* Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, (CP 1121), Buenos Aires, Argentina;
Facultad de Medicina, Universidad Abierta Interamericana, (CP 1147), Buenos Aires, Argentina; and
Ciclo Básico Común, Universidad de Buenos Aires, (CP 1053), Buenos Aires, Argentina
Received July 25, 2003; accepted September 16, 2003
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ABSTRACT |
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Key Words: hexachlorobenzene; rat liver; ODC activity; PTK activity; polyamine content; proto-oncogene levels.
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INTRODUCTION |
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Most tumor promoters induce ornithine decarboxylase (ODC) in vivo (Kitchin and Brown, 1989). ODC catalyzes the formation of putrescine and CO2 from ornithine and is the first and key regulatory enzyme in polyamine biosynthesis. ODC and polyamines are known to play a pivotal role in cell proliferation and to contribute to the development of cancer (Heby and Persson, 1990
; Pegg, 1988
). Celano et al. (1989)
suggested that individual polyamines may play a selective role in the expression of specific growth-related genes. In normal cells, the activity of ODC shows a rapid, transient increase upon stimulation by growth factors (Heby and Persson, 1990
; Pegg, 1988
), whereas the cells transformed by chemical carcinogens and oncogenes such as v-src, neu, and ras seem to have a constitutively elevated ODC activity (Gilmour et al., 1986
; Hölttä et al., 1994
). Recently, it has been postulated that the ODC gene may act as an oncogene, because the overexpression of this gene is essential for cell transformation (Shanz and Pegg, 1994
).
ODC activity has been shown to have multiple levels of control ranging from transcriptional regulation (Katz and Kahana, 1987) to posttranslational modification (Ray et al., 1999
). A relevant signal associated with cell growth, differentiation, and malignant transformation is the induction of a group of immediate-early (IE) genes that encode the transcriptional activator proteins c-Fos, c-Jun, and c-Myc (Schütte et al., 1989
; Vogt and Bos, 1989
). Interaction of c-Fos with any one of the various members of the Jun family forms the activating protein-1 (AP-1) transcription factor required for cell proliferation (Abate et al., 1990
).
Transcription regulatory factors encoded by early-response genes might control the expression of delayed-early response genes, such as ODC (Desiderio et al., 1998). Tyrosine-kinase receptors trigger intracellular signals by coupling to multiple signal transduction pathways. Mitogenic activated protein kinases (MAPK) phosphorylate and regulate protein kinases involved in the control of gene expression (Whitmarsh and Davis, 1996
). Ornithine decarboxylase activity can be regulated by phosphorylation, confirming the pivotal role of ODC in cell-signaling pathways triggered at the cell surface (Desiderio et al., 1998
).
As HCB is a nongenotoxic tumor promoter, it is reasonable to ask whether it can initiate the signals associated with cell growth and transformation. We have previously demonstrated that HCB induces time- and dose-dependent alterations, both in rat hepatic microsomal protein phosphorylation and protein tyrosine kinase (PTK) activity, during the early stages of HCB administration (Randi et al., 1998). We have also reported that HCB changes epidermal growth factor (EGF) affinity for its membrane receptor, induces EGFR internalization and stimulates EGFR-tyrosine kinase activity (Randi et al., 1998
, 2003
). Other subcellular mechanistic studies have shown that HCB exposure induced alterations in intercellular gap junctional communication in female rat liver (Mally and Chipman, 2002
; Plante et al., 2002
).
The aim of this study was to investigate if alterations of ODC activity and proto-oncogenes proteins (c-Fos, c-Jun, and c-Myc) could be involved in the early stages of HCB exposure. Our results showed, for the first time, that HCB in vivo treatment elicited very early changes in the induction of a group of immediate-early genes and ODC activity that could be relevant in the early molecular events involved in HCB tumor promoter activity in rat liver.
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MATERIALS AND METHODS |
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Animals and treatment.
