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. 2001 Jul;49(1):119–130. doi: 10.1136/gut.49.1.119

After portal branch ligation in the rat, cellular proliferation in associated with selective induction of c-Ha-ras, p53, cyclin E, and Cdk2

P Starkel 1, L Lambotte 1, C Sempoux 1, C De Saeger 1, A Saliez 1, D Maiter 1, Y Horsmans 1
PMCID: PMC1728374  PMID: 11413120

Abstract

BACKGROUND—In liver regeneration after portal branch ligation we previously showed that early cellular changes are observed in both the proliferating and atrophying liver lobes. They are therefore not indicative of future proliferative response. In this study we attempted to define precisely, in the same model, the time at which the cellular processes diverge between the lobes by measuring various parameters associated with cellular proliferation. We also investigated the possible role of inhibitors of cell proliferation in the absence of progression towards the S phase in the atrophying lobes.
AIMS—Expression of p53, c-Ha-ras, cyclin E, cyclin dependent kinase (Cdk2), transforming growth factor (TGF)-β, and interleukin (IL)-1α and IL-1β were assessed in relation to their potential role in proliferating and atrophying cellular phenomenons.
METHODS—Immunohistochemistry, northern blotting, western blotting, and reverse transcription-polymerase chain reaction were performed, mainly at time points corresponding to mid-G1/S phase progression (8-24 hours after surgery).
RESULTS—The common and thus most likely non-specific response was still evident 5-8 hours after surgery and included an increase in IL-1 mRNA as well as p53 and cyclin E proteins. From 12 hours onwards, p53, c-Ha-ras, cyclin E, and Cdk2 were selectively induced in proliferating lobes whereas IL-1β was predominantly activated in atrophying lobes. No changes in TGF-β or IL-1α expression were observed at the same time points in any of the liver lobes.
CONCLUSIONS—The initial response to portal branch ligation and thus probably to partial hepatectomy seems to be non-specific for at least eight hours. Thereafter, p53, c-Ha-ras, cyclin E, and Cdk2 seem to drive cellular proliferation while IL-1β is associated with cellular atrophy. In contrast, TGF-β and IL-1α do not seem to play a role in determining the commitment of cells towards atrophy or proliferation.


Keywords: portal branch ligation; liver regeneration; delayed early proto-oncogenes; cytokines; cyclin dependent kinase; rat

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Figure 1  .

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Northern blot analysis of p53 and c-Ha-ras mRNA. (A) Quantification of c-Ha-ras mRNA by densitometric analysis of the blots. The adjusted values did not reveal any significant rise in c-Ha-ras mRNA in the anterior lobes. In the posterior lobes, a significant increase in c-Ha-ras level was noticed from 12 hours after portal branch ligation (PBL). **p<0.01, ***p<0.001. (B) Quantification of p53 mRNA by densitometric analysis of the blots. After adjustment for the respective 28S ribosomal signals, no elevation above baseline levels was observed in atrophying (Ant) lobes whereas p53 mRNA was significantly upregulated (**p<0.01) in regenerating (Post) lobes at 12 hours after PBL. (C) Northern blots of p53, c-Ha-ras, and 28S mRNAs extracted from control animals (lanes 1-3 and 10-12) as well as from the anterior and posterior lobes of PBL animals (lanes 4-9 and 13-18, respectively). Total liver mRNA (20 µg) was separated by electrophoresis on agarose gels, hybridised with their respective 32P labelled cDNAs, and blotted. Signals were analysed by densitometry and adjusted for their 28S signals.

Figure 2  .

