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Telomerase activity in experimental animal tumors
Exp Toxic Patho11998; 50: 371-374 Gustav Fischer Verlag
Department of Oncological Pathology, Cancer Center, Nara Medical University, Nara, Japan
Telomerase activity in experimental animal tumors* TOSHIFUMI TSUJIUCHI, MASAHIRO TSUTSUMI, and Y OICHI KONISHI With 2 tables Address for correspondence: Dr. YOICHI KONISHI, Department of Oncological Pathology, Cancer Center, Nara Medical University, 840 Shijocho, Kashihara, Nara 634, Japan. Key words: Telomerase, TRAP assay, rat, hamster, carcinogenesis.
Introduction
Telomerase activities in animal tumors
Telomeres are tandem arrays of guanine-rich repetitive sequences at the ends of chromosomes. They have been highly conserved throughout evolution and are defi.n~d functionally as necessary for chromosomal stabIlIty (BLACKBURN 1991; COUNTER et al. 1992). Telomere reduction has been reported in several human cancers, such as colorectal, ovarian, renal cell and hepatocellular carcinomas (BACCHETTI and COUNTER 1995; RHYU 1995). Telomerase is a ribonucleoprotein which catalyzes the formation of telomeric repeats, represented by TT AGGG at the end of chromosomes in vertebrates (BLACKBURN 1991; COUNTER et al. 1992). Recently, KIM et al. (1994) have established a new, highly sensitive assay for the detection of telomerase activity, which has been designated the TRAP (telomeric repeat amplification protocol) assay. Telomerase activation has been reported in several human cancers using the TRAP assay. These findings suggest that telomerase might play an important role in the progression and maintenance of the malignant state in tumor cells (BACCHETTI and COUNTER 1995; RHYU 1995; KIM et al. 1994). Recently, it has also been reported that there is increased telomerase activity in experimental animal tumors. We assayed telomerase activity in animal tumors by a modification of original TRAP method. The range of tumors examined included hepatocellular carcinomas, osteosarcomas and brain tumors of rats and pancreatic duct carcinomas of hamsters (TSUJIUCHI et al. 1996; KIDO et al. 1997; SAKITANI et al. 1998 (in press); KOBITSU et al. 1997). In this review, we summarize data from the several reports and outline the importance of experimental animal models for investigating the biological significance of telomerase activation.
Liver tumors
* Dedicated to Prof. em. Dr. Dres. h.c. FRANZ BOLeK on the occasion of his 80th birthday on September 15,1998.
It is now widely accepted that hepatocarcinogenesis basically consists of qualitatively different multi steps in humans and animals. The prolonged feeding of rats with choline deficient L-amino acid defined (CDAA) diet, which is useful for studies of endogenous hepatocarcinogenesis, can induce unequivocal liver tumors without treatment with chemical carcinogens (NAKAE et al. 1992). Possible mechanisms underlying hepatocarcinogenesis by the CDAA diet have been proposed to be as follows; liver necrosis and regeneration, oxidative DNA damage and lipid peroxidation, and production of gene alterations. Recently, telomerase activity during hepatocarcinogenesis induced by the CDAA diet in rats has been investigated (TSUJIUCHI et al. 1996). Male, 6-week-old, F344 rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) were given the CDAA or choline supplemented Lamino acid defined (CSAA) diet (Dyets, PA, USA). Animals were sacrificed at 10, 50 and 75 weeks after the beginning of the experiment. The whole Ii ver was used from 6-week-old control rats and also from rats on diets for 10 weeks. Unequivocal liver nodules and cancers of rats killed at 50 and 75 weeks were separated from non-tumorous areas before being assayed for telomerase activity. Histologically, livers of rats fed the CDAA diet for 10 weeks showed fatty cirrhosis with putative preneoplastic foci. After 50 and 75 weeks, hyperplastic nodules and well differentiated hepatocellular carcinomas (HCCs) were observed, respectively. The protein was extracted from each sample and the TRAP assay was performed. Normal liver showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver. Exp Toxic Pathol50 (1998) 4-6
371
We found that telomerase activity was already increased in hyperplastic lesions during hepatocarcinogenesis induced by the CDAA diet in rats. In the next study, since the change in telomerase activity during the cell cycle had not been explored, we measured telomerase activity in hepatocytes regenerating after partial hepatectomy (PH) in rats (TSUJIUCHI et al. 1998). Male, 6-week-old, F344 rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) received two-thirds PH by the method of HIGGINS and ANDERSON (HIGGINS and ANDERSON 1931) and were killed at 0, 6, 16, 24, 36 or 72 h after PH. The livers were removed and immediately frozen for use in the TRAP assay. It was found that telomerase activity was increased significantly from 6 to 36 h but had decreased to close to the normal levels by 72 h. DNA synthesis reached a maximum 24 h after PH. We also examined the expression oftelomerase RNA by Northern blot analysis and found that its expression level did not change during regeneration. These results suggest that the regulation of telomerase activation might differ between normal regenerating cells and cancer cells.
