This grant has been continuously funded for more than nineteen years to study REGULATION OF DIFFERENTIATION IN HEPATOCYTES IN VITRO. The applicant has established two in vitro culture systems. (1) A long term culture system for primary hepatocytes in which the cells are maintained in a chemically-defined medium supplemented with dimethylsulfoxide (DMSO), and (2) A series of immortalized hepatocyte cell lines, transformed cell lines and tumor cell lines that retain the ability to express liver-specific functions, some at liver-like levels. During the current funding period, she has utilized both systems to make several important findings. She has demonstrated that hepatocytes in long-term DMSO culture: (1) Maintain the potential not only to undergo DNA synthesis but also to proliferate, (2) Can differentiate into bile duct-like cells, and (3) Can express specific oncogenes and growth factors in a similar pattern to what is observed in regenerating liver, when induced to synthesize DNA. Using immortalized hepatocyte cell lines and transformed and tumor cell lines derived from them, the applicant has made several contributions with regard to the effect of transformation on expression of differentiated functions, regulation of albumin and alpha 1 integrin expression, factors that suppress the transformed phenotype in transformed hepatocytes and differential responses of premalignant and malignant cells to TGF-beta. She has also made several important technical advances in gene delivery that will enable her to carry out both gain and loss of function studies in both culture systems. In the current application, the applicant proposes to pursue specific findings made during the past funding period and focus her studies on regulation of alpha 1 integrin expression in hepatocytes, the relationship between alpha 1 integrin expression and growth control in hepatocytes and TGF-beta 1 induced suppression of the transformed phenotype. She hypothesizes that transformation in hepatocytes is accompanied by loss of alpha 1 integrin expression which may be mediated by increased expression of ras, and that suppression of the transformed phenotype in hepatocytes induced by TGF-beta may be mediated, at least in part, by restoration of alpha 1 integrin expression.
The specific aims are: (1) To examine regulation of expression of alpha 1 integrin in hepatocytes and hepatocyte cell lines; (2) To determine whether alpha 1 integrin expression plays an active role in growth control; (3) To elucidate key players in the signal transduction pathway used by TGF-beta to suppress the transformed phenotype; (4) To determine whether the type II/type I TGF-beta receptor ratio is related to the differential responses to TFG-beta observed in hepatocytes and hepatocyte cell lines.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA023931-22
Application #
2894498
Study Section
Special Emphasis Panel (ZRG2-SSS-1 (04))
Program Officer
Mohla, Suresh
Project Start
1978-09-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
22
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Starkey, Jason L; Chiari, Estelle F; Isom, Harriet C (2009) Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro. J Gen Virol 90:115-26
Heipertz Jr, Richard A; Starkey, Jason L; Miller, Thomas G et al. (2009) trans-Complementation of HBV rtM204I mutant replication by HBV wild-type polymerase. Virology 388:57-67
Shan, Weiwei; Palkar, Prajakta S; Murray, Iain A et al. (2008) Ligand activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) attenuates carbon tetrachloride hepatotoxicity by downregulating proinflammatory gene expression. Toxicol Sci 105:418-28
Heipertz Jr, Richard A; Miller, Thomas G; Kelley, Colleen M et al. (2007) In vitro study of the effects of precore and lamivudine-resistant mutations on hepatitis B virus replication. J Virol 81:3068-76
Bilello, John P; Cable, Edward E; Isom, Harriet C (2003) Expression of E-cadherin and other paracellular junction genes is decreased in iron-loaded hepatocytes. Am J Pathol 162:1323-38
Bilello, J P; Cable, E E; Myers, R L et al. (2003) Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes. Gene Ther 10:733-49
Abdelhamed, Ayman M; Kelley, Colleen M; Miller, Thomas G et al. (2003) Comparison of anti-hepatitis B virus activities of lamivudine and clevudine by a quantitative assay. Antimicrob Agents Chemother 47:324-36
Iocca, Heather A; Isom, Harriet C (2003) Tumor necrosis factor-alpha acts as a complete mitogen for primary rat hepatocytes. Am J Pathol 163:465-76
Stoehr, Stephanie A; Isom, Harriet C (2003) Gap junction-mediated intercellular communication in a long-term primary mouse hepatocyte culture system. Hepatology 38:1125-35
Malecki, Elise A; Cable, Edward E; Isom, Harriet C et al. (2002) The lipophilic iron compound TMH-ferrocene [(3,5,5-trimethylhexanoyl)ferrocene] increases iron concentrations, neuronal L-ferritin, and heme oxygenase in brains of BALB/c mice. Biol Trace Elem Res 86:73-84

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