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Effect of folic acid on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy
Biochimica et Biophysica Acta 1379 Ž1998. 289–296
Effect of folic acid on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy Masami Komatsu, Ikuyo Tsukamoto
)
Department of Food Science and Nutrition, Nara Women’s UniÕersity, Nara 630, Japan Received 27 March 1997; revised 29 August 1997; accepted 29 August 1997
Abstract The effects of folic acid on liver regeneration after partial hepatectomy were investigated. The injection of folic acid inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 h after partial hepatectomy, with a concomitant reduction in DNA content. Northern blot analysis showed that this inhibition was due to the delay of the elevation of the mRNA levels of thymidylate synthase and thymidine kinase after partial hepatectomy. At 48 and 72 h, after partial hepatectomy, the thymidylate synthase activities in the folic acid injected rats increased to about 1.9- and 1.7-fold the corresponding control level, respectively, while thymidine kinase activities were similar to the control. Immunoblotting assay indicated that the increases in the thymidylate synthase activity at 48 and 72 h after partial hepatectomy were caused by a three fold increase in its protein level. Folic acid suppressed chymotryptic hydrolysis of thymidylate synthase. These suggest that folic acid increases the protein level of thymidylate synthase, at least in part, through protection against proteolysis. q 1998 Elsevier Science B.V. Keywords: Folic acid; Thymidylate synthase; Thymidine kinase; DNA synthesis; Liver regeneration; ŽRat.
1. Introduction Folic acid plays essential roles in several major cellular processes and the requirement for folate coenzymes for the synthesis of purines and thymidylate has led to the development of a large number of drug therapies. These antifolate drugs are widely used in the treatment of cancer to inhibit cell proliferation. Antifolates disrupt the nucleotide biosynthesis by the inhibition of the folate Ž tetrahydrofolate. -dependent enzymes such as thymidylate synthase, glycinamide
Abbreviations: TS, thymidylate synthase; TK, thymidine kinase; PH, partial Ž70%. hepatectomy; Dig, digoxigenin ) Corresponding author. Fax: q81 742 20 3499.
ribonucleotide formyltransferase and aminoimidazole carboxamide ribonucleotide formyltransferase. Folic acid, not reduced coenzyme form, also inhibits these enzymes like antifolates in vitro. However, it is not known whether folic acid itself can inhibit cell proliferation. The action of folic acid on the regenerative response in the liver has not yet been elucidated. The regenerative responses of liver following removal of 70% of its mass provide a suitable in vivo model of cell proliferation. The early stages of the regenerative responses in rats consist of a hypertrophic, prereplicative phase, lasting approximately 12–16 h, during which the rate of protein synthesis increases, and a subsequent hyperplastic phase that is characterized by the onset of DNA synthesis at about 18 h, peaking at about 24 h, followed by a mitosis
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M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
wave 6–8 h later w1,2x. A second peak of DNA synthesis emerges at 48 h. The proliferative activation process is quite synchronized w3x. Using this system, the action of folic acid on cell proliferation was investigated in vivo. The proliferative response was evaluated by the activities and the levels of the protein and the mRNA of TS ŽEC 2.1.1.45. and TK ŽEC 2.7.1.21., in addition to gross parameters such as DNA, RNA, and protein contents of the liver. TS and TK catalyze the formation of thymidylate via the de novo and salvage pathways, respectively. We previously described that these are the rate-determining enzymes of DNA synthesis and that the capacity of liver regeneration is estimated by the induction of these enzymes w4–13x. We show here that folic acid retards the regenerative responses by delaying increases in TS and TK at the mRNA level and that folic acid increases the TS protein level at 48 and 72 h after PH by protecting against proteolysis.
2. Materials and methods 2.1. Materials The reagents were purchased as follows: w5-3 HxDeoxyuridine monophosphateŽ 14.8 Cirmmol., from Amersham; wmethyl-3 Hx thymidine Ž 60 Cirmmol., ICN Radiochemicals; Durapore membrane filter, Millipore; goat anti-Ž rabbit IgG. antibody conjugated to horseradish-peroxidase, Cappel Laboratories; DIG RNA labeling kit and DIG luminescent detection kit, Boehringer Manheim Biochemica; OligoŽdt.cellulose Žtype 3., Collaborative Biomedical Products; Gene screen nylon membrane, Dupont. All other reagents were of analytical grade. 2.2. Animals Male Wistar rats weighing 170–190 g were used for all experiments. The animals were kept in temperature-controlled rooms with 12 h alternating light and dark cycles and given commercial laboratory chow ŽMF, Oriental Yeast, Osaka, Japan. and water ad libitum at all times. Two-thirds partial hepatectomy ŽPH. was performed under diethyl ether anesthesia by the procedure of Higgins and Anderson w14x. Folic acid Ž200 mgrkg body weight., dissolved in 0.1 M