Osteosarcomas Osteosarcomas are highly malignant bone tumors which develop in young people. They have a poor prognosis because of frequent lung metastasis. Previously, we have established two lines of transplantable osteosarcomas in rats. One was derived from rats treated with 4hydroxyanimo quinoline I-oxide (4-HAQO) and is referred to as carcinogen-induced osteosarcoma (COS). The other is from a spontaneous lesion (SOS). Highly metastatic tumor lines (C-SLM and S-SLM) were established by serially transplanting lung metastatic lesions into ssubcutaneous tissue. We investigated telomerase activites in subcutaneously transplanted osteosarcomas and their lung metastases and compared levels with their metastatic potential (KIDO et al. 1997). Telomerase activity was found to be elevated in all four osteosarcoma lines and their metastatic lung lesions. However, increased activity was not correlated with metastatic potential. These results suggest that activation of telomerase occurs in rat osteosarcomas but that it is not directly involved in determining their metastatic potential.
Pancreatic duct carcinomas Pancreatic duct carcinoma has a very poor prognosis since the vast majority of cases are incurable. Experimentally, a high incidence of pancreatic duct carcinomas closely resembling human counterparts can be induced in the Syrian golden hamster by treatment with N-nitrosobis(2-oxopropyl)amine (BOP) as described by MIZUMOTO et al. (1988). Transplantable and cultured cell lines have been established from this model. To clarify whether telomerase activities change during pancreatic carcinogenesis in hamsters, we investigated this parameter in primary carcinomas, in transplantable carcinomas and in cell lines. Female Syrian golden hamsters (Shizuoka Animal Laboratory Center, Shizuoka, Japan) weighing approximately 100 g each were used. Pancreatic duct adenocarcinomas were induced by the rapid-production model. This consists of an initial subcutaneous injection of 70 mg/kg body weight BOP (Nakarai Tesque Inc, Kyoto, Japan) followed 12 days later by three cycles of ethionine-methionine rescue-induced pancreatic regeneration. During this time, hamsters were maintained on a choline-deficient diet. Each switch to methionine was followed after 2 days by an injection of 20 mg/kg BOP, and the cycles were separated from one another by an interval of 10 days. All hamsters were killed 10 weeks after the beginning of the experiment. Macroscopically detected tumors of the pancreas were removed and used for the TRAP assay (KOBITSU et al. 1997). In the primary and transplantable pancreatic carcinomas and cell lines, telomerase activity was significantly increased compared to the levels in normal spleen and pancreas. These results indicate that increased telomerase activation in hamster pancreatic duct carcinoma cells might play an important role in progression and maintenance of the malignant state. 372
Exp Toxic Pathol 50 (1998) 4-6
Brain tumors Gliomas are the most common human brain tumors. Experimentally, oligo-astrocytomas can be induced by transplacental administration of N-ethyl-N-nitrosourea (ENU) in rats (DRUCKREY et al. 1996). Pregnant F344 rats at their 20th day of gestation were treated with ENU at a dose of 75 mg/kg body weight intravenously. Their offspring were sacrificed at 8 and 30 weeks after birth and brains were removed. Histologically, both oligo-astrocytomas and early neoplastic lesions composed of oligodendrocytes, astrocytes and cells of uncertain origin were detected. The TRAP assay was performed and telomerase activity in these abnormal samples was compared to normal brain (SAKIT ANI et al. 1998). Telomerase activity levels of oligo-astrocytomas, including early neoplastic lesions, were significantly increased as compared to normal brain. These results indicate that the activation of telomerase occurs during astrocytoma carcinogenesis and contributes to the development of brain tumors.
