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Changes in Cyclic Adenosine Monophosphate-Responsive Element Binding Proteins in Rat Hepatomas

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Changes in Cyclic Adenosine Monophosphate-Responsive Element Binding Proteins in Rat Hepatomas (CANCER RESEARCH 51. 528-535. January 15. 199I| Changes in Cyclic Adenosine Monophosphate-responsive Element Binding Proteins in Rat Hepatomas Joanna Kwast-Welfeld, Ian de Belle, P. Roy Walker, Richard J. Isaacs, James F. VVhitfield, and Marianna Sikorska1 Molecular Cell Biology Group, Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K 1.4 OR6 ABSTRACT associated with normal liver function (7). In some cases (i.e., hepatoma 5123tc), these enzymes are expressed at constitu- We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine tively high levels (8). Recent developments in our understanding monophosphate (cAMP)-responsive element (CRE) binding (CREB) pro of the mechanisms of transcriptional activation of cAMP-re- teins in rapidly growing, chemically induced 5123tc and 5123D Morris sponsive genes has permitted us to reexamine the basis for the hepatomas. Using the CRK sequences from the r-/«.v.E2A, and somato- alterations in these responses to cAMP in hepatoma cells. statin gene promoters, we identified in the nuclear proteins from normal In the search for the molecular mechanism by which cAMP unstimulated or proliferating rat liver cells six different protein factors controls gene expression, a as-acting DNA regulatory sequence of M, 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of named the CRE has been discovered (9, 10). The CRE consen binding to the element. The M, 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene sus sequence found in the promoter region of a number of cAMP-inducible genes is an 8-base pair palindrome, 5'- expression. We could not lind the M, 47,000 CREB protein in the 5123tc TGACGTCA-3'. After that, a number of nuclear protein factors and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering ranging in MTfrom 38,000 to 55,000 that are capable of binding the protocol for extracting nuclear proteins, or (<•)attempting to restore to this sequence have been either predicted to exist or actually its DNA-binding property by phosphorylation [with endogenous protein identified (11-17). These transcription factors provided the kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and missing link between the elevation of the intracellular level of protein kinase C/dephosphorylation (with alkaline phosphatase)) »ere unsuccessful. The loss of the M, 47,000 CREB protein from solid tumors cAMP and changes in gene expression. of the Morris hepatoma is likely to be related to the neoplastic properties Several genes whose products are essential for liver cell of the tumor cell rather than to cell growth because the level of this function [such as phosphoenolopyruvate carboxykinase (18), protein remained unchanged during a 6-day period of liver regeneration. láclatedehydrogenase (15), glutamine synthetase (19), tyrosine The nuclear extract from the Morris hepatoma that did not have the M, aminotransferase (20), and c-fos (21, 22)] contain a functional 47,000 CRE-binding factor contained proteins immunologically related CRE sequence within their promoters. Of these genes, c-fos has to the CREB, c-Jun, and c-Fos proteins. We conclude that the M, 47,000 factor represents a distinct member of the CRE-binding protein family attracted the most attention in recent years because it belongs and that its absence from the hepatomas may lead to aberrant expression to a family of genes that respond rapidly to extracellular signals of cAMP-inducible genes. (23, 24) and because its product, the Fos protein, is itself a transcription factor involved in the transcriptional control of INTRODUCTION other genes (25). Therefore, abnormal expression of c-fos could Transient changes in the intracellular level of cAMP2 are initiate a cascade of events leading to, or contributing to, cell characteristic features of proliferating hepatocytes (1). They transformation (26). stimulate events leading to the initiation of DNA replication We showed recently that tissues in vivo and cultured cells in and mitosis, suggesting that cAMP-responsive genes are essen vitro contain multiple CREB proteins that are involved in tial for the proliferation of normal liver cells (2-4). A loss of conferring cAMP-responsiveness on genes.' We identified five these cAMP-mediated cell cycle controls could deregulate the different CREB factors with M, 34,000, 36,000,40,000,47,000, cell cycle and contribute to the generation of tumor cells that and 56,000 in nuclear extracts from rat liver using the CREs of have different growth