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Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats
Vol. 153, No. 2,1988 June 16,1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 576-582
%~dMOR NECROSIS FACTOR STIMUIATES DNA SYNTHESIS IN THE LIVER OF INTACT RATS Kenneth R. Feingold, Mounzer Soued and Carl Grunfeld With the Technical Assistance of Arthur H. Moser, Jennifer A. Verdier and Heather G. DoVale Department of Medicine, University of California, San Francisco, CA,
and
Metabolism Section, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, CA 94121 Received April 8, 1988
Summary: TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [ H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [ H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is i0 ug/200 g rat with a maximal increase occuring with 25 ug/200 g, considerably less than the dose re~u.ired for maximally increasing plasma triglycerides. The increase in [ H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals,
© 1988 Academic Press, Inc.
Tumor necrosis factor (TNF) is secreted by macrophages in response to endotoxin and produces hemorrhagic necrosis of tumors (1-3). Sugarman, et al, have shown that TNF is directly cytotoxic to a variety of tumor cell lines (4). These investigators also observed that TNF enhances growth in some non-transformed diploid human cell lines.
Others have also shown that TNF
stimulates normal fibroblast division (5). In addition TNF has a large number of other biological actions including being a prime mediator of the metabolic effects of sepsis, especially hyperlipidemia (1-3,6,7). Recently studies have demonstrated that TNF regulates hepatic function. Perlmutter, et al, and Darlington, et al, have shown that TNF stimulates the production of acute phase proteins by hepatoma cells (8,9). Our laboratory has demonstrated that TNF stimulates beth hepatic fatty acid and cholesterol synthesis in intact animals (i0). Durir~ these studies we observed that TNF 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
576
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
administration produces a 21% increase in liver mass which could not be accounted for by an increase in either lipid accumulation or glycogen storage. In this manuscript we demonstrate that TNF administration stimulates DNA synthesis in the liver of intact rats. MATERIAIS AND METHODS Materials: [3H-Methyl]thymidine (78.8 Ci/ mmole) was purchased from New England Nuclear. Male Sprague-Dawley rats were purchased from Simonsen animal vendors. Aphidicolin and calf thymus DNA Type I were purchased from Sigma Chemical Company. 7Human tumor necrosis factor alpha (TNF) with a specific activity of 5 x i0 units/mg was provided by Dr. Shepard of Genentech, Inc. Methods: On the day prior to study animals were injected intravenously with TNF in 0.9% saline or saline alone. Animals were fasted after injection. The next day the rats were injected intraperitoneally with 50 uCi of [ H]thymid~_e. One hour later the rats were killed and the liver excised and weighed. [ H]thymidine incorporation into DNA and total DNA was determined according to the method of Schneider (Ii). Briefly, a 250 mg portion of liver was homogenized in 1 ml of H_O after which 5 ml of cold 10% TCA was added. Ten minutes later the material was centrifuged at 2,000 g at 4° for 5 rain. The supernatant was discarded and the precipitant material washed 2 times with 2 ml of 5% cold TCA followed by centrifugation 6 The precipitated material was then suspended in 2 ml, 5% TCA, heated at 90 C in a water bath for 15 rain. and centrifuged. An aliquod of the supernatant containing the hydrolyzed DNA was counted by liquid scintillation. Total DNA was then determined by incubating 1 ml of the supernatant with 2 ml diphenylamine reagent (i g diphenylamine ~lus i00 ml glacial acetic acid plus 2.75 ml concentrated sulfuric acid) at i00 C for 15 min. After cooling, the absorbance was determined at 600 nm. . ~ polymerase alpha activity was determined by measuring the incorporation of [ H]thymidine into DNA in liver slices incubated in the presence or absence of aphidicolin, a specific inhibitor of DNA polymerase alpha (12,13). Twentyfour hours after TNF administration the animals were killed and 150 mg of liver slices were incubated in a 1 ml Krebs phosphate buffer, pH 7.4 with or w i ~ o u t 25 ug/ml aphidicolin. The mixture was preincubated for 5 m ~ u t e s at 37 C in a shaking water bath followed by the addition of 20 uCi of [~H]thymidine with further incubation for 30 minutes and incorporation determined as above. As a positive control, 60-70% hepatectomy was carried out in another set of animals 24 hours prior to study. Values are mean + S.E.M. Statistical significance was determined by using a two-tailed Student' s t-test. RESULTS The time course of effect of the administration of TNF (25 ug/200 gm rat) on the incorporation of tritiated thymidine into ENA in the liver of intact animals is shown in Figure i.