Other tumors Recently, YOSHIMI et al. (1996) reported that telomerase activity increased in colon adenocarcinomas induced by methylazoxymethanol in rats. BEDNAREK et al. (1995) reported that increased telomerase activities were detected in papillomas and carcinomas from skin carcinogenesis studies in mice. It is especially interesting that telome rase activity already increased in early neoplastic lesions during mouse skin carcinogenesis as well as in our CDAA-induced liver tumors in rats.
Screened inhibitors for increased telomerase activity in hamster pancreatic duct carcinoma cell lines A recent important discovery is that telomerase inhibitors may be useful in treating cancers by inhibiting their proliferative activity. We therefore investigated the effects of several potential telomerase inhibitors in hamster pancreatic duct carcinoma cell lines which had been established from hamster pancreatic duct carcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP). The experimental strategy was as follows: (1) TRAP assay with the mixture of protein extracted from hamster pancreatic duct carcinoma cell lines and test compounds; (2) compounds which showed inhibitory effects on telomerase activity by TRAP assay were tested to confirm that they had no inhibitory effects on Taq polymerase; (3) application of the compound to hamster pancreatic duct carcinoma cell lines directly prior to the TRAP assay; and (4) test any compound found to be effectice in steps I through 3 by in vivo study in hamsters with pancreatic duct carcinomas. According to the above strategy, 55 compounds have been tested so far, including antibiotics, anticancer drugs,
Table 1. Compounds tested in hamster pancreatic duct carcinoma cell line. Antibiotics Anticancer drugs Antiinflammatory drugs Antioxidants Carcinogens and promoters Herbal medicines Vitamins Others
erythromycin, chloramphenicol, neomycin CPA, CDDP, DXR, MTX, THP-ADR, TSPA, VCR aspirin, dexamethasone, ibuprofen, indomethacin, lysozyme, piroxycam, QU DPPD, l3-carotene AFB I , B[a]p, ENU, EtBr, ethionine, PB astragali radix, ginseng radix , scutellariae radix, sho-saiko-to ascorbic acid, CF-36JJ , vitamin K ABA, 4-acetamidephenol, achidicolin, BrdU, caffeine, colchicine, cyclohexamide, FeCI 2, green tea, K232a, LN, AGM1470, novobiocin , nordihydroguaiaretic acid, saponin
CPA: cyclophosphamide; CDDP: cis-diamine-dichloroplatinum; DXR: doxorubicin; MTX: methotrexate; THPADR: THP-adriamycin; TSPA: thio-TEPA; VCR: vincristine; QU: quercetin; DPPD: NN' -diphenyl-p-phenylenediamine; AFB\: aflatoxin B I ; B[a]P: benzo(a)pyrene; ENU: N-ethyl-N-nitrosourea; EtBr: ethidiumbromide; PB: phenobarbital; CV -3611: 2-0-octadecyl ascorbic acid; ABA: 3-aminobenzamide; BrdU: 5-bromo-2' -deoxyuridine; LN: lead nitrate; AGM1470: o-(choloroacetyl-carbamoyI)fumagillol.
antiinflammatory drugs, antioxidants, carcinogens, herbal medicines, vitamins and others (table l). None of these compounds to date has shown specific inhibition of the increased in telomerase activity associated with tumors. Further screening using this strategy is underway.
