Takashi Sugimura

Presence of nitrosable mutagen precursors in cooked meat and fish

Broiled chicken, pork, mutton, beef and sun-dried sardine were found to yield direct-acting mutagenicity after nitrite treatment. When 50% methanol extracts of cooked foods were treated with 50 mM nitrite at pH 3 for 1 h at 37 degrees C, they induced 3800-17,900 revertants of Salmonella typhimurium TA100 and 15,000-43,600 revertants of TA98 per g. In contrast, raw meat and uncooked sun-dried sardine showed little or no mutagenicity after nitrite treatment. Treatment of broiled chicken with 0.5-3 mM nitrite, which is a physiologically feasible concentration in the human stomach under some conditions, induced direct-acting mutagenicity. When broiled chicken was treated with 1 mM nitrite at pH 3 for 1 h at 37 degrees C, its mutagenicities on TA100 and TA98 without S9 mix were 7100 and 5400 revertants/g, respectively.

Extensive Purification of Nuclear Poly(ADP-ribose) Glycohydrolase

Catabolism of poly(ADP-tibose) attached to specific chromosomal proteins has been shown to occur during distinct nuclear processes such as DNA replication, repair and transcription (1). Thus, de-poly(ADP-ribosyl)ation is an important response of nuclei that reflect various cellular signals. Two different types of enzymes have been thought to be involved in de-poly(ADP-ribosyl)ation of chromosomal proteins. One enzyme, poly(ADP-ribose) glycohydrolase catalyzes hydrolysis of glycosidic (1“-2’) linkages of poly(ADP-ribose) to give mono(ADP-ribosyl)-protein and free ADP-ribose (2-9). A second type is ADPribosyl-protein lyase, which is capable of splitting mono(ADP-ribose)-protein linkages (10). The glycohydrolase has been purified from nuclei (4) and post-nuclear fractions (cytoplasm) (6-9) of several tissues and cultured cells. To distinguish the nuclear glycohydrolase from the cytoplasmic glycohydrolase, the nuclear enzyme is designated as poly(ADP-ribose) glycohydrolase I, and the cytoplasmic enzyme, poly(ADP-ribose) glycohydrolase II (5, 7, 9). The biological relationship between the two forms of glycohydrolase remains to be determined. As yet no procedure that makes available nuclear poly(ADP-ribose) glycohydrolase with sufficient purity and quantity to determine amino acid composition or sequence has been reported. Here, we report a reproducible and efficient method for extensive purification of the major poly(ADP-ribose) glycohydrolase present in mammalian cell nuclei and characterization of its properties.

Molecular Cloning of cDNA for Human Placental Poly(ADP-Ribose) Polymerase and Decreased Expression of its Gene during Retinoic Acid-Induced Granulocytic Differentiation of HL-60 Cells

Poly(ADP-ribose) is suggested to be involved in various physiological phenomena, such as DNA repair, sister chromatid exchanges, differentiation, proliferation and transformation of eukaryotic cells (1). To find a means of clarifying the biological function of poly(ADP-ribose), we purified poly(ADP-ribose) polymerase from human placenta. We also isolated the cDNA for poly(ADP-ribose) polymerase and determined the nucleotide sequence. Using the cDNA probe we found a decrease in the amount of transcript for the poly(ADP-ribose) polymerase gene during the retinoic acid-induced granulocytic differentiation of HL-60 cells.

Analysis of an RNA adduct formed from aminophenylnorharman

The endogenous mutagenic/carcinogenic 9- (4-aminophenyl) -9H- pyrido [3,4-b] indole (aminophenylnorharman, APNH) is formed from norharman and aniline in the presence of cytochrome P-450s. The major APNH-DNA adduct has been reported to be 2-deoxyguanosin-8-yl-aminophenylnorhaman (dG-C8-APNH). In addition, demonstrated formation of APNH-RNA adduct and conducted a structural analysis using various spectrometric approaches. The compound produced from guanosine (Guo) and N-acetoxy-APNH, an ultimate mutagenic form of APNH, was concluded to be guanosin-8-yl-APNH (Guo-C8-APNH) on the basis of various spectroscopic analysis. The same adduct was found in the livers of rats administered APNH. The total adduct levels of APNH-RNA were six times higher than total APNH-DNA adducts in the same rat liver samples.

A Gene Therapy for Pancreatic Cancer

Pancreatic cancer is often resistant to conventional treatment, and the development of a new therapeutic strategy has been eagerly awaited. Characteristically, K-ras point mutation is observed at a high incidence in human pancreatic cancer. To determine if it is feasible to suppress the growth of pancreatic cancer by counteracting mutated K-ras, we constructed a plasmid vector expressing antisense K-ras RNA and transfected into human pancreatic cancer cells by lipofection. The in vitro growth was significantly suppressed for the antisense K-ras-transfected pancreatic cancer cells, but not for the sense K-ras-transfected cells. Immunoblot analysis showed a reduction of up to 20% of K-ras specific p21 protein the antisense K-ras-transfected cells. There was no evidence of the induction of a massive apoptosis or the presence of a bystander effect. In an in vivo treatment model for peritoneal dissemination, the AsPC-1 pancreatic cancer cells were transplanted to the peritoneal cavity of nude mice at day 1. At day 4, the antisense K-ras-vector /lipopolyamine (DOGS) complex was injected intra-peritoneally 3 times every 12hrs. At day 28, 9 of the 10 sense K-ras-injected mice developed peritoneal dissemination and/or solid tumor formation on the pancreas,or liver; in contrast, only 2 of the 12 mice treated with the antisense K-ras vector showed any evidence of intraperitoneal tumors. Although PCR screening indicated that the injected DNA was distributed to various organs except the brain, treatment-related toxicity was observed neither macroscopically nor microscopically. This study showed that the liposome-mediated in vivo gene transfer of antisense K-ras construct may be a useful therapeutic strategy for a subset of pancreatic cancer.

Altered Oncogene Expression in Hepatocellular Carcinomas Developing Spontaneously in LEC Rats

LEC rats spontaneously suffer hepatitis at around 4 months after birth, enter the chronic phase of hepatitis, and spontaneously develop hepatocellular carcinomas (HCCs) between 1 – 1.5 years after birth [1]. Although dominant oncogene activation in chemically induced HCCs in rats is generally understood to be relatively rare, for the LEC rat, in which an endogenous causative agent appears to be involved, the situation remains unclear. Ha-ras activation has been found with a very high frequency in both chemically induced and spontaneously induced liver carcinomas of B6C3 F1 mice [2–5], but, with the exception of those induced by aflatoxin B1 [6, 7], any type of activated ras was only rarely observed in chemically induced rat liver tumors [4, 8, 9]. We examined HCCs in LEC rats for Ha-, Ki- and N-ras gene mutations. The Polymerase chain reaction (PCR) was adopted for amplifying discrete specific DNA fragments [10, 11], and the sequences were determined directly.

Two-DimensionaL Electrophoretic Analysis of Cellular Polypeptides from Livers of LEC Rats

Cancer development in the liver, and most probably in all other organs and tissues as well, is a multistep process occurring over extended periods of time, and which ultimately leads to the frank development of hepatocellular carcinoma. This process has operationally been divided into the stages of initiation, promotion, and progression although the molecular mechanisms responsible for these stages of carcinogenesis are far from being adequately defined [1, 2]. In man, it is generally accepted that multiple stages, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma, also exist during the development of liver cancer, especially due to HB virus infection [3]. Although numerous rodent models have been developed for studies on multistage hepatocarcinogenesis induced by chemical carcinogens, attempts to define stages during spontaneous tumor formation in the livers of rats have been few, due to the extremely low natural occurrence of liver tumors in most rat strains [4, 5]. The LEC rat, however, offers a very valuable animal model for such studies, in particular, for defining the relationship between the pathogenesis of hepatitis and hepatocellular carcinogenesis [6–8].

ADP-Ribosylation of Human c-Ha- ras Protein by Hen Liver ADP-Ribosyl Transferase

A portion of the amino acid sequence of human c-Ha-ras protooncogene product p21 is highly homologous with the corresponding region of a family of guanine binding membrane proteins, G-proteins, that are involved in signal transduction (1). Therefore, an analogous function is suggested for ras proteins and G-proteins. Although G-proteins are ADP- ribosylated by cholera toxin (2) or pertussis toxin (3), there has been no data of ADP-ribosylation of c-Ha-ras product by bacterial toxins. On the other hand, both Tsai et al. (4) and our group (5) found ADP-ribosylation of Escherichia coli synthesized c-Ha-ras protein by eukaryotic ADP-ribosyl transferases. Here we identify the amino acid residue which is ADP- ribosylated by a purified hen liver enzyme.