Volume 1, Issue 4, Pages 358-365 (April 2000) Hepatocyte Transplantation Improves Survival in Mice with Liver Toxicity Induced by Hepatic Overexpression of Mad1 Transcription Factor  S. Gagandeep, Rana Sokhi, Sanjeev Slehria, Giridhar R. Gorla, Joseph Furgiuele, Ronald A. DePinho, Sanjeev Gupta  Molecular Therapy  Volume 1, Issue 4, Pages 358-365 (April 2000) DOI: 10.1006/mthe.2000.0051 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 1 Effect of AdMad on cultured primary rat hepatocytes. (A) DNA synthesis measurements with a 1-h pulse of [3H]thymidine showing that hHGF stimulated cell proliferation. However, cells lost their proliferative ability following AdMad treatment. (B) Flow cytometry showing cell cycle states. Panels show untreated control cells and cells treated with Adβgal or AdMad at doses of 1 and 10 m.o.i. The cell number is plotted on the Y axis and DNA content on the × axis. 2C, 4C, and 8C refer to diploid, tetraploid, and octaploid (+greater) DNA content, respectively. As shown here, the normal rat or mouse liver typically contains more tetraploid than diploid cells. Although the relative proportions of diploid and tetraploid cells were unchanged following treatments (see table), G0/G1 peaks became broadened following exposure to 10 m.o.i. AdMad. Also, the octaploid hepatocyte fraction decreased (see table). Cells were cultured for 48 h and all analyses were in triplicate at least. Molecular Therapy 2000 1, 358-365DOI: (10.1006/mthe.2000.0051) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 2 Evidence for liver injury following AdMad treatment. (A) X-gal staining showing lacZ expression 14 days after treatment of a mouse with Adβgal. (B) Tissue from an animal not treated with Adβgal showing absence of lacZ activity. (C) Tissue from an animal that died 10 days after AdMad treatment showing extensive cell dropout (thick straight arrow), fatty change (curved arrow), and small pyknotic nuclei, compared with the size of normal hepatocyte nuclei (open arrow at left bottom). (D) TUNEL-positive nuclei (arrow) in the same tissue as in (C) showing apoptosis. (E) Tissue showing no TUNEL positivity when Tdt was omitted from the reaction. (F) Mouse liver 6 weeks after AdMad treatment showing megalonuclei (arrows). A, B, C, and F: hematoxylin and eosin counterstaining. Molecular Therapy 2000 1, 358-365DOI: (10.1006/mthe.2000.0051) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 3 Liver repopulation with transplanted hepatocytes. (A) Normal distribution of hepatic DPPIV activity (red) in a mouse treated 2 weeks previously with Adβgal. (B) Liver from a mouse 6 weeks after Adβgal administration and cell transplantation showing occasional transplanted cells with DPPIV–, ATPase+ bile canaliculi (brown, arrows) adjacent to a portal area (p). Host cells contain DPPIV+ bile canaliculi (red). (C and D) Low and high power views showing areas devoid of DPPIV activity (marked by Xs). These DPPIV– areas were adjacent to portal areas (p) and contained transplanted cells. (E) Dual staining for DPPIV and ATPase activities showing the former in host hepatocytes and the latter in transplanted cells (arrows). Networks of hybrid ATPase+ and DPPIV+ bile canaliculi among adjacent transplanted and host hepatocytes indicate integration of cells in the liver parenchyma. (F) Actuarial analysis of survival in animals treated with AdMad alone (yellow line) or with AdMad followed by cell therapy (red line). The overall survival of animals treated with cells improved significantly. Molecular Therapy 2000 1, 358-365DOI: (10.1006/mthe.2000.0051) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 4 Western blot analysis showing AdMad expression. The samples in various lanes are as follows: lane 1, untreated primary rat hepatocytes after culture for 24 h; lane 2, primary rat hepatocytes after treatment with 20 m.o.i. AdMad for 24 h; lanes 3 and 4, liver from untreated SCID mice; lanes 5 and 6, liver from mice after treatment with AdMad for 5 days. The arrow indicates the expected position of Mad1 protein in the blot. Molecular Therapy 2000 1, 358-365DOI: (10.1006/mthe.2000.0051) Copyright © 2000 American Society for Gene Therapy Terms and Conditions