Female Wistar rats (160180 g at the onset of the experiment) were purchased from the Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. The rats were fed Purine 3 rat chow (Cabeca S.C.A, Argentina) and water ad libitum. Environmental conditions consisted of a 12-h light-dark cycle, 2024°C, and 4575% humidity. Following a 7-day acclimation period, HCB (1000 mg/kg body weight) and other doses as indicated in the text, were administered daily through a stomach tube. HCB (40 mg/ml) was suspended in water containing Tween 20 (0.5 ml/100 ml). Control animals received equal volumes of appropriate solvent by the same route. For time-course studies, the animals received HCB (1000 mg/kg body weight) for 1, 2, and 10 days. The acute exposure assay was carried out at 6, 12, 18, and 24 h posttreatment. This dose of HCB was chosen based on our recent results that showed that the maximum increase in rat liver EGFR-tyrosine kinase activity was reached at this concentration (Randi et al., 2003). For the dose-response assay, HCB was administered for 24 h at 10, 100, and 1000 mg/kg body weight. ODC and PTK activities and free polyamine content were evaluated in the same group of six animals.
To inhibit ODC activity in vivo, we used DFMO, an irreversible specific suicide inhibitor and analogue of ornithine (Prakash et al., 1980). This soluble product was administered to rats, in water beverage at a concentration of 2%, 24 h before starting the treatment with HCB (1000 mg/kg body weight). Following an overnight fast, the animals were killed by decapitation, and the livers perfused through the portal vein with 0.154 M NaCl.
The general health of the animals was not affected by the doses of HCB employed, as evaluated by the behavior and appearance of the rats, including examination of their coats, mucous membranes, and body weights, and their food and water consumption.
Hepatic microsomal fraction preparation.
Liver microsomal membranes were isolated from a crude homogenate as described by Randi et al.(2003).
Protein tyrosine kinase assay.
PTK activity assay in rat liver membranes was based on that described by Tremblay and Beliveau (1984). Phosphorylation of the synthetic peptide, poly-[Glu80 Na, Tyr20] (poly GT), was carried out in a reaction mixture containing 80 µg/100 µl of particulate proteins, 500 µg/100 µl of poly GT, 8 µCi of -32P-ATP (40 µM), 10 mM MnCl2, 20 mM MgCl2, 0.2 mM Na3VO4, and 25 mM HEPES-Tris (pH 7.0).
Preparation of nuclear protein extracts.
Nuclear proteins were isolated from rat liver according to the procedure of Gorski et al.(1986) with modifications. Briefly, the tissue was homogenized in a Dounce-type glass homogenizer with a Teflon pestle in three volumes of homogenization buffer (0.25 M sucrose, 10 mM NaCl, 5 mM MgCl2, 50 mM Tris, pH 7.5, and 0.2% Triton) and filtered through moistened gauze. The filtrate was centrifuged for 4 min at 1,225 x g, and the pellet was resuspended in 2 volumes of homogenization buffer without Triton and centrifuged at 1,225 x g for 4 min. The resulting pellet was resuspended in the same buffer and centrifuged at 100,000 x g for 60 min on a 2 M sucrose layer in homogenization buffer. Pure nuclei were treated with lysis buffer (0.3 M KCl, 1 mM MgCl2, 20 mM TrisHCl, 2mM DTT, pH 8.0) for 40 min and centrifuged at 1,075 x g for 15 min. The supernatant containing the nuclear protein extract was stored at -80°C. Protein concentration was determined by the method of Bradford (1976)
, using bovine serum albumin (BSA) as a standard.
Western blot analysis.
Nuclear protein extracts were electrophoresed in a 10% SDSPAGE and transferred to polyvinylidene difluoride membrane (PVDF, Bio-Rad). Filters were blocked with 1% fat-free dried milk, 1% BSA in TBST (10 mM Tris, 150 mM NaCl and 0.05% Tween 20, pH 8.0). Blocked membranes were washed five times with TBST for 10 min and incubated with the first antibody (1:400) in TBST overnight. The membranes were washed five times with TBST and incubated with the suitable horseradish peroxidase-conjugated anti-species-specific antibody. Immunoblots were then washed five times with TBST and once with TBS. Proteins were visualized employing the enzyme-linked enhanced chemoluminescence kit (ECL, Amersham Biosciences, Inc., UK) and quantified by scanning laser densitometry in a Fotodyne (Foto/Analyst), Gel-Pro Analyzer 3.1.
