This experimental approach provides quantitative and mechanistic data regarding the role of specific oncogenes

during hepatocarcinogenesis. BrdU, bromodeoxuridine; CHeGA, comparative hepatocyte growth assay; EO, extreme outlier; hPAP, human placental alkaline phosphatase; lacZ, Selleckchem Opaganib β-galactosidase; TAg, simian virus 40 T antigen; TGFα, transforming growth factor alpha; uPA, urokinase-type plasminogen activator. Mice were housed and maintained according to The Guide for the Care and Use of Laboratory Animals in Association for Assessment and Accreditation of Laboratory Animal Care–accredited facilities. All experimental procedures were approved by the Institutional Animal Care and Use Committee. The transgenic lines used in selleck these studies have been assigned the following genetic designations: major urinary protein uPA line 350-2, TgN(MupPlau)1Eps; hsMT-nLacZ line 379-4, TgN (Mt1nLacZ)4Eps; R26-hPAP line 808-6, TgN(R26ALPP)5Eps; AL-TAg line 522-8, TgN(Alb1SV)46Bri; MT-TGFα line 1745-8, TgN(Mt1Tgfa)149Bri;

AL-c-myc line 741-3, TgN(Alb1Myc)82Bri.3, 5, 14 For these studies, mice were of the FVB, C57BL/6, or (FVB6)F1 strain background. One group of recipient mice were athymic Swiss nu/nu. Transplant recipient mice carrying metallothionein (MT)-TGFα donor hepatocytes were administered 25 mM zinc sulfate in drinking water starting at the time of transplant to induce transgene expression.5 β-Galactosidase (LacZ)-marked or human placental alkaline phosphatase (hPAP)-marked donor hepatocytes were isolated from 2-week-old to 5-week-old donor mice using a modified two-step ethylenediaminetetra-acetic acid/collagenase A protocol.14 In all cases, mice were excluded as donors if they displayed focal lesions visible on gross examination. Transgenic mouse livers lacking see more these alterations contain few or no areas of parenchyma that would be microscopically

diagnosed as neoplastic, although dysplastic cells may be present.3, 5, 6, 12 The concentration of viable large cells (hepatocytes) was determined by trypan blue exclusion using a hemacytometer. Cells were maintained at 4°C until transplanted. Hepatocytes were transplanted surgically in 10 μl of L15 medium (Life Technologies, Rockville, MD) via intrasplenic injection into histocompatible recipients within 6 hours of isolation.14 Recipient mice were administered 0.1 mg/kg cadmium sulfate intraperitoneally to induce expression of the MT-nLacZ transgene, then 16 to 24 hours later liver was collected and a portion was fixed in 4% paraformaldehyde at 4°C for 1 hour then transferred to 70% ethanol. β-Galactosidase- and hPAP-expressing hepatocyte foci were identified as described,14 by incubating separate pieces of liver with an appropriate enzyme-selective substrate. Transgene-expressing cells displayed a blue reaction product.