Adenomatous polyposis coli-mediated hypersensitivity of mouse embryonic fibroblast cell lines to methylmethane sulfonate treatment: implication of base excision repair pathways.

The role of adenomatous polyposis coli (APC) has been implicated in various cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation and apoptosis. Recently, we discovered a novel role of APC in DNA base excision repair (BER) and showed that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 and interferes long-patch base excision repair (LP-BER) by blocking strand displacement synthesis. Many times, the chemotherapeutic drugs induce DNA alkylation damage, which is primarily repaired by the BER pathway. Thus, the efficacy of such drugs can be increased by APC resulting in the blockage of LP-BER. In the present study, we tested this hypothesis by using isogenic wild-type and Pol-beta-knockout mouse embryonic fibroblast (MEF) cell lines in which the Apc gene was knocked down by the small interfering RNA technique and treated with methylmethane sulfonate (MMS). The MEF-Apc(WT)/Polbeta-/- cells were hypersensitive to MMS treatment compared with the MEF-Apc(WT)/Polbeta+/+ cells. However, once the Apc gene was knocked down, these cells became more resistant to MMS treatment, suggesting that the MMS-induced hypersensitivity was associated with Apc. We then determined whether the hypersensitivity of MEF-Apc(WT)/Polbeta-/- and MEF-Apc(WT)/Polbeta+/+ cell lines were due to decreased Pol-beta-independent and Pol-beta-dependent LP-BER pathways, respectively. The results of in vivo and in vitro LP-BER assays supported our findings. Furthermore, Apc-mediated hypersensitivity to MMS treatment was correlated with increased apoptosis of MEF-Apc(WT)/Polbeta-/- and MEF-Apc(WT)/Polbeta+/+ as compared with MEF-Apc(KD)/Polbeta-/- and MEF-Apc(KD)/Polbeta+/+ cells. These results suggest that an increased level of Apc can increase the efficacy of DNA-alkylating drugs used as a curative therapy.

Chemopreventive effect of S-methylmethane thiosulfonate and sulindac administered together during the promotion/progression stages of colon carcinogenesis.

S-methylmethane thiosulfonate (S-MMTS), isolated from cauliflower and having antiproliferative activity, and the non-steroidal anti-inflammatory drug sulindac have been shown to inhibit chemically induced colon carcinogenesis when they are administered during the initiation and/ or post-initiation stages. The present study was designed to investigate the chemopreventive efficacy of 80 p.p.m. S-MMTS administered during the initiation and post-initiation stages and of S-MMTS and sulindac administered together at low doses (40 and 160 p.p.m., respectively) during the promotion/progression phases (late in the premalignant stage) of colon carcinogenesis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0 (control diet) or 80 p.p.m. S-MMTS. At 7 and 8 weeks of age all rats except those in the vehicle-treated groups were given s.c. injections of 15 mg/kg body wt azoxymethane (AOM). Rats receiving the control diet and intended for the study of inhibition of colon carcinogenesis during the promotion/progression phases were continued on the control diet for 14 weeks after the second AOM treatment; they were then switched to experimental diets containing 80 p.p.m. S-MMTS, 160 p.p.m. sulindac or 40 p.p.m. S-MMTS plus 160 p. p.m. sulindac. The rats were maintained on their respective dietary regimens until 52 weeks after carcinogen treatment and were then killed. Colon tumors were evaluated histopathologically. Administration of 80 p.p.m. S-MMTS alone during the initiation and post-initiation stages and promotion/progression stages had no significant effect on colon tumor inhibition. In contrast, the administration of 160 p.p.m. sulindac during the promotion/progression stages did significantly inhibit total colon tumor multiplicity (P < 0.05). Moreover, co-administration of 40 p.p. m. S-MMTS with 160 p.p.m. sulindac during the promotion/progression stages suppressed the incidence and multiplicity of non-invasive adenocarcinomas (P < 0.05-0.01) and multiplicity of invasive and total adenocarcinomas of the colon to a significant degree (P < 0. 05-0.01). These findings have potential clinical implications.

Effects of protocatechuic acid, S-methylmethanethiosulfonate or 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone(KYN-54) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary carcinogenesis in mice.