Polyamine determination.
Putrescine, spermidine, and spermine were extracted with 0.2 N perchloric acid at 4°C. Supernatants were obtained after centrifugation at 1,075 x g for 15 min and treated with dansyl-chloride according to Seilers method (Seiler, 1970).
Determination of ODC activity.
ODC activity was determined by measuring 14C-CO2 release from L-1-14C-ornithine. Liver was homogenized in a cold extraction buffer containing 50 mM TrisHCl (pH 8.0), 1 mM dithiothreitol (DTT), 1 mM pyridoxal phosphate, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). The reaction mixture contained 50 mM TrisHCl buffer (pH 7.5), 2 mM EDTA, 2 mM DTT, 50 µM pyridoxal phosphate, 1 mM PMSF, 1 mM ornithine, and 0.2 µCi L-1-14C-ornithine, and the enzyme preparation (0.40.8 mg) in a final volume of 0.8 ml. The incubation was carried out at 37°C for 1 h and stopped by adding 30% trichloracetic acid (TCA) and incubated for an additional 30 min. 14CO2 released was trapped on hyamine-soaked Whatman 1 filter paper and counted for radioactivity in a scintillation counter. Blanks did not contain enzyme preparation. The enzyme activity was expressed in terms of nmols/mg protein/h.
Statistical analysis.
Data from dose and time-course effects were analyzed by one-way ANOVA, followed by Tukeys post-hoc test. Other statistical analyses were performed using Students t-test, as indicated in the text. Differences between control and treated animals were considered significant when p values were < 0.05.
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RESULTS |
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As shown in Figure 3, the immunodetectable proto-oncogene levels showed time-dependent changes, with a maximum increase on the second day. It is noteworthy that c-Jun showed a remarkable increase (5-fold) at this time, when compared with c-Fos and c-Myc. The protein levels of c-Fos/c-Jun remained significantly elevated at 10 days. c-Myc exhibited a transient increase at 2 days and returned to basal values later.
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ODC activity increased in a dose-dependent manner. This increase was significant at doses of at least 100 mg/kg body weight; a maximum effect was observed at HCB 1000 mg/kg body weight. Conversely, liver microsomal PTK activity decreased significantly at the same concentrations (Fig. 4).
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The following experimental groups were used: Control (rats received water containing Tween 20 by gavage 24 h before sacrifice); DFMO (rats received 2% DFMO in drinking water until sacrifice, 48 h later; 24 h before sacrifice they received water containing Tween 20 by gavage); HCB (rats received 1000 mg/kg body weight of HCB by gavage and were sacrificed 24 h later); and HCB + DFMO (rats received 2% DFMO in drinking water until sacrifice 48 h later; 24 h before sacrifice they were administered HCB at a dose of 1000 mg/kg body weight by gavage).
Our results showed that PTK activity was reduced significantly both in Control and HCB-treated rats, when ODC activity was knocked out by DFMO, suggesting that there is no direct link between these two enzymes (Fig. 6).
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DISCUSSION |
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The early significant increase in hepatic immunodetectable proto-oncogene levels (6 h) followed by the induction of ODC activity at 18 h, suggests that the enhancement in ODC activity may be the consequence of the induction of one or several proto-oncogenes. Another possibility is that upregulation of c-Fos/c-Jun and ODC follow two independent pathways, as proposed by Jansen et al.(1999). ODC activity may be only induced by c-Myc, in agreement with Arora et al.(2000)
, who reported that c-Myc is an important positive regulator of cell proliferation in rat liver. Bartolome et al.(1999)
showed that insulin-stimulated ODC mRNA expression and enzymatic activity in the rat liver are accompanied by a concomitant increase in the expression of c-Myc and Max mRNAs, which are known to act physiologically as transcriptional activators of the ODC gene (Bello-Fernández et al., 1993
). Pereira et al.(2001)
reported that tumor promoters increased mRNA expression of c-Myc in liver. Further studies are necessary to understand whether the observed increase in ODC activity found in this work is related to the enhancement in the proto-oncogene/s levels.