A significant increase in [3H]thymidine incor-
poration is first seen 20 hours following TNF administration. increase in E~A synthesis is observed at 24 hours. returns to baseline.
The maximal
By 30 hours DNA synthesis
TNF administration did not alter [3H]thymidine incorpo-
ration into DNA in the heart or small intestine, indicating that the TNF 577
Vol. 153, No. 2, 1988
=~
~,
(b
25-
/I\
20-
0,.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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o:o
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~
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o
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30
40
50
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Fig. 1. Time co~rse of the effect of TNF on ~ synthesis in the liver. At ~.e times indicated after TNF administration, animals were injected with H-thymidine, One hour later incorporation of thymidine into ~ was determined. Closed circles - TNF animals (N=5 for each point). Open circles - control animals (N=5 for each point), *p <. 05. FiG. 2. The effect of various doses of TNF on ~ synthesis. Animals were administered the indicated dose of TNF and 24 hours later the incorporation of thymidine into E~A determined. TNF animals (N=5 for each point), 0 point
(N=20).
induced increase in E~A synthesis in the liver is not a generalized nonspecific phenc~enon. Figure 2 illustrates the dose response curve for the stimulation of [3H]thymidine incorporation into DNA in the liver of intact animals 24 hours following TNF.
The lowest dose of TNF that significantly increases DNA
synthesis in the liver is i0 ug/200 g rat (1.7 fold increase).
The maximal
increase occurs with the administration of 25 ug/200 g rat (4.8 fold increase).
Higher doses of TNF (50 ug/200 g rat) resulted in only a 2.5 fold
increase in DNA synthesis.
These results thus indicate that relatively small
quantities of TNF stimulate hepatic DNA synthesis in vivo. E~A polymerase alpha is associated with replication of DNA whereas DNA polymerase beta plays a role in repair of E~A (14,15).
DNA polymerase alpha
can be specifically inhibited by aphidicolin and, thus, incorporation of [3H]thymidine into E~A in the presence or absence of aphidicolin can be used to quantitate the activity of DNA polymerase alpha and by ialolication, DNA replication (12,13).
The incorporation of thymidine into DNA that is not
inhibited by aphidicolin is representative of DNA polymerase beta and garm~ activity.
To validate this assay we studied animals who had undergone a 578
60
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12 /
I0
D
O3
__J
B c ~5 E ~
8
--
4
O
=~
6
E 2
c mf total
,iii
c tnf c tnf alpha beta & gamma DNA polymerase
Fiq. 3. Effect of TNF on E~A polymerase alpha, beta, and garana. Animals were injected with 25 ug TNF. Twenty-four hours later the incorporation of Hthymidine into E~A was determined in liver slices incubated in the presence or absence of 25 ug/ml of aphidicolin. Controls (N=7), TNF animals (N=6) 60-70% hepatectomy 24 hours prior to study.
Incorporation of [3H]thymidine
into DNA was, as expected, markedly increased in the hepatectomized animals (control (N=7) 1.03 + .09 vs. hepatectamy (N=3) 34.7 + 4.33 pmoles incorporated/ug DNA, p <.001).
Moreover, DNA polymerase alpha activity was 40 fold in-
creased in hepatectomized animals (control 0.853 + .091 vs. hepatectomy 34.1 + 4.31 pmoles/incorporated/ug DNA, p <.001).
In contrast, the activity of DNA
polymerase beta plus gar~a was only slightly increased (control 0.176 _+ .020 vs. hepatectomy 0.593 + .070 pmoles/incorporated/ug DNA, p <.001).
These
results indicate, as expected, that partial hepatectomy leads to a marked increase in hepatic DNA replication. Figure 3 presents the effect of TNF administration on [3H]thymidine incorporation in liver slices mediated by DNA polymerase alpha and DNA polymerase beta plus gamma.
