells. The results suggest the possibility that early-passage RT101 cells contained two subpopulations, one TPA-sensitive and one TPA-resistant population. Conventional subcultivation may have selected for the former subpopulation. The sensitive subpopulation may have been irreversibly inhibited as a result of TPA-induced cell killing, possibly apoptosis.
Assuntos
Divisão Celular/efeitos dos fármacos , Dano ao DNA , Acetato de Tetradecanoilforbol/farmacologia , Animais , Peróxido de Benzoíla/farmacologia , Catalase/metabolismo , Linhagem Celular Transformada , DNA/genética , DNA/isolamento & purificação , Fator de Crescimento Epidérmico/farmacologia , Genes fos/efeitos dos fármacos , Genes jun/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Toxinas de Lyngbya/farmacologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA/genética , RNA/isolamento & purificação , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
To better understand the molecular mechanism of multistage carcinogenesis, we have attempted to identify putative oncogenes and/or tumor suppressor genes involved in preneoplastic-to-neoplastic progression of mouse epidermal JB6 variants. The JB6 variants consist of P- (promotion resistant), P+ (promotion sensitive), and Tx [transformed; both apoptosis-sensitive (A(s)) and apoptosis-resistant (Ar)] cells, representing progression from early to late stages of carcinogenesis. By using the newly developed differential mRNA display technique, we have isolated five clones from these JB6 variants. The isolated clones were uniquely expressed either in P-/P+ cells or in Tx (A(s)/Ar) cells or showed highly differential expression among the variants. The expression pattern shown by differential mRNA display was confirmed by Northern blot analysis. DNA sequencing followed by computer search against Genbank and EMBL DNA databases indicates that three clones are novel and two have high homology with recorded genes. One of the clones (C1.14), which detects expression in preneoplastic not neoplastic JB6 cells, was used as a probe for complementary DNA library screening. The corresponding gene, named sun for specifically unexpressed in neoplastic JB6 cells, was isolated and sequenced. The longest open reading frame of the sun clone predicts a peptide showing 96% amino acid sequence identity to the recorded sequence of human tissue inhibitor of metalloproteinases-3, one of a family of genes implicated in tumorigenesis and tumor invasion. This is the first report, to our knowledge, of the simultaneous display of mRNAs of four phenotypically distinct cell variants and of the isolation of five clones which may be associated with specific stages of tumor promotion and/or progression and apoptosis.
Assuntos
Proteínas de Neoplasias/genética , Lesões Pré-Cancerosas/genética , RNA Mensageiro/genética , Neoplasias Cutâneas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA de Neoplasias/genética , Bases de Dados Factuais , Epitélio/patologia , Estudos de Avaliação como Assunto , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/isolamento & purificação , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Neoplasias Cutâneas/patologia , Inibidor Tecidual de Metaloproteinase-3RESUMO
The potent skin tumor promoter (12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulates epidermal macromolecular synthesis as well as proliferation, but little is known of specific functional aberrations produced by TPA. This report presents results of a study on the effects of TPA on epidermal histidase (L-histidine ammonia lyase), an enzyme found in normal epidermis but not in dermis or in mouse squamous cell carcinomas. Histidase activity was assayed on postmitochondrial supernatants obtained from hairless mouse epidermis after removal by keratotome. Topical TPA treatment at doses active in tumor promotion (1.7 to 17.0 nmoles/application) produced dose-dependent decreases in epidermal histidase specific activity at 19 hr posttreatment. The onset of the decrease occurred at 12 hr with recovery to control level specific activity by 5 days, showing kinetics similar to those obtained for stimulation of DNA synthesis. This decrease in histidase could not be attributed to a general inhibition of soluble protein synthesis or to the appearance of an inhibitor of histidase activity. The strong promoter TPA produced a greater histidase decrease than did the moderate promoter and mitogen 12,13-didecanoyl phorbol at equimolar dose, while phorbol, a nonpromoter and nonmitogen, produced no effects on histidase. The relationship of this histidase depression to tumor promotion and not initiation is further indicated by the finding that (a) Tween 60, a structurally unrelated tumor promotor, also produced a decrease in histidase; and (b) the tumor initiator urethan and an initiating dose of 9,10-dimethybenz(a)anthracene showed no effects on histadase activity.