Conclusion The data on telomerase activity in experimental animal tumors are summarized in table 2. In humans, activation of telomerase is not detectable in normal tissues except for germ cells, lymphocytes, hematopoietic progenitor cells and epidermis. In contrast to humans, normal tissues in mice, rats and hamsters do show telomerase activity. The relative ease of immortalization of murine primary cells, in contrast to human cells, or the longer length of telomeres in rodents might be due to this difference in telomerase activation (PROWSE and GREIDER 1995). Increased tel orne rase activities, however, occur both in human tumors and in experimentally-induced tumors in animals. It therefore is important to clarify differences in telomerase activation between normal cells of humans and rodents.
Table 2. Summary of telomerase activities in animal tumors. Species
Normal tissues Tumors
Rats
liver (+) muscle (+)
Hamsters Mice
brain (+) colon (+) pancreas (-) skin (-)
hypef]?lastic nodule t HCCI transplantable osteosarcoma t oligo-astrocytoma t adenocarcinoma tal
pancreatic duct carcinoma t papilloma t squamous cell carcinomab) t
(-): not detected (+): weakly or moderately l increased YOSHIMI N, et al. : Mol Carcinog 1996; 16: 1-5. b) BEDNAREK A, et al.: Cancer Res 1995; 55: 4566-4569.
a)
Telomeres might be a valuable target for anti-tumor therapy and tumor models in experimental animals provide ample opportunity for exploring this strategy. Acknowledgement: This work was supported by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture, Grant-in-Aid for Scientific Expenses for Health and Welfare Programs, 2nd-Term Comprehensive IO-year Strategy for Cancer Control, and Grant-in-Aid from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare in Japan. Exp Toxic Pathol 50 ( 1998) 4-6
373
References BACCHETTI S and COUNTER CM: Telomeres and telomerase in human cancer (Review). Int J Onco11995; 7: 423-432. BEDNAREK A, BUDUNOVA I, SLAGA TJ, et al.: Increased telomerase activity in mouse skin premalignant progression. Cancer Res 1995; 55: 4566-4569. BLACKBURN EH: Structure and function of telomeres. Nature 1991; 350: 569-573. COUNTER CM, AVILLON AA, LEFEUVRE CE, et al.: Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1992; 11: 1921-1929. DRUCKREY H, IVANKOVIC R, PREUSSMANN R, et al.: Teratogenic and carcinogenic effects in the offspring after a single injection of ethylnitrosourea to pregnant rats. Nature 1996; 210: 1378-1379. HIGGINS GM and ANDERSON RM: Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12: 186-202. KIDO A, TSUJIUCHI T, TSUTSUMI M, et al.: Increased telomerase activity is not directly related to metastatic potential in rat transplantable osteosarcomas. Cancer Lett 1997; 117: 67-71. KOBITSU K, TSUTSUMI M, TSUJIUCHI T, et al.: Shortened telomerase length and increased telomerase activity in hamster pancreatic duct adenocarcinomas and cell lines. Mol Carcinog 1997; 18: 153-159.
