Tetsuya Tsukamoto

Adenoviral-mediated gene transfer into primary human and mouse mammary epithelial cells in vitro and in vivo.

Primary mammary epithelial cells from both the human and mouse mammary glands can be genetically altered under a variety of situations using the replication-defective adenoviral vector containing a marker gene encoding the E. coli beta-galactosidase. Primary human and mouse mammary epithelial cells in monolayer culture and in three-dimensional collagen gel culture systems were transduced by adenovector at high efficiency. Successful gene transfer was also accomplished in situ and in vivo. In the mouse mammary gland, anatomically restricted gene transfer and expression was demonstrated by micro-injection of adenoviral vector directly into the main duct of the mammary gland. Injection of adenoviral vector directly into the human mammary tissues from reduction mammoplasty specimens, into the mouse mammary gland-free fat pad containing the previously transplanted dissociated human mammary epithelial cells, and intratumorally into the human breast cancer xenografts in nude mice, all resulted in successful gene transfer to human mammary epithelial cells. High efficiency introduction of genetic material into primary mammary epithelial cells is important in the study of mammary carcinogenesis and potentially for gene therapy of human breast cancer.

Expression of MAT1/PEA-15 mRNA isoforms during physiological and neoplastic changes in the mouse mammary gland.

MAT1 is a novel transforming gene which was cloned from a mouse mammary tumor induced by N-methyl-N-nitrosourea in vitro in the presence of lithium as a mitogen. Later, it was found to be identical to the 3 untranslated region (UTR) of the 2.5 kb isoform of PEA-15 (phosphoprotein enriched in astrocytes-15 kDa). We re-cloned MAT1/PEA-15 cDNAs and showed 2.5, 2.0 and 1.8 kb isoforms and confirmed MAT1 localization as reported. The 2.0 and 1.8 kb isoforms were produced by alternative splicing and alternative polyadenylation at the 3 UTR, respectively. To analyze the role of MAT1/PEA-15, we examined the expression of MAT1/PEA-15 mRNA in normal mammary tissues and in mammary tumors. The mammary gland during pregnancy, lactation and weaning showed weak but stable expression. Compared with normal mammary gland, mammary tumors showed stronger expression. Aberrant expression of MAT1/PEA-15 isoforms was found in mouse mammary epithelial cell lines, FSK7 and TM6, which lost the 2.5/2.0 and 2.5 kb isoforms, respectively. In contrast to other oncogenes like c-myc, MAT1/PEA-15 mRNA was extremely stable after actinomycin D and cycloheximide treatments suggesting that other protein expression is prerequisite for degradation of MAT1/PEA-15 mRNA. It evoked the possibility of the 3 UTR of MAT1/PEA-15 (designated as MAT1-T) as a riboregulator in mammary tumorigenesis and necessity for further analysis of human breast cancers as well as mouse mammary tumors.

Distinction of carcinogens from mutagens by induction of liver cell foci in a model for detection of initiation activity

Initiating activities of 26 chemicals were investigated in an in vivo 5 week initiation assay model with evaluation of the induction of glutathione S-transferase placental form (GST-P) positive foci as end-point lesions. With the five genotoxic hepatocarcinogens (diethylnitrosamine, dimethylnitrosamine, 2-acetylaminofluorene, N-bis(2-hydroxypropyl)-nitrosamine and safrole) and 11 genotoxic non-hepatocarcinogens, (2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, benzo[a]pyrene, N-butyl-N-(4-hydroxybutyl)nitrosamine, 7,12-dimethylbenz[a]anthracene, 1,2-dimethylhydrazine, N-ethyl-N-hydroxyethylnitrosamine, 3-methylcholanthrene, N-methyl-N-nitrosourea, N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide and 8-hydroxyquinoline), the numbers of GST-P positive foci were significantly higher than in the controls. On the other hand, the mutagenic non-carcinogens (quercetin, p-phenylenediamine dihydrochloride, 2-chloroethanol and 6-hydroquinoline) did not cause a significant increase. Similarly, non-genotoxic hepatocarcinogens of the hepatopromotor class and promotors which target organs other than the liver did not induce GST-P positive foci. The specificity was thus remarkable. Moreover, regardless of the target organ, mutagenic carcinogens were detected by this in vivo 5 week initiation assay, which therefore constitutes a powerful method for screening for carcinogenic potential, especially in the initiation stage of carcinogenesis.

Summation of initiation activities of low doses of the non-hepatocarcinogen 1,2-dimethylhydrazine in the liver after carbon tetrachloride administration

Summation of initiation by low doses of the indirect-acting non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) after proliferative stimulation with a necrogenic dose of carbon tetrachloride (CCl4) was investigated in terms of the induction of glutathione S-transferase placental form (GST-P) positive liver cell foci. Cell kinetics of liver after CCl4 i.g. treatment were examined with bromodeoxyuridine (BrdU) labeling (experiment I). To assess the correlation between cell proliferation and induction of liver cell foci, DMH (10 mg/kg i.g.) was administrated to 7-week-old male F344 rats at 12, 24, 36, 48, 60, 96 h after CCl4 i.g. and initiated populations expanded using the resistant hepatocyte model (experiment IIA). Subsequently, effects of repeated administration (10 mg/kg, four times, i.g.) of DMH were compared with the results of a single administration (40 mg/ kg, i.g.) with the same total dose (experiment IIB). In experiments I and IIA, the numbers and areas of GST-P-positive foci increased with the BrdU labeling index at the time of DMH treatment (maximum after 60 h). In experiment HB, repeated exposure of DMH at 10 mg/kg, four times resulted in significant (P<0.05) increase in number and area of GST-P-positive foci compared with the single administration (40 mg/kg). Thus, multiple low dose treatments during cell proliferation might be most effective for detection of weak initiation activity.