Modifying effects of dietary exposure of protocatechuic acid (PCA), a natural monophenolic compound, S-methylmethanethiosulfonate (MMTS), an organosulfur compound newly isolated from cauliflower, and 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone (KYN-54), a novel retinoidal butenolide compound, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (10 micromol, [corrected] single i.p. injection)-induced pulmonary carcinogenesis were examined in female A/J mice. Each of the test chemicals was given in diets during initiation or post-initiation phases (PCA, 1000 ppm; MMTS, 100 ppm; KYN-54, 200 ppm). All of these which had been proved to be chemopreventive mainly in digestive-organs carcinogenesis did not exert any preventive effect in this model when the incidence or multiplicity of pulmonary tumors (adenomas) of mice given NNK and the test chemical at the termination of the experiment (4 months) was compared to that of mice exposed to the carcinogen alone. In contrast, the multiplicity of lung tumors of mice receiving KYN-54 during the post-initiation phase was significantly larger than of the animals with NNK alone (P < 0.05), showing that KYN-54 has a promoting effect on pulmonary carcinogenesis in mice. These data indicate an organotropic activity of these compounds and suggest that candidate compounds for cancer chemoprevention need to be carefully examined for effectiveness in multiple organs by different models.

Suppressive effects of S-methyl methanethiosulfonate on promotion stage of diethylnitrosamine-initiated and phenobarbital-promoted hepatocarcinogenesis model.

Modifying effects of S-methyl methanethiosulfonate (MMTS) on diethylnitrosamine (DEN)-initiated and phenobarbital (PB)-promoted hepatocarcinogenesis were examined in rats. Five-week-old male F344 rats were divided into 8 groups. After a week, groups 1-5 were given DEN (100 mg/kg body weight, i.p.) once a week for 3 weeks, whereas groups 6-8 received vehicle treatment. Group 2 was given 100 ppm MMTS containing diet in the initiation phase. From 4 weeks after the start of experiment, groups 3 and 5 were fed MMTS, and groups 1-3 and 7 received drinking water containing 500 ppm PB. Group 6 was given MMTS diet alone throughout the experiment (24 weeks). The incidences of hepatocellular adenoma and total liver tumors were significantly smaller in group 3 than those of group 1. The average numbers of hepatocellular adenoma, carcinoma and total tumors in group 3 were significantly smaller than in group 1. Glutathione S-transferase placental form-positive foci were also significantly decreased by MMTS treatment in the promotion phase. MMTS treatment in the initiation or promotion phase reduced ornithine decarboxylase activity in the liver of rats given DEN. The antioxidant activity against lipid peroxidation of MMTS was confirmed in tests with rabbit erythrocyte membrane ghosts or rat hepatocytes. These results suggest that MMTS is a promising chemopreventive agent for liver neoplasia when concurrently administered with PB.

A negative phase II trial methylene dimethane sulphonate in advanced ovarian cancer (Cancer Research Campaign Phase I/II Trials Committee).

Methylene dimethane sulphonate (MDMS), the first member of the homologous series of dimethane sulphonic acid esters, was administered to 19 patients with advanced epithelial ovarian cancer. All patients had received prior chemotherapy and in addition 3 had received prior radiotherapy. MDMS was given as an i.v. bolus injection at a dose of 125mg m-2 and repeated in a q35 day schedule. Ten patients received only one course, six two courses, two three courses and one four courses. The major toxicity was thrombocytopenia which was cumulative. Serious neutropenia did not occur and no infective episodes requiring i.v. antibiotics were seen. Seven patients experience hair loss and four nausea and vomiting. Sixteen patients were evaluable for response but no objective remissions were seen although three patients had stable disease lasting at least 8 weeks. MDMS is therefore not recommended for further trial in epithelial ovarian carcinoma.

Phase I clinical trial of methylene dimethane sulfonate.

