Effects of antioxidant capacity on micronucleus induction by cigarette smoke in mammalian cells.

We have compared micronucleus (MN) induction by cigarette smoke in the L5178Y, TK6, and CHL/IU cell lines. The test sample was total particulate matter of 3R4F reference cigarette smoke, suspended in DMSO. After 3-h treatment, with or without a rat liver S9 metabolic activation system, followed by 24-h recovery, dose-dependent MN increases were seen in all cell lines. However, CHL/IU and TK6 cells were more resistant than L5178Y cells (comparison by Benchmark Doses with PROAST software). 3R4F smoke generates reactive oxygen species (ROS). Therefore, we explored the relationship between the sensitivities to 3R4F smoke and the antioxidant capacities of the cell lines. While the total antioxidant capacities were not significantly different among the cell lines, cellular glutathione (GSH) was higher in CHL/IU cells than in L5178Y cells. Pretreatment of CHL/IU cells with a GSH precursor, N-acetylcysteine (NAC), reduced the genotoxicity/cytotoxicity of 3R4F, whereas an inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO), enhanced it. The effects of NAC and BSO were also seen after treatment with allyl isothiocyanate, a ROS-generating chemical, but not with mitomycin C, a ROS-independent genotoxicant. Pretreatment with NAC increased cellular thiol levels. From the present results, the genotoxicity and cytotoxicity of cigarette smoke differs among these cell lines in a manner that may be related to their antioxidant thiol levels.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). RESULTS: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. CONCLUSIONS: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

[A case of recurrent malignant melanoma of esophagus responsive to combined chemotherapy, radiotherapy and cellular immunotherapy].

Treatment of primary malignant melanoma of the esophagus remains challenging. We treated a 53-year-old man with pT4N2M0, Stage IVa malignant melanoma of the esophagus with esophagectomy followed by adjuvant chemotherapy. Six months later, computed tomography revealed a 12 cm disseminated tumor of the mesenterium, multiple peritoneal dissemination, and a large amount of ascites. We administered chemotherapy consisting of dacarbazine combined with cisplatin and nimustine, and radiotherapy(50 Gy)was applied to the disseminated mesenteric tumor. At another clinic, the patient was administered synchronous cellular immunotherapy consisting of dendritic cells pulsed with autologous tumor lysates and lymphokine-activated killer cells. The mesenteric tumor was extremely responsive to this trimodal treatment. Because recurrence occurred later within the left orbita muscle, we added 50 Gy of radiation to prevent blindness. The patient responded to this treatment and survived another 6 months with high quality of life. It is difficult to treat advanced malignant melanoma of the esophagus, and patient prognosis is extremely poor. In this patient, the recurrent tumors responded well to trimodal therapy consisting of chemotherapy, radiotherapy and cellular immunotherapy.

IGFBP3 and BAG1 enhance radiation-induced apoptosis in squamous esophageal cancer cells.

Identification of reliable markers of radiosensitivity and the key molecules that enhance the susceptibility of esophageal cancer cells to anticancer treatments would be highly desirable. To identify molecules that confer radiosensitivity to esophageal squamous carcinoma cells, we assessed the radiosensitivities of the TE-5, TE-9 and TE-12 cloneA1 cell lines. TE-12 cloneA1 cells showed significantly greater susceptibility to radiotherapy at 5 and 10Gy than either TE-5 or TE-9 cells. Consistent with that finding, 24h after irradiation (5Gy), TE-12 cloneA1 cells showed higher levels of caspase 3/7 activity than TE-5 or TE-9 cells. When we used DNA microarrays to compare the gene expression profiles of TE-5 and TE-12 cloneA1 cells, we found that the mRNA and protein expression of insulin-like growth factor binding protein 3 (IGFBP3) and Bcl-2-associated athanogene 1 (BAG1) was five or more times higher in TE-12 cloneA1 cells than TE-5 cells. Conversely, knocking down expression of IGFBP3 and BAG1 mRNA in TE-12 cloneA1 cells using small interfering RNA (siRNA) significantly reduced radiosensitivity. These data suggest that IGFBP3 and BAG1 may be key markers of radiosensitivity that enhance the susceptibility of squamous cell esophageal cancer to radiotherapy. IGFBP3 and BAG1 may thus be useful targets for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.

Regenerating gene I regulates interleukin-6 production in squamous esophageal cancer cells.

Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Ialpha and Ibeta and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Ialpha and Ibeta led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-gamma, tumor necrosis factor-alpha, granulocyte-colony stimulating factor or transforming growth factor-beta1. The elevated IL-6 expression seen in REG Ialpha transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology.