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Expression of GCIP in Transgenic Mice Decreases Susceptibility to Chemical Hepatocarcinogenesis

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Expression of GCIP in Transgenic Mice Decreases Susceptibility to Chemical Hepatocarcinogenesis Oncogene (2006) 25, 4207–4216 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Expression of GCIP in transgenic mice decreases susceptibility to chemical hepatocarcinogenesis WMa1,2,3,4, X Xia5, LJ Stafford1,2,3,CYu1,2,3, F Wang1,2,3, G LeSage5 and M Liu1,2,3 1Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX, USA; 2Center for Cancer Biology and Nutrition, Texas A&M University System Health Science Center, Houston, TX, USA; 3Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston, TX, USA; 4The Graduate School of Biomedical Sciences, The University of Texas-Houston Health Science Center, Houston, TX, USA and 5Department of Internal Medicine, The University of Texas-Houston Health Science Center, Houston, TX, USA Transcription factors with helix–loop–helix (HLH) motif Introduction play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, Liver cancer is the third most common causes for cancer proliferation, and tumorigenesis. To examine whether death in the world. In 2005, a total of 15 420 estimated the newly identified HLH protein GCIP/CCNDBP1 deaths by liver/intrahepatic bile duct cancer are expected modulates cell fate determination and plays a role in in the US, including 10 330 male cases and 5090 female hepatocyte growth, proliferation, and hepatocarcino- cases (Jemal et al., 2005). Although lots of research has genesis, we generated transgenic mice with human GCIP been performed to study the mechanism of hepatocarci- gene driven by a liver-specific albumin promoter. We nogenesis, few of them are related to the early stage of demonstrated that in GCIP transgenic mice, the overall liver tumor development. Helix–loop–helix (HLH) liver growth and regeneration occurred normally after proteins are key regulators of cell growth and differ- liver injury induced by carbon tetrachloride (CCl4). In the entiation in embryonic and adult tissues. They have diethylnitrosamine (DEN)-induced mouse hepatocarcino- been demonstrated to play critical roles in regulation of genesis, we demonstrated that overexpression of GCIP in gene expression, cell cycle control, cell lineage commit- mouse liver suppressed DEN-induced hepatocarcinogen- ment and numerous developmental processes (Blackwell esis at an early stage of tumor development. The number et al., 1990; Kreider et al., 1992; Zebedee and Hara, of hepatic adenomas at 24 weeks was significantly lower 2001). From the yeast to humans, over 240 HLH or not detected in GCIP transgenic male mice compared proteins have been identified to date (Massari and to the control mice under the same treatment. Although Murre, 2000). Based upon the presence or absence of GCIP has little inhibition on the number of hepatic tumors DNA-binding domain and other additional functional at later stages (40 weeks), hepatocellular tumors in GCIP domains, HLH proteins can be classified into five major transgenic mice are smaller and well-differentiated classes, basic HLH (bHLH) proteins, basic HLH compared to the poorly differentiated tumors in wild- Per-AhR-Arnt-Sim (bHLH-PAS) proteins, basic HLH type mice. Furthermore, we demonstrate that GCIP func- leucine zipper (bHLH-LZ) proteins, dominant-negative tions as a transcriptional suppressor, regulates the HLH (dnHLH) proteins, and HLH leucine zipper expression of cyclin D1, and inhibits anchorage-indepen- (HLH-LZ) proteins. In multicellular organisms, bHLH dent cell growth and colony formation in HepG2 cells, proteins are required for many important developmental suggesting a significant role of GCIP in tumor initiation processes, including neurogenesis (Lee et al., 1995; Ma and development. et al., 1996; Sun et al., 2001), myogenesis (Nabeshima Oncogene (2006) 25, 4207–4216. doi:10.1038/sj.onc.1209450; et al., 1993; Molkentin et al., 1995; Spicer et al., 1996), published online 27February 2006 hematopoiesis (Kreider et al., 1992; Zhao and Aplan, 1999; Barndt et al., 2000), and pancreatic development Keywords: GCIP; CCNDBP1; DIP1/HHM; transgenic (Jensen et al., 2000; Pin et al., 2001). On the basis of mouse; hepatocarcinogenesis; diethylnitrosamine; tissue distribution and dimerization capabilities, bHLH HLH-Zipper proteins protein can be further divided into two subgroups. One subgroup also known as the E proteins and includes E12, E47, E2-5, E2-2, HEB, and Daughterless. These proteins are ubiquitously expressed in many tissues and capable of forming either homo- or heterodimers Correspondence: Dr M Liu, Alkek Institute of Biosciences and (Zhuang et al., 1992; Shirakata and Paterson, 1995). Technology, Texas A&M University System Health Science Center, The DNA-binding specificity of E proteins is limited to 2121 W Holcombe Blvd, Houston, TX 77030, USA. E-mail: [email protected] the E-box site, which have the consensus sequence Received 28 September 2005; revised 27December 2005; accepted 27 CANNTG (Cordle et al., 1991; Voronova and Lee, December 2005; published