NaHCO 3 , was injected intraperitoneally immediately after PH. For the 48 h- and 72 h-regenerating liver, the injection of folic acid was repeated 24 and 48 h after PH. Control rats received the same quantity of 0.1 M NaHCO 3 as did the experimental animals. The rats were killed and their livers were excised at 24, 48, or 72 h after PH. All liver samples were dissected in half: one part was immediately stored at y808C and used for RNA isolation, the other half was used for the determinations of the enzyme activity and the contents of DNA, RNA and protein, and for the immunoblotting assay. 2.3. Determinations of the enzymatic actiÕity and contents of DNA, RNA and protein The excised liver was homogenized with 5 volumes of 50 mM Tris–HCl buffer Ž pH 7.3. containing 0.25 M sucrose, 10 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. The DNA and RNA contents of the liver were measured by the diphenylamine w15x and orcin w16x reactions, respectively, after extraction with trichloroacetic acid according to the procedure of Schneider w16x. The protein concentration was measured by the method of Lowry et al. w17x with bovine serum albumin as the standard. After a 20% liver homogenate was centrifuged at 36 000 = g for 30 min at 48C, the supernatant fraction was used for determining the enzymatic activity. The activities of TS and TK were determined as described previously w4x and expressed as pmols of product formed per min per mg protein at 378C. Statistical analyses of data were done with Student’s t-test with Welch’s correction. 2.4. Isolation and Northern blot analysis of RNA Total RNA was extracted from the liver in 4 M guanidium isothiocyanate w18x and fractionated by affinity chromatography on oligoŽ dT. -cellulose columns to obtain polyŽ A.-rich RNA w19x. The concentrations of RNA samples were measured by absorbance at 260 nm. The purities of RNA samples were determined by a ratio of A 260 nmrA 280 nm Ž) 1.8. and by eletrophoresis in formaldehyde-agarose gels stained with ethidium bromide. The RNA preparations were denatured and electrophoresed on 1.2% agaroser2.2 M formaldehyde gels. After separation,
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
the RNA was transferred to Gene screen membranes by capillary blotting. Hybridization was carried out using Dig-labelled RNA probes and the chemiluminescent signals were quantitated by a densitometer ŽShimadzu dual wavelength chromato-scanner, type CS-930. as described previously w20x. 2.5. Preparation of RNA probe The pstI fragment Ž0.7 kb. from the mouse TS cDNA clone pMTS3 and the BamHI fragment Ž1.1 kb. from the mouse TK cDNA clone PMTK4 ŽATCC 37556. were subcloned into the plasmid pGEM 3Z ŽPromega.. After linearization of the plasmid, T7 RNA-polymerase was employed to obtain run-off transcripts of the antisense strands. Transcription and labeling were performed utilizing the commercial DIG RNA labeling system according to the manufacture’s instructions. 2.6. Antibodies and immunoblotting assay Anti-polyclonal rat TS antibodies and anti-polyclonal rat TK antibodies were prepared as described previously w21,22x. The supernatant fractions of the 20% liver homogenates were separated under denaturing conditions on polyacrylamide gels and electrophoretically transferred to Durapore membrane filters. After overnight blocking with 5% skim milk, the filter was incubated in anti-Žrat TS. rabbit serum or anti-Žrat TK. rabbit serum diluted 1:70 or 1:500, respectively, for 2 h and washed 4 times. The immunoblot was visualized by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase and the color development with 0.025% 3,3X-diaminobenzidine tetrahydrochloride and 0.03% H 2 O 2 . The reflectance of the bands was assayed quantitatively using the densitometer as described previously w21x. 2.7. Proteolysis Proteolysis was carried out by adding chymotrypsin Ž a proteinrchymotrypsin ratio of 100:1. to the supernatant fractions of the 24 h-regenerating liver of the control. The digestion mixtures were incubated at 378C and aliquots were withdrawn at 0, 10 and
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30 min to determine the residual TS protein levels by immunoblotting assay. 3. Results 3.1. Effects of folic acid on the actiÕities of TS and TK, the contents of DNA, RNA and protein, and the liÕer weight of regenerating liÕer The activities of TS and TK after PH were shown in Table 1. The TS activity increased by about 5 times compared with the normal activity Ž resting in Go state; just after PH. at 24 h. The injection of folic acid inhibited the activity of TS to 46% of the control value at 24 h after PH. The TK activity was also depressed by the injection of folic acid to 11% of the control, which was comparable to the normal level. At 48 h after PH, the TS activity was similar to that at 24 h in the control group. In the folic acid-treated group, the TS activity at 48 h increased four times compared with that at 24 h. This activity was 1.9-fold the corresponding control value. The TK activity at 48 h in the folic acid-treated group was similar to that of the corresponding control. At 72 h after PH, the TS activity in folic acid-injected group increased by 1.7 times compared with the control, whereas the TK activity was similar to the control. The increases in the DNA contents of the folic acid-injected 24 h-, 48 h-, and 72 h-regenerating liver were significantly inhibited to 80%, 75% and 69% of the corresponding control levels, respectively. The contents of RNA and protein and the liver weight of folic acid treated rats were lower than the control levels at 72 h after PH. These results indicated that folic acid delayed the increases in TS and TK activities and inhibited the regeneration of the liver after PH. 3.2. Effect of folic acid on mRNA leÕels of TS and TK TS mRNA started to elevate and reached a maximum level at 24 h after PH as shown in Fig. 1Ž A. . The TS mRNA level at 48 and 72 h decreased to 43% and 17% of the 24 h control level, respectively Ž Fig. 1ŽA. and Table 2.. The injection of folic acid inhibited the increase in 1.3 kb TS mRNA level at 24 h after PH. At 48 h after PH, the TS mRNA level of folic acid-injected rats increased to 79% of the 24 h control level and then decreased to 19% at 72 h after
292
Treatment
Time after PH Žh.
Enzymatic activity Žunitsrmg of protein. TS
Total liver content Žmg. DNA
RNA
6.70 " 0.38 9.20 " 0.34 12.87 " 0.46
37.9 " 1.1 55.8 " 3.8 76.4 " 3.3
Protein
Liver weight Žg.
565.9 " 35.3 834.8 " 31.4 1113.8 " 56.0
3.3 " 0.08 4.3 " 0.13 5.6 " 0.20
TK
Control
24 48 72
57.2 " 3.9 56.2 " 4.4 47.0 " 2.6
333.8 " 20.8 196.8 " 17.5 157.7 " 13.8
Folic acid
24 48 72
26.3 " 4.0 a 104.6 " 15.9 a 82.2 " 10.2
36.4 " 7.5 a 194.7 " 27.7 132.5 " 19.3
5.39 " 0.34 a 6.86 " 0.58 a 8.82 " 0.55 a
38.0 " 2.6 47.7 " 1.3 63.4 " 2.9 a
521.0 " 11.2 783.9 " 39.1 886.8 " 52.0 a
3.3 " 0.25 3.7 " 0.26 4.5 " 0.08 a
11.7 " 1.1
30.4 " 4.2
4.98 " 0.16
29.4 " 2.2
484.4 " 11.7
2.5 " 0.04
Normal ŽGo, just after PH.
Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately just after PH and repeated at every 24 h. At 24, 48 or 72 h after PH, the activities of TS and TK, liver contents of DNA, RNA and protein, and liver weight were determined as described in Section 2. Values are shown as means " S.E. of 4 to 9 rats. a Significant differences from the corresponding control. Ž P - 0.05..
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
Table 1 The effect of folic acid on liver regeneration after partial hepatectomy
Treatment
Time after
TS
PH Žh.
Activity Žunitsrmg of protein.
TK Protein level Ž%.
mRNA level Ž%.
Activity Žunitsrmg of protein.
Protein level Ž%.
mRNA level % 2.6 kb
1.1 kb
100 43.4 " 8.3 17.3 " 4.0
353.3 " 37.5 214.7 " 51.5 157.7 " 30.9
100 54.0 " 3.2 38.2 " 13.4
100 43.2 " 6.6 33.6 " 7.7
100 57.4 " 9.4 39.9 " 8.7
Control
24 48 72
60.0 " 8.5 63.2 " 9.4 50.3 " 4.1
100 97.6 " 19.9 104.2 " 26.6
Folic acid
24 48 72
25.3 " 9.2 a 101.8 " 22.7 a 77.6 " 23.3 a
55.3 " 14.4 a 278.3 " 25.7 a 273.0 " 37.6 a
20.5 " 5.4 a 78.5 " 8.2 a 19.3 " 2.9
42.8 " 20.5 a 147.0 " 27.1 118.1 " 33.1
11.4 " 4.3 a 42.0 " 7.0 35.8 " 9.6
12.0 " 4.9 a 102.0 " 30.2 a 35.2 " 6.3
11.0 " 4.0
14.2 " 1.0
18.4 " 3.5
33.5 " 8.8
7.8 " 2.0
4.9 " 2.1
Normal ŽGo, just after PH.
4.7 " 4.2 a 98.4 " 25.0 a 32.1 " 9.3
10.0 " 3.3
Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately just after PH and repeated at every 24 h. At 24, 48 or 72 h after PH, the activities, the protein and the mRNA levels of TS and TK were determined as described in Section 2. Levels of the protein and the mRNA are shown as percentages of the values of the 24 h-control and expressed as means " S.D. of four rats. a Significant differences from the corresponding control Ž P - 0.05..
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
Table 2 The effects of folic acid on the activity, the protein and the mRNA levels of TS and TK in regenerating liver
293
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PH. The increases in the TK mRNA levels at 24 h after PH were almost completely inhibited by the injection of folic acid. At 48 h after PH, the levels of the two species of TK mRNA in the folic acid-treated group elevated to a maximum which was similar to that of 24 h-control. These results indicated that the increases in the TK mRNA levels by PH were also delayed about 24 h by folic acid. 3.3. Effect of folic acid on the protein leÕels of TS and TK To investigate the mechanism of stimulation of TS activity at 48 and 72 h after PH by folic acid, TS protein level was determined by immunoblot analysis. The TS protein reacted as a 35 kDa protein band on the immunoblots of the control and folic acidtreated liver as shown in Fig. 2. Scanning densitometry demonstrated that TS protein in the rats injected with folic acid increased to 2.8- and 2.7-fold of the control at 48 and 72 h after PH, respectively ŽFig. 2 and Table 2.. These results clearly indicated that folic acid stimulated the increase in TS protein content of the regenerating liver. However, this increase was not proportional to the TS activity. The observed smaller increase in the activity was caused by the inhibitory effect of folic acid on TS activity. In our assay Fig. 1. Northern blot analysis of TS mRNA ŽA. and TK mRNA ŽB.. Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately after PH and repeated at every 24 h. Poly ŽA.-rich RNA Ž8 mg. from the normal and regenerating liver of the control and folic acid-injected rats were fractionated in agarose-formaldehyde gel, blotted to membrane, and hybridized with Dig-labeled RNA probes of TS or TK as described in Section 2. Lane 1, normal; lane 2, control 24 h-; lane 3, control 48 h-; lane 4, control 72 h-; lane 5, control 24 h-; lane 6, folic acid-injected 24 h-; lane 7, folic acid-injected 48 h-; lane 8, folic acid-injected 72 h-regenerating liver. The photographs of the gel exhibiting ethidium bromide flurescence are shown in the lower panel, demonstrating equal loading of RNA samples. The results presented are typical of eight separate experiments.
PH ŽTable 2.. The peak of the TS mRNA level of the folic acid-injected rats was found to be delayed for about 24 h. TK mRNA was detected as a 2.6 and 1.1 kb band as previously reported w22x. These two species of TK transcripts were similarly changed as shown in Fig. 1ŽB. and Table 2. The 2.6 and 1.1 kb TK mRNA were barely detectable in normal liver and increased markedly to a maximum at 24 h after
Fig. 2. Immunoblot analysis of TS protein Župper. and TK protein Žlower.. Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately after PH and repeated at every 24 h. At 24, 48 and 72 h after PH, the supernatant fraction Ž40 mg of protein. of the liver homogenate was separated on SDS polyacrylamide gel. The gel was then blotted onto a membrane and the resulting blot was reacted with anti-ŽTS. serum Župper. or anti ŽTK. serum Žlower., as described in Section 2. Lanes contained the supernatant fractions of the normal liver Žlane N.; control regenerating liver at 24 h Žlane 1., 48 h Žlane 2. and 72 h Žlane 3.; folic acid-injected regenerating liver at 24 h Žlane 4., 48 h Žlane 5. and 72 h Žlane 6.. The results presented are typical of eight separate experiments.
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
system, folic acid inhibited TS activity by 30% and 40% at a concentration of 0.1 and 1 mM, respectively. The immuno-reacted TK protein of 26 kDa was barely detectable in the normal and the 24 h-regenerating liver of folic acid-injected rats Ž Fig. 2.. The TK protein level of the treated rats was 11%, 42% and 36% of the 24 h-control at 24, 48 and 72 h after PH, respectively. These levels were closely correlated with enzymatic activities as observed in the 48 h- and 72 h-regenerating liver of the control group. 3.4. ProtectiÕe effect of folic acid on chymotryptic proteolysis of TS To understand the increase of TS protein content in folic acid-injected rats, the effect of folic acid on the degradation rate of TS was examined. When the supernatant fractions of the regenerating liver were incubated with chymotrypsin at 378C for 10 and 30 min, the TS protein levels were reduced to 20 and 3% of the native value, respectively, as shown in Fig. 3. The addition of 1 mM folic acid to the chymotryptic digestion mixtures suppressed the loss of TS protein to about 50 and 20% of the native value at 10 and 30 min, respectively. These results indicated that folic acid protected TS protein from chymotryptic hydrolysis.
