Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha.

Abstract

Hepatocytes are extensively used in studies of gene regulation but cannot be maintained in long-term culture as replicating, differentiated cells while remaining nontumorigenic. We have derived two hepatocyte lines from livers of transgenic mice overexpressing transforming growth factor a, a potent hepatocyte mitogen, which overcome these limitations. The transgenic hepatocytes were maintained for .2 months in serum-supplemented primary culture and gave rise to cell lines, of which two (AML12 and AML14) have been cultured for >1.5 years (>80 passages). Both lines have typical hepatocyte features such as peroxisomes and bile canalicular-like structures, do not grow in soft agar, and are nontumorigenic in nude mice. Like normal hepatocytes, AML cells express high levels of mRNA for serum (albumin, al-antitrypsin, and transferrin) and gap junction (connexins 26 and 32) proteins, secrete albumin, and contain solely isozyme 5 of lactate dehydrogenase. After extensive passaging, AML12 cells continue to strongly coexpress hepatocyte connexin mRNAs but do not display nonparenchymal cell markers. Although mRNA levels for some serum proteins progressively fall, high expression in late AML12 cultures may be regained by passage in serum-free medium. The AML14 line loses expression of both differentiated markers and transgene mRNA with extended passaging, and hepatocytic traits are only partially restored by passage in serum-free medium. These differentiated, nontumorigenic cell lines should serve as models in which to study hepatocyte growth and differentiation.Mammalian hepatocytes have long been used to investigate mechanisms of cell growth, differentiation, and tumorigenesis and as vectors for gene therapy. These studies have been hampered because dissociated hepatocytes rapidly lose their cell-specific functions, are short-lived, and have a limited ability to replicate in culture. Longer-term differentiated cultures can be maintained with specialized media and substrata or by coculture with nonparenchymal cells, but under these conditions the cells remain nonproliferative (1-3).Hepatocyte lines which express some tissue-specific markers have been propagated in vitro from hepatocellular tumors. More recently, well-differentiated lines have been derived by introducing viral oncogenes into hepatocytes. While useful, these lines have the drawback of either being fully transformed or undergoing transformation with repeated passaging (3)(4)(5)(6).Transforming growth factor a (TGFa) is a polypeptide that regulates normal growth in epithelial tissues, and its overproduction in cells possessing epidermal growth factor receptors (EGFRs) is often correlated with malignant transformation (7-10). TGFa is expressed in the liver during embryogenesisThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and transiently increases during liver regeneration after partial hepa...

Sorafenib inhibits transforming growth factor β1-Mediated Epithelial-Mesenchymal Transition and apoptosis in mouse hepatocytes

“…To test this hypothesis we used mouse AML12 cells, a normal cell line that exhibits typical hepatocyte features, such as peroxisomes and bile canaliculi-like structures. 27 To date, this cell line has been used extensively as an ideal model to assess TGF-b-induced EMT on the resultant cellular phenotypes and gene expression profiles in vitro. 8,18,24 When exposed to TGF-b1 for 48 hours, AML12 cells underwent EMT, in which cells lost their epithelial honeycomb-like morphology and obtained a spindle-like shape ( Fig.…”

Stabilized β-catenin promotes hepatocyte proliferation and inhibits TNFα-induced apoptosis

“…The cells grow indefinitely but without anchorage-independent growth potential, such as colony formation in soft agar or tumor development in immune-deficient mice. 32 Using this cell line, we could determine whether mutant b-catenin is a transforming oncogene. We first established a cell line that stably expresses mutant b-catenin.…”

“…31 It is critical to investigate the molecular pathways that are involved in the tumorigenesis, in order to design better treatment for this fatal disease. A murine hepatocyte cell line AML12, established by Wu et al from a human transforming growth factor a (TGFa) transgenic mouse, 32 has been used as a cell model to study the tumorigenic activity of hepatitis B viral pX protein. 33 In this study, the investigators demonstrated that the pX protein was capable of transforming the immortalized hepatocytes as determined by anchorage-independent cell growth assay.…”

Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line

“…We employed hepatocytes obtained from mice transgenic for human TGF-␣ (hTGF-␣) (26,46). hTGF-␣-transformed hepatocytes (designated AML12 hepatocytes) are well differentiated and can be passaged for many times without loss of differentiation (46). Evidence has been accumulating that AML12 hepatocytes represent an adequate and reproducible model for long-term studies of hepatocyte proliferation, survival, and neoplastic transformation (5,40).…”

“…We employed hepatocytes obtained from mice transgenic for human TGF-␣ (hTGF-␣) (26,46). hTGF-␣-transformed hepatocytes (designated AML12 hepatocytes) are well differentiated and can be passaged for many times without loss of differentiation (46).…”