In agreement with our in vivo observations, total thymidine
incorporation into ~
is i n c h e d
9 fold in animals administered TNF.
Of
more iai0ortance, DNA polymerase alpha activity is increased 10.5 fold in the TNF treated animals whereas the activity of beta plus gamma DNA polymerase is increased only 1.6 fold.
These results indicate that the increased incorpora-
tion of [3H]thymidine into E~A in animals administered TNF is due almost entirely to an increase in DNA replication. 579
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION Studies have demonstrated that hormones can stimulate [3H]thymidine incorporation into DNA in the liver.
For exani01e, triiodothyronine (I00 ug/160 g
rat) leads to a 7 fold increase in the incorporation of thymidine into DNA in vivo (16). similarly EGF (34 ug/200 g rat) results in 3 fold increase in hepatic thymidine incorporation (17). In addition, glucagon, insulin and vasopressin have all been reported to enhance hepatic DNA synthesis (17,18). In the present manuscript we report the first evidence that the cytokine TNF can stimulate DNA synthesis in the liver. As illustrated in Figure 2, the administration of small doses of TNF (I0 ug/200 g rat) can stimulate hepatic DNA synthesis.
The dose of TNF that
increases DNA synthesis is considerably less than the dose that half maximally increases plasma triglycerides, a well described biological effect of TNF (7,10). TNF.
A maximal increase in DNA synthesis (4.8 fold) is observed with 25 ug
This dose is approximately one-quarter of that shown to produce tumor
necrosis in vivo (19). The increase in the incorporation of thymidine into I~A following TNF could result from injury to ENA followed by DNA repair, or alternatively might represent a stimulation of de novo DNA replication.
Obviously the signifi-
cance of the TNF effect could be quite different depending on which process was responsible.
Our study also presents evidence indicating that this
stimulation is due to an increase in ENA polymerase alpha activity.
DNA
polymerase alpha activity is associated with the replication of DNA and is increased 10.5 fold in the TNF animals.
In contrast, the activity of DNA
polymerase beta, which is believed to play a role in the repair of DNA and I~A polymerase gaam~, is increased only 1.6 fold in the animals administered TNF. These results indicate that the increased incorporation of thymidine into DNA in the liver of TNF treated animals is aecounted for by an increase in DNA replication.
Furthermore, the ability of TNF to stimulate hepatic DNA repli-
cation is not a generalized non-specific phenomenon, as 3H-thymidine incorporation was not increased in the heart and small intestine. 580
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Of interest is that the time course of the stimulation of E~A synthesis following TNF (Fig. i) is very similar to that observed following hepatic resection (20).
In this light, Cornell has demonstrated that endotoxin can
stimulate thymidine incorporation into hepatic DNA (21). Endotoxin stimulates the release of TNF by macrophages and it is therefore possible that the endotoxin effect could be mediated by TNF (1-3). More significantly, Cornell has also demonstrated that the inhibition of the usual increase in circulating endotoxin concentrations that occurs following hepatic resection prevents the characteristic increase in hepatic DNA synthesis that occurs in response to liver resection (22). His observations suggest that endotoxin (and possibly TNF) may play a role in mediating liver regeneration following hepatectomy. The mechanism by which TNF increases hepatic DNA synthesis and the specific cell types stimulated have not yet been elucidated.
Whether TNF acts directly
or via inducing the release of other cytokines (such as interleukin-i or interferon-beta2) remains to be determined (23-26). cytokines will also affect hepatic DNA synthesis.
It is possible that other In conclusion, the present
study provides the first evidence that the cytokine TNF stimulates DNA replication in the liver.
ACKNOWLEDG~NTS: We thank J. Patton, H.M. Shepard, M.D. Siperstein, and M. Joe for their support. This work was supported by grants from the Veterans Administration and the NIH (AM-37102, CA 15979). Dr. Feingold and Dr. Grunfeld are the recipients of clinical Investigator Awards from the V.A.
