Inhibition of rat liver regeneration after partial hepatectomy and induction of ERK phosphorylation by Cpd 5, a K vitamin-based anticancer compound

Siddhartha Kar, Meifang Wang, Kathryn S. Rosi1, Craig S. Wilcox1 and Brian I. Carr2

Department of Surgery, Liver Cancer Center, Starzl Transplantation Institute and 1 Department of Chemistry, University of Pittsburgh, E1552 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15260, USA

2 To whom correspondence should be addressed. Tel: +1 412 624 6684; Fax: +1 412 624 6666; Email: carrbi{at}upmc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thioalkyl K vitamin derivatives, like 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone (Cpd 5), have been shown to inhibit both hepatoma cell growth and DNA synthesis in rat hepatocytes in vitro. We have here examined the tissue distribution, in vivo tolerance and growth inhibitory effects of a single injected dose of Cpd 5 in rats. Cpd 5 administered i.p. was sufficient to cause a 90% inhibition of the peak in DNA synthesis in rat liver 24 h after two-thirds partial hepatectomy (PH). However, DNA synthesis in post-PH, Cpd 5-treated rat livers did occur, but with a delay of 36 h. Dual phosphorylation of ERK2 was induced in rat liver dose-dependently as early as 0.5 h, but gradually returned to almost basal levels by 6 h after Cpd 5 treatment. The MEK1/2 inhibitor PD098059, administered in vivo 1 h prior to Cpd 5 treatment, antagonized both induction of ERK2 phosphorylation and inhibition of DNA synthesis in rat liver. Liver protein lysates post-PH exhibited protein phosphatase activity for phospho-ERK2, which was inhibited by Cpd 5. These results show that induction of ERK2 phosphorylation is likely involved in the mechanism by which Cpd 5 inhibits PH-induced DNA synthesis, probably as a result of its ability to inhibit the activity of ERK phosphatase(s).

Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; BrdU, bromodeoxyuridine; Cpd 5, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone; EGF, epidermal growth factor; ERK, extracellularly regulated kinase; H&E, hematoxylin and eosin; LD50, 50% lethality dose; LDH, lactate dehydrogenase; OMFP, 3-O-methylfluorescein phosphate; PH, partial hepatectomy; PTP, protein tyrosine phosphatase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Liver regeneration in rodents has provided a powerful model for studying the regulation of cell proliferation in vivo (1). A two-thirds partial hepatectomy (PH) of mice or rats induces the remainder of the liver to undergo a synchronous first wave of DNA synthesis, followed by several rounds of mitosis (24). After PH, the liver remnant remains mitotically quiescent for 12–16 h before it undergoes a rapid and synchronous first wave of DNA synthesis (5). This first wave of DNA synthesis occurs between 20 and 24 h after PH (4). Mitosis begins 6 h later and peaks at 30 h. The original DNA content of the regenerating liver remnant is normally restored within 96 h after PH (6). The capacity of the liver to regain its original weight within 2 weeks indicates that both the onset of cell proliferation and its inhibition are tightly regulated.

We previously synthesized a series of K vitamin analogs which are potent inhibitors of cell growth (7,8). The prototype of these, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone (Cpd 5), has been studied extensively in hepatoma cells (810). Cpd 5 has been shown to inhibit the activity of protein tyrosine phosphatases in vitro and in cells in culture, particularly the dual specificity phosphatase Cdc25A (9,11). This resulted in tyrosine phosphorylation of its cellular substrate Cdk4 and consequent loss of its activity in phosphorylating its substrate, Rb (12,13). It also strongly induces ERK1/2 phosphorylation, which is likely involved in its mechanisms of growth inhibition (12,14). Cpd 5 was found to be a potent growth inhibitor of transplantable rat hepatoma and it also inhibited the formation of pre-tumor enzyme-altered foci in rat livers in response to the chemical carcinogen diethylnitrosamine (15).

We have also recently found that Cpd 5 inhibits the induction of the first wave of DNA synthesis that occurs in vivo after two-thirds PH in rat (13). Cpd 5 could be injected at several time points up to 6 h after PH to induce this inhibition. However, when injected at later times its growth inhibitory activity was lost. In the experiments reported here we extend our previous studies on the in vivo action of Cpd 5 on rat liver regeneration after PH to examine the likely mechanism(s) of Cpd 5 action in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Two-month-old male Fischer F344 rats was obtained from Hilltop Laboratories (Scottdale, PA). Cpd 5 was synthesized as previously described (8).