295
the expression of TS and TK which occurs at G1 to S transition during liver regeneration, although a direct effect of folic acid on the regulation of transcription of TS and TK is not excluded. The regenerative response took place 48 h after about 24 h delay in folic acid-injected rats. The injection of folic acid stimulated the TS activity at 48 and 72 h after PH as shown in Table 1. Immunoblotting assay clearly demonstrated that this stimulation was caused by an increase in TS protein levels Ž Table 2. . However, the extent of TS activity was not directly proportional to the level of TS protein in folic acidinjected rats, while a close correlation was observed in the control group. This appears to be due to the inhibition of TS activity by folic acid accumulated in the liver of folic acid-injected rat. In fact, TS activity was inhibited to 60% by 1 mM folic acid in our assay system. The stimulation of the TS protein level at 48
4. Discussion The results of this study showed that folic acid inhibited cell proliferation by the delay of the induction of TS and TK by about 24 h during liver regeneration after PH. This is the first evidence of the inhibitory effect on cell proliferation of folic acid. An antifolate, methotrexate had no inhibitory effect on the activities of TS and TK and liver regeneration at 24 h after PH as previously reported w23x. Methotrexate inhibited regenerative response at 48 and 72 h after PH. These results suggest that folic acid inhibits liver regeneration by a different mechanism from that of methotrexate. The northern blot analysis indicated that the increases in TS and TK mRNA levels at 24 h after PH were depressed to a similar to the normal level by folic acid ŽTable 2.. This suggested that folic acid inhibited some G1 events required to stimulate
Fig. 3. Protective effect of folic acid on chymotryptic proteolysis of TS. The supernatant fraction of liver homogenate Ž18.5 mgrml. was incubated at 378C without chymotrypsinŽI. or with chymotrypsin Žproteinrchymotrypsin ratio; 100:1. in the absence Žl. or presence of 1 mM ŽB. folic acid. An aliquot Ž30 ml. of the incubation mixture was withdrawn at 0, 10 and 30 min and used for the determination of TS protein by immunoblotting. The TS protein levels are shown as percentages of the native value Žno incubation without chymotrypsin. and expressed as means " S.D. of four experiments. The photograph was the typical immunoblot of the incubation mixtures; lanes left to right: without chymotrypsin at 0, 10 and 30 min, with chymotrypsin in the absence of folic acid at 0, 10 and 30 min, with chymotrypsin in the presence of 1 mM folic acid at 0, 10 and 30 min.
296
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
and 72 h after PH by folic acid could not be fully explained by the TS mRNA level as shown in Table 2. The results of proteolysis suggested a posttranslational mechanism. The experiment of chymotryptic digestion showed that folic acid protected TS protein from proteolysis ŽFig. 3.. This is in agreement with the result that methotrexate suppressed tryptic hydrolysis of Lactobacillus casei TS w24x. It was also reported that folate analog caused major conformational changes in Escherichia coli TS w25x. The formation of the folate-bound enzyme in the liver and the consequent ligand-induced conformational changes may increase in the stability to proteolysis. The TK activity closely correlated to the protein level in both the control and folic acid-injected rats. However, the levels of TK protein and TK activity at 48 h in folic acid-injected rats did not reach those of 24 h-regenerating liver in the control, while the TK mRNA level increased to the 24 h-control level. A posttranscriptional andror translational control might be involved. The regulatory mechanism of the changes in TK protein level during liver regeneration in folic acid-injected rats appears to be different form that of TS protein. In conclusion, folic acid inhibited the liver regeneration by retarding increases in TS and TK at the mRNA level at 24 h after PH and increased TS protein levels at 48 and 72 h after PH. Further study is needed to clarify the molecular mechanism of the actions of folic acid on the expression of these particular genes during liver regeneration.
5. Acknowledgements We are grateful to Dr. L.F. Johnson ŽOhio State University, Ohio. for providing the cDNA clone pMTS3.
References w1x J.W. Grisham, Cancer Res. 22 Ž1962. 842–849. w2x H.M. Rabes, R. Wirshing, H.V. Tuczek, G. Iselar, Cell. Tissue Kin. 6 Ž1976. 517–532. w3x M.R. Alison, Physiol. Rev. 66 Ž1986. 499–541. w4x R. Nakata, I. Tsukamoto, M. Nanme, S. Makino, M. Miyoshi, S. Kojo, Eur. J. Pharmacol. 114 Ž1985. 355–360. w5x I. Tsukamoto, S. Kojo, Chem. Lett. Ž1987. 2313–2316. w6x I. Tsukamoto, S. Kojo, Biochem. Pharmacol. 36 Ž1987. 2871–2872. w7x I. Tsukamoto, S. Kojo, Eur. J. Pharmacol. 144 Ž1987. 159–162. w8x R. Nakata, I. Tsukamoto, M. Miyoshi, S. Kojo, Clin. Sci. 72 Ž1987. 455–461. w9x I. Tsukamoto, S. Kojo, Gut 30 Ž1989. 387–390. w10x I. Tsukamoto, S. Kojo, Biochim. Biophys. Acta 1009 Ž1990. 191–193. w11x I. Tsukamoto, S. Kojo, Biochim. Biophys. Acta 1033 Ž1990. 287–290. w12x I. Tsukamoto, S. Kojo, Biochim. Biophys. Acta 1074 Ž1991. 52–55. w13x I. Tsukamoto, Y. Yoshida, Y. Kitamura, S. Nomura, Biochem. Pharmacol. 52 Ž1996. 879–884. w14x G.M. Higgins, R.M. Anderson, Arch. Pathol. 12 Ž1931. 186–202. w15x K. Burton, Biochem. J. 62 Ž1956. 315–322. w16x W.C. Schneider, Methods Enzymol. 3 Ž1957. 680–684. w17x O.H. Lowry, N.J. Rosebrrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 Ž1951. 265–275. w18x P. Chomczynski, N. Sacchi, Anal. Biochem. 162 Ž1987. 156–159. w19x R.E. Kingston, Curr. Protocols Mol. Biol. 1 Ž1992. 4.5.1– 4.5.3. w20x M. Wakabayashi, R. Nakata, I. Tsukamoto, Biochem. Mol. Biol. Int. 34 Ž1994. 345–350. w21x I. Tsukamoto, R. Nakata, M. Miyoshi, S. Taketani, S. Kojo, Biochim. Biophys. Acta 946 Ž1988. 254–259. w22x I. Tsukamoto, M. Wakabayashi, K. Takebayashi, S. Nomura, Biochim. Biophys. Acta 1290 Ž1996. 267–272. w23x I. Tsukamoto, S. Kojo, Clin. Sci. 82 Ž1992. 181–184. w24x Al-W.A. Mohsen, J.L. Aull, D.M. Payne, H.H. Daron, Biochemistry 34 Ž1995. 1669–1677. w25x A. Kamb, J. Finer-Moore, R.M. Stroud, Biochemistry 31 Ž1992. 12876–12884.