Differential Effects of Partial Hepatectomy and Carbon Tetrachloride Administration on Induction of Liver Cell Foci in a Model for Detection of Initiation Activity

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CC14) administration on induction of glutathione S‐transferase placental form (GST‐P)‐positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative‐quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) and CYP 2E1 apoprotein amount by immuno‐blotting (experiment I) after PH or CC14 administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CC14 administration and induction of liver cell foci, the non‐hepatocarcinogen, 1,2‐dimethylhydrazine (DMH) was administered to 7‐week‐old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CC14 administration were drastically decreased at the 12‐h tune point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CC14 group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST‐P‐positive foci was related to cell kinetics in the PH group, with about a 6‐h time lag between tune for carcinogen administration giving greatest induction of GST‐P‐positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CC14 administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.

N-Ethyl-N-nitrosourea induces mammary cancers in the pituitary-isografted mouse which are histologically and genotypically distinct from those induced by N-methyl-N-nitrosourea

N-Ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) are alkylating agents which respectively ethylate or methylate nucleophilic centers in the cell such as DNA. In vitro studies with naked DNA and bacterial mutagenesis assays suggest that these two compounds induce different spectra of genetic lesions. In addition, the ethyl-DNA adducts induced by ENU persist longer than the methyl-DNA adducts induced by MNU. Since MNU is a known mammary carcinogen in the pituitary-isografted mouse, these data suggest that ENU may be an even more potent carcinogen than MNU. The purpose of this study was to determine whether ENU was a mammary carcinogen in the pituitary-isografted mouse and if so, to compare the genotype and phenotype of ENU-induced mammary tumors with those induced by MNU. Fifteen adult female virgin BALB/c mice were isografted with two pituitaries and subsequently treated with a single intravenous injection of ENU (50 micrograms/g body weight). Mammary adenocarcinomas arose in all of the survivors (n=12) with a median latency of 27 weeks and a mean frequency of 1.4 cancers per mouse. When tumor DNA was analyzed for mutations in the 12th and 61st codons of c-Ki-ras or c-Ha-ras protooncogenes, only wild type sequences were found. This is in contrast to MNU which causes a G to A transition mutation in the 12th codon of the c-Ha-ras proto-oncogene in about one of five mammary cancers induced in pituitary-isografted mice. Furthermore, the ENU-induced tumors were solid viable papillary adenocarcinomas, whereas MNU induced tumors are highly necrotic adenocarcinomas with squamous metaplasia. These results demonstrate that, in the pituitary-isografted mouse, ENU is as potent a mammary carcinogen as MNU and suggest that oncogenes other than c-Ki-ras or c-Ha-ras may be involved in ENU-induced mammary cancers.

Synergistic effect of MNU and DMBA in mammary carcinogenesis and H-ras activation in female Sprague–Dawley rats

The combined application of N-methyl-N-nitrosourea (MNU) and 7,12-dimethylbenz[alpha]anthracene (DMBA) was compared with the administration of each carcinogen alone as to the effectiveness of the induction of mammary carcinomas and the influence of H-ras oncogene activation in female Sprague-Dawley rats. At 50 days of age, group 1 received 30 mg/kg MNU intraperitoneally (i.p.), group 2 received 30 mg/kg DMBA i.p., group 3 received 60 mg/kg MNU i.p., group 4 received 60 mg/ kg DMBA i.p., group 5 received 30 mg/kg MNU followed by 30 mg/kg DMBA i.p., group 6 received 30 mg/kg MNU i.p. and then 30 mg/kg DMBA intravenously (i.v.) and group 7 remained untreated. Animals were killed when the largest mammary tumor reached 1-2 cm in diameter or were necropsied when they were 30 weeks of age. MNU i.p. produced no deaths (groups 1 and 3), however, the i.p. administration of DMBA induced death due to peritonitis (groups 2, 4 and 5), whereas the i.v. administration of DMBA suppressed the death (group 6). All of the tumors produced by MNU were adenocarcinomas of mammary origin. In contrast, DMBA produced tumors of other than mammary origin. The combined treatment with DMBA and MNU increased the mammary carcinogenic effect; it significantly increased the mean number of mammary cancers per rat. With either carcinogen alone and in combination, the mammary carcinomas produced identical adenocarcinoma histology. Of the mammary carcinomas induced by the combined application of MNU and DMBA (group 6), all 11 tumors from five rats showed the GGA to GAA transitional mutation in H-ras codon 12 (38%) and all 18 tumors from the other 10 rats remained as wild-type. An H-ras point mutation at codon 61 was not detected.

Summation of initiation activities in the liver after partial hepatectomy

Summation of initiation activities of different carcinogens in the liver after partial hepatectomy (PH) was investigated with reference to induction of glutathione S-transferase placental form (GST-P) positive foci. Firstly, effects of repeated administration of 1,2-dimethylhydradine (DMH) were compared with the results of a single administration of the same total dose (Expt. I). Subsequently, we studied summation of initiation potential with serial administration of DMH with diethylnitrosamine (DEN) or N-bis (2-hydroxpropyl)-nitrosamine (DHPN). In Expt. I, induction of GST-P-positive foci by multiple low-dose administration was equal to that with the single large-dose treatment. In order to avoid toxicity in hepatectomized rats, the low repeated-dose approach appeared superior. In Expt. II, the numbers of GST-P-positive foci in the groups treated with DMH plus DHPN or DMH plus DEN were significantly higher than those in the groups receiving the carcinogens singly. It is concluded that there is summation of initiation potential with doses of a single or multiple carcinogens. These results suggest that the present initiation assay model is useful to investigate summation of initiation activities of various environmental chemicals.