Assuntos
Amônia-Liases/metabolismo , Histidina Amônia-Liase/metabolismo , Forbóis/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Pele/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Benzo(a)Antracenos/farmacologia , Carcinógenos/farmacologia , Depressão Química , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/enzimologia , Polissorbatos/farmacologia , Pele/efeitos dos fármacos , Neoplasias Cutâneas/enzimologia , Uretana/farmacologiaRESUMO
1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, was shown previously to enhance chemically induced transformation of BALB 3T3 cells and Syrian hamster embryo cells. This report demonstrates that 1 alpha,25(OH)2D3, like phorbol ester tumor promoters, induces anchorage-independent growth of mouse JB6 epidermal cells. When plated on agar plates containing 1 alpha,25(OH)2D3 at concentrations higher than 0.05 ng/ml or 0.12 nM, JB6 cells formed colonies on the surface of agar plates dose dependently. This anchorage-independent growth was further confirmed by stimulation of DNA synthesis after liquefying the agar layer with NaI. A phorbol-ester resistant variant of JB6 cells was also resistant to 1 alpha,25(OH)2D3 in terms of induction of anchorage independency. Induction of anchorage-independent growth was specific for 1 alpha,25(OH)2D3: other derivatives of vitamin D3 also induced colony formation on agar plates but only at a higher concentration (500 ng/ml) and to much less extent than did 1 alpha,25(OH)2D3. JB6 cells were found to contain a receptor specific for 1 alpha,25(OH)2D3 with a Kd of 55.7 pM and Nmax of 102.5 fmol/mg protein, suggesting a receptor-mediated mechanism of the induction. The clone that was resistant to 1 alpha,25(OH)2D3 also contained the receptor. DNA-cellulose chromatography showed that a 1 alpha,25(OH)2D3-receptor complex interacted with DNA. In contrast to 1 alpha,25(OH)2D3, retinoic acid did not induce anchorage-independent growth of JB6 cells, but it inhibited the induction by 1 alpha,25(OH)2D3 when applied with it.
Assuntos
Calcitriol/farmacologia , Carcinógenos/farmacologia , Células Epidérmicas , Animais , Calcitriol/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Camundongos , Receptores de Calcitriol , Receptores de Esteroides/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologiaRESUMO
Cross-coupling of active protein-1 (AP-1) and nuclear factor (NF)-kappaB has been reported. In the present study, we investigated the possibility that both of these two transcription factors might contribute to the process of tumor promoter-induced transformation. To establish a stable reporter cell system, two reporter genes were stably transfected into a JB6 mouse tumor promotion-sensitive (P+) cell line: a luciferase reporter controlled by a collagenase AP-1 sequence and a chloramphenicol acetyltransferase reporter controlled by an interleukin 6 NF-kappaB sequence. This double-reporter cell line maintained the phenotype of tumor promotion sensitivity and was able to report basal or induced AP-1 and NF-kappaB transactivation. The cytokine tumor promoter tumor necrosis factor (TNF)-alpha transactivated NF-kappaB and AP-1 for both DNA binding and transcriptional activity. Pyrrolidine dithiocarbamate, an antioxidant that acts as an NF-kappaB inhibitor, efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or TNF-alpha induced NF-kappaB as well as AP-1 transactivation and cell transformation, suggesting dependency of transformation on both transcription factors. The AP-1 transrepressing-retinoid SR11302 transrepressed AP-1 and cell transformation when these were TPA induced but not when TNF-alpha induced, indicating different signaling pathways for TNF-alpha and TPA. Supershift electrophoresis mobility shift assay revealed that Jun B and c-Jun were absent from the AP-1/DNA complex following TNF-alpha but present following TPA treatment. Together, these results suggest that both AP-1 and NF-kappaB activation may be required for transformation whether induced by TPA or by TNF, and the differential sensitivity of TPA and TNF-alpha-induced transformation to inhibition by a retinoid might be explained by differences in the composition of the DNA-bound AP-1 complexes.
Assuntos
Transformação Celular Neoplásica/genética , NF-kappa B/efeitos dos fármacos , Fator de Transcrição AP-1/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Animais , Anticarcinógenos/farmacologia , Carcinógenos/antagonistas & inibidores , Carcinógenos/farmacologia , Células Cultivadas/efeitos dos fármacos , DNA/metabolismo , Genes Reporter , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Pirrolidinas/farmacologia , Retinoides/farmacologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Tiocarbamatos/farmacologia , Fatores de Tempo , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Both retinoic acid (RA) treatment and dominant-negative c-Jun mutant expression effectively inhibit phorbol ester-induced AP-1 activity and induced neoplastic transformation in mouse epidermal JB6 cells. However, both reagents also target non-AP-1 molecules in addition. Because liganded retinoic acid receptors interact with and transactivate RA response elements (RAREs) on DNA, as well as interact with Jun protein to block AP-1 activity, the question arises as to which of these two activities of retinoids is responsible for antitumor-promoting activity. To address this question we generated JB6 promotion-sensitive (P+) cell lines that are stably transfected with a construct containing the collagenase promoter bearing one AP-1-binding site that drives a luciferase reporter gene. The stable collagenase-luciferase-transfected cell lines showed 1.5-3.5-fold enhanced AP-1 activity when treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA). Up to 90% of TPA-induced AP-1 activity was blocked by retinoids SR11238, SR11302, or trans-RA, but not by retinoid SR11235. Of these retinoids, only RA and SR11235 were able to transactivate RARE-dependent gene expression. Transrepression of TPA-induced AP-1 and transactivation of RARE by RA, SR11238, and SR11302 were concentration dependent at 10(-10) to 10(-6) M retinoid. When tested for activity in inhibiting tumor promoter-induced transformation in JB6 P+ cells, the retinoids specific for AP-1 transrepression were inhibitory, whereas SR11235, which only activated RARE, showed little effect. We thus conclude that the AP-1-blocking activity of retinoids is likely to be responsible for the antitumor-promoting activity. This result, together with the observation that dominant-negative Jun blocks transformation, argues for a requirement of induced AP-1 in the tumor promoter-induced transformation process.