Effect of folic acid on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy Masami Komatsu, Ikuyo Tsukamoto
)
Department of Food Science and Nutrition, Nara Women’s UniÕersity, Nara 630, Japan Received 27 March 1997; revised 29 August 1997; accepted 29 August 1997
Abstract The effects of folic acid on liver regeneration after partial hepatectomy were investigated. The injection of folic acid inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 h after partial hepatectomy, with a concomitant reduction in DNA content. Northern blot analysis showed that this inhibition was due to the delay of the elevation of the mRNA levels of thymidylate synthase and thymidine kinase after partial hepatectomy. At 48 and 72 h, after partial hepatectomy, the thymidylate synthase activities in the folic acid injected rats increased to about 1.9- and 1.7-fold the corresponding control level, respectively, while thymidine kinase activities were similar to the control. Immunoblotting assay indicated that the increases in the thymidylate synthase activity at 48 and 72 h after partial hepatectomy were caused by a three fold increase in its protein level. Folic acid suppressed chymotryptic hydrolysis of thymidylate synthase. These suggest that folic acid increases the protein level of thymidylate synthase, at least in part, through protection against proteolysis. q 1998 Elsevier Science B.V. Keywords: Folic acid; Thymidylate synthase; Thymidine kinase; DNA synthesis; Liver regeneration; ŽRat.
1. Introduction Folic acid plays essential roles in several major cellular processes and the requirement for folate coenzymes for the synthesis of purines and thymidylate has led to the development of a large number of drug therapies. These antifolate drugs are widely used in the treatment of cancer to inhibit cell proliferation. Antifolates disrupt the nucleotide biosynthesis by the inhibition of the folate Ž tetrahydrofolate. -dependent enzymes such as thymidylate synthase, glycinamide
Abbreviations: TS, thymidylate synthase; TK, thymidine kinase; PH, partial Ž70%. hepatectomy; Dig, digoxigenin ) Corresponding author. Fax: q81 742 20 3499.
ribonucleotide formyltransferase and aminoimidazole carboxamide ribonucleotide formyltransferase. Folic acid, not reduced coenzyme form, also inhibits these enzymes like antifolates in vitro. However, it is not known whether folic acid itself can inhibit cell proliferation. The action of folic acid on the regenerative response in the liver has not yet been elucidated. The regenerative responses of liver following removal of 70% of its mass provide a suitable in vivo model of cell proliferation. The early stages of the regenerative responses in rats consist of a hypertrophic, prereplicative phase, lasting approximately 12–16 h, during which the rate of protein synthesis increases, and a subsequent hyperplastic phase that is characterized by the onset of DNA synthesis at about 18 h, peaking at about 24 h, followed by a mitosis
0304-4165r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 7 . 0 0 1 1 1 - 6
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M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
wave 6–8 h later w1,2x. A second peak of DNA synthesis emerges at 48 h. The proliferative activation process is quite synchronized w3x. Using this system, the action of folic acid on cell proliferation was investigated in vivo. The proliferative response was evaluated by the activities and the levels of the protein and the mRNA of TS ŽEC 2.1.1.45. and TK ŽEC 2.7.1.21., in addition to gross parameters such as DNA, RNA, and protein contents of the liver. TS and TK catalyze the formation of thymidylate via the de novo and salvage pathways, respectively. We previously described that these are the rate-determining enzymes of DNA synthesis and that the capacity of liver regeneration is estimated by the induction of these enzymes w4–13x. We show here that folic acid retards the regenerative responses by delaying increases in TS and TK at the mRNA level and that folic acid increases the TS protein level at 48 and 72 h after PH by protecting against proteolysis.