374
Exp Toxic Pathol 50 (1998) 4-6
MlZUMOTO K, TSUTSUMI M, DENDA A, et al.: Rapid production of pancreatic carcinoma by initiation with N-nitrosobis(2-oxopropyl)amine and repeated augmentation pressure in hamsters. J Natl Cancer Inst 1998; 80: 1564-1567. NAKAE D, YOSHIJI H, MIZUMOTO Y, et al.: High incidence of hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats. Cancer Res 1992; 52: 5042-5045. PROWSE KR and GREIDER CW: Developmental and tissuespecific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci USA 1995; 92: 4818-4822. RHYU MS: Telomeres, telomerase, and immortality. J Natl Cancer Inst 1995; 87: 884-894. SAKITANI H, TSUJIUCHI T, KOBITSU K, et al.: Increased telomerase activity and absence of p53 mutation in oligoastrocytomas induced by N-ethyl-N-nitrosourea in rats. Cancer Lett 1998 (in press). TSUJIUCHI T, TSUTSUMI M, KIDO A, et al.: Increased telomerase activity in hyperplastic nodules and hepatocellular carcinomas induced by a choline dificient L-amino acid defined diet in rats. Jpn J Cancer Res 1996; 87: 1111-1115. TSUJIUCHI T, TSUTSUMI M, KIDO A, et al.: Induction oftelomerase activity during regeneration after partial hepatectomy in the rat. Cancer Lett 1998; 122: 115-120. YOSHIMI N, INO N, SUZUI M, et al.: Telomerase activity of normal tissues and neoplasms in rat colon carcinogenesis induced by methylazoxymethanol acetate and its difference from that of human colonic tissues. Mol Carcinog 1996; 16: 1-5.
Department of Oncological Pathology, Cancer Center, Nara Medical University, Nara, Japan
Telomerase activity in experimental animal tumors* TOSHIFUMI TSUJIUCHI, MASAHIRO TSUTSUMI, and Y OICHI KONISHI With 2 tables Address for correspondence: Dr. YOICHI KONISHI, Department of Oncological Pathology, Cancer Center, Nara Medical University, 840 Shijocho, Kashihara, Nara 634, Japan. Key words: Telomerase, TRAP assay, rat, hamster, carcinogenesis.
Introduction
Telomerase activities in animal tumors
Telomeres are tandem arrays of guanine-rich repetitive sequences at the ends of chromosomes. They have been highly conserved throughout evolution and are defi.n~d functionally as necessary for chromosomal stabIlIty (BLACKBURN 1991; COUNTER et al. 1992). Telomere reduction has been reported in several human cancers, such as colorectal, ovarian, renal cell and hepatocellular carcinomas (BACCHETTI and COUNTER 1995; RHYU 1995). Telomerase is a ribonucleoprotein which catalyzes the formation of telomeric repeats, represented by TT AGGG at the end of chromosomes in vertebrates (BLACKBURN 1991; COUNTER et al. 1992). Recently, KIM et al. (1994) have established a new, highly sensitive assay for the detection of telomerase activity, which has been designated the TRAP (telomeric repeat amplification protocol) assay. Telomerase activation has been reported in several human cancers using the TRAP assay. These findings suggest that telomerase might play an important role in the progression and maintenance of the malignant state in tumor cells (BACCHETTI and COUNTER 1995; RHYU 1995; KIM et al. 1994). Recently, it has also been reported that there is increased telomerase activity in experimental animal tumors. We assayed telomerase activity in animal tumors by a modification of original TRAP method. The range of tumors examined included hepatocellular carcinomas, osteosarcomas and brain tumors of rats and pancreatic duct carcinomas of hamsters (TSUJIUCHI et al. 1996; KIDO et al. 1997; SAKITANI et al. 1998 (in press); KOBITSU et al. 1997). In this review, we summarize data from the several reports and outline the importance of experimental animal models for investigating the biological significance of telomerase activation.
Liver tumors
* Dedicated to Prof. em. Dr. Dres. h.c. FRANZ BOLeK on the occasion of his 80th birthday on September 15,1998.
