mezerein and Cell-Transformation--Neoplastic

In this paper, recent studies on the role of cell communication in cancer induction, particularly in two-stage carcinogenesis, were reviewed. Cell communication has been proposed to play an important role in cell growth and differentiation since its discovery. The recent finding that tumor promoters inhibit cell communication supports this possibility. The inhibition of cell communication by phorbol ester tumor promoters was also shown to correlate with enhancement of in vitro carcinogenesis in Balb/c 3T3 cells. This strongly suggests that the blocked cell communication may play a crucial causative role in the process of carcinogenesis. Accumulated evidence indicates that phorbol ester may induce blockage of cell communication through binding to its membrane receptor which is presumably Ca2+/phospholipid-dependent kinase. cAMP enhances cell communication and protects its inhibition by phorbol ester, presumably through activating cAMP-dependent kinase. This indicates the possibility that the two kinases may be key elements for physiological regulation of cell communication. It is proposed that the disturbance of the kinase systems by endogenous and exogenous factors may be responsible for the promotion phase of cancer induction. However, the true physiological role of cell communication in carcinogenesis remains to be demonstrated more directly. Especially, what kinds of molecules can pass through the gap junction and regulate cell functions in a cell community must be challenged in future. Some such molecules were speculatively described in this review.

Topics: Animals; Antigens, Surface; Calcium; Carcinogens; Carrier Proteins; Cell Adhesion Molecules; Cell Communication; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Diterpenes; Humans; Intercellular Junctions; Mice; Mice, Inbred BALB C; Permeability; Phorbol Esters; Phospholipids; Protein Kinases; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase; Terpenes; Tetradecanoylphorbol Acetate

To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been questioned. In vitro cell transformation assays (CTAs) could represent an interesting alternative to animal models as it has the advantage of detecting both genotoxic and non-genotoxic transforming chemicals. Among them, Bhas 42 CTA uses a cell line that has been transfected with the oncogenic sequence v-Ha-ras. This sequence confers an "initiated" status to these cells and makes them particularly sensitive to non-genotoxic agents. In a previous work, transcriptomic analysis revealed that the treatment of Bhas 42 cells with transforming silica (nano)particles and 12-O-tetradecanoylphorbol-13-acetate (TPA) commonly modified the expression of 12 genes involved in cell proliferation and adhesion. In the present study, we assess whether this signature would be the same for four other soluble transforming agents, i.e. mezerein, methylarsonic acid, cholic acid and quercetin. The treatment of Bhas 42 cells for 48 h with mezerein modified the expression of the 12 genes of the signature according to the same profile as that of the TPA. However, methylarsonic acid and cholic acid gave an incomplete signature with changes in the expression of only 7 and 5 genes, respectively. Finally, quercetin treatment induced no change in the expression of all genes but exhibited higher cytotoxicty. These results suggest that among the transforming agents tested, some may share similar mechanisms of action leading to cell transformation while others may activate different additional pathways involved in such cellular process. More transforming and non-transforming agents and gene markers should be tested in order to try to identify a relevant gene signature to predict the transforming potential of non-genotoxic agents.

Topics: Animals; BALB 3T3 Cells; Butylated Hydroxyanisole; Carcinogenicity Tests; Carcinogens; Cell Transformation, Neoplastic; Cholic Acid; Mice; Quercetin; Reproducibility of Results; Tetradecanoylphorbol Acetate; Transcriptome