2. Materials and methods 2.1. Materials The reagents were purchased as follows: w5-3 HxDeoxyuridine monophosphateŽ 14.8 Cirmmol., from Amersham; wmethyl-3 Hx thymidine Ž 60 Cirmmol., ICN Radiochemicals; Durapore membrane filter, Millipore; goat anti-Ž rabbit IgG. antibody conjugated to horseradish-peroxidase, Cappel Laboratories; DIG RNA labeling kit and DIG luminescent detection kit, Boehringer Manheim Biochemica; OligoŽdt.cellulose Žtype 3., Collaborative Biomedical Products; Gene screen nylon membrane, Dupont. All other reagents were of analytical grade. 2.2. Animals Male Wistar rats weighing 170–190 g were used for all experiments. The animals were kept in temperature-controlled rooms with 12 h alternating light and dark cycles and given commercial laboratory chow ŽMF, Oriental Yeast, Osaka, Japan. and water ad libitum at all times. Two-thirds partial hepatectomy ŽPH. was performed under diethyl ether anesthesia by the procedure of Higgins and Anderson w14x. Folic acid Ž200 mgrkg body weight., dissolved in 0.1 M
NaHCO 3 , was injected intraperitoneally immediately after PH. For the 48 h- and 72 h-regenerating liver, the injection of folic acid was repeated 24 and 48 h after PH. Control rats received the same quantity of 0.1 M NaHCO 3 as did the experimental animals. The rats were killed and their livers were excised at 24, 48, or 72 h after PH. All liver samples were dissected in half: one part was immediately stored at y808C and used for RNA isolation, the other half was used for the determinations of the enzyme activity and the contents of DNA, RNA and protein, and for the immunoblotting assay. 2.3. Determinations of the enzymatic actiÕity and contents of DNA, RNA and protein The excised liver was homogenized with 5 volumes of 50 mM Tris–HCl buffer Ž pH 7.3. containing 0.25 M sucrose, 10 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. The DNA and RNA contents of the liver were measured by the diphenylamine w15x and orcin w16x reactions, respectively, after extraction with trichloroacetic acid according to the procedure of Schneider w16x. The protein concentration was measured by the method of Lowry et al. w17x with bovine serum albumin as the standard. After a 20% liver homogenate was centrifuged at 36 000 = g for 30 min at 48C, the supernatant fraction was used for determining the enzymatic activity. The activities of TS and TK were determined as described previously w4x and expressed as pmols of product formed per min per mg protein at 378C. Statistical analyses of data were done with Student’s t-test with Welch’s correction. 2.4. Isolation and Northern blot analysis of RNA Total RNA was extracted from the liver in 4 M guanidium isothiocyanate w18x and fractionated by affinity chromatography on oligoŽ dT. -cellulose columns to obtain polyŽ A.-rich RNA w19x. The concentrations of RNA samples were measured by absorbance at 260 nm. The purities of RNA samples were determined by a ratio of A 260 nmrA 280 nm Ž) 1.8. and by eletrophoresis in formaldehyde-agarose gels stained with ethidium bromide. The RNA preparations were denatured and electrophoresed on 1.2% agaroser2.2 M formaldehyde gels. After separation,
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
the RNA was transferred to Gene screen membranes by capillary blotting. Hybridization was carried out using Dig-labelled RNA probes and the chemiluminescent signals were quantitated by a densitometer ŽShimadzu dual wavelength chromato-scanner, type CS-930. as described previously w20x. 2.5. Preparation of RNA probe The pstI fragment Ž0.7 kb. from the mouse TS cDNA clone pMTS3 and the BamHI fragment Ž1.1 kb. from the mouse TK cDNA clone PMTK4 ŽATCC 37556. were subcloned into the plasmid pGEM 3Z ŽPromega.. After linearization of the plasmid, T7 RNA-polymerase was employed to obtain run-off transcripts of the antisense strands. Transcription and labeling were performed utilizing the commercial DIG RNA labeling system according to the manufacture’s instructions. 2.6. Antibodies and immunoblotting assay Anti-polyclonal rat TS antibodies and anti-polyclonal rat TK antibodies were prepared as described previously w21,22x. The supernatant fractions of the 20% liver homogenates were separated under denaturing conditions on polyacrylamide gels and electrophoretically transferred to Durapore membrane filters. After overnight blocking with 5% skim milk, the filter was incubated in anti-Žrat TS. rabbit serum or anti-Žrat TK. rabbit serum diluted 1:70 or 1:500, respectively, for 2 h and washed 4 times. The immunoblot was visualized by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase and the color development with 0.025% 3,3X-diaminobenzidine tetrahydrochloride and 0.03% H 2 O 2 . The reflectance of the bands was assayed quantitatively using the densitometer as described previously w21x. 2.7. Proteolysis Proteolysis was carried out by adding chymotrypsin Ž a proteinrchymotrypsin ratio of 100:1. to the supernatant fractions of the 24 h-regenerating liver of the control. The digestion mixtures were incubated at 378C and aliquots were withdrawn at 0, 10 and
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30 min to determine the residual TS protein levels by immunoblotting assay. 3. Results 3.1. Effects of folic acid on the actiÕities of TS and TK, the contents of DNA, RNA and protein, and the liÕer weight of regenerating liÕer The activities of TS and TK after PH were shown in Table 1. The TS activity increased by about 5 times compared with the normal activity Ž resting in Go state; just after PH. at 24 h. The injection of folic acid inhibited the activity of TS to 46% of the control value at 24 h after PH. The TK activity was also depressed by the injection of folic acid to 11% of the control, which was comparable to the normal level. At 48 h after PH, the TS activity was similar to that at 24 h in the control group. In the folic acid-treated group, the TS activity at 48 h increased four times compared with that at 24 h. This activity was 1.9-fold the corresponding control value. The TK activity at 48 h in the folic acid-treated group was similar to that of the corresponding control. At 72 h after PH, the TS activity in folic acid-injected group increased by 1.7 times compared with the control, whereas the TK activity was similar to the control. The increases in the DNA contents of the folic acid-injected 24 h-, 48 h-, and 72 h-regenerating liver were significantly inhibited to 80%, 75% and 69% of the corresponding control levels, respectively. The contents of RNA and protein and the liver weight of folic acid treated rats were lower than the control levels at 72 h after PH. These results indicated that folic acid delayed the increases in TS and TK activities and inhibited the regeneration of the liver after PH. 3.2. Effect of folic acid on mRNA leÕels of TS and TK TS mRNA started to elevate and reached a maximum level at 24 h after PH as shown in Fig. 1Ž A. . The TS mRNA level at 48 and 72 h decreased to 43% and 17% of the 24 h control level, respectively Ž Fig. 1ŽA. and Table 2.. The injection of folic acid inhibited the increase in 1.3 kb TS mRNA level at 24 h after PH. At 48 h after PH, the TS mRNA level of folic acid-injected rats increased to 79% of the 24 h control level and then decreased to 19% at 72 h after
292
Treatment
Time after PH Žh.