14C-labeled Cpd 5
Radiolabeled compound was synthesized following the procedure of Williams et al. (16). A solution of 2-methyl[14C]-1,4-naphthoquinone and imidazole in tetrahydrofuran was treated with 2-mercaptoethanol (3 equiv.) at room temperature. The resulting solution was stirred for 2 h, after which time it was left open to the air for 2–3 h. The resulting solution was taken into ether, acidified and extracted with water. After evaporating the ether, the product was purified on silica gel using 1/1 ethyl acetate/hexane as eluent.

Partial hepatectomy
Higgins and Anderson two-thirds PH was performed (3). Hepatic DNA synthesis and nuclear labeling with bromodeoxyuridine (BrdU) were done as previously described (5,6).

Western blots and immunoprecipitation
Western blots and immunoprecipitation were done following our standard protocol (16).

Protein tyrosine phosphatase (PTP) activity assay
PTP activity of liver protein lysate was measured by using the substrate 3-O-methylfluorescein phosphate (OMFP) as previously described (13).

Phospho-ERK2 dephosphorylation assay
Phospho-ERK1/2 was obtained from Cell Signaling Technologies (Beverly, MA). Liver lysate was immunoprecipitated with anti-ERK2 antibodies and cleared of endogenous ERK2 proteins by centrifugation. Phospho-ERK2 was incubated with ERK2-cleared cell lysate in phosphatase buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol) for 30 min at 37°C in the presence or absence of Cpd 5. The phosphatase reaction was terminated by the addition of an equal volume of 2x sample buffer (1.52% Tris, 2% SDS, 2% 2-mercaptoethanol, 20% glycerol, adjusted to pH 6.8). The proteins were separated by 10% SDS–PAGE and transferred to a western blot that was probed with phospho-ERK1/2 and ERK2 antibodies.

Cpd 5 administration to rats
Cpd 5 was dissolved in DMSO at a stock concentration of 40 mg/ml. Dilutions were made in DMSO before i.p. injection. An equal volume of injected DMSO served as a control.

PD098059 treatment in vivo
An aliquot of 5 ml of a solution containing PD098059 (75 µM in 0.37% DMSO) or 0.37% DMSO alone (18) was injected into rats via a thigh vein 1 h before PH and Cpd 5 injection.

In vivo toxicity
Cpd 5 was injected i.p. at various doses in rats with or without PH and survival was determined after 1 day. Serum levels of the enzyme markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and total bilirubin in PH rats were determined with or without i.p. administration of 30 mg/kg Cpd 5. Normal rats were also injected with 110 mg/kg Cpd 5 and the same serum markers were assayed for toxicity. Tissue sections collected from rat heart, lungs, kidneys, spleen, pancreas, intestine, stomach, liver, colon and brain 1 day after Cpd 5 treatment or from untreated rats were stained with hematoxylin and eosin (H&E).

Statistical analysis
Statistical analysis of significance was determined by the t-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cpd 5 pharmacokinetics and toxicity
14C-labeled Cpd 5 was injected i.p. into rats and its distribution in several organs and blood was determined by measuring the radioactivity in selected organs. High levels of Cpd 5 were seen in liver, lung, heart, intestine and colon initially at 6 h after Cpd 5 administration. However, the amounts of Cpd 5 in these organs declined after 24 h, except in the colon (Figure 1A). Blood levels of Cpd 5 rose sharply 0.5 h after administration and remained high at 24 h (Figure 1B).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Cpd 5-mediated toxicity and distribution in rats. (A) 14C-labeled Cpd 5 was injected i.p. and radioactivity measured in heart, lung, liver, intestine and colon at 6 and 24 h after injection. (B) 14C-labeled Cpd 5 levels were measured in rat blood at various times up to 24 h after i.p. administration. (C) Cpd 5-mediated toxicity was determined by measuring rat survival 24 h after Cpd 5 injection with or without PH. (D) Levels of ALT, AST, LDH and total bilirubin were measured in rat blood 24 h after i.p. injection of Cpd 5 in normal or PH rats and the values were compared with untreated controls. Three rats were used for each measurement in (A), (B) and (D) and 6 rats were used in (C).