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Immunohistochemistry of p53 protein expression after portal branch ligation (PBL). (A-F) Histology and p53 nuclear staining. Slides were stained immunohistochemically with a polyclonal anti-p53 antibody. No staining is observed in the anterior and posterior lobes of control animals (A, B). At five hours, nuclear staining (indicated by arrows) is slightly enhanced in both parts of the liver (E, F). At 30 hours, no further increase in p53 staining is noticed in the anterior lobes (C) whereas marked upregulation in positive staining nuclei is seen in the posterior lobes (D). (G) Quantification of p53 labelled nuclei. The labelled nuclei were counted in the anterior (Ant) and posterior (Post) lobes of control (0 hours) and PBL animals at one, five, and 30 hours. The final result was adjusted for the surface (mm2) of the section. A similar and significant increase in p53 positive nuclei was noticed in the regenerating (Post) and atrophying (Ant) lobes at five hours after PBL (**p<0.01 and ***p<0.001, respectively) whereas at 30 hours a further rise in p53 positive nuclei was only shown in the regenerating lobes. At this time point a significant difference between the lobes was observed (*p<0.05).

Figure 3  .

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Expression of cyclin E mRNA in the atrophying and regenerating lobes after portal branch ligation (PBL). (A) Quantification of cyclin E mRNA by densitometric analysis. Total liver RNA obtained from the atrophying (Ant) and regenerating (Post) lobes was subjected to reverse transcription-polymerase chain reaction (RT-PCR), as described in materials and methods. The results were adjusted for their respective RPL19 levels and then compared with a standard dilution curve obtained by amplification of a cyclin E mRNA sample in the same PCR procedure. A significant induction of cyclin E mRNA was only observed in the regenerating lobes at 24 hours after PBL (*p<0.05). (B) RT-PCR of cyclin E mRNA. Representation of RT-PCR of RPL19 and cyclin E mRNA in control animals (lanes 5 and 6) and in the atrophying (Ant) and regenerating (Post) lobes at various time points after PBL (lanes 7-12). Total liver RNA was subjected to RT-PCR as described in materials and methods. The results were adjusted for their respective RPL19 levels and compared with a standard dilution curve obtained by amplification of liver mRNA from lipopolysaccharide treated rats in the same PCR procedure (lanes 1-4).

Figure 4  .

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Western blot of cyclin E protein. (A) Cytoplasmic proteins (40 µg) were resolved using 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted with an anti-cyclin E polyclonal antibody. Samples from control animals (0 hours) and from the atrophying (Ant) and regenerating (Post) lobes of animals at various times after portal branch ligation (PBL) were analysed. The antibody recognised two bands of approximately 52 and 54 kDa in control and PBL animals. A representative blot of three control (lanes 2-4) and three PBL cytosol extracts at 12 hours (lanes 5-10) and 24 hours (lanes 11-16) after surgery is shown. KNRK nuclear extracts were used as a positive control (C+, lane 1). (B) Quantification of cyclin E protein in the cytoplasm by densitometry. Cyclin E protein was rapidly upregulated in both lobes within the first hours after PBL. A further significant increase was observed in the regenerating lobes between 8 and 24 hours after PBL (**p<0.01, ***p<0.001, respectively). In contrast, cyclin E protein expression decreased in the atrophying lobes during the same time without, however, reaching control levels. (C) Nuclear proteins (40 µg) were subjected to western blot analysis. Very low amounts of cyclin E protein were detected in the nuclear fraction of control livers (Con). A faint band of approximately 52 kDa was found in nuclear fractions of both lobes at all investigated time points which increased selectively in the regenerating lobes (Post) from 12 hours onwards (lane 3). A slower migrating approximately 54 kD band, initially weakly present in both parts, was strongly upregulated in the nuclear fractions from the regenerating (Post) lobes at 24 hours (lane 5).

Figure 5  .

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Western blot of cyclin dependent kinase (Cdk2) protein in the cytoplasm. (A) Cytoplasmic proteins (30 µg) were resolved using 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted with an anti-Cdk2 polyclonal antibody. Samples from control animals (0 hours) and from the atrophying (Ant) and regenerating (Post) lobes of animals at various times after portal branch ligation (PBL) were analysed. An approximately 34 kDa band was found in control livers (Con, lane 2) and in both lobes after PBL. Increased expression was observed in the regenerating (Post) lobes mainly at 12 and 24 hours after PBL (lanes 8 and 10, respectively). KNKR nuclear extracts were used as a positive control (Con+, lane 1). (B) Quantification of Cdk2 protein in the cytoplasm by densitometry. Cdk2 was similarly increased in both lobes during the very first hours after PBL but returned to control levels at eight hours. Thereafter, Cdk2 increased again significantly (*p<0.05;**p<0.01) only in the regenerating (Post) lobes until 24 hours after PBL.