Enzymatic activity Žunitsrmg of protein. TS
Total liver content Žmg. DNA
RNA
6.70 " 0.38 9.20 " 0.34 12.87 " 0.46
37.9 " 1.1 55.8 " 3.8 76.4 " 3.3
Protein
Liver weight Žg.
565.9 " 35.3 834.8 " 31.4 1113.8 " 56.0
3.3 " 0.08 4.3 " 0.13 5.6 " 0.20
TK
Control
24 48 72
57.2 " 3.9 56.2 " 4.4 47.0 " 2.6
333.8 " 20.8 196.8 " 17.5 157.7 " 13.8
Folic acid
24 48 72
26.3 " 4.0 a 104.6 " 15.9 a 82.2 " 10.2
36.4 " 7.5 a 194.7 " 27.7 132.5 " 19.3
5.39 " 0.34 a 6.86 " 0.58 a 8.82 " 0.55 a
38.0 " 2.6 47.7 " 1.3 63.4 " 2.9 a
521.0 " 11.2 783.9 " 39.1 886.8 " 52.0 a
3.3 " 0.25 3.7 " 0.26 4.5 " 0.08 a
11.7 " 1.1
30.4 " 4.2
4.98 " 0.16
29.4 " 2.2
484.4 " 11.7
2.5 " 0.04
Normal ŽGo, just after PH.
Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately just after PH and repeated at every 24 h. At 24, 48 or 72 h after PH, the activities of TS and TK, liver contents of DNA, RNA and protein, and liver weight were determined as described in Section 2. Values are shown as means " S.E. of 4 to 9 rats. a Significant differences from the corresponding control. Ž P - 0.05..
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
Table 1 The effect of folic acid on liver regeneration after partial hepatectomy
Treatment
Time after
TS
PH Žh.
Activity Žunitsrmg of protein.
TK Protein level Ž%.
mRNA level Ž%.
Activity Žunitsrmg of protein.
Protein level Ž%.
mRNA level % 2.6 kb
1.1 kb
100 43.4 " 8.3 17.3 " 4.0
353.3 " 37.5 214.7 " 51.5 157.7 " 30.9
100 54.0 " 3.2 38.2 " 13.4
100 43.2 " 6.6 33.6 " 7.7
100 57.4 " 9.4 39.9 " 8.7
Control
24 48 72
60.0 " 8.5 63.2 " 9.4 50.3 " 4.1
100 97.6 " 19.9 104.2 " 26.6
Folic acid
24 48 72
25.3 " 9.2 a 101.8 " 22.7 a 77.6 " 23.3 a
55.3 " 14.4 a 278.3 " 25.7 a 273.0 " 37.6 a
20.5 " 5.4 a 78.5 " 8.2 a 19.3 " 2.9
42.8 " 20.5 a 147.0 " 27.1 118.1 " 33.1
11.4 " 4.3 a 42.0 " 7.0 35.8 " 9.6
12.0 " 4.9 a 102.0 " 30.2 a 35.2 " 6.3
11.0 " 4.0
14.2 " 1.0
18.4 " 3.5
33.5 " 8.8
7.8 " 2.0
4.9 " 2.1
Normal ŽGo, just after PH.
4.7 " 4.2 a 98.4 " 25.0 a 32.1 " 9.3
10.0 " 3.3
Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately just after PH and repeated at every 24 h. At 24, 48 or 72 h after PH, the activities, the protein and the mRNA levels of TS and TK were determined as described in Section 2. Levels of the protein and the mRNA are shown as percentages of the values of the 24 h-control and expressed as means " S.D. of four rats. a Significant differences from the corresponding control Ž P - 0.05..
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Table 2 The effects of folic acid on the activity, the protein and the mRNA levels of TS and TK in regenerating liver
293
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PH. The increases in the TK mRNA levels at 24 h after PH were almost completely inhibited by the injection of folic acid. At 48 h after PH, the levels of the two species of TK mRNA in the folic acid-treated group elevated to a maximum which was similar to that of 24 h-control. These results indicated that the increases in the TK mRNA levels by PH were also delayed about 24 h by folic acid. 3.3. Effect of folic acid on the protein leÕels of TS and TK To investigate the mechanism of stimulation of TS activity at 48 and 72 h after PH by folic acid, TS protein level was determined by immunoblot analysis. The TS protein reacted as a 35 kDa protein band on the immunoblots of the control and folic acidtreated liver as shown in Fig. 2. Scanning densitometry demonstrated that TS protein in the rats injected with folic acid increased to 2.8- and 2.7-fold of the control at 48 and 72 h after PH, respectively ŽFig. 2 and Table 2.. These results clearly indicated that folic acid stimulated the increase in TS protein content of the regenerating liver. However, this increase was not proportional to the TS activity. The observed smaller increase in the activity was caused by the inhibitory effect of folic acid on TS activity. In our assay Fig. 1. Northern blot analysis of TS mRNA ŽA. and TK mRNA ŽB.. Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately after PH and repeated at every 24 h. Poly ŽA.-rich RNA Ž8 mg. from the normal and regenerating liver of the control and folic acid-injected rats were fractionated in agarose-formaldehyde gel, blotted to membrane, and hybridized with Dig-labeled RNA probes of TS or TK as described in Section 2. Lane 1, normal; lane 2, control 24 h-; lane 3, control 48 h-; lane 4, control 72 h-; lane 5, control 24 h-; lane 6, folic acid-injected 24 h-; lane 7, folic acid-injected 48 h-; lane 8, folic acid-injected 72 h-regenerating liver. The photographs of the gel exhibiting ethidium bromide flurescence are shown in the lower panel, demonstrating equal loading of RNA samples. The results presented are typical of eight separate experiments.