 
The oxicity of Cpd 5 was determined by measuring the survival of rats after Cpd 5 treatment with or without partial hepatectomy. The dose for 50% lethality (LD50) was 110 mg/kg for normal rats and 65 mg/kg for the PH rats (Figure 1C). A 4-fold increase in serum LDH was found in normal rats treated with 110 mg/kg Cpd 5. However, there was no toxicity at 30 mg/kg Cpd 5 in either normal or PH rats. Serum levels of the enzymes ALT, AST and LDH and total bilirubin were measured 24 h after Cpd 5 (30 mg/kg) administration in PH rats and was compared with untreated PH rats. No differences in these serum markers between the PH and untreated rats were observed after 30 mg/kg Cpd 5 (Figure 1D). The same serum markers were also assayed after treating normal rats at 110 mg/kg Cpd 5. Tissue sections from rat heart, lungs, kidneys, spleen, pancreas, intestine, stomach, liver, colon and brain were also examined by a pathologist after H&E staining. No differences were observed either in the serum markers or stained sections between the untreated and Cpd 5-treated rats (Figure 1D).

Cpd 5 inhibits DNA synthesis in rat livers after partial hepatectomy (PH)
Male Fischer F344 rats were subjected to two-thirds PH and Cpd 5 was injected i.p. at a dose of 30 mg/kg just after the PH procedure. DNA synthesis was assayed after injecting 50 µCi [3H]thymidine at 22 h and harvesting the livers at 24 h after PH. DNA was extracted from the rat livers and [3H]thymidine incorporation were determined with a scintillation counter. Total DNA concentration was determined by measuring its absorbance at 260 nm. The peak of DNA synthesis at 24 h after PH was found to be inhibited by 90% (P < 0.001) in the Cpd 5-treated rat livers (Figure 2A). This inhibition has been previously shown to be dependent on the dose of Cpd 5 and the time of its administration. Cpd 5 inhibited DNA synthesis when injected up to 6 h after PH, but not at times beyond (13). Liver DNA synthesis after PH was also assayed by a second method. Instead of [3H]thymidine, BrdU was injected i.p. at 22 h after PH and the rats were killed at 24 h. Liver sections were stained histologically with anti-BrdU antibody and the percentage of stained cells was counted under a microscope. The number of stained cells reflects those undergoing DNA synthesis. Cpd 5 treatment reduced the number of BrdU-positive cells by ~80% (P < 0.001) (Figure 2B).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Cpd 5 mediates the inhibition of rat liver DNA synthesis after PH. (A) Rat liver DNA synthesis was measured 24 h after PH in Cpd 5 (30 mg/kg)-treated or untreated rats by incorporation of [3H]thymidine at 22–24 h. Liver DNA was extracted and [3H]thymidine incorporated was measured by scintillation counter. (B) Rat liver DNA synthesis was also measured by incorporation of BrdU in newly synthesized DNA 22–24 h after PH in rats treated or untreated with Cpd 5 (30 mg/kg).

 
Cpd 5 delays the peak of DNA synthesis in regenerating liver
Since a single dose of Cpd 5 was found to cause such a dramatic reduction in the peak DNA synthesis after PH, we next examined whether this reduction was transient or not. DNA synthesis after PH was determined every 12 h, using a 2 h pulse-label with BrdU, in both untreated and Cpd 5 treated rat livers. Cpd 5 treatment was found to delay the peak in DNA synthesis by 36 h. Cpd 5-treated livers of PH rats showed a peak at 60 h, instead of the observed peak at 24 h after PH in untreated animals. The peak height was, however, similar to that of the Cpd 5-untreated PH animals. The percentage of BrdU stained cells was indistinguishable between the treated and untreated livers at 96 h after PH (Figure 3).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Cpd 5 mediates a delay in the peak of DNA synthesis during rat liver regeneration. Rat liver regeneration after PH was measured by assaying BrdU incorporation into hepatocytes at various times after PH. BrdU was injected at 12 h intervals up to 96 h after PH and incorporation into newly synthesized DNA was allowed for 2 h. The number of BrdU stained hepatocytes at each time point were counted under a light microscope.