It is now widely accepted that hepatocarcinogenesis basically consists of qualitatively different multi steps in humans and animals. The prolonged feeding of rats with choline deficient L-amino acid defined (CDAA) diet, which is useful for studies of endogenous hepatocarcinogenesis, can induce unequivocal liver tumors without treatment with chemical carcinogens (NAKAE et al. 1992). Possible mechanisms underlying hepatocarcinogenesis by the CDAA diet have been proposed to be as follows; liver necrosis and regeneration, oxidative DNA damage and lipid peroxidation, and production of gene alterations. Recently, telomerase activity during hepatocarcinogenesis induced by the CDAA diet in rats has been investigated (TSUJIUCHI et al. 1996). Male, 6-week-old, F344 rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) were given the CDAA or choline supplemented Lamino acid defined (CSAA) diet (Dyets, PA, USA). Animals were sacrificed at 10, 50 and 75 weeks after the beginning of the experiment. The whole Ii ver was used from 6-week-old control rats and also from rats on diets for 10 weeks. Unequivocal liver nodules and cancers of rats killed at 50 and 75 weeks were separated from non-tumorous areas before being assayed for telomerase activity. Histologically, livers of rats fed the CDAA diet for 10 weeks showed fatty cirrhosis with putative preneoplastic foci. After 50 and 75 weeks, hyperplastic nodules and well differentiated hepatocellular carcinomas (HCCs) were observed, respectively. The protein was extracted from each sample and the TRAP assay was performed. Normal liver showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver. Exp Toxic Pathol50 (1998) 4-6
371
We found that telomerase activity was already increased in hyperplastic lesions during hepatocarcinogenesis induced by the CDAA diet in rats. In the next study, since the change in telomerase activity during the cell cycle had not been explored, we measured telomerase activity in hepatocytes regenerating after partial hepatectomy (PH) in rats (TSUJIUCHI et al. 1998). Male, 6-week-old, F344 rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) received two-thirds PH by the method of HIGGINS and ANDERSON (HIGGINS and ANDERSON 1931) and were killed at 0, 6, 16, 24, 36 or 72 h after PH. The livers were removed and immediately frozen for use in the TRAP assay. It was found that telomerase activity was increased significantly from 6 to 36 h but had decreased to close to the normal levels by 72 h. DNA synthesis reached a maximum 24 h after PH. We also examined the expression oftelomerase RNA by Northern blot analysis and found that its expression level did not change during regeneration. These results suggest that the regulation of telomerase activation might differ between normal regenerating cells and cancer cells.
Osteosarcomas Osteosarcomas are highly malignant bone tumors which develop in young people. They have a poor prognosis because of frequent lung metastasis. Previously, we have established two lines of transplantable osteosarcomas in rats. One was derived from rats treated with 4hydroxyanimo quinoline I-oxide (4-HAQO) and is referred to as carcinogen-induced osteosarcoma (COS). The other is from a spontaneous lesion (SOS). Highly metastatic tumor lines (C-SLM and S-SLM) were established by serially transplanting lung metastatic lesions into ssubcutaneous tissue. We investigated telomerase activites in subcutaneously transplanted osteosarcomas and their lung metastases and compared levels with their metastatic potential (KIDO et al. 1997). Telomerase activity was found to be elevated in all four osteosarcoma lines and their metastatic lung lesions. However, increased activity was not correlated with metastatic potential. These results suggest that activation of telomerase occurs in rat osteosarcomas but that it is not directly involved in determining their metastatic potential.