c-Myc increases transiently at very early times (6, 12 h), while c-Jun is remarkably increased at 2 days. Conversely c-Fos increases steadily from the beginning of HCB exposure. These results suggest that HCB deregulates each proto-oncogene in a different manner. Future experiments are necessary to determine whether the elevated c-Fos and c-Jun levels encountered at 10 days are biologically relevant. It is interesting that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known proven tumor promoter and teratogen, also induced ODC activity (Raunio and Perlkonen, 1983) and the expression of c-fos, c-jun, jun-B and jun-D proto-oncogenes at early exposure times (Puga et al., 1992
).
We have previously shown that chronic treatment with HCB produced downregulation of EGFR and dose-dependent increases in EGFR-tyrosine kinase activity in rat liver (Randi et al., 2003). Our results showing an early increase in PTK activity (12 h), followed by a peak in ODC activity at 18 h, suggest that ODC could be regulated by phosphorylation. The growth factor signal transduction pathway may be involved in the activation of "primary response genes" through a series of phosphorylations and dephosphorylations of cytosolic and eventually nuclear proteins, as suggested by Matsumura (1994)
.
Our studies demonstrate, for the first time, that HCB stimulated early increases in proto-oncogene levels, ODC activity, and free polyamine content in rat liver. The induction of proto-oncogenes may be relevant to the early molecular events involved in HCB tumor promoter activity. Although our results are obviously far from establishing a direct connection between the induction of proto-oncogene and such an activity, they provide the groundwork for further promising research in this area.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Arora, V., Knapp, D. C., Smith, B. L., Statdfield, M. L., Stein, D. A., Reddy, M. T., Weller, D. D., and Iversen, P. L. (2000). c-Myc antisense limits rat liver regeneration and indicates role for c-Myc in regulating cytochrome P-450 3A activity. J. Pharmacol. Exp. Ther. 292, 921928.
Bartolome, J. V., Wang, S., Schanberg, S. M., and Bartolome, M. B. (1999). Involvement of C-Myc and C- Max in CNS ß-endorphin modulation of hepatic ornithine decarboxylase responsiveness to insulin in rat pups. Life Sci. 64, 8791.
Bello-Fernández, C., Packham, G., and Cleveland, J. L. (1993). The ornithine decarboxylase gene is a transcriptional target of c-Myc. Proc. Natl Acad. Sci. U.S.A. 90, 78047808.
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254.[CrossRef][ISI][Medline]
Cabral, J. R. P., and Shubik, P. (1986). Carcinogenic activity of hexachlorobenzene in mice and hamsters. In Hexachlorobenzene: Proceedings of an international symposium. (Morris, C. R., and Cabral, J. R. P., Eds.), pp. 411416. IARC Scientific Publications, Lyon.
