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Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models
Cancer Letters 143 (1999) 245±247
Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models Snorri S. Thorgeirsson*, Doug-Young Ryu, Valerie Weidner, Elizabeth G. Snyderwine Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Received 3 November 1998; received in revised form 8 February 1999; accepted 9 February 1999
Abstract Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c±myc cDNA and mouse metallothionein1 promoter-human TGFa cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c±myc and TGFa transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (Muta TMMice) and double transgenic c±myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with speci®c genes during the neoplastic process. q 1999 Published by Elsevier Science Ireland Ltd. Keywords: Transgenic mouse models; Heterocyclic amines; Carcinogenicity; Mutagenicity
1. Introduction The nuclear oncogene c±myc and transforming growth factor (TGF) a , a ligand for the epidermal growth factor receptor tyrosine kinase, are frequently co-expressed in human tumors including hepatocellular carcinoma (HCC) (reviewed in [1]), indicating that their interaction may represent a critical step in malignant growth. To investigate this interaction, we * Corresponding author. LEC, Building 37, Room 3C28, National Cancer Institute, 37 Convent Drive MSC4255, Bethesda, MD 20892-4255 USA. Tel.: 301-496-1935; fax: 301-496-0734. E-mail address: [email protected] (S.S. Thorgeirsson)
recently generated double-transgenic mice overexpressing c±myc and TGFa in the liver and demonstrated that these two genes synergize to generate a dramatic enhancement of neoplastic development in this organ [2,3]. The progression from early and severe liver cell dysplasia and preneoplastic focal lesions to HCC occurred in 4 months in the c±myc/ TGFa double transgenic mice, resulting in striking liver enlargement, 100% frequency of HCCs before 8 months, and survival reduced to a year [3]. In contrast, both the single-transgenic mouse lines were characterized by a slower neoplastic development, with HCCs appearing only after one year of age and
0304-3835/99/$ - see front matter q 1999 Published by Elsevier Science Ireland Ltd. PII: S 0304-383 5(99)00133-0
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S.S. Thorgeirsson et al. / Cancer Letters 143 (1999) 245±247
at much lower incidence [3,4]. Potent oncogenic cooperation between c±myc and TGFa transgenes has also been demonstrated in pancreas, salivary glands and breast of transgenic mice [5,6], and when these two genes have been overexpressed in rat liver epithelial cells [7,8]. In the present study we have used this transgenic mouse model to analyze the role of mutagenesis in MeIQx induced hepatocarcinogenesis. The results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction of these compounds with speci®c genes during the neoplastic process. 2. Results and discussion The synergistic effect of c±myc and c±myc/TGFa on the neoplastic development in the liver has been shown to disrupt regulation of both cell cycle and apoptosis [9]. Both transgenic lines displayed dramatic increases of mitotic and apoptotic rates before the onset of HCC, but only c±myc/TGFa livers showed signi®cant levels of net proliferation (mitosis minus apoptosis). Subsequently, mitosis declined in peritumorous tissues, concomitant with the previously reported induction of TGFb 1, whereas c±myc and c± myc/TGFa HCCs maintained mitotic hyperactivity. The c±myc/TGFa HCCs were also characterized by a particularly strong expression of TGFa and very low apoptotic index in contrast to high levels of apoptosis in peritumorous tissues and c±myc HCCs. The differential levels of cell proliferation in noncancerous and cancerous tissues correlated with a stronger induction of cyclin D1 mRNA and protein in c±myc/TGFa and c±myc HCCs associated with intense pRb hyperphosphorylation [9]. Severe deregulation of G1/S transition was also demonstrated by up-regulation, particularly in the HCCs, of pRb-free E2F1- and E2F2-DP1 transcription factor heterodimers. The enhanced E2F activity during hepatocarcinogenesis was also shown by the transcriptional induction of putative E2F target genes involved in cell cycle progression, including endogenous c±myc, cyclin A, Cdc2 and E2F itself. Cdc2 overexpression and the elevated mitotic indices in the HCCs correlated also with induction of cyclin B steady-state
levels. Based on these data we have postulated that coexpression of c±myc and TGFa leads to selective growth advantage for hepatic (pre)neoplastic cells by disrupting the pRb/E2F pathway and by TGFa mediated reduction of apoptosis. To investigate the role of mutagenesis in the carcinogenic process, 2 - amino -3,8-dimethyl-imidazo [4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/ lacZ (Muta TMMice) and double transgenic c±myc/ IacZ mice that carry the lacZ mutation reporter gene [10]. Mice from both strains were weaned onto an AIN-76-based diet containing 0.06% MeIQx. Control mice received AIN-76 diet only. After 30 and 40 weeks on the diet, mutagenicity in the lacZ reporter gene and tumor incidence were examined in the livers of mice. After 40 weeks on diet, the incidence of HCC was 100% (17%) and 44% (0%) in male c±myc/lacZ and C57BL/lacZ mice given MeIQx (or control) diet, respectively. The lacZ mutant frequency was signi®cantly lower in both strains of mice on control diet (i.e. approximately 45±135 £ 1026 compared to 2000±8000 £ 1026 plaque forming units). Thus MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results indicate that the synergistic effect of MeIQ and c±myc may involve MeIQ-induced mutations in critical genes that accelerate c±myc induced hepatocarcinogenesis.