Pancreatic duct carcinomas Pancreatic duct carcinoma has a very poor prognosis since the vast majority of cases are incurable. Experimentally, a high incidence of pancreatic duct carcinomas closely resembling human counterparts can be induced in the Syrian golden hamster by treatment with N-nitrosobis(2-oxopropyl)amine (BOP) as described by MIZUMOTO et al. (1988). Transplantable and cultured cell lines have been established from this model. To clarify whether telomerase activities change during pancreatic carcinogenesis in hamsters, we investigated this parameter in primary carcinomas, in transplantable carcinomas and in cell lines. Female Syrian golden hamsters (Shizuoka Animal Laboratory Center, Shizuoka, Japan) weighing approximately 100 g each were used. Pancreatic duct adenocarcinomas were induced by the rapid-production model. This consists of an initial subcutaneous injection of 70 mg/kg body weight BOP (Nakarai Tesque Inc, Kyoto, Japan) followed 12 days later by three cycles of ethionine-methionine rescue-induced pancreatic regeneration. During this time, hamsters were maintained on a choline-deficient diet. Each switch to methionine was followed after 2 days by an injection of 20 mg/kg BOP, and the cycles were separated from one another by an interval of 10 days. All hamsters were killed 10 weeks after the beginning of the experiment. Macroscopically detected tumors of the pancreas were removed and used for the TRAP assay (KOBITSU et al. 1997). In the primary and transplantable pancreatic carcinomas and cell lines, telomerase activity was significantly increased compared to the levels in normal spleen and pancreas. These results indicate that increased telomerase activation in hamster pancreatic duct carcinoma cells might play an important role in progression and maintenance of the malignant state. 372
Exp Toxic Pathol 50 (1998) 4-6
Brain tumors Gliomas are the most common human brain tumors. Experimentally, oligo-astrocytomas can be induced by transplacental administration of N-ethyl-N-nitrosourea (ENU) in rats (DRUCKREY et al. 1996). Pregnant F344 rats at their 20th day of gestation were treated with ENU at a dose of 75 mg/kg body weight intravenously. Their offspring were sacrificed at 8 and 30 weeks after birth and brains were removed. Histologically, both oligo-astrocytomas and early neoplastic lesions composed of oligodendrocytes, astrocytes and cells of uncertain origin were detected. The TRAP assay was performed and telomerase activity in these abnormal samples was compared to normal brain (SAKIT ANI et al. 1998). Telomerase activity levels of oligo-astrocytomas, including early neoplastic lesions, were significantly increased as compared to normal brain. These results indicate that the activation of telomerase occurs during astrocytoma carcinogenesis and contributes to the development of brain tumors.
Other tumors Recently, YOSHIMI et al. (1996) reported that telomerase activity increased in colon adenocarcinomas induced by methylazoxymethanol in rats. BEDNAREK et al. (1995) reported that increased telomerase activities were detected in papillomas and carcinomas from skin carcinogenesis studies in mice. It is especially interesting that telome rase activity already increased in early neoplastic lesions during mouse skin carcinogenesis as well as in our CDAA-induced liver tumors in rats.
Screened inhibitors for increased telomerase activity in hamster pancreatic duct carcinoma cell lines A recent important discovery is that telomerase inhibitors may be useful in treating cancers by inhibiting their proliferative activity. We therefore investigated the effects of several potential telomerase inhibitors in hamster pancreatic duct carcinoma cell lines which had been established from hamster pancreatic duct carcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP). The experimental strategy was as follows: (1) TRAP assay with the mixture of protein extracted from hamster pancreatic duct carcinoma cell lines and test compounds; (2) compounds which showed inhibitory effects on telomerase activity by TRAP assay were tested to confirm that they had no inhibitory effects on Taq polymerase; (3) application of the compound to hamster pancreatic duct carcinoma cell lines directly prior to the TRAP assay; and (4) test any compound found to be effectice in steps I through 3 by in vivo study in hamsters with pancreatic duct carcinomas. According to the above strategy, 55 compounds have been tested so far, including antibiotics, anticancer drugs,
Table 1. Compounds tested in hamster pancreatic duct carcinoma cell line. Antibiotics Anticancer drugs Antiinflammatory drugs Antioxidants Carcinogens and promoters Herbal medicines Vitamins Others
erythromycin, chloramphenicol, neomycin CPA, CDDP, DXR, MTX, THP-ADR, TSPA, VCR aspirin, dexamethasone, ibuprofen, indomethacin, lysozyme, piroxycam, QU DPPD, l3-carotene AFB I , B[a]p, ENU, EtBr, ethionine, PB astragali radix, ginseng radix , scutellariae radix, sho-saiko-to ascorbic acid, CF-36JJ , vitamin K ABA, 4-acetamidephenol, achidicolin, BrdU, caffeine, colchicine, cyclohexamide, FeCI 2, green tea, K232a, LN, AGM1470, novobiocin , nordihydroguaiaretic acid, saponin
CPA: cyclophosphamide; CDDP: cis-diamine-dichloroplatinum; DXR: doxorubicin; MTX: methotrexate; THPADR: THP-adriamycin; TSPA: thio-TEPA; VCR: vincristine; QU: quercetin; DPPD: NN' -diphenyl-p-phenylenediamine; AFB\: aflatoxin B I ; B[a]P: benzo(a)pyrene; ENU: N-ethyl-N-nitrosourea; EtBr: ethidiumbromide; PB: phenobarbital; CV -3611: 2-0-octadecyl ascorbic acid; ABA: 3-aminobenzamide; BrdU: 5-bromo-2' -deoxyuridine; LN: lead nitrate; AGM1470: o-(choloroacetyl-carbamoyI)fumagillol.
