1. C-kit Inhibition by Imatinib Mesylate Attenuates Progenitor Cell Expansion and Inhibits Liver Tumor Formation in Mice 
Belinda Knight, Janina E.E. Tirnitz–Parker, John K. Olynyk 
Gastroenterology 
Volume 135, Issue 3, Pages e1 (September 2008)
DOI: /j.gastro
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2. Figure 1
Expression of IM target genes in CDE- and hepatitis B-damaged liver. RNA preparations from whole liver tissue from animals fed a control or CDE diet for 2 weeks were analyzed for the presence of c-Abl, c-kit, or PDGF-C mRNA by quantitative RT-PCR (A). Sections of archival human liver from 3 control (normal) subjects and 10 subjects with chronic hepatitis B were immunohistochemically stained to identify cells positive for c-kit (B) or c-Abl (C). Specimens from hepatitis B patient biopsies were divided into 2 groups according to their Metavir score for fibrosis grade (Metavir 0, 2 = mild; Metavir 3, 4 = severe). Cells positive for c-kit (D) or c-Abl (E) were quantified in each liver specimen by manual cell counting, and the average number of positive cells present per field in each experimental group was determined. Data represent mean ± SEM, n = 5 or n = 3 (normal liver tissue). Asterisk (*) denotes a significant difference between groups (Mann–Whitney U test, P < .05).
Gastroenterology  , e1DOI: ( /j.gastro )
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3. Figure 2
IM is antiproliferative to LPC lines via effects mediated through c-kit. Three LPC lines (BMOL, PIL-1, PIL-2) were analyzed for c-kit expression by RT-PCR (A). Phosphorylation of c-kit or Akt was examined by Western blotting in control cultures of BMOL cells or BMOL cells treated with either recombinant stem cell factor (SCF 50 ng/mL) or IM (10 μmol/L) (B). Proliferation/viability of cell lines was analyzed by MTT assay; PIL-1 or PIL-2 cells were exposed to various concentrations of IM (or vehicle), and dose response was examined (C). BMOL cells were treated with 0–100 μmol/L Akt inhibitor in the presence or absence of IM at 2 or 20 μmol/L, and viability was determined (D). Knockdown of either c-kit or Akt by siRNA transfection altered the effect of IM treatment on BMOL cells when compared with cultures transfected with a scrambled (untargeted) vector control (E). Data represent mean ± SEM, n = 4. Asterisk (*) denotes a significant difference from untreated cultures by Mann–Whitney U test (A–D) or 2-way ANOVA (E), P < .05.
Gastroenterology  , e1DOI: ( /j.gastro )
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4. Figure 3
Effect of IM administration on the liver progenitor cell response to the CDE diet. Mice fed the CDE diet for up to 21 days were administered either normal saline (placebo) or IM at 25 mg/kg/day. Liver sections were prepared and stained for the presence of liver progenitor cells using immunohistochemistry for the oval cell antigens A6 (A–C) or MPK (D). A6 staining depicts the reduced numbers of LPCs present in the livers of mice treated with IM (B) compared with placebo (A). Cell count data illustrate that, following 14 days on the diet, a significant reduction in the number of both A6 (C) and MPK (D) positive oval cells was observed in mice administered IM (Mann–Whitney U test, P < .05). A time course experiment (E) showed that IM-treated mice have a similar pattern of induction of progenitor cells to placebo-treated mice; however, the rate of expansion of the cells was reduced, leading to a significant reduction in numbers of progenitor cells present at 14 and 21 days (P < .01) and a difference of borderline significance at 7 days (P = .07). Data represent mean ± SEM, n = 6 (C and D) or n = 4 (E).
Gastroenterology  , e1DOI: ( /j.gastro )
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5. Figure 4
Effect of IM administration on liver fibrosis induced by the CDE diet. Liver sections from mice administered the CDE diet for 14 days were stained for Sirius red (A and B) or α-smooth muscle actin (C and D) to identify fibrosis and activation of hepatic stellate cells, respectively. A reduction in the presence of both markers was evident in animals that received IM (B and D) vs those that received placebo (A and C). Illustrated are representative photomicrographs for each experimental group. Scale bars represent 100 μM.
Gastroenterology  , e1DOI: ( /j.gastro )
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6. Figure 5
Intervention therapy with IM reduces the LPC response but not fibrosis induced by the CDE diet. Animals were fed a CDE diet for 16 days, during which time one third received continuous placebo treatments, one third received continuous IM treatments (25 mg/kg/day), and the remaining one third received 8 days of placebo treatments followed by 8 days of IM treatments (intervention group). At the end of the experiment, the livers were examined for the presence of LPCs by A6 staining (A) and for fibrosis by Sirius red staining and digital quantification (B). Numbers of A6-positive LPCs were significantly reduced in IM intervention animals compared with placebo, but fibrosis was not (Mann–Whitney U test; **P < .01; ***P < .001) Data represent mean ± SEM, n = 6 (C and D) or n = 4 (E).
Gastroenterology  , e1DOI: ( /j.gastro )
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7. Figure 6
Pathologies present in long-term CDE-fed animals. Animals were fed the CDE diet for up to 14 months. By 9 months, mild pericellular fibrosis was apparent in the livers of placebo-treated animals (A). This was absent in the IM-treated group (B). Quantification of Sirius red staining confirmed a highly significant reduction in the extent of fibrosis at 9 months between groups (C) (Mann–Whitney U test, ***P < .001). At 14 months, a significant difference was apparent in the body weights of the animals in treatment groups. IM-administered animals had higher body weight compared with those receiving placebo alone (D) (Student t test, P < .05). Data represent mean ± SEM, n = 12. Liver damage of various etiologies was present in all 14-month samples, regardless of drug treatment or the presence of tumors. Typical histology of nontumorous placebo- (E) and IM-treated (F) liver is presented to illustrate this. Note the presence of both micro- and macrovesicular steatosis, hepatocyte dysplasia, and loss of chord arrangement in livers of both treatment groups.
Gastroenterology  , e1DOI: ( /j.gastro )
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8. Figure 7
Analysis of 14-month CDE-induced liver tumors. Liver lesions were identified at the time of sacrifice as white, nodular, subcapsular lesions of varying sizes (A). Serial sections were used to examine the phenotype of a small lesion (B–E). Histologically, lesions had a circular appearance in cross section and were composed of highly packed hepatocyte-like cells with extensive steatosis and dysplasia (B). Lesions were encapsulated with collagen as evidenced by Sirius red staining (C) and contained a high proportion of α-fetoprotein-positive cells (D). The proportion of proliferating cells in these lesions was very high, as evidenced by Ki67 staining (E).
Gastroenterology  , e1DOI: ( /j.gastro )
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