 
Cpd 5 induces ERK2 phosphorylation in regenerating liver
We previously observed a robust induction of ERK1/2 phosphorylation due to the action of Cpd 5, both in various tumor cell lines and in primary cultures of normal rat hepatocytes (13,17,19). The degree of ERK1/2 phosphorylation induced by Cpd 5 correlated with the potency of growth inhibition in different cell lines and in many cell lines EGFR was also found to be tyrosine phosphorylated on Cpd 5 treatment (8,20). However, only ERK1/2 phosphorylation was found to be necessary for the Cpd 5-mediated growth inhibitory effects (14). We therefore examined EGFR tyrosine phosphorylation and ERK1/2 phosphorylation at T202 and Y204 in the livers of Cpd 5-treated PH rats, using an antibody that recognizes the dually phosphorylated protein. We found that ERK2 was phosphorylated as early as 0.5 h after Cpd 5 treatment. The phosphorylation gradually decreased with time and returned almost to the untreated level at 6 h after Cpd 5 administration. However, no ERK2 phosphorylation was observed in the rat livers undergoing regeneration after PH without Cpd 5 treatment. In contrast, EGFR tyrosine phosphorylation was only slightly induced during PH in both Cpd 5-treated and control livers (Figure 4A). ERK2 phosphorylation was also examined in rat livers after 0.5 h with increasing doses of Cpd 5 treatment at the time of PH. Induction of ERK2 phosphorylation was found to be dose dependent and reached a maximum at a dose of 10 mg/kg. No further induction was seen in vivo at doses above this (Figure 4B).



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 4. Cpd 5 induces ERK phosphorylation in rat livers after partial hepatectomy. (A) Rat livers were collected 0.25, 0.5, 1, 2, 3 and 6 h after PH with or without i.p. injection of Cpd 5 (30 mg/kg) immediately after PH. Western blots of the liver lysate proteins were probed with anti-phospho-ERK2 (ERK-P) and control ERK2 (ERK) antibodies. EGFR was immunoprecipitated from the liver lysates and probed with anti-PY antibody, to determine EGFR phosphorylation, and control EGFR antibody. (B) Increasing doses of Cpd 5 were injected i.p. immediately after PH and rat livers were collected 0.5 h after Cpd 5 injection. Phospho-ERK levels in the liver lysates were determined by western blotting with pERK and ERK2 (control) antibodies.

 
Inhibition of ERK2 phosphorylation in vivo antagonizes the growth delaying action of Cpd 5
ERK1/2 phosphorylation is catalyzed by its upstream kinase MEK1/2. PD098059 is an extensively used MEK1/2 inhibitor. It has been previously shown that treatment of hepatocytes in vitro with PD098059 can effectively antagonize both Cpd 5-induced ERK1/2 phosphorylation as well as its growth-inhibiting effects (13). Therefore, we used PD098059 to examine whether it inhibited Cpd 5-mediated ERK2 phosphorylation in vivo. PD098059 (5 ml of 75 µM in 0.37% DMSO) was injected i.v. prior to PH and Cpd 5 treatment. ERK2 phosphorylation was assayed by western blotting at 0.5 h after Cpd 5 (30 mg/kg) treatment and BrdU incorporation into newly synthesized DNA was assayed at 24 h after PH and Cpd 5 treatment. Both the induction of ERK2 phosphorylation and inhibition of DNA synthesis mediated by Cpd 5 in vivo were antagonized by PD098059 pretreatment (Figure 5A and B), suggesting that in vivo as in vitro ERK phosphorylation is important in mediating Cpd 5 growth inhibitory actions.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Antagonism of ERK phosphorylation and growth inhibition induced by Cpd 5. (A) Phosphorylation of ERK1/2 in livers of Cpd 5-treated rat 0.5 h after PH was determined by western blotting. Lane a, normal liver; lane b, liver from PD098059-treated (5 ml of 75 µM in 0.37% DMSO) PH rats; lane c, liver from Cpd 5-treated (30 mg/kg) PH rats; lane d, liver from PD098059- (5 ml of 75 µM in 0.37% DMSO) and Cpd 5-treated (30 mg/kg) PH rats. ERK2 was probed on the same western blot as a control. (B) The numbers of BrdU-labeled hepatocytes were measured 24 h after PH in Cpd 5-treated rats with or without PD098059 pretreatment.