3. Conclusion Extensive studies in transgenic mouse models have demonstrated that overexpression of c±myc as a transgene induces tumor formation in a variety of tissues. In addition, c±myc cooperates with a variety of other genes to accelerate tumorigenesis. In addition, regulation of c±myc is disrupted in the majority of human tumors. The results from the present study indicate that transgenic mouse models, in particular those involving targeted overexpression of c±myc, may provide a valuable model to examine both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with speci®c genes during the neoplastic process.
S.S. Thorgeirsson et al. / Cancer Letters 143 (1999) 245±247
References [1] J.W. Grisham, Interspecies comparison of liver carcinogenesis: implications for cancer risk assessment, Carcinogenesis 18 (1997) 5981. [2] H. Murakami, N. Sanderson, P. Nagy, P. Marino, G.T. Merlino, S.S. Thorgeirsson, Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: interaction of c±myc and transforming growth factor a in hepatic oncogenesis, Cancer Res. 53 (1993) 1719±1723. [3] E. SantoniRugiu, P. Nagy, M.R. Jensen, V.M. Factor, S.S. Thorgeirsson, Evolution of neoplastic development in the liver of transgenic mice coexpressing c±myc and transforming growth factor a, Am. J. Pathol. 149 (1996) 407±428. [4] G-H. Lee, G. Merlino, N. Fausto, Development of liver tumors in transforming growth factor a transgenic mice, Cancer Res. 52 (1992) 5162±5170. [5] E.P. Sandgren, N.C. Luetteke, T.H. Qiu, R.D. Palmiter, R.L. Brinster, D.C. Lee, Transforming growth factor alpha dramatically enhances oncogeneinduced carcinogenesis in transgenic mouse pancreas and liver, Mol. Cell. Biol. 13 (1993) 320±330.
247
[6] L.T. Amundadottir, M.D. Johnson, G. Merlino, G.H. Smith, R.B. Dickson, Synergistic interaction of transforming growth factor a and c±myc in mouse mammary and salivary gland tumorigenesis, Cell Growth Diff. 6 (1995) 737±748. [7] L.W. Lee, V.W. Raymond, M-S. Tsao, D.C. Lee, H.S. Earp, J.W. Grisham, Clonal segregation of tumorigenicity with overexpression of c±myc and transforming growth factor-a genes in chemically transformed rat liver epithelial cells, Cancer Res. 51 (1991) 5238±5244. [8] S. Collins Presnell, M.T. Thompson, S.C. Strom, Investigation of the cooperative effects of transforming growth factor-a and c±myc overexpression in rat liver epithelial cells, Mol. Carcinog. 13 (1995) 233±244. [9] E. Santoni-Rugiu, M.R. Jensen, S.S. Thorgeirsson, Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c±myc and transforming growth factor a, Cancer Res. 58 (1998) 123± 134. [10] C.D. Davis, E.J. Dacquel, H.A.J. Schut, S.S. Thorgeirsson, E.G. Snyderwine, In vivo mutagenicity and DNA adduct levels of heterocyclic amines in MutateMice and c±myc/ lacZ double transgenic mice, Mutat. Res. 356 (1996) 287± 296.
Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models Snorri S. Thorgeirsson*, Doug-Young Ryu, Valerie Weidner, Elizabeth G. Snyderwine Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Received 3 November 1998; received in revised form 8 February 1999; accepted 9 February 1999
Abstract Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c±myc cDNA and mouse metallothionein1 promoter-human TGFa cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c±myc and TGFa transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (Muta TMMice) and double transgenic c±myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with speci®c genes during the neoplastic process. q 1999 Published by Elsevier Science Ireland Ltd. Keywords: Transgenic mouse models; Heterocyclic amines; Carcinogenicity; Mutagenicity
1. Introduction The nuclear oncogene c±myc and transforming growth factor (TGF) a , a ligand for the epidermal growth factor receptor tyrosine kinase, are frequently co-expressed in human tumors including hepatocellular carcinoma (HCC) (reviewed in [1]), indicating that their interaction may represent a critical step in malignant growth. To investigate this interaction, we * Corresponding author. LEC, Building 37, Room 3C28, National Cancer Institute, 37 Convent Drive MSC4255, Bethesda, MD 20892-4255 USA. Tel.: 301-496-1935; fax: 301-496-0734. E-mail address: [email protected] (S.S. Thorgeirsson)
recently generated double-transgenic mice overexpressing c±myc and TGFa in the liver and demonstrated that these two genes synergize to generate a dramatic enhancement of neoplastic development in this organ [2,3]. The progression from early and severe liver cell dysplasia and preneoplastic focal lesions to HCC occurred in 4 months in the c±myc/ TGFa double transgenic mice, resulting in striking liver enlargement, 100% frequency of HCCs before 8 months, and survival reduced to a year [3]. In contrast, both the single-transgenic mouse lines were characterized by a slower neoplastic development, with HCCs appearing only after one year of age and
0304-3835/99/$ - see front matter q 1999 Published by Elsevier Science Ireland Ltd. PII: S 0304-383 5(99)00133-0
246
S.S. Thorgeirsson et al. / Cancer Letters 143 (1999) 245±247
at much lower incidence [3,4]. Potent oncogenic cooperation between c±myc and TGFa transgenes has also been demonstrated in pancreas, salivary glands and breast of transgenic mice [5,6], and when these two genes have been overexpressed in rat liver epithelial cells [7,8]. In the present study we have used this transgenic mouse model to analyze the role of mutagenesis in MeIQx induced hepatocarcinogenesis. The results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction of these compounds with speci®c genes during the neoplastic process. 2. Results and discussion The synergistic effect of c±myc and c±myc/TGFa on the neoplastic development in the liver has been shown to disrupt regulation of both cell cycle and apoptosis [9]. Both transgenic lines displayed dramatic increases of mitotic and apoptotic rates before the onset of HCC, but only c±myc/TGFa livers showed signi®cant levels of net proliferation (mitosis minus apoptosis). Subsequently, mitosis declined in peritumorous tissues, concomitant with the previously reported induction of TGFb 1, whereas c±myc and c± myc/TGFa HCCs maintained mitotic hyperactivity. The c±myc/TGFa HCCs were also characterized by a particularly strong expression of TGFa and very low apoptotic index in contrast to high levels of apoptosis in peritumorous tissues and c±myc HCCs. The differential levels of cell proliferation in noncancerous and cancerous tissues correlated with a stronger induction of cyclin D1 mRNA and protein in c±myc/TGFa and c±myc HCCs associated with intense pRb hyperphosphorylation [9]. Severe deregulation of G1/S transition was also demonstrated by up-regulation, particularly in the HCCs, of pRb-free E2F1- and E2F2-DP1 transcription factor heterodimers. The enhanced E2F activity during hepatocarcinogenesis was also shown by the transcriptional induction of putative E2F target genes involved in cell cycle progression, including endogenous c±myc, cyclin A, Cdc2 and E2F itself. Cdc2 overexpression and the elevated mitotic indices in the HCCs correlated also with induction of cyclin B steady-state
levels. Based on these data we have postulated that coexpression of c±myc and TGFa leads to selective growth advantage for hepatic (pre)neoplastic cells by disrupting the pRb/E2F pathway and by TGFa mediated reduction of apoptosis. To investigate the role of mutagenesis in the carcinogenic process, 2 - amino -3,8-dimethyl-imidazo [4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/ lacZ (Muta TMMice) and double transgenic c±myc/ IacZ mice that carry the lacZ mutation reporter gene [10]. Mice from both strains were weaned onto an AIN-76-based diet containing 0.06% MeIQx. Control mice received AIN-76 diet only. After 30 and 40 weeks on the diet, mutagenicity in the lacZ reporter gene and tumor incidence were examined in the livers of mice. After 40 weeks on diet, the incidence of HCC was 100% (17%) and 44% (0%) in male c±myc/lacZ and C57BL/lacZ mice given MeIQx (or control) diet, respectively. The lacZ mutant frequency was signi®cantly lower in both strains of mice on control diet (i.e. approximately 45±135 £ 1026 compared to 2000±8000 £ 1026 plaque forming units). Thus MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results indicate that the synergistic effect of MeIQ and c±myc may involve MeIQ-induced mutations in critical genes that accelerate c±myc induced hepatocarcinogenesis.