Cabral, R., Hoshiya, T., Hakoi, K., Hasegawa, R., and Ito, N. (1996). Medium-term bioassay for the hepatocarcinogenicity of hexachlorobenzene. Cancer Lett. 100, 223226.[CrossRef][ISI][Medline]
Celano, P., Berchtold, C. M., Giardello, R. A., and Casero, R. A., Jr. (1989). Modulation of growth gene expression by selective alteration of polyamines in human colon carcinoma cells. Biochem. Biophys. Res. Commun. 165, 384390.[ISI][Medline]
Courtney, K. D. (1979). Hexachlorobenzene: A review. Environ. Res. 20, 225246.[ISI][Medline]
Desiderio, M. A., Pogliaghi, G., and Dansi, P. (1998). Hepatocyte growth factor-Induced expression of ornithine decarboxylase, c-met, and c-myc is differently affected by protein kinase inhibitors in human hepatoma cells HepG2. Exp. Cell Res. 242, 401409.[CrossRef][ISI][Medline]
Gilmour, S. K., Aglow, E., and OBrien, T. G. (1986). Heterogeneity of ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin and epidermal tumours. Carcinogenesis 7, 943947.[Abstract]
Gorski, K., Carneiro, M., and Schibler, U. (1986). Tissue-specific in vitro transcription from the mouse albumin promoter. Cell 47, 767776.[ISI][Medline]
Grimalt, J. O., Sunyer, J., Moreno, V., Amaral, O. C., Sala, M., Rosell, A., Anto, J. M., and Albaiges, J. (1994). Risk excess of soft-tissue sarcoma an thyroid cancer in a community exposed to airbone organochlorinated compound mixtures with a high hexachlorobenzene content. Int. J. Cancer 56, 200203.[ISI][Medline]
Hahn, M. E., Goldstein, J. A., Linko, P., and Gasiewicz, T. (1989). Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Arch. Biochem. Biophys. 270, 344355.[ISI][Medline]
Heby, O., and Persson, L. (1990). Molecular genetics of polyamine synthesis in eukaryotic cells. Trends Biochem. Sci. 15, 153158.[CrossRef][ISI][Medline]
Hölttä, E., Auvinen, M., Paasinen, A., Kangas, A., and Anderson, L. C. (1994). Ornithine decarboxylase-induced cellular transformation: the involvement of protein tyrosine kinase(s) and pp130. Biochem. Soc. Trans. 22, 853859.[ISI][Medline]
Iscan, M., Coban, T., Cok, I., Bulbul, D., Eke, B. C., and Burgaz, S. (2002). The organochlorine pesticide residues and antioxidant enzyme activities in human breast tumors: Is there any association? Breast Cancer Res. Treat. 72, 173182.[CrossRef][ISI][Medline]
Jänne, J., Alhonen, L., and Leinonen, P. (1991). Polyamines: From molecular biology to clinical applications. Ann. Med. 23, 241259.[ISI][Medline]
Jansen, A. P., Colburn, N. H., and Verma, A. K. (1999). Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation. Oncogene 18, 58065813.[CrossRef][ISI][Medline]
Kashiwagi, K., Kobayashi, H., and Igarashi, K. (1986). Apparently unidirectional polyamine transport by proton motive force in polyamine-deficient Escherichia coli. J. Bacteriol. 165, 972977.[ISI][Medline]
Katz, A., and Kahana, C. (1987). Transcriptional activation of mammalian ornithine decarboxylase during stimulated growth. Mol. Cell. Biol. 7, 26412643.[ISI][Medline]
Kitchin, K. T., and Brown, J. L. (1989). Biochemical studies of promotes of carcinogenesis in rat liver. Teratog. Carcinog. Mutagen. 9, 273285.[ISI][Medline]
Kleiman de Pisarev, D. L., Ríos de Molina, M. del C., and San Martín de Viale, L. C. (1990). Thyroid function and thyroxine metabolism in hexachlorobenzene induced porphyria. Biochem. Pharmacol. 39, 817825.[CrossRef][ISI][Medline]
Mally, A., and Chipman J. K. (2002). Non-genotoxic carcinogens: early effects on gap junctions, cell proliferation and apoptosis in the rat. Toxicology 180, 233248.[CrossRef][ISI][Medline]
Matsumura, F. (1994). How important is the protein phosphorylation pathway in the toxic expression of dioxin- type chemicals? Biochem. Pharmacol. 48, 215224.[CrossRef][ISI][Medline]
Pegg, A. E. (1988). Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48, 759774.[Abstract]
Pereira, M. A., Kramer, P. M., Conran, P. B., and Tao, L. (2001). Effect of chloroform on dichloroacetic acid and trichloroacetic acid-induced hypomethylation and expression of the c-myc gene and on their promotion of liver and kidney tumours in mice. Carcinogenesis 22, 15111519.
