Kohji Yamakage

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice.

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.

Growth control of 3T3 fibroblast cell lines established from connexin 43–deficient mice

Connexins are considered to be involved in cell growth control, on the basis of studies mainly with tumorigenic cells. To study the role of connexin genes in normal cell growth control, we established fibroblast cell lines from connexin 43 (Cx43)–deficient mice and characterized their growth. Embryonic fibroblasts from wild‐type mice (Cx43+/+) and those with heterozygous (Cx43+/–) and homozygous (Cx43–/–) deficiencies of the Cx43 gene were cultured and passaged by a 3T3 protocol (every 3 d, 3 × 105 cells/60‐mm dish). All cell lines showed a growth crisis during passages 6–15 and then started to grow well. All cell lines grew at similar rates under the 3T3 protocol, but Cx43‐deficient (Cx43–/–) cell lines tended to grow faster when they were plated at 105 cells per dish. Cx43–/– cells did not express Cx43 and showed little gap‐junctional intercellular communication (GJIC), confirming that Cx43 is the major connexin responsible for GJIC of these fibroblasts. While all Cx43+/+ and Cx43+/– cell lines expressed Cx43 protein, some of them showed very little GJIC. Those cell lines with high GJIC showed higher levels of the P2 form of Cx43 protein, and more Cx43 was localized in the plasma membrane than in cell lines with lower GJIC levels. We investigated effects of serum concentration on cell growth in these cell lines. Although different cell lines responded differentially to these agents, there was no clear relationship between Cx43 expression and cell growth stimulation by them. This suggests that Cx43 expression alone is not a strong regulator of mouse fibroblast growth. Mol. Carcinog. 23:121–128, 1998.

In vitro clastogenicity and phototoxicity of fullerene (C60) nanomaterials in mammalian cells

Carbon nanomaterials such as carbon nanotubes, graphene, and fullerenes (C(60)) are widely used in industry. Because of human health concerns, their toxic potential has been examined in vivo and in vitro. Here we used mammalian cells to examine the in vitro clastogenicity as well as the phototoxicity of C(60). While C(60) induced no structural chromosome aberrations in CHL/IU cells at up to 5mg/ml (the maximum concentration tested), it significantly induced polyploidy at 2.5 and 5mg/ml with and without metabolic activation. In BALB 3T3 cells, C(60) showed no phototoxic potential but the anatase form of titanium oxide did. Since insoluble nanomaterials cause polyploidy by blocking cytokinesis rather than by damaging DNA, we concluded that the polyploidy induced by C(60) in CHL/IU cells was probably due to non-DNA interacting mechanisms.

Comparative studies of MCL-5 cells and human lymphocytes for detecting indirect-acting clastogens.

The MCL-5 cell line was established from human lymphoblastoid TK+/- cells transfected with cDNAs of human cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4) and microsomal epoxide hydrolase. The TK+/- cells constitutively express a relatively high level of endogenous CYP1A1. To study metabolic activities to indirect-acting clastogens, MCL-5 cells were treated with four clastogens, i.e. aflatoxin B1 (AFB1), diethylnitrosamine (DEN), cyclophosphamide (CPA), and 7,12-dimethylbenz[a]anthracene (DMBA). Human lymphocytes from peripheral blood were used as control cells under the assay conditions with or without induced rat liver metabolic activation (S9). All chemicals tested without S9 induced chromosomal aberrations (CA) in MCL-5 cells but not in human lymphocytes. All chemicals induced CA in both cell types in the presence of S9.

In vivo comet assay of acrylonitrile, 9-aminoacridine hydrochloride monohydrate and ethanol in rats

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, we examined the ability of acrylonitrile, 9-aminoacridine hydrochloride monohydrate (9-AA), and ethanol to induce DNA damage in the liver and glandular stomach of male rats. Acrylonitrile is a genotoxic carcinogen, 9-AA is a genotoxic non-carcinogen, and ethanol is a non-genotoxic carcinogen. Positive results were obtained in the liver cells of male rats treated with known genotoxic compounds, acrylonitrile and 9-AA.

Genotoxic potential and in vitro tumour-promoting potential of 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, two radiolytic products of fatty acids.

The DNA-damaging and tumour-promoting effects of two 2-alkylcyclobutanones (2-ACBs), which are found in irradiated fat-containing foods, were investigated by use of the comet assay and in an azoxymethane (AOM)-induced colon-carcinogenesis study in rats, respectively. We conducted genotoxicity tests of 2-dodecylcyclobutanone (2-dDCB) and 2-tetradecylcyclobutanone (2-tDCB) according to the test guidelines for chemicals or drugs. In addition, a cell-transformation assay with Bhas 42 cells was performed to investigate their promoting potential in vitro. The Salmonella typhimurium mutagenicity assay (Ames test), conducted with five tester strains, revealed that neither 2-dDCB nor 2-tDCB possessed mutagenic activity. Moreover, both in the in vitro chromosomal aberration test on CHL/IU cells and the in vivo bone-marrow micronucleus test where mice were given 2-dDCB and 2-tDCB (orally, up to 2000 mg/kg bw/day), we did not detect any clastogenic effects. Furthermore, DNA strand-breaks were not detected in the in vitro comet assay with CHL/IU cells, and DNA adducts derived from 2-dDCB and 2-tDCB were not detected in the colon tissues of the mice used for the micronucleus tests, in rats from a repeated dose 90-day oral toxicity test (0.03% 2-tDCB in the diet), or in rats from the AOM-induced carcinogenesis study (0.025% 2-tDCB in the diet). An in vitro tumour-promotion assay with Bhas 42 cells revealed that the number of transformed foci increased significantly following treatment of cells in the stationary phase with 2-dDCB or 2-tDCB for 10 days. Our results indicate that neither 2-dDCB nor 2-tDCB were genotoxic chemicals. However, they exhibited promoting activity, at least in vitro, when Bhas 42 cells were continuously exposed to these chemicals at toxic doses.

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats

A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.