[CANCER RESEARCH 45, 1845-1849, April 1985] Induction of Rat Pancreatic B-Cell Tumors by the Combined Administration of Streptozotocin or Alloxan and Poly(Adenosine Diphosphate Ribose) Synthetase Inhibitors1 Takashi Yamagami, Atsuo Miwa, Shin Takasawa, Hiroshi Yamamoto, and Hiroshi Okamoto2 Departments of Biochemistry [T. Y., S. T., H. Y., H. 0.] and Pathology [A. M.], Toyama Medical and Pharmaceutical University School of Medicine, Toyama 930-01, Toyama, and Department of Biochemistry, Tohoku University School of Medicine, Sendai 980, Miyagi [H. O.], Japan ABSTRACT Streptozotocin and alloxan were administered to Wistar rats in combination with poly(adenosine diphosphate ribose) synthe- tase inhibitors. Ten to 16 months after the injection of Strepto zotocin (50 mg/kg body weight i.v.) and 3-aminobenzamide (345 mg/kg i.v.), Streptozotocin (50 mg/kg) and nicotinamide (350 mg/ kg i.p.), Streptozotocin (50 mg/kg) and picolinamide (250 mg/kg i.p.), alloxan (40 mg/kg i.v.) and nicotinamide (350 mg/kg), alloxan (40 mg/kg) and 3-aminobenzamide (345 mg/kg), and alloxan (40 mg/kg) and picolinamide (250 mg/kg), pancreatic islet cell tumors developed in 100, 98, 60, 26, 22, and 20% of surviving rats, respectively. However, after the single injection of Streptozotocin and alloxan, islet cell tumors developed in 42 and 11% of surviving rats, respectively. The tumors were rich in B-granules on electron micrographs and contained as large amounts of proinsulin messenger RNA as normal pancreatic islets. The results indicate that poly(adenosine diphosphate ribose) synthe- tase inhibitors enhance the tumorigenic effect of Streptozotocin and alloxan on islet B-cells. INTRODUCTION The induction of pancreatic islet cell tumors in rats by a combined administration of Streptozotocin and nicotinamide was first reported by Rakieten ef al. (22) in 1971. Histochemical and biochemical properties of the tumors have been described (12, 14, 23). Our recent study revealed that the Streptozotocin- and nicotinamide-induced tumor cells contain as much proinsulin mRNA as do normal pancreatic islets (9,11,19). Islet cell tumors were also found in rats given Streptozotocin and picolinamide, an isomer of nicotinamide, and in rats given alloxan and nicotin amide (13, 14). However, how the combined administrations cause the islet tumors is not yet understood. Recently, we found that both Streptozotocin and alloxan cause DMA strand breaks in islet B-cells to activate poly(ADP-ribose)3 synthetase (31) and that nicotinamide and picolinamide are po tent inhibitors of islet poly(ADP-ribose) synthetase (26, 29). The present study was designed to investigate whether the combined administration of Streptozotocin or alloxan with various poly(ADP-ribose) synthetase inhibitors to rats induces islet B-cell tumors and to determine the proinsulin mRNA content of the 1 Supported in part by grants-in-aid for Cancer Research and for Scientific Research from the Ministry of Education, Science and Culture, Japan. 2 To whom all correspondence should be addressed, at Department of Biochem istry, Tohoku University School of Medicine, Sendai 980, Miyagi, Japan. 3The abbreviations used are: poly(ADP-ribose), poly(adenosine diphosphate ribose); cDNA, complementary DMA; SDS, sodium dodecyl sulfate; SSPE, 0.18 M NaCI, 10 mw sodium phosphate buffer (pH 7.7), and 1 mm EDTA. Received 1/17/84; accepted 12/4/84. tumors induced. As a result, we found that, in any combination examined, islet B-cell tumors containing significant amounts of proinsulin mRNA sequences as well as B-granules were induced with a high incidence. A possible mechanism of the B-cell tu- morigenesis is discussed. MATERIALS AND METHODS Animals and Chemicals. Male Wistar rats were purchased from Shizuoka Laboratory Animal Center, Hamamatsu, Japan; Streptozotocin was from The Upjohn Co., Kalamazoo, Ml; alloxan and nicotinamide were from Wako Pure Chemical Industries, Osaka, Japan; picolinamide was from Tokyo Kasei Kogyo Co., Tokyo, Japan; [«-32P]dCTP(3000 Ci/ mmol) from Amersham International, Amersham, Buckinghamshire, Eng land; aminophenyl thioether papers from Schleicher and Schuell, Inc., Keene, NH. 3-Aminobenzamide-HCI was donated from Research Labo ratories, Chugai Pharmaceutical Co., Tokyo, Japan. Induction and Morphological Examination of Tumors. Rats weighing 150 g were divided into 8 groups; Streptozotocin group; streptozotocin- nicotinamide group; streptozotocin-picolinamide group; streptozotocin- 3-aminobenzamide group; alloxan group; alloxan-nicotinamide group; alloxan-picolinamide group; and alloxan-3-aminobenzamide group. Each group consisted of 17 to 99 rats (see Table 1). All chemicals were dissolved in 0.9% NaCI solution. Streptozotocin and alloxan were dis solved just before injection and were administered i.v. via the tail vein into ether-anesthetized rats at doses of 50 and 40 mg/kg body weight, respectively (13,16, 22, 32). Nicotinamide (350 mg/kg) was injected i.p. into rats 10 min before and 3 hr after the administration of Streptozotocin or alloxan (22). Picolinamide (250 mg/kg) was injected i.p. 10 min before Streptozotocin or alloxan injection (14). 3-Aminobenzamide-HCI (345 mg/ kg) was injected i.v. via the tail vein 30 min before Streptozotocin or alloxan injection. Rats were allowed free access to standard rat chow and water. After 10 to 16 months, rats were anesthetized with pento- barbital sodium (45 mg/kg body weight i.p.), and laparotomy was carried out under sterile conditions. Grossly visible tumors were extirpated and halved; one half was immediately fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for histological evaluations, and the other half was stored at -80° until RNA isolation. For an ultrastructural study, fixed segments of tumors were postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4), dehydrated in graded ethanol solutions, embedded in Epon 812, and sectioned on a LKB Ultratome 8000. The sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H-300 electron microscope. Northern Blot Hybridization. RNA was isolated as described previ ously (9) from tumors induced by Streptozotocin and alloxan with or without poly(ADP-ribose) synthetase inhibitors and from various tissues of untreated rats including pancreatic islets, liver, kidney, and spleen. Islets were isolated by the collagenase method as described previously (20). RNA was denatured by glyoxylation, electrophoresed on a 1% agarose gel at 90 V for 100 min, and blotted to a diazophenyl thioether paper (an active form of aminophenyl thioether paper) by the method of Alwine ef a/. (1). Proinsulin cDNA insert, the hybridization probe, was cut off from a recombinant plasmid and nick-translated in the presence of CANCER RESEARCH VOL. 45 APRIL 1985 1845 Research. on September 29, 2021. © 1985 American Association for Cancer cancerres.aacrjournals.org Downloaded from