Plante, I., Charbonneau, M., and Cyr, D. G. (2002). Decreased gap junctional intercellular communication in hexachlorobenzene-induced gender-specific hepatic tumor formation in the rat. Carcinogenesis 23, 12431249.
Prakash, N. J., Schechter, P. J., Mamont, P. S., Grove, J., Koch-Weser, J., and Sjoerdsma, A. (1980). Inhibition of EMT 6 tumor growth by interference with polyamine biosynthesis; effects of alphadifluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase. Life Sci. 26, 181194.[CrossRef][ISI][Medline]
Puga, A., Nebert, D. W., and Carrier, F. (1992). Dioxin Induces Expression of c-fos and c-jun proto- oncogenes and a large increase in transcription factor AP-1. DNA Cell Biol. 4, 269281.
Randi, A., Sancovich, H. A., Ferramola de Sancovich, A. M., Loaiza, A., Kölliker-Frers, R., Spinelli, F., and Kleiman de Pisarev, D. L. (2003). Effect of in vivo administered hexachlorobenzene on Epidermal Growth Factor Receptor levels, protein tyrosine kinase activity and phosphotyrosine content in rat liver. Biochem. Pharmacol. 65, 14951506.[CrossRef][ISI][Medline]
Randi, A., Sancovich, H., Ferramola de Sancovich, A. M., Loaiza, A., Krawiec, L., and Kleiman de Pisarev, D. L. (1998). Hexachlorobenzene-induced alterations of rat hepatic microsomal membrane functions. Toxicology 125, 8394.[CrossRef][ISI][Medline]
Raunio, H., and Pelkonen, O. (1983). Effect of polycyclic aromatic compounds and phorbol esters on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in mouse liver. Cancer Res. 43, 782786.[Abstract]
Ray, R. M., Viar, M. J., Patel, T. B., and Johnson, L. R. (1999). Interaction of asparagine and EGF in the regulation of ornithine decarboxylase in IEC-6 cells. Am. J. Physiol. 276, G773G780.[ISI][Medline]
Schütte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., and Minna, J. (1989). Jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun. Cell 59, 12501256.
Seiler, N. (1970). Use of the dansyl reaction in biochemical analysis. In Methods of Biochemical Analysis, (Glick, D., Ed.), pp. 259337. Interscience Publ., New York.
Shanz, L. M., and Pegg, A. E. (1994). Overproduction of ornithine decarboxylase caused by relief of translational repression is associated with neoplastic transformation. Cancer Res. 54, 23132316.[Abstract]
Sopena de Kracoff, Y. E., Ferramola de Sancovich, A. M., Sancovich, H. A., and Kleiman de Pisarev, D. L. (1994). Effect of thyroidectomy and thyroxine on hexachlorobenzene induced porphyria. J. Endocrinol. Invest. 17, 301305.[ISI][Medline]
Stewart, F. P., Manson, M. M., Cabral, J. R. P., and Smith, A. G. (1989). Hexachlorobenzene as a promoter of diethylnitrosamine-initiated hepatocarcinogenesis in rats and comparison with induction of porphyria. Carcinogenesis 10, 12251230.[Abstract]
Tabib, A., and Bachrach, U. (1999). Role of polyamines in mediating malignant transformation and oncogene expression. Int. J. Biochem. Cell Biol. 31, 12891295.[CrossRef][ISI][Medline]
Tremblay, L., and Beliveau, R. (1994). Protein tyrosine phosphorylation in normal rat tissues. Int. J. Biochem. 26, 2934.[CrossRef][ISI][Medline]
Vogt, P. K., and Bos, T. J. (1989). The oncogene jun and nuclear signaling. Trends Biol. Sci. 14, 172175.[CrossRef]
Whitmarsh, A. J., and Davis, G. D. (1996). Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathway. J. Mol. Med. 74, 589607.[CrossRef][ISI][Medline]
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