online 27February 2006 1994). The other subgroup bHLH proteins show a GCIP suppresses hepatocarcinogenesis in transgenic mouse WMaet al 4208 tissue-restricted pattern of expression and regulate liver injury induced by carbon tetrachloride (CCl4). tissue-specific cell growth and differentiation. Examples In the mouse model of hepatocarcinogenesis induced by include the myogenic bHLH family of proteins (MyoD, DEN, we observed that overexpression of GCIP in Myogenin, Myf-5, MRF4), the cardiogenic family of transgenic mice suppressed the number of chemical- proteins (eHand and dHand), the neurogenic family of induced hepatocarcinogenesis at the early stage of tumor proteins (Neurogenin, NeuroD, Mash-1, Mash-2, Hes, development. At the late stage, hepatocellular tumors NSCL), and the haemopoietic family of bHLH proteins in GCIP transgenic mice are smaller and well- (SCL/TAL-1, Lyl-1, and ABF-1) (Murre et al., 1989; differentiated compared to the poorly differentiated Lassar et al., 1991; Hu et al., 1992; Kee et al., 2000). tumors in wild-type mice. We further demonstrated that With few exceptions, they are incapable of forming GCIP functions as a general transcriptional suppressor. homodimers and preferentially heterodimerize with the Overexpression of GCIP inhibited anchorage-indepen- E proteins. The heterodimers can bind both canonical dent cell growth and colony formation whereas down- (CANNTG) and noncanonical (CACNAG) E-box sites regulation of GCIP promotes colony formation in (Gaubatz et al., 1994). Dominant-negative HLH pro- HepG2 cells. These data suggest that GCIP may teins, including Id proteins (Id1, Id2, Id3, Id4) and emc, function as an important negative regulator in tumor are small proteins lack a basic region. Owing to a lack initiation and progression but does not alter the of the basic region, the Id proteins appear to function proliferation of normal hepatocytes following liver as dominant-negative regulators of bHLH proteins injury. These findings suggest GCIP maybe an attractive through the formation of ‘nonfunctional’ Id/bHLH target for prevention of hepatocellular carcinoma. heterodimers, thus quench the activity of the E proteins, inhibit cell differentiation and regulate tumorigenesis (Benezra et al., 1990; Sun et al., 1991; Peppelenbosch et al., 1995; Yokota and Mori, 2002; Perk et al., 2005). Results Mice deficient in the Id family of proteins have profound defects in neurogenesis, angiogenesis, vasculari- Generation of transgenic mice overexpressing GCIP under zation of tumor xenografts, impaired immune responses, the control of the albumin promoter and tumorigenesis (Lyden et al., 1999; Rivera et al., The full-length human GCIP was cloned from a human 2000; Rivera and Murre, 2001; Ruzinova and Benezra, bone marrow cDNA library (clontech) (Xia et al., 2000). 2003; Sikder et al., 2003). Human GCIP cDNA fragment (1.1 kb) containing the GCIP (CCNDBP1) is a HLH leucine zipper (HLH- entire coding region was inserted into the EcoRI site LZ) proteins without basic DNA-binding region, which of an albumin promoter-driven expression vector was identified and characterized in our lab and others to (Figure 1a) and a chicken insulator is inserted at interact with cyclinD1 (DIP1) and as a human homo- 30-end of the transgene to ensure the expression of logue of MAID protein (HHM) (Hwang et al., 1997; target gene. Two GCIP transgenic lines were developed Terai et al., 2000; Xia et al., 2000; Yao et al., 2000). and utilized for characterization. Transcription of GCIP gene is localized on chromosome 15q15, a region human GCIP was identified by Northern blot analysis frequently deleted or undergoes loss of heterozygosity of liver mRNA using a 32P-labeled human GCIP cDNA (LOH) in different tumors, including colorectum, probe. Expected transcripts of the human GCIP breast, lung, and bladder tumor (Natrajan et al., transgene were found only in GCIP transgenic mice 2003). Previous data demonstrate that GCIP is ex- but not in control wild-type mice (Figure 1b), whereas pressed specifically in foci of the 2-acetylaminofluorene/ transcript of the mouse GCIP were found in both partial hepatectomy (AAF/PH) model, which is used to transgenic mice lines and control wild-type mice. The monitor the differentiation of rat hepatic stem cells into expression of GCIP mRNA in transgenic mice was hepatocytes, suggesting GCIP may be involved in the approximately two to five folds higher than that of the hepatic stem cell development and differentiation endogenous murine GCIP gene. RT–PCR with primers processes (Terai et al., 2000). GCIP was also showed specific to human GCIP also showed the presence of to regulate the transcription of hepatocyte nuclear human GCIP mRNA in the liver of both Tg1 and Tg2 factor 4 (HNF4), which has E-box sequences in its GCIP transgenic mice, with higher levels of expression promoters, thus suggesting that GCIP might be involved in Tg1 GCIP transgenic animals, which is consistent in the liver growth and differentiation (Terai et al., 2000; with our results obtained from Northern blot analysis Takami et al., 2005). High-level expression of GCIP was (Figure 1b and c).
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