REFERENCES i. Old, L.J. (1984) Science 230,630-632. 2. Shalaby, M.R., Pennian, D., and Palladino, M.A., Jr. (1986) se~in. Immunopathol. 9,33-37. 3. Beutler, B., and Cerami, A. (1986) Nature 320,584-588. 4. Sugarman, B.J., Aggarwal, B.B., Hass, P.E., et al (1985) Science 230, 943-945. 5. Vilcek, J., Palcm%bella, V.J., Henriksen-DeStefano, D., et al (1986) J. Exp. Med. 163,632-643. 6. Nathan, C.F. (1987) J. clin. Invest. 79,319-326. 7. Beutler, B., and Cerami, A. (1987) New Engl. J. M~d. 316,379-385. 8. Perl/m/tter, D.H, Dinarello, C.A., Punsal, P.I., and Colten, H.R. (1986) J. clin. Invest. 78,1349-1354. 9. Darlington, G.J., Wilson, D.R., and lachman, L.B. (1986). J. Cell Biol. 103,787-793. i0. Feingold, K.R., and Grunfeld, C. (1987) J. clin. Invest. 80,184-190. 581
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ii. Schneider, W.C. (1957) In Methods of Enzymology (S.P. Colowiek, and N.O. Eaplan), Vol. III, pp. 680-684. Academic Press, Inc., New York, New York. 12. Pedrali-Noy, G., and Spadari, S. (1979) Biochem. and Biophys. Res. Co~m. 88,1194-1202. 13. Hubealmm~, J.A. (1981). Cell 23,647-648. 14. Keir, H.M., Craig, R.K., and McLennan, A.G. (1977) Biochem. Soc. Syrup. 42,37-54. 15. Fry, M., and Loeb, L.A. (1986) CRC Publication #6507. 16. Short, J., Brown, R.F., Husakova, A., et al (1972) J. Biol. C~em. 247, 1757-1766. 17. Bucher, N.L.R., Patel, U., and Cohen, S. (1978) Advances in Enzyme Regulation 16,205-213. 18. Leffert, H.L., Koch, K.S., Moran, T., and Rubalcava, B. (1979) Gastroenterology 76,1470-1482. 19. Patton, J.S., Peters, P.M., McCabe, J., Crase, D., Hansen, S., C~en, A.B., and Liggitt, D. (1987) J. clin. Invest. 80,1587-1596. 20. Bucher, N.L.R., and Swatfield, M.N. (1975) In Advances in Enzyme Regulation 13,281-293. 21. Cornell, R.P. (1985) Am. J. Physiol. 249 (Regulatory Integrative Comp. Physiol 18) ,R551-562. 22. Cornell, R.P. (1985) Am. J. Physiol. 249 (Regulatory Integrative Comp. Physiol 18) ,R563-569. 23. Libby, P., Ordovas, J.M., Auger, K.R., Robbins, A.H., Birinyi, L.K., and Dinarello, C.A. (1986) Am. J. Pathol. 124,179-185. 24. Nawroth, P.P., Bank, I., Handley, D., Cassimeris, J., Chess, L., and Ster~, D. (1986) J. Exp. Med. 163,1363-1375. 25. Zilberstein, A., Ruggieri, R., Korn, J.H., and Revel, M. (1986) Embo. J. 5,2529-2537. 26. Kohase, M., Henriksen-DeStefano, D., May, L.T., Vilcek, J., and Sehgal, P.B. (1986) Cell 45,659-666.
582
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 576-582
%~dMOR NECROSIS FACTOR STIMUIATES DNA SYNTHESIS IN THE LIVER OF INTACT RATS Kenneth R. Feingold, Mounzer Soued and Carl Grunfeld With the Technical Assistance of Arthur H. Moser, Jennifer A. Verdier and Heather G. DoVale Department of Medicine, University of California, San Francisco, CA,
and
Metabolism Section, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, CA 94121 Received April 8, 1988
Summary: TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [ H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [ H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is i0 ug/200 g rat with a maximal increase occuring with 25 ug/200 g, considerably less than the dose re~u.ired for maximally increasing plasma triglycerides. The increase in [ H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals,
© 1988 Academic Press, Inc.
Tumor necrosis factor (TNF) is secreted by macrophages in response to endotoxin and produces hemorrhagic necrosis of tumors (1-3). Sugarman, et al, have shown that TNF is directly cytotoxic to a variety of tumor