Assuntos
Anticarcinógenos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico , Retinoides/farmacologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Fator de Transcrição AP-1/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Sequência de Bases , Carcinógenos/toxicidade , Células Cultivadas , Expressão Gênica , Genes Reporter , Luciferases/biossíntese , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sensibilidade e Especificidade , Pele/efeitos dos fármacos , Pele/patologia , Acetato de Tetradecanoilforbol/toxicidade , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/fisiologia , TransfecçãoRESUMO
A previous report demonstrated that mouse JB6 cells transformed to promotion sensitive (P+) phenotype by transfection with an activated promotion sensitivity (pro) gene showed both evidence for the presence of the transfected gene and sensitivity to phorbol ester induced transformation similar to that observed in parental P+ cells. In addition, pro-1 and pro-2 transfectants were similar to each other in phorbol ester response. The current report extends these findings to ask whether pro-1 or pro-2 transfectants are also sensitive to promotion of transformation by other classes of tumor promoters such as epidermal growth factor (EGF), lanthanides, and phthalate esters and to inhibition of phorbol ester promoted transformation by several classes of antipromoters. The results showed that both pro-1 and pro-2 transfectants resembled parental P+ cells in sensitivity to promotion of anchorage independent transformation by lanthanides and by diethylhexylphthalate. In addition both pro-1 and pro-2 transfectants showed inhibition of phorbol ester induced transformation by antipromoters ganglioside GT1b, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and forskolin. Thus the pathways implicated by these inducers and inhibitors of transformation appear similar to those implicated for parental P+ cells and similar when controlled by pro-1 or pro-2. The single differential response was that of EGF-induced transformation. pro-2 transfectants but not pro-1 transfectants were sensitive to EGF-induced neoplastic transformation. The nonresponsiveness could not be attributed to lack of EGF receptors since 125I-EGF binding to pro-1 transfectants was similar to that for pro-2 transfectants and parental P+ cells. Thus pro genes transfer responsiveness to a C-kinase mediated promotion of transformation pathway and to putatively non-C kinase pathways triggered by lanthanides or phthalate esters, but not necessarily to an EGF receptor kinase mediated pathway.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Transfecção , Animais , Linhagem Celular , Dietilexilftalato/toxicidade , Ácido Egtázico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase C/fisiologia , Acetato de TetradecanoilforbolRESUMO
Transfection of activated promotion sensitivity genes (pro genes) confers on insensitive (P-) cells susceptibility to induction of anchorage-independent growth by tumor-promoting phorbol esters. Promotion-sensitive (P+) JB6 cell variants, from which activated pro-1 and pro-2 were cloned, respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and various nonphorbol tumor promoters with anchorage-independent transformation that is irreversible 60-80% of the time. Anchorage-independent (Tx) clonal lines derived from these TPA-induced agar colonies were also tumorigenic in nude mice. This report has addressed the question of whether the phenotypes associated with parental P+ cells are transferred by transfection of activated pro-1 and pro-2. Clonal lines were established after transfection of JB6 P- cells with activated pro-1 or pro-2, induction of anchorage-independent colony formation by TPA, and growth of individual agar colonies to yield clonal transfectant lines. The lines so derived from transfected populations included Tx, P+, and P- lines, reflecting irreversible neoplastic transformation and greater and lesser degrees of preneoplastic progression, respectively. The anchorage-independent transfectants were found to be tumorigenic. Since untransfected P- cells subjected to the same single-cycle TPA treatment and cloning in agar yielded no anchorage-independent and few P+ transfectants, the appearance of P+ and Tx transfectants after pro-1 and pro-2 transfection is therefore likely to be due to the transfected pro genes. Indirect assay of pro gene uptake by quick-blot hybridization of transfectant cell DNA with the vectors into which pro genes had been cloned confirmed the association of transferred P+ and Tx phenotypes with the presence of the transfected DNA. Finally, assay of the sensitivity of P+ pro-1 and pro-2 transfectants to transformation by TPA at various concentrations showed that transfection with pro-1 or pro-2 conferred about equal responses that were somewhat lower than those observed with parental P+ controls. Taken together these data indicate that promotion-insensitive JB6 cells need only an activated pro gene and TPA exposure to become neoplastically transformed.