Figure 6  .

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Western blot of cyclin dependent kinase (Cdk2) protein in the nuclear fraction. (A) Cytoplasmic proteins (30 µg) were resolved using 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted with an anti-Cdk2 polyclonal antibody. Samples from control animals (Con) and from the atrophying (Ant) and regenerating (Post) lobes of animals at various times after portal branch ligation (PBL) were analysed. Low levels of Cdk2 were found in control livers (Con, lane 1) and in the atrophying (Ant) lobes after PBL. Increased expression was observed in the regenerating (Post) lobes from 16 hours onwards, reaching a peak at 24 hours after PBL (lanes 7 and 9, respectively). (B) Quantification of Cdk2 protein in the nuclear fraction by densitometry. No significant increase in Cdk2 expression compared with control levels was seen in the atrophying (Ant) lobes after PBL. In the regenerating (Post) lobes however Cdk2 levels started to rise from 12 hours after PBL and reached a significant peak (***p<0.001) at 24 hours after PBL.

Figure 7  .

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Cyclin dependent kinase (Cdk2) associated activity in cytoplasmic and nuclear fractions. Cdk2 protein was immunoprecipitated and a histone H1 kinase assay was performed followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. (A) Low constitutive activity was detected in the cytosol of control livers (C) and in the atrophying (Ant) and regenerating (Post) lobes after portal branch ligation (PBL) (lanes 2-7) which did not significantly vary during the experiment. (B) In contrast, no Cdk2 activity was observed in the nuclear fractions of control (C, lane 1) and PBL livers (lanes 2-6) until 24 hours. At 24 hours after PBL a sharp increase in Cdk2 activity was selectively found in the regenerating lobes (lane 7).

Figure 8  .

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Quantification of transforming growth factor (TGF)-β1, -β2, and -β3 mRNA by densitometric analysis. Total liver RNA was subjected to reverse transcription-polymerase chain reaction, as described in materials and methods using TGF-β1 (A), TGF-β2 (B), and TGF-β3 (C) specific primers. The results were adjusted for their respective RPL19 levels and compared with a standard dilution curve obtained by amplification of liver mRNA from lipopolysaccharide treated rats in the same polymerase chain reaction procedure. Compared with control levels (0 hours), no significant differences in TGF-β mRNA levels were noted at any of the investigated time points after portal branch ligation (eight, 12, or 24 hours).

Figure 9  .

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Expression of interleukin (IL)-1β in atrophying and regenerating lobes after portal branch ligation (PBL). (A) Quantification of IL-1β mRNA by densitometric analysis. IL-1β mRNA was significantly induced in both lobes during the first hours (1-5) after PBL. From eight hours after PBL, IL-1β mRNA returned to control levels in the regenerating lobes (Post) whereas significant activation of IL-1 persisted in the atrophying lobes (Ant) (*p<0.05, **p<0.01). (B) Reverse transcription-polymerase chain reaction (RT-PCR) of IL-1β mRNA. Representation of a RT-PCR of RPL19 and IL-1β mRNA in control animals (lanes 5-6) and in the atrophying (Ant) and regenerating (Post) lobes at various time points after portal branch ligation (lanes 7-18). Total liver RNA was subjected to RT-PCR, as described in materials and methods. The results were adjusted for their respective RPL19 levels and compared with a standard dilution curve obtained by amplification of liver mRNA from lipopolysaccharide treated rats in the same PCR procedure (lanes 1-4). (C) Western blot of IL-1β protein in liver homogenates. At high protein load (up to 300 µg), an approximately 31 kDa band (IL-1β precursor) was observed in the atrophying lobes at eight (lanes 1, 2), 12 (lanes 5-7), and 24 hours (lanes 11, 12) after PBL. No signal was detected in the proliferating lobes at the same time points (lanes 3, 4, 8-10, 13, 14, respectively).

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