Retinoic acid (RA) was topically applied to the skin of Sencar mice during the promotion phase of specific tumor induction protocols that produce papillomas at low (12-O-tetradecanoylphorbol-13-acetate promoted, TPA) or high (mezerein-promoted) risk for premalignant progression and malignant conversion. RA consistently reduced the yield of papillomas and carcinomas in both protocols, but the frequency of malignant conversion in papillomas that emerged during RA treatment was not reduced. When TPA was reapplied after cessation of RA treatment, the number of papillomas increased 2-fold, suggesting that RA had not eliminated initiated cells. In vitro, RA prevented the emergence of transformed keratinocytes in an assay that mimics malignant conversion, suggesting that RA can suppress conversion if applied during the stage of premalignant progression. Examination of tumor markers at weeks 14 and 22 of the tumor-induction experiments in vivo indicated that papillomas evolving during RA treatment exhibited a phenotype of high progression risk, even in the TPA-promoted groups. In the majority of these tumors, the alpha6beta4 integrin and retinoid X receptor alpha transcripts were detected suprabasally, indicating an advanced state of premalignant progression. RA-treated tumors also expressed higher levels of transcripts for transforming growth factor (TGF)-beta1 and localized TGF-beta1 peptide in the basal portions of the tumor fronds. Because up-regulated expression of TGF-beta1 suppresses papilloma formation, these studies suggest a mechanism whereby RA can prevent papilloma eruption via a TGF-beta intermediate, but papillomas resistant to RA may have altered TGF-beta signaling and progress to carcinomas at an increased frequency.

Topics: Administration, Topical; Animals; Anticarcinogenic Agents; Antineoplastic Agents; Biomarkers, Tumor; Carcinogens; Carcinoma, Basal Cell; Cell Transformation, Neoplastic; Disease Progression; Diterpenes; Female; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Inbred SENCAR; Papilloma; Phenotype; Precancerous Conditions; Receptors, Retinoic Acid; Retinoid X Receptors; Risk Factors; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor beta; Tretinoin

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01-0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene.

Topics: Alkyl and Aryl Transferases; Animals; Carcinoma; Cell Compartmentation; Cell Membrane; Cell Transformation, Neoplastic; Cytosol; Diterpenes; Enzyme Inhibitors; Mice; Papilloma; Proto-Oncogene Proteins p21(ras); Skin; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Transferases; Tretinoin

Mezerein (MEZ) has been described as a weak complete tumor promoter but an effective stage II promoter in the mouse skin initiation-promotion tumor model. In this study MEZ produced a strong transformation response when tested under code in the Syrian hamster embryo (SHE) clonal morphological transformation assay. Using a standard 7-day exposure protocol designed to detect complete carcinogens, MEZ was active at non-toxic concentrations, producing a linear response between 0.3-10 ng/ml in a log-log plot of transformation activity versus concentration. These concentrations were in the same range as those which had been shown to elicit promotion activity in several in vitro cell culture systems. Our data suggest that the SHE assay has the ability to detect some tumor promoters under the same conditions used to test for complete carcinogens. The possibility that MEZ may possess in vivo carcinogenic activity cannot be excluded.

Topics: Animals; Benzo(a)pyrene; Carcinogenicity Tests; Carcinogens; Cell Transformation, Neoplastic; Cricetinae; Diterpenes; Embryo, Mammalian; Mesocricetus; Terpenes

We have compared the responses of normal human cervical keratinocytes (HCE) to diethylstilboestrol (DES), and the promoting agents, phorbol-12-myristate-13-acetate (PMA) and mezerein using the loss of cloning efficiency as a measure of terminal differentiation in vitro. Dose-response studies showed that normal HCE are growth inhibited by chronic exposure to DES at concentrations greater than or equal to 2.5 X 10(-5) M, to PMA at concentrations greater than 10(-8) M and mezerein at concentrations greater than 10(-9) M. Compared to acetone controls, promoter or DES-treated cells exhibited a 10- to 12-fold increase in cornified-envelope formation. Normal HCE exhibit a heterogeneous response to PMA in that 85-90% of colony-forming cells lose their colony-forming ability after a 24-h exposure to 10(-6) M PMA. The PMA-resistant subpopulation, PMAR, remains constant and is not reduced even after 96 h chronic exposure to PMA. In contrast, the colony-forming ability of normal HCE is almost totally suppressed after 24 h exposure to 10(-6) M mezerein. After 24 h incubation with 5 X 10(-5) M DES, 20% of normal HCE are capable of colony formation but this resistant fraction is eliminated after 96 h chronic exposure. Cornified-envelope formation was negligible in malignant cervical keratinocytes grown in the presence of DES or promotors and these cells were characterised by a very large PMAR fraction - 85 - 90% of cells retained colony-forming ability after exposure to 10(-6) M PMA for 24 h. Furthermore, 90-100% of malignant cervical keratinocytes retained their colony-forming capacity after exposure to 10(-6) M mezerein. However, colony-forming ability declined steadily in the presence of 5 X 10(-5) M DES and after 96 h only a tiny fraction, 1% of malignant cervical keratinocytes could form colonies on replating. The mechanisms by which DES inhibits growth and induces cornified-envelope formation in HCE would appear to be distinct from those activated by PMA and mezerein.

Topics: Carcinogens; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cervix Uteri; Clone Cells; Diethylstilbestrol; Diterpenes; Female; Humans; Keratins; Kinetics; Phorbols; Terpenes; Tetradecanoylphorbol Acetate

beta-All-trans-retinoic acid (RA) inhibited the anchorage-independent growth of JB6 cells induced by either mezerein or alpha-epidermal growth factor (alpha-EGF) (a purified fraction of epidermal growth factor). The inhibition was dose dependent for alpha-EGF as well as for RA. Mezerein-induced growth in soft agar was inhibited to a greater extent by RA than was alpha-EGF-induced growth in soft agar, at similar colony yields. The extent of inhibition of anchorage-dependent growth induced by RA was similar for nontransformed JB6 cells and for alpha-EGF-transformed cells, so that transformation was shown not to influence the sensitivity of cells to retinoid inhibition of anchorage-dependent growth. RA was as effective at inhibiting anchorage-independent growth when it was applied after promoter-induced transformation as when it was applied during promoter-induced transformation. Therefore, the antiproliferative effect of RA, without an additional antitransformation effect, was sufficient to account for the reduced colony yield. These results suggest that the antipromoting action of retinoids in JB6 cells may occur by limiting proliferation, the regulation of which may be coupled with the state of differentiation of cells.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Epidermal Growth Factor; Epidermis; Mice; Skin Neoplasms; Terpenes; Tretinoin

Alterations in NMRI mouse skin induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in "stage I of tumor promotion" are slowly reversible, and this reversibility has a half-time of 10 to 12 weeks. The tumor response observed in the course of an initiation-promotion experiment in vivo is independent of whether stage I of promotion occurs before or after initiation. Since the time interval between treatment with the promoter, and subsequent initiation can be extended up to at least 6 weeks, an enhancement of initiation because of promoter-induced cellular DNA synthesis seems to be unlikely. This result may be inconsistent with the two-stage model of tumor promotion because it indicates that in skin the existence of initiated cells is not required for the induction of cellular alterations that are essential for the stage of skin tumorigenesis called stage I of promotion.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Transformation, Neoplastic; Diterpenes; Female; Mice; Phorbol Esters; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Time Factors

The complete tumour promoter phorbol, 12-myristate, 13-acetate (PMA) induces terminal differentiation in the majority of normal cultured human and mouse keratinocytes but a subpopulation exists which is resistant to this effect (PMAR). We have compared with PMA the effects of mezerein (Mez) and phorbol, 12-retinoate, 13-acetate (PRA) on the ability of normal and transformed human and mouse keratinocytes to terminally differentiate in an attempt to elucidate why the latter two compounds are inefficient complete tumour promoters but are effective as second-stage promoters when given after PMA in the two-stage promotion regimen. Both PMA and Mez increased cornified envelope formation in a similar way in normal and transformed keratinocyte cultures inducing a 20- to 25-fold increase over the solvent controls in normal keratinocytes but only a 2-fold increase in line SCC-27 (a cell line derived from a human squamous cell carcinoma). However, while quantitative dose response studies of the effect of phorbol esters on colony forming ability revealed a proportion of normal human and mouse keratinocytes which were resistant to PMA, no normal keratinocytes were resistant to Mez or PRA. In contrast, cell lines derived from papillomas and squamous cell carcinomas showed a resistant fraction of similar size with all three compounds. Furthermore, when Mez or PRA were mixed with PMA the survival of line SCC-27 was the same as when the cultures were treated with the compounds individually indicating that the keratinocytes which were resistant to PRA or Mez were also the PMAR subpopulation. A non-tumorigenic subclone of line SCC-12 (clone F.2), previously shown to possess all known properties of transformed keratinocytes except defective terminal differentiation in suspension culture responded to PMA and Mez in a similar way to normal keratinocytes, suggesting that resistance of the PMAR subpopulation to second-stage promoters requires the expression of a defect in the keratinocyte terminal differentiation programme.

Topics: Carcinogens; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Drug Resistance; Epidermal Cells; Epidermis; Humans; Keratins; Phorbol Esters; Phorbols; Terpenes; Tetradecanoylphorbol Acetate

The effects of the tumor promoter -12-O-tetradecanoyl phorbol-13-acetate, its analogues, and other tumor promoters, on the proliferation of human skin fibroblasts (SF) have been investigated. We have previously shown (Kopelovich and Bias, 1979), that TPA caused a biphasic (upward concave) dose effect in the cloning efficiency assay of normal and mutant human fibroblastic cell strains. Here we report that the biphasic dose response pattern, consisting of an inhibitory phase and a stimulatory phase, was shared by the active analogues of TPA, e.g., phorbol-12,13-dibutyrate and phorbol-12,13-dibenzoate. This biphasic dose effect relationship, however, was not seen with phorbol-12,13-diacetate or the inactive analogues of TPA such as phorbol and 4-O-methyl-12-O-tetradecanoyl phorbol-13-acetate, nor was it seen with mezerein, teleocidin, or bile-acid derivatives of humans. An analysis of the cloning efficiency data by the median-effect equation (Chou and Talalay, 1981) showed that in low-density cultures both the inhibitory phase and the stimulatory phase of the dose-effect relationships of TPA, its analogues, mezerein, and teleocidin exhibited a linear median-effect plot and thus closely followed the basic mass-action principle. The median-effect plot of these data allowed quantitative determination of growth curve characteristic such as regression coefficient, slope (a measure of sigmoidicity), median-effect concentrations such as I50 for the inhibitory effect, and A50 for the stimulatory effect (i.e., the relative potency of the analogues) and the transition point of the biphasic phenomenon in the case of the phorbol esters. In addition, we have demonstrated a relationship between the dose response effect of TPA on the proliferation of various human cells and tumor progression in vitro.

Topics: Caenorhabditis elegans Proteins; Carcinogens; Carrier Proteins; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Fibroblasts; Humans; Lyngbya Toxins; Protein Kinase C; Receptors, Drug; Receptors, Immunologic; Terpenes; Tetradecanoylphorbol Acetate

The effect of the plant diterpenes, phorbol derivatives and mezerein, on differentiation of various human myeloid leukemic cells to macrophages was determined. The results indicate that, within the group of phorbol esters tested, a correlation exists between the potency of the compounds as inducers of differentiation and their reported potency as tumor promoters. However, mezerein and 12-O-retinoylphorbol 13-acetate, which promote tumors only weakly or not at all, were found to be efficient inducers. The efficiency of all the active phorbol derivatives, including the weak inducers, also known to be weak promoters, could be potentiated by pretreatment of the cells with retinoids, compounds which have been reported to inhibit tumor promotion. Similar results were obtained in 3 different established cell lines, as well as in short-term cultures of cells obtained from patients with acute myeloid leukemia. The results suggest that the activities of the diterpenes as tumor promoters and inducers of differentiation are not necessarily linked. Moreover, certain conditions which are unfavorable for tumor promotion may not affect or even potentiate induction of differentiation.

Topics: Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Drug Interactions; Humans; Leukemia, Myeloid, Acute; Macrophages; Phorbol Esters; Phorbols; Retinoids; Structure-Activity Relationship; Terpenes

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.

Topics: Animals; Carbon Radioisotopes; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Epidermal Growth Factor; Gangliosides; Glucosamine; Mice; Phorbol Esters; Phorbols; Skin; Terpenes; Tetradecanoylphorbol Acetate