 
Cpd 5 inhibits both PTP and ERK2 phosphatase activities in vivo
Cpd 5 has been previously shown to be a PTP inhibitor (11,13). Therefore, we assayed PTP activity in liver lysates before and after Cpd 5 (30 mg/kg) treatment using OMFP as substrate. PTP activity remained unchanged at various times up to 6 h after PH in control rat livers without Cpd 5 treatment. In contrast, PTP activity was inhibited as early as 0.5 h after Cpd 5 treatment. The activity recovered almost to its normal levels by 6 h after Cpd 5 treatment (Figure 6A). In order to characterize this PTP activity in liver lysates, we used Cpd 5-treated liver lysates in an ERK2 dephosphorylation assay. The endogenous ERK2 protein in the lysates was removed prior to the assay by immunoprecipitation with ERK2 antibody and centrifugation. Exogenous phospho-ERK2 protein was incubated with Cpd 5-treated or untreated liver lysates from PH rats which had been cleared of endogenous ERK2 protein and the phosphorylation status of phospho-ERK2 was determined by western blotting using phospho-ERK antibody. Untreated, but not Cpd 5-treated, PH rat liver lysates dephosphorylated the phospho-ERK substrate (Figure 6B). These results show that rat liver contains a potent phospho-ERK2 phosphatase(s), the activity of which is inhibited by Cpd 5 action in vivo.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6. Cpd 5 inhibits PTP activity of liver lysates in vivo. (A) PTP activity of lysates from normal liver and livers 0.5, 1, 3 and 6 h after PH, with or without Cpd 5 (30 mg/kg) treatment. (B) Endogenous ERK was cleared from the liver lysates by immunoprecipitation with anti-ERK2 antibody. The ERK2-cleared lysate proteins were used as a source of phosphatase for exogenous pERK2. Proteins were run on western blots and probed with pERK2 and ERK2 control antibodies. Lane 1, exogenous pERK2 control; lanes 2–9, pERK2 incubated with ERK2-cleared liver lysates 0.5, 1, 3 and 6 h after PH, without (lanes 2–5) and with Cpd 5 (lanes 6–9), respectively.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously shown that Cpd 5 potently inhibits the growth of several tumor cell lines in vitro (9,17,19). Subsequently, we showed that it also inhibited epidermal growth factor (EGF)-induced DNA synthesis in primary rat hepatocytes in culture, as well as the first wave of DNA synthesis at 24 h in rat livers after a 67% PH (13). Inhibition of both in vitro and in vivo DNA synthesis occurred when Cpd 5 was present for the first 6 h after addition of EGF in vitro or PH in vivo. Inhibition did not occur when Cpd 5 was present at later times. Cpd 5 would therefore seem to be acting in the G1 phase of the cell cycle.

After i.p. injection, Cpd 5 was found to accumulate in several organs, including liver, and blood levels remained high for at least 24 h. No toxicity was observed at a dose of 30 mg/kg. When Cpd 5 was injected into normal rats at a dose of 110 mg/kg, a 4-fold elevation of LDH was observed. However, there was no difference in the enzyme markers in the PH rats, either treated or untreated with 30 mg/kg Cpd 5. Pathological examination of tissue sections of various rat organs in the Cpd 5-treated (30 mg/kg) normal or PH rats revealed no cytotoxicity.

In the experiments reported here we found that the peak of DNA synthesis after PH was delayed in Cpd 5-treated rats. Instead of the observed peak at 24 h after PH in the untreated rat livers, the Cpd 5-treated rat livers showed a peak at 60 h after PH. The duration of G1 phase was increased due to Cpd 5 action. However, the peak height was similar to the Cpd 5 untreated PH rat livers. These results thus showed a transient inhibitory effect of Cpd 5 in vivo on hepatic DNA synthesis.

