MYC Overexpression Does Not Contribute to Oval Cell Expansion.

<p>A6 Immunohistochemistry of: (A) a normal adult liver, (B) an adult liver in which MYC has been activated for 30 days, (C) an adult liver treated with DDC for 7 days, (D) an adult liver that overexpressed MYC in conjunction with DDC treatment for 7 days, (E) an adult liver treated with DDC for 17 days, (F) an adult liver that overexpressed MYC in conjunction with DDC treatment for 17 days, (G) an adult liver treated with DDC for 28 days, and (H) an adult liver with adjacent tumor foci that stemmed from MYC and DDC treatment for 28 days. A6 positive cells are evident in the periportal areas but not in the tumors. Representative data is shown.</p

DDC and CCl₄ Cooperate with MYC to Induce Accelerated Liver Tumorigenesis.

<p>Shown are Kaplan-Meier survival curves for 6–8 w adult mice that overexpressed MYC and were simultaneously treated with: nothing (filled black square), DDC (filled blue circle), or CCl₄ (filled blue diamond). Adult mice treated with hepatotoxins in the absence of MYC overexpression (MYC OFF) are displayed with the corresponding open shape: nothing (empty black square), with DDC (empty blue circle), or CCl₄ (empty blue diamond). Also shown are survival curves of neonatal mice in which MYC was either overexpressed (filled red triangle), or remained inactive (empty red triangle) starting at birth. Cohorts consisted of 5–10 mice. MYC transgene expression was activated by removal of doxycycline from the mouse drinking water on the day of hepatotoxin treatment initiation. Mice were sacrificed when moribund with tumor burden.</p

Hepatotoxins Induce Liver Toxicity and Cooperate with MYC to Induce Diffuse HCC's Arising from Periportal Areas.

<p>(A) Histology of a normal adult liver. (B) Histology of a MYC-induced microcarcinoma that developed in a periportal area of the liver. Inner panel is a magnified view of the microcarcinoma. (C) Histology of a MYC-induced, multifocal adult liver tumor. (D) Histology of an adult liver 3 w after continuous DDC treatment. The liver lobule exhibits oval cell expansion radiating from the periportal area, typical of DDC-induced injury. (E) Histology of a MYC and DDC-induced microcarcinoma that stems from the periportal area. Inner panel is a magnified view of the microcarcinoma. (F) Histology of a MYC and DDC-induced diffuse liver tumor. (G) Histology of an adult liver after 4 weeks of continuous CCl₄ treatment. Microvesicular lipidosis can be seen in the centrilobular area of the liver lobule. (H) Histology of a MYC and CCl4-induced microcarcinoma that stems from the periportal area. Inner panel is a magnified view of the microcarcinoma. (I) Histology of a MYC and CCl₄-induced diffuse liver tumor. Representative data is shown.</p

Examining the Impact of DDC and CCl₄ Damage on MYC-Induced HCC.

<p>(A) Schematic of the liver lobule illustrating that DDC causes liver damage associated with the emergence and proliferation of oval cells in the periportal area of the hepatic lobule, while CCl₄ causes damage associated with the destruction of hepatocytes near the central vein and triggers mature hepatocytes to proliferate. (B) Schematic of experimental design. Eight different cohorts of mice were monitored for tumorigenesis. MYC was activated (MYC ON) or kept inactive (MYC OFF) in mice: at birth, in 6–10 week old adult, in 6–10 w old adult mice treated with DDC, or in 6–10 w old adult mice treated with CCl₄. Mice were sacrificed when moribund with tumor burden.</p

Hepatotoxins Facilitate MYC-Induced Proliferation Irrespective of p53 Status.

<p>(A) Immunofluorescence for Ki67 expression as a marker of liver cell proliferation is presented. (B) Bar graphs illustrating a ratio of Ki67 positive hepatocytes to total hepatocytes as a relative measure of hepatocyte proliferation are shown. (C) Bar graphs illustrate a ratio of phospho-Histone-H3 positive hepatocytes to total hepatocytes as a relative measure of hepatocyte mitotic division. Gray bar graphs represent normal adult livers in which MYC was either overexpressed (MYC ON/Adult), or kept inactive (MYC OFF/Adult). Red bar graphs represent DDC-treated livers in which MYC was either overexpressed (MYC ON/DDC), or kept inactive (MYC OFF/DDC). Blue bar graphs represent CCl₄-treated livers in which MYC was either overexpressed (MYC ON/ CCl₄), or kept inactive (MYC OFF/ CCl₄). T represents a liver tumor. Results are based on an average of 4 samples per time-point. Statistical significance was measured using a Mann Whitney test: graph in (A) (* P<0.05, ** P<0.05), and graph in (B) (* P<0.05). (D) Western blot analysis for p53 protein expression after different durations of MYC overexpression in: Normal, DDC-treated, and CCl₄-treated livers. As a positive control, a lymphoma cell line that overexpressed a mutant p53 was used. Normal FVB/N adult liver was used as a negative control.</p

MYC-induced Proliferation and Tumorigenesis in Hepatotoxin-Treated Livers is Associated with Increased Cyclin B1 Expression.

<p>Real-time quantitative PCR of: (A) human c-MYC mRNA, (B) mouse Ornithine Decarboxylase (ODC) mRNA, (C) mouse Nucleolin mRNA, and (D) mouse Cyclin B1 mRNA. Statistical significance was measured using a Mann Whitney test (* P<0.05, ** P<0.05, *** P<0.05). Gray bar graphs represent normal adult livers in which MYC was overexpressed for the times indicated. White bar graphs represent normal neonatal livers in which MYC was overexpressed. Red bar graphs represent DDC-treated livers in which MYC was either overexpressed (MYC ON/DDC) or kept inactive (MYC OFF/DDC). Blue bar graphs represent CCl₄-treated livers in which MYC was either overexpressed (MYC ON/ CCl₄) or kept inactive (MYC OFF/ CCl₄). mRNA was quantitated from four different liver samples per time-point.</p