3. Conclusion Extensive studies in transgenic mouse models have demonstrated that overexpression of c±myc as a transgene induces tumor formation in a variety of tissues. In addition, c±myc cooperates with a variety of other genes to accelerate tumorigenesis. In addition, regulation of c±myc is disrupted in the majority of human tumors. The results from the present study indicate that transgenic mouse models, in particular those involving targeted overexpression of c±myc, may provide a valuable model to examine both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with speci®c genes during the neoplastic process.
S.S. Thorgeirsson et al. / Cancer Letters 143 (1999) 245±247
References [1] J.W. Grisham, Interspecies comparison of liver carcinogenesis: implications for cancer risk assessment, Carcinogenesis 18 (1997) 5981. [2] H. Murakami, N. Sanderson, P. Nagy, P. Marino, G.T. Merlino, S.S. Thorgeirsson, Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: interaction of c±myc and transforming growth factor a in hepatic oncogenesis, Cancer Res. 53 (1993) 1719±1723. [3] E. SantoniRugiu, P. Nagy, M.R. Jensen, V.M. Factor, S.S. Thorgeirsson, Evolution of neoplastic development in the liver of transgenic mice coexpressing c±myc and transforming growth factor a, Am. J. Pathol. 149 (1996) 407±428. [4] G-H. Lee, G. Merlino, N. Fausto, Development of liver tumors in transforming growth factor a transgenic mice, Cancer Res. 52 (1992) 5162±5170. [5] E.P. Sandgren, N.C. Luetteke, T.H. Qiu, R.D. Palmiter, R.L. Brinster, D.C. Lee, Transforming growth factor alpha dramatically enhances oncogeneinduced carcinogenesis in transgenic mouse pancreas and liver, Mol. Cell. Biol. 13 (1993) 320±330.
247
[6] L.T. Amundadottir, M.D. Johnson, G. Merlino, G.H. Smith, R.B. Dickson, Synergistic interaction of transforming growth factor a and c±myc in mouse mammary and salivary gland tumorigenesis, Cell Growth Diff. 6 (1995) 737±748. [7] L.W. Lee, V.W. Raymond, M-S. Tsao, D.C. Lee, H.S. Earp, J.W. Grisham, Clonal segregation of tumorigenicity with overexpression of c±myc and transforming growth factor-a genes in chemically transformed rat liver epithelial cells, Cancer Res. 51 (1991) 5238±5244. [8] S. Collins Presnell, M.T. Thompson, S.C. Strom, Investigation of the cooperative effects of transforming growth factor-a and c±myc overexpression in rat liver epithelial cells, Mol. Carcinog. 13 (1995) 233±244. [9] E. Santoni-Rugiu, M.R. Jensen, S.S. Thorgeirsson, Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c±myc and transforming growth factor a, Cancer Res. 58 (1998) 123± 134. [10] C.D. Davis, E.J. Dacquel, H.A.J. Schut, S.S. Thorgeirsson, E.G. Snyderwine, In vivo mutagenicity and DNA adduct levels of heterocyclic amines in MutateMice and c±myc/ lacZ double transgenic mice, Mutat. Res. 356 (1996) 287± 296.