The failure of Cpd 5 to permanently suppress hepatocyte proliferation after PH could be due to a number of causes. The dose might not have been adequate to maintain the suppressive effect. Alternatively, the hepatocytes after PH might be less sensitive to Cpd 5 action. This type of temporal change in sensitivity of hepatocytes to a growth inhibitor like transforming growth factor ß has been reported (21). We have previously found that a single or chronic s.c. dose of Cpd 5 was able to efficiently inhibit growth of transplanted tumor cells in rat liver (15). Tumor growth remained inhibited up to 2 weeks after transplantation. In contrast, in the experiments reported here we found that growth inhibition by Cpd 5 was transient in normal liver regeneration. Thus, Cpd 5 may have differential activities on normal and neoplastic hepatocytes and could provide a useful therapeutic margin for treatment of primary liver tumors.

We also found that Cpd 5-mediated growth inhibition in various tumor cells and primary rat hepatocytes in vitro closely correlated with a strongly prolonged induction of phosphorylation and activation of ERK1/2. Other K vitamin analogs which were not potent growth inhibitors did not induce phosphorylation of ERK1/2 (19,20). We observed here the same effect of Cpd 5 in regenerating rat liver in vivo as we previously found in vitro. ERK2 phosphorylation was induced 0.5 h after Cpd 5 administration to PH rats. We did not find ERK2 phosphorylation in control rats without Cpd 5 treatment. This was in contrast to previously published reports, in which ERK1/2 phosphorylation was observed in mice and rats after PH (18,22). Two peaks of phospho-ERK1/2 were found in female Sprague–Dawley rats, 2 and 10.5 h after two-thirds PH (22). A single peak of phospho-ERK1/2 at 2 h was also reported in mice after PH (18). The differences in these results could be due to differences in rat strain or gender.

The degree of phospho-ERK2 induction after PH was found to be dependent on the dose of Cpd 5. This correlated with the previously reported dose dependence of DNA synthesis inhibition in Cpd 5-treated rat livers after PH (13). Moreover, when phospho-ERK1/2 induction was inhibited in cultured cells by the MEK inhibitors PD098059 and U0126, the growth inhibitory effects of Cpd 5 were also antagonized (14,17,23). We inhibited phospho-ERK2 induction in Cpd 5-treated PH rats by pretreatment with the MEK1/2 inhibitor PD098059 and found that it antagonized both phospho-ERK2 induction and DNA synthesis inhibition induced by Cpd 5. These results showed that phospho-ERK induction was correlated with the growth inhibitory action of Cpd 5. We did not observe a significant decrease in DNA synthesis after PH in rat livers pretreated with PD098059 alone compared with untreated rats. This lent further support to the idea that ERK1/2 was probably not phosphorylated after PH in our experimental model.

Tyrosine phosphorylation of EGFR and its downstream protein ERK1/2 is generally associated with cellular proliferation (24). However, in some cases ERK1/2 activation has also been associated with growth inhibition (25,26). The duration and intensity of ERK1/2 activation seem to correlate with the proliferative or inhibitory pathway. Transient activation may lead to cell cycle progression, whereas a sustained activation may result in cell cycle arrest (27,28). Transient or sustained ERK activation might differentially regulate downstream genes involved in cell growth. It might also affect nuclear localization of different signal transduction components. Sustained activation of ERK might also induce the Cdk inhibitor proteins p21 and p16 and inhibit CdK2 activity similarly to primary hepatocyte culture (29).

In order to investigate the possible mechanism of sustained ERK2 phosphorylation in vivo, we assayed for phospho-ERK2 dephosphorylating activities in liver lysates. Untreated PH liver lysates were found to contain phosphatase activity which dephosphorylated an exogenous phospho-ERK2 protein target. However, this phosphatase activity was found to be inhibited in the PH liver lysates from Cpd 5-treated rats. The phosphatase activity of the lysate was a minimum 0.5 h after Cpd 5 treatment and increased with time and correlated with the decrease in phospho-ERK2 seen in Cpd 5-treated PH livers. These results show that inhibition of phospho-ERK2 phosphatase activity in the PH liver lysates might be the underlying mechanism for induction of phospho-ERK2 in Cpd 5-treated PH livers. Thus inhibition of the activity of phospho-ERK2 phosphatase(s) by Cpd 5 in PH livers was likely responsible for the inhibition of DNA synthesis and delay in liver generation after PH, due to prolonged ERK1/2 activation. MAPK phosphatases such as MPK1 play an important role in negatively regulating ERK activity (30). When activated these phosphatases dephosphorylate ERK and thereby inactivate it. Inhibitors of the ERK phosphatase(s) are expected to cause induction of ERK phosphorylation and activation. Cpd 5 most likely inhibits these phosphatases and causes a prolonged activation and phosphorylation of ERK. The ERK phosphatase(s) that is inhibited by Cpd 5 in rat liver cells has not yet been identified. The transient nature of the growth inhibition could then be attributed to synthesis of new phosphatase(s) enzyme.