PH ŽTable 2.. The peak of the TS mRNA level of the folic acid-injected rats was found to be delayed for about 24 h. TK mRNA was detected as a 2.6 and 1.1 kb band as previously reported w22x. These two species of TK transcripts were similarly changed as shown in Fig. 1ŽB. and Table 2. The 2.6 and 1.1 kb TK mRNA were barely detectable in normal liver and increased markedly to a maximum at 24 h after
Fig. 2. Immunoblot analysis of TS protein Župper. and TK protein Žlower.. Folic acid Ž200 mgrkg body weight. was injected intraperitoneally immediately after PH and repeated at every 24 h. At 24, 48 and 72 h after PH, the supernatant fraction Ž40 mg of protein. of the liver homogenate was separated on SDS polyacrylamide gel. The gel was then blotted onto a membrane and the resulting blot was reacted with anti-ŽTS. serum Župper. or anti ŽTK. serum Žlower., as described in Section 2. Lanes contained the supernatant fractions of the normal liver Žlane N.; control regenerating liver at 24 h Žlane 1., 48 h Žlane 2. and 72 h Žlane 3.; folic acid-injected regenerating liver at 24 h Žlane 4., 48 h Žlane 5. and 72 h Žlane 6.. The results presented are typical of eight separate experiments.
M. Komatsu, I. Tsukamotor Biochimica et Biophysica Acta 1379 (1998) 289–296
system, folic acid inhibited TS activity by 30% and 40% at a concentration of 0.1 and 1 mM, respectively. The immuno-reacted TK protein of 26 kDa was barely detectable in the normal and the 24 h-regenerating liver of folic acid-injected rats Ž Fig. 2.. The TK protein level of the treated rats was 11%, 42% and 36% of the 24 h-control at 24, 48 and 72 h after PH, respectively. These levels were closely correlated with enzymatic activities as observed in the 48 h- and 72 h-regenerating liver of the control group. 3.4. ProtectiÕe effect of folic acid on chymotryptic proteolysis of TS To understand the increase of TS protein content in folic acid-injected rats, the effect of folic acid on the degradation rate of TS was examined. When the supernatant fractions of the regenerating liver were incubated with chymotrypsin at 378C for 10 and 30 min, the TS protein levels were reduced to 20 and 3% of the native value, respectively, as shown in Fig. 3. The addition of 1 mM folic acid to the chymotryptic digestion mixtures suppressed the loss of TS protein to about 50 and 20% of the native value at 10 and 30 min, respectively. These results indicated that folic acid protected TS protein from chymotryptic hydrolysis.
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the expression of TS and TK which occurs at G1 to S transition during liver regeneration, although a direct effect of folic acid on the regulation of transcription of TS and TK is not excluded. The regenerative response took place 48 h after about 24 h delay in folic acid-injected rats. The injection of folic acid stimulated the TS activity at 48 and 72 h after PH as shown in Table 1. Immunoblotting assay clearly demonstrated that this stimulation was caused by an increase in TS protein levels Ž Table 2. . However, the extent of TS activity was not directly proportional to the level of TS protein in folic acidinjected rats, while a close correlation was observed in the control group. This appears to be due to the inhibition of TS activity by folic acid accumulated in the liver of folic acid-injected rat. In fact, TS activity was inhibited to 60% by 1 mM folic acid in our assay system. The stimulation of the TS protein level at 48
4. Discussion The results of this study showed that folic acid inhibited cell proliferation by the delay of the induction of TS and TK by about 24 h during liver regeneration after PH. This is the first evidence of the inhibitory effect on cell proliferation of folic acid. An antifolate, methotrexate had no inhibitory effect on the activities of TS and TK and liver regeneration at 24 h after PH as previously reported w23x. Methotrexate inhibited regenerative response at 48 and 72 h after PH. These results suggest that folic acid inhibits liver regeneration by a different mechanism from that of methotrexate. The northern blot analysis indicated that the increases in TS and TK mRNA levels at 24 h after PH were depressed to a similar to the normal level by folic acid ŽTable 2.. This suggested that folic acid inhibited some G1 events required to stimulate
Fig. 3. Protective effect of folic acid on chymotryptic proteolysis of TS. The supernatant fraction of liver homogenate Ž18.5 mgrml. was incubated at 378C without chymotrypsinŽI. or with chymotrypsin Žproteinrchymotrypsin ratio; 100:1. in the absence Žl. or presence of 1 mM ŽB. folic acid. An aliquot Ž30 ml. of the incubation mixture was withdrawn at 0, 10 and 30 min and used for the determination of TS protein by immunoblotting. The TS protein levels are shown as percentages of the native value Žno incubation without chymotrypsin. and expressed as means " S.D. of four experiments. The photograph was the typical immunoblot of the incubation mixtures; lanes left to right: without chymotrypsin at 0, 10 and 30 min, with chymotrypsin in the absence of folic acid at 0, 10 and 30 min, with chymotrypsin in the presence of 1 mM folic acid at 0, 10 and 30 min.
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and 72 h after PH by folic acid could not be fully explained by the TS mRNA level as shown in Table 2. The results of proteolysis suggested a posttranslational mechanism. The experiment of chymotryptic digestion showed that folic acid protected TS protein from proteolysis ŽFig. 3.. This is in agreement with the result that methotrexate suppressed tryptic hydrolysis of Lactobacillus casei TS w24x. It was also reported that folate analog caused major conformational changes in Escherichia coli TS w25x. The formation of the folate-bound enzyme in the liver and the consequent ligand-induced conformational changes may increase in the stability to proteolysis. The TK activity closely correlated to the protein level in both the control and folic acid-injected rats. However, the levels of TK protein and TK activity at 48 h in folic acid-injected rats did not reach those of 24 h-regenerating liver in the control, while the TK mRNA level increased to the 24 h-control level. A posttranscriptional andror translational control might be involved. The regulatory mechanism of the changes in TK protein level during liver regeneration in folic acid-injected rats appears to be different form that of TS protein. In conclusion, folic acid inhibited the liver regeneration by retarding increases in TS and TK at the mRNA level at 24 h after PH and increased TS protein levels at 48 and 72 h after PH. Further study is needed to clarify the molecular mechanism of the actions of folic acid on the expression of these particular genes during liver regeneration.
5. Acknowledgements We are grateful to Dr. L.F. Johnson ŽOhio State University, Ohio. for providing the cDNA clone pMTS3.
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