rates and are blocked at various stages of the c-fos and somatostatin genes as probes. Among these fac differentiation (5). Moreover, alterations in the cells' ability to tors, the MT47,000 CREB protein showed the highest specific respond to changes in cAMP are likely to be involved in the ity for the CRE sequence, suggesting that this protein could development of the phenotypic diversity that is observed in the play a very important physiological role in mediating the effect Morris series of experimental hepatomas (6). For example, the of cAMP on gene expression. The M, 47,000 protein band amino acid transport systems and cAMP-inducible gluconeo- observed here was originally identified by Kwast-Welfeld et al. genic enzymes (such as tyrosine aminotransferase, phosphoen- (15) in rat ovarian tissue, rat liver, and C6 glioma cells as a olpyruvate carboxykinase, and glutamine synthetase) do not protein that specifically binds to the lactate dehydrogenase A undergo the normal periodic oscillations in activity that are subunit gene promoter, which contains a CRE octamer. To better understand the changes in cAMP-controlled gene Received 5/9/90: accepted 10/26/90. The costs of publication of this article were defrayed in part by the payment expression in tumors we analyzed the nuclear proteins from of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rapidly growing neoplastic cells of chemically induced 5123tc 1To whom requests for reprints should be addressed, at Institute for Biological and 5123D Morris hepatomas for their ability to bind to the Sciences, National Research Council. M-54 Montreal Road. Ottawa K1A OR6. DNA fragments containing the CRE sequence using the South Canada. : The abbreviations used are: cAMP. cyclic AMP; CRE. cAMP-responsive western hybridization technique and gel retardation assays. element: CREB. CRE-binding; HPX. hepatectomized: poly(dl-dC). alternating copolymer of deoxyinosine and deoxycytidine; SDS-PAGE. sodium dodecyl 'J. Kwast-Welfeld. P. R. Walker. J. F. Whitfield, and M. Sikorska. Influence sulfate-polyacrylamide gel electrophoresis; TPA, 12-0-tetradecanoylphorbol 13- of flanking sequences on DNA-protein interactions at the cyclic AMP-responsive acctate. element. J. Biol. Chem.. submitted for publication. 528 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1991 American Association for Cancer Research. CYCLIC AMP-RESPONSIVE ELEMENT BINDING PROTEINS MATERIALS AND METHODS Synthesis of Peptides, Production of Antibodies, and Immunoblotting of Nuclear Proteins. The amino acid sequence of human placentai Tissue Sources. Normal liver tissue was obtained from 200-250-g CREB (11), mouse c-Jun (34), and mouse c-Fos (35) proteins were male, specific pathogen-free Sprague-Dawley rats bred in this labora scanned for likely antigenic sites using the University of Wisconsin tory. Partial hepatectomy (70% HPX) was performed as previously Genetic Computer Group software package and the algorithm of Jame described (27). Hepatomas 5123D and 5123tc were propagated by s.c. son and Wolf (36). The peptides. representing amino acids 134-150 of inoculation into the inguinal regions of 200-300-g male Buffalo rats CREB, 301 -314 of c-Jun, and 77-90 of c-Fos proteins, were synthesized (Harlem Sprague Dawley Co., Indianapolis, IN) as described by by the simultaneous multiple peptide synthesis method (37). New MacManus (28). The original malignant (metastasized to lung) 5123 Zealand rabbits were immunized with unconjugated peptides according hepatoma was produced in a female Buffalo rat that had been fed a to the following schedule. The first i.m. injection of 0.5 mg peptide slowly acting carcinogen, 7V-2-fluorenylphthalamic acid (29). The emulsified with complete Freund's adjuvant (1:1 v/v) was followed by 5123tc hepatoma originated from 5123 tumor cells that had been two consecutive s.c. injections of peptides emulsified with incomplete passaged in tissue culture, and the 5123D hepatoma is a slower-growing adjuvant at 1-wk intervals. The rabbits were test bled at 7 and 14 days subline of the 5123 tumor after the 16th serial transplant generation. after the last vaccination, and the titers of their sera were determined Preparation of Nuclear Protein Extracts. Nuclei were isolated and by enzyme-linked immunosorbent assay. Nuclear proteins were resolved purified following the low speed centrifugation and Triton X-100 by 10% SDS-PAGE, electrotransfered onto nitrocellulose membranes washing procedure as described previously by Sikorska and Whitfield (Hybond C, Amersham), and immunoblotted with the appropriate (30). Proteins were extracted from nuclei with 25 mM 4-(2-hydroxy- antisera. The antigen-antibody complexes were visualized by autoradi
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