antiinflammatory drugs, antioxidants, carcinogens, herbal medicines, vitamins and others (table l). None of these compounds to date has shown specific inhibition of the increased in telomerase activity associated with tumors. Further screening using this strategy is underway.
Conclusion The data on telomerase activity in experimental animal tumors are summarized in table 2. In humans, activation of telomerase is not detectable in normal tissues except for germ cells, lymphocytes, hematopoietic progenitor cells and epidermis. In contrast to humans, normal tissues in mice, rats and hamsters do show telomerase activity. The relative ease of immortalization of murine primary cells, in contrast to human cells, or the longer length of telomeres in rodents might be due to this difference in telomerase activation (PROWSE and GREIDER 1995). Increased tel orne rase activities, however, occur both in human tumors and in experimentally-induced tumors in animals. It therefore is important to clarify differences in telomerase activation between normal cells of humans and rodents.
Table 2. Summary of telomerase activities in animal tumors. Species
Normal tissues Tumors
Rats
liver (+) muscle (+)
Hamsters Mice
brain (+) colon (+) pancreas (-) skin (-)
hypef]?lastic nodule t HCCI transplantable osteosarcoma t oligo-astrocytoma t adenocarcinoma tal
pancreatic duct carcinoma t papilloma t squamous cell carcinomab) t
(-): not detected (+): weakly or moderately l increased YOSHIMI N, et al. : Mol Carcinog 1996; 16: 1-5. b) BEDNAREK A, et al.: Cancer Res 1995; 55: 4566-4569.
a)
Telomeres might be a valuable target for anti-tumor therapy and tumor models in experimental animals provide ample opportunity for exploring this strategy. Acknowledgement: This work was supported by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture, Grant-in-Aid for Scientific Expenses for Health and Welfare Programs, 2nd-Term Comprehensive IO-year Strategy for Cancer Control, and Grant-in-Aid from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare in Japan. Exp Toxic Pathol 50 ( 1998) 4-6
373
References BACCHETTI S and COUNTER CM: Telomeres and telomerase in human cancer (Review). Int J Onco11995; 7: 423-432. BEDNAREK A, BUDUNOVA I, SLAGA TJ, et al.: Increased telomerase activity in mouse skin premalignant progression. Cancer Res 1995; 55: 4566-4569. BLACKBURN EH: Structure and function of telomeres. Nature 1991; 350: 569-573. COUNTER CM, AVILLON AA, LEFEUVRE CE, et al.: Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1992; 11: 1921-1929. DRUCKREY H, IVANKOVIC R, PREUSSMANN R, et al.: Teratogenic and carcinogenic effects in the offspring after a single injection of ethylnitrosourea to pregnant rats. Nature 1996; 210: 1378-1379. HIGGINS GM and ANDERSON RM: Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12: 186-202. KIDO A, TSUJIUCHI T, TSUTSUMI M, et al.: Increased telomerase activity is not directly related to metastatic potential in rat transplantable osteosarcomas. Cancer Lett 1997; 117: 67-71. KOBITSU K, TSUTSUMI M, TSUJIUCHI T, et al.: Shortened telomerase length and increased telomerase activity in hamster pancreatic duct adenocarcinomas and cell lines. Mol Carcinog 1997; 18: 153-159.
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