Methylene dimethane sulfonate is the first member of the homologous series of straight-chain diol sulfonates. It was selected for phase I testing because of high activity in the rat Yoshida sarcoma system and a possible novel site of alkylation due to its small molecular size. Methylene dimethane sulfonate was administered as a rapid iv bolus infusion to 39 patients at doses ranging from 14 to 225 mg/m2. Nausea and vomiting were not severe but total alopecia occurred in the majority of patients at doses greater than 100 mg/m2. The dose-limiting toxic effect was thrombocytopenia, which was cumulative with lower, more prolonged nadirs following successive courses. The median time to platelet count nadir was 21 days, with recovery by 35 days. A minor response was seen in one patient with an adenocarcinoma of the lung. The recommended dose for phase II studies is 125 mg/m2 for patients who have received prior chemotherapy and 150 mg/m2 for those who have not, in an every 35-day schedule.

DNA repair synthesis in fibroblast strains from patients with actinic keratosis, squamous cell carcinoma, basal cell carcinoma, or malignant melanoma after treatment with ultraviolet light, N-acetoxy-2-acetyl-aminofluorene, methyl methanesulfonate, and N-methyl-N-nitrosourea.

Fibroblast strains derived from skin biopsies of patients with actinic keratosis (6), malignant melanoma (18), squamous cell carcinoma (11), and basal cell carcinoma (12) were investigated for DNA repair synthesis, with 16 fibroblast strains for normal donors as controls. Cells were exposed to UV light, the "UV-like" carcinogen (Ac)2ONFln, and the methylating carcinogens MeSO2OMe and MeNOUr. Dose-response experiments, which included 10 dose levels, were performed, the data analyzed by linear regression, and the slope of the regression line (term: G0) used as a measure of DNA repair synthesis. The mean experimental variability of G0 of individual fibroblast strains was 9.5%-15.4%, depending upon exposure. For comparison of all cell strains belonging to the same skin malignancy group with those of the control group, G0 values of the individual strains were combined to yield group-specific weighted mean G0 values. In addition, the capacity to incise UV-damaged DNA was measured in 24 cell strains from patients with skin tumors using the alkaline elution technique. For quantitating DNA-incising capacity, the initial velocities of the elution curves were plotted versus the UV dose, and the slope of the resulting regression line was used to obtain the characteristic value E0. The mean experimental variability of E0 of individual strains was +/- 22%. These E0 values were combined to yield weighted mean values of groups. The fibroblast strains in the groups of patients with actinic keratosis and malignant melanoma were found to have normal mean G0 values when DNA repair synthesis was challenged with UV light or one of the three carcinogens. However, the squamous cell carcinoma group exhibited significantly lower mean G0 values after treatment with UV light (82% that of normal donors), (Ac)2ONFln (70%), MeSO2OMe (70%), and MeNOUr (69%). The basal cell carcinoma group showed significantly diminished repair synthesis upon treatment with UV light (81% that of normal donors) and MeSO2OMe (67%). In contrast to these findings, in no skin malignancy group was post UV DNA-incising capacity (E0) significantly diminished, although it should be noted that group sizes were only half as large as for G0 determinations. These data may be interpreted as indicating that DNA excision repair is impaired in fibroblast strains from patients with squamous cell carcinoma and-to a lesser extent-basal cell carcinoma.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of formaldehyde in methylene dimethanesulphonate-induced DNA cross-links and its relevance to cytotoxicity.

The interaction of the antitumour dialkanesulphonate ester, methylene dimethanesulphonate (MDMS) with the DNA of Yoshida sarcoma cells has been studied using the technique of alkaline elution. A marked retention which was reversed by proteolytic treatment indicated the presence of DNA-protein cross-links. A small proteinase-resistant retention was also observed which indicated that a low level of DNA-DNA interstrand cross-links also occurred. Treatment of cells with the amounts of formaldehyde expected by hydrolysis of the drug showed proteinase-reversible DNA-protein cross-linking which on proteolytic removal revealed a small number of single-strand breaks. Thus, MDMS causes both DNA-protein and DNA-DNA interstrand cross-links, the former component being attributed totally to the formaldehyde produced on the drug's hydrolysis. Cell survival data showed formaldehyde to be comparatively non-cytotoxic, providing evidence that DNA-DNA cross-links observable by this technique may be associated with the cytotoxicity of MDMS.