    Acknowledgments
 
We thank Dr Mike Nalesnik M.D. for examining the H&E stained tissue sections. This work was supported in part by National Institutes of Health grant CA82723 (B.I.C.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Michalopoulos,G.K. and DeFrances,M.C. (1997) Liver regeneration. Science, 276, 60–66.[Abstract/Free Full Text]
  2. Harkness,R.D. (1957) Regeneration of liver. Br. Med. Bull., 13, 87–93.[ISI]
  3. Higgins,G.M. and Anderson,R.M. (1931) Experimental pathology of the liver: restoration of the liver following partial hepatectomy. Arch. Pathol., 12, 186–202.
  4. Bucher,N.L.R. (1963) Regeneration of mammalian liver. Int. Rev. Cytol., 15, 245–300.[ISI]
  5. Grisham,J. (1964) A morphogenetic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating liver: autoradiography with thymidine-H3. Cancer Res., 22, 842–849.
  6. Russell,W.R. and Bucher,N.L. (1983) Vasopressin modulates liver regeneration in Brattleboro rats. Am. J. Physiol., 245, G321–G324.[ISI][Medline]
  7. Kerns,J., Naganathan,S., Dowd,P., Finn,F. and Carr,B.I. (1995) Thioalkyl derivatives of vitamin K3 and vitamin K3 oxide inhibit growth of Hep3B and Hep G2 cells. Bioorg. Chem., 2, 101–108.[CrossRef]
  8. Nishikawa,Y., Carr,B.I., Wang,Z., Kar,S., Finn,F., Dowd,P., Zheng,Z.B., Kerns,J. and Naganathan,S.J. (1995) Growth inhibition of hepatoma cells induced by vitamin K and its analogs. J. Biol. Chem., 270, 28304–28310.[Abstract/Free Full Text]
  9. Nishikawa,Y., Wang,Z., Kerns,J., Wilcox,C. and Carr,B.I. (1999) Inhibition of hepatoma cell growth in vitro by arylating and non-arylating K vitamin analogs: significance of protein tyrosine phosphatase inhibition. J. Biol. Chem., 274, 34803–34810.[Abstract/Free Full Text]
  10. Ni,R., Nishikawa,Y. and Carr,B.I. (1998) Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation. J. Biol. Chem., 273, 9906–9911.[Abstract/Free Full Text]
  11. Tamura,K., Southwick,E.C., Kerns,J., Rosi,K., Carr,B.I., Wilcox,C. and Lazo,J.S. (2000) Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. Cancer Res., 60, 1317–1325.[Abstract/Free Full Text]
  12. Wang,Z., Southwick,E.C., Wang,M. et al. (2001) Involvement of Cdc25A phosphatase in Hep3B hepatoma cell growth inhibition induced by novel K vitamin analogs. Cancer Res., 61, 7211–7216.[Abstract/Free Full Text]
  13. Carr,B.I., Wang,Z., Wang,M., Kar,S., Wilcox,C.S., Rosi,K., Southwick,E. and Lazo,J.S. (2003) A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo. J. Mol. Biol., 326, 721–735.[CrossRef][ISI][Medline]
  14. Adachi,T., Kar,S., Wang,M. and Carr,B.I. (2002) Transient and sustained ERK phosphorylation and nuclear translocation in growth control. J. Cell. Physiol., 192, 151–159.[CrossRef][ISI][Medline]
  15. Kar,S., Wang,M., Wilcox,C.S. and Carr,B.I. (2003) Antitumor and anticarcinogenic action of Cpd 5: a new class of protein phosphatase inhibitor. Carcinogenesis, 24, 411–416.[Abstract/Free Full Text]
  16. Williams,W., Sun,X. and Jebaratnam,D. (1997) Synthetic studies on the kinamycin family of antibiotics: synthesis of 2-(diazobenzyl)-p-naphthoquinone 1,7-dideoxy-3-demethylprekinamycin and 1-deoxy-3-demethylprekinamycin. J. Org. Chem., 62, 4364–4369.[CrossRef][ISI][Medline]
