The heat map from 1 mouse liver per treatment group (group1 = 43; group 2 = 11; group 3 = 90; group 4 = 105) is shown in . The gene expression changed in a large number of genes when DDC was fed for 10 weeks and again when DDC was refed after a 4 week interval where DDC was withheld from the diet. Gene expression returned to control levels after one month withdrawal of the drug DDC. DDC refed for 7 days after withdrawal caused a return of gene expression seen after the initial 10 weeks of DDC feeding. When mouse 90 (DDC primed mouse) was compared to mouse 43 (control mouse), the expression of 2300 genes was changed using a + or – 1.6 cut off. When mouse 11 (DDC fed 10 weeks) was compared with mouse 43, the expression of over 5300 genes was changed. When mouse 105 (DDC refed) was compared with mouse 90, the expression of over 4600 genes was changed. To further analyze the data, the number of genes analyzed was reduced by changing the cut off fold change.
Fig. 1 Heat map of 3147 genes clustered from the comparison between 00–43 (Control), 00–11 (DDC fed mouse), 00–90 (DDC fed and withdrawn mouse), and 00–105 (DDC fed mouse, withdrawn and refed DDC mouse). Mouse MB Model In vivo (more ...)
When the changes were analyzed by Venn to determine the shared gene expressions when the three mice treated with DDC were compared with the control or the mouse withdrawn from DDC for 5 weeks, 649 out of 9319 changes in gene expression were identified (). The 649 changed genes identified, constituted the gene expression memory present in the DDC withdrawn mice livers.
Fig. 2 Venn diagram of gene expression, when the control was compared with the DDC treated mice or the mouse fed DDC 10 wk and then DDC withdrawn for 5 weeks. The change in gene expression in the same direction shared by the DDC treated mice numbers 625 with (more ...)
These changes in gene expression may explain how hepatocytes are able to spontaneously form MBs 7 days after refeeding DDC compared to the mice that were initially fed DDC for 10 weeks. The 649 genes common to all three comparisons in the Venn diagram () were grouped according to functional pathways ().
Functional pathways where the expression of 3343 genes changed when DDC treated mice were compared with the control or the DDC withdrawn mouse (n=1)..
The genes most changed were those involved in the regulation of the actin cytoskeleton. MAPK signaling and focal adhesion function genes were the next most commonly affected. In all cases, more genes where up regulated than down regulated. The % total of gene expression changes is illustrated in .
The % of the gene expression changes by functional pathways is shown when the DDC treated mice were compared to the control or the DDC withdrawn mice (625 genes) (n=1).
When all the changes in gene expression were analyzed, a few changes stood out as potentially being involved in the memory of MB formation because the changes are in the same direction. For instance the gene for ubiquitin D was up regulated ().
Ubiquitin D is involved in protein degradation by the 26S proteasome. Alpha fetoprotein (AFP) was up regulated, especially in the DDC withdrawn mouse (the mouse which has the memory of how to form MBs when DDC was refed). Connective tissue growth factor (CTGF), and growth arrest and DNA-damage—inducible 45 gamma (Gadd45g) were especially up regulated in the memory mouse (). Integrin beta 2 (Itgb2) was up regulated (). Integrin is involved in MB formation in vitro
(Wu et al, 2005
). Glutathione S-transferase (GSTm4) expression was up regulated when DDC was fed but was down regulated by DDC withdrawal. GST isoforms are involved in drug detoxification and are induced by DDC feeding (Roomi et al, 2006
). Platelet derived growth factor, alpha (Pdgfα) was marginally up regulated (). Adrenomedulin (Adm) was up regulated (). Adm is up regulated by HIF1α (Li et al, 2004
). However, HIF1α was down regulated in the nuclear extract when DDC was fed or refed ().
Gene expression of growth factors changed when in DDC withdrawn mice (Memory cells that form MB's) compared to the control mouse liver. Note the increased expression of Gadd45g and Ctgf (n=1).
HIF1 alpha expression in nuclear extracts from the livers of mice fed DDC (Mean +/− SEM, n=3).
When the epigenetic memory of the mouse withdrawn from DDC for a month was compared with the control mouse, Dnmt3b was markedly down regulated suggesting that gene methylation was reduced (). The levels of methyl cytosine and dimethylated histone 3 lysine 9 were not changed by the DDC treatment ().
DDC Withdrawal Vs Control (90 vs. 43)
The protein levels of Dnmt3b and dimethyl histone 3 lysine 9 were unchanged in the liver homogenates by DDC feeding and refeeding (Mean +/− SEM, n=3).
The reduction in methylation by Dnmt3b () could be a mechanism for the up regulation of gene expression observed. The expression of the acetyl transferase p300 was decreased by DDC withdrawal (see ). When the DDC refed mouse was compared with the DDC withdrawn mouse, down regulation of the deacetylases Sirt1 and 3 was observed ().
Gene expression changes when DDC was refed
When measured by Western blot, HDAC levels were also reduced by DDC feeding but not in the DDC refed mice (), which does not support the significance of HDAC action in MB induction. Deacetylation of histones, therefore, may have been reduced during DDC feeding which could further enhance epigenetic up regulation of gene expression without involvement of signal transduction. To support this possibility, acetylation of H3 lysine 9 was measured by Western blot. Acetylation of histone 3 lysine 9 was increased when DDC was fed (). When the HDAC level is reduced, the level of H3K9 acetylation would logically increase. However when DDC was refed, the level of HDAC did not change, as shown by Western blot, suggesting mechanisms other than HDAC activity are regulating the level of H3K9 acetylation.
The protein level of histone deacetylase (HDAC) is decreased in the liver homogenates of DDC fed mice (Mean +/− SEM, n=3).
The protein levels of acetylated histone 3 lysine 9 are increased in the liver homogenates of DDC fed and refed mice (Mean +/− SEM, n=3).
There were no significant changes in the level of Dnmt3b or dimethyl H3 lysine 9 (H3K9) measured by western blot (). This data has to be confirmed by measuring the nuclear fraction as well.
The protein levels of Gadd45g were decreased by DDC feeding and refeeding, supporting a negative role for this important protein in the formation of MBs (). The protein levels of DDC W vs. control did not correlated with the gene expression levels (). The increase in Gadd45g and CTGF expression seen by microarray analysis was confirmed using RT PCR ( ).
The protein levels of Gadd45g in the liver homogenates were decreased by DDC feeding and DDC refeeding (Mean +/− SEM, n=3).
Fig. 11 MB formation in vivo was associated with an increased expression of Gadd45g (SEM +/−, n=3; C vs DDC p=0.004; C vs DDC W p= 0.016). MB formation was also associated with an increased expression of CTGF (C vs DDC W p=0.03; C vs DDC R p= 0.014). (more ...)