mezerein and Carcinoma--Squamous-Cell

Hypermethylation of CpG sites within the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene occurs frequently in human cancer, preventing both MGMT expression and repair of alkylation damage. To assess the role of MGMT in the development of mouse skin tumors induced by initiation-promotion protocols, methylation of the MGMT promoter was examined in tumor DNA using methylation-specific PCR. To determine whether MGMT promoter methylation was affected by the tumor induction protocol, tumors were initiated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein. Although the MGMT promoter was not methylated in normal skin, promoter methylation was found in 56 of 136 papillomas (41.2%) and in 19 of 37 squamous cell carcinomas (51.4%). When methylation of the MGMT promoter was compared in the 4 treatment groups, hypermethylation was found more frequently in tumors initiated by DMBA and promoted by mezerein, a protocol associated with a high frequency of malignant conversion. Methylation was found in some tumors as early as 5 weeks after initiation, but the methylation frequency increased with time. MGMT promoter methylation reduced MGMT expression as determined by immunohistochemistry. Although MGMT promoter methylation was not generally correlated with ras mutations, the frequency of MGMT methylation was higher in MNNG-initiated, mezerein-promoted papillomas with mutations in Ha-ras compared to papillomas with Ki-ras. Methylation of the MGMT promoter, associated with reduced MGMT expression, is found in nearly half of mouse skin tumors, but varies with both the tumor initiator and tumor promoter, and may be a key step in the progression from papillomas to carcinomas.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Diterpenes; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Methylnitronitrosoguanidine; Mice; Mutation; O(6)-Methylguanine-DNA Methyltransferase; Papilloma; Promoter Regions, Genetic; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

We earlier showed that a polyphenolic fraction isolated from green tea (GTP) affords protection against tumor promotion and tumor progression in SENCAR mouse skin. The present study was designed to further evaluate the protective effect of GTP against the induction and subsequent progression of papillomas to squamous cell carcinomas (SCCs) in experimental protocols where papillomas were developed with a low or high probability of their malignant conversion. Topical application of GTP (6 mg/animal) 30 min prior to that of 12-O-tetradecanoylphorbol-13-acetate (TPA) either once a week for 5 weeks (high risk TPA protocol) or once a week for 20 weeks (low risk TPA protocol) or mezerein (MEZ) twice a week for 20 weeks (high risk MEZ protocol) in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin resulted in significant protection against skin tumor promotion in terms of tumor incidence (32-60%), multiplicity (49-63%) and tumor volume/mouse (73-90%) at the termination of the experiment at 20 weeks. In three separate malignant progression experiments when papilloma yield in DMBA-initiated and TPA or MEZ promoted low and high risk protocols was stabilized at 20 weeks, animals were divided into two subgroups. These animals were either topically treated twice weekly with acetone (0.2 ml/animal, spontaneous malignant conversion group) or with GTP (6 mg/animal in 0.2 ml acetone) for an additional period of 31 weeks. During these treatment regimens, all suspected carcinomas were recorded and each one was verified histopathologically either at the time when tumor-bearing mouse died/moribund or at the termination of the experiment at 51 weeks. GTP resulted in significant protection against the malignant conversion of papillomas to SCC in all the protocols employed. At the termination of the experiment at 51 weeks, these protective effects were evident in terms of mice with carcinomas (35-41%), carcinomas per mouse (47-55%) and percent malignant conversion of papillomas to carcinomas (47-58%). The kinetics of malignant conversion suggest that a subset of papillomas formed in the early phase of tumor promotion in all the protocols had a higher probability of malignant conversion into SCCs because all the positive control groups (acetone treated) produced nearly the same number of carcinomas (33-38 in a group of 20 animals) at the end of the progression period. In the GTP-treated group of animals the number of carcinomas formed was less (14-20 in a group of 20 anima

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anticarcinogenic Agents; Antioxidants; Carcinoma, Squamous Cell; Catechin; Cocarcinogenesis; Disease Progression; Diterpenes; Flavonoids; Mice; Mice, Inbred SENCAR; Papilloma; Phenols; Plant Extracts; Polymers; Skin Neoplasms; Tea; Terpenes

To develop mouse strains useful for studies of susceptibility and resistance to the induction of skin tumors, three new inbred SENCAR strains were independently derived by random inbreeding of outbred SENCAR mice. Characterization of these mice for sensitivity to skin tumor development indicated that mice of all three strains displayed increased sensitivity to initiation by 7,12-dimethylbenz[a]anthracene (DMBA), urethane, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Promotion by mezerein as well as carcinogenesis by repeated treatment with DMBA or MNNG produced papillomas with a high frequency of conversion to squamous cell carcinomas (SCCs). Compared with outbred SENCAR mice, development of both squamous papillomas and carcinomas was increased at least two-fold by all protocols tested. The F1 hybrid between SENCARA/Pt males and resistant BALB/cAnPt females was resistant to the induction of both papillomas and SCCs after initiation by 2 microg of DMBA and promotion by 20 weekly applications of 2 microg of TPA. Papillomas developed in all of the SENCARA/Pt mice, none of the BALB/cAnPt mice, and 12% of the F1 progeny. Thus, at these doses of initiator and promoter, resistance was incompletely dominant in the F1 hybrid. However, the responsiveness of the F1 mice could be increased substantially by increasing the dose of the promoter.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cocarcinogenesis; Disease Susceptibility; Diterpenes; Female; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Inbred SENCAR; Papilloma; Phenotype; Sensitivity and Specificity; Skin Neoplasms; Terpenes; Urethane

The present study was designed to further evaluate the growth and progression of papillomas to squamous cell carcinomas (SCCs) in groups of animals receiving initiating doses of 7,12-dimethylbenz[a]anthracene (DMBA) producing relatively low papilloma yields following long term promotion (60 weeks) with 12-O-tetradecanoylphorbol-13-acetate (TPA). For comparison, groups of animals were initiated with various doses of DMBA and then promoted with mezerein (MEZ), benzoyl peroxide (BzPo) and chrysarobin (CHRY). Following initiation, groups of female SENCAR mice received the following promoter doses: TPA (1.0 or 2.0 micrograms per mouse); MEZ (2.0 micrograms per mouse); BzPo (20.0 mg per mouse); and CHRY (52.8 micrograms per mouse). The maximum papilloma to SCC conversion ratio obtained with TPA in the current study was 0.32. This value was in the range of maximum conversion ratios obtained with the other compounds: MEZ, 0.40; CHRY, 0.32 and BzPo, 0.19. In general, the highest papilloma to SCC conversion ratios observed with TPA as the promoter were obtained in groups that received the lowest doses of DMBA and had relatively low papilloma burdens. A comparison of papilloma to SCC conversion in groups of mice promoted with TPA, MEZ or CHRY and having similar papilloma yields, revealed very similar conversion ratios. Comparison of the BzPo group with a similar papilloma yield indicated that the conversion ratio was slightly lower with this promoter. The present results indicate that in mice promoted with TPA and having relatively low papilloma numbers, a larger proportion of these papillomas progress to SCCs during continued promoter treatment. Furthermore, the results suggest that papillomas behave similarly in their ability to progress to SCCs regardless of the promoter used when comparing groups of mice with similar tumor numbers. The data are discussed in terms of possible mechanisms for the observed results.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anthracenes; Benzoyl Peroxide; Carcinoma, Squamous Cell; Diterpenes; Dose-Response Relationship, Drug; Female; Mice; Papilloma; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Time Factors

The present work was undertaken with the basic objective to compare the effect of related tumour promoters phorbol-12-myristate-13-acetate (PMA), benzoyl peroxide (BP) and mezerein (M) on poly ADP-ribosylation in HeP2 cells. PMA, BP and mezerein showed an increase in the enzyme activity to the extent of 66, 89 and 35% respectively. This was accompanied by a drop in NAD levels of 22% with PMA, 24% with BP and 49% with mezerein, PMA-treated cells showed a decrease in the enzyme activity of 42% with superoxide dismutase (SOD) and catalase (CAT), 18% with butylated hydroxy toluene (BHT), 52% with indomethacin and 32% with 3-aminobenzamine (3AB). However, for BP the inhibition was 16% with CAT, 41% with SOD, 18% with BHT, 34% with 3AB and there was no inhibition with indomethacin. The involvement of active oxygen species was supported by a 2.5-fold increase in superoxide anion levels with PMA and BP and no change with mezerein. There was a twofold and a 2.5-fold increase in Ca2+ influx after PMA and BP treatment respectively. It is concluded that PMA and BP mediate their effect on poly ADP-ribosylation through active oxygen species which in turn stimulates the influx of Ca2+ into cells and this may play an important role in signal transduction.

Topics: Benzoyl Peroxide; Carcinogens; Carcinoma, Squamous Cell; Diterpenes; Enzyme Activation; Humans; NAD; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Terpenes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

This study evaluated the skin tumor-promoting activity of mezerein in SENCAR mice. The effect of initiation dose of 7,12-dimethylbenz(a)anthracene (DMBA) on tumor promotion by mezerein was examined. Excellent dose-response relationships were observed for initiation with DMBA at 0.2-20 micrograms per mouse with mezerein as a complete promoter. None of the mezerein-only promotion groups had papilloma responses similar to those of the corresponding groups receiving two-stage promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) followed by mezerein, even when a 40-micrograms initiating dose of DMBA was used. The effect delaying promotion with mezerein for 10 weeks was also examined in mice initiated with either 0.2, 2, 20, or 40 micrograms of DMBA per mouse. The 10-week delay led to a slight increase in the number of papillomas per mouse in some but not all treatment groups. Again, none of the delayed-mezerein-treatment groups had papilloma responses similar to those of the corresponding two-stage promotion (TPA-mezerein) groups at any corresponding initiating dose of DMBA. Finally, the progression of papillomas to carcinomas during promotion with mezerein was examined in groups of mice initiated with either 2 or 20 micrograms of DMBA. Higher ratios of carcinomas to papillomas were observed in mice promoted with mezerein than in mice receiving TPA promotion or two-stage promotion (TPA-mezerein). However, the presence of two to four times more papillomas in some mezerein-treated groups did not lead to greater numbers of carcinomas than in the groups with fewer papillomas. The data do not support the idea that spontaneous stage I promotion can be induced by delaying mezerein treatment for 10 weeks. Furthermore, the data suggest that the higher ratio of carcinomas to papillomas observed with mezerein promotion may be a function of the lower tumor burdens obtained after promotion with this compound rather than a specific property of the chemical.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Diterpenes; DNA; Female; Mice; Papilloma; Skin; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate

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