  17. Kar,S. and Carr,B.I. (2000) Growth inhibition and protein tyrosine phosphorylation in MCF7 breast cancer cells by a novel K-vitamin. J. Cell. Physiol., 185, 386–393.[CrossRef][ISI][Medline]
  18. Leu,J.I., Crissey,M.A., Craig,L.E. and Taub,R. (2003) Impaired hepatocyte DNA synthetic response posthepatectomy in insulin-like growth factor binding protein 1-deficient mice with defects in C/EBP beta and mitogen-activated protein kinase/extracellular signal-regulated kinase regulation. Mol. Cell. Biol., 23, 1251–1259.[Abstract/Free Full Text]
  19. Osada,S., Osada,K. and Carr,B.I. (2001) Tumor cell growth inhibition and extracellular signal-regulated kinase (ERK) phosphorylation by novel K vitamins. J. Mol. Biol., 314, 765–772.[CrossRef][ISI][Medline]
  20. Wang,Z., Wang,M., Lazo,J.S. and Carr,B.I. (2002) Identification of epidermal growth factor receptor as a target of Cdc25A protein phosphatase. J. Biol. Chem., 277, 19470–19475.[Abstract/Free Full Text]
  21. Carr,B.I., Hayashi,I., Branum,E.L. and Moses,H.L. (1986) Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor. Cancer Res., 46, 2330–2334.[Abstract]
  22. Talarmin,H., Rescan,C., Cariou,S., Glaise,D., Zanninelli,G., Bilodeau,M., Loyer,P., Guguen-Guillouzo,C. and Baffet,G. (1999) The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes. Mol. Cell. Biol., 19, 6003–6011.[Abstract/Free Full Text]
  23. Kar,S., Adachi,T. and Carr,B.I. (2002) EGFR-independent activation of ERK1/2 mediates growth inhibition by a PTPase antagonizing K-vitamin analog. J. Cell. Physiol., 190, 356–364.[CrossRef][ISI][Medline]
  24. Misra-Press,A., Rim,C.S., Yao,H., Roberson,M.H. and Stork,P.J. (1995) A novel mitogen-activated protein kinase phosphatase. J. Biol. Chem., 270, 14587–14596.[Abstract/Free Full Text]
  25. Pumiglia,K.M. and Decker,S.T. (1997) Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc. Natl Acad. Sci. USA, 94, 448–452.[Abstract/Free Full Text]
  26. Bromberg,J.F., Fan,Z., Brown,C., Mendelson,J. and Darnell,L.E.,Jr (1998) Epidermal growth factor-induced growth inhibition requires Stat 1 activation. Cell Growth Differ., 9, 505–512.[Abstract]
  27. Marshall,C.J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell, 80, 179–185.[ISI][Medline]
  28. Traverse,S., Gomez,N., Paterson,H., Marshall,C. and Cohen,P. (1992) Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem. J., 288, 351–355.[ISI][Medline]
  29. Tombes,R.M., Auer,K.L., Mikkelsen,R., Valerie,K., Wymann,M.P., Marshall,C.J., McMahon,M. and Dent,P. (1998) The mitogen activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. Biochem. J., 330, 1451–1460.[ISI][Medline]
  30. Wu,G.-S. (2004) The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol. Ther., 3, 156–161.[ISI][Medline]
Received June 10, 2004; revised July 22, 2004; accepted August 2, 2004.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
25/12/2345    most recent
bgh262v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Kar, S.
Articles by Carr, B. I.
PubMed
PubMed Citation
Articles by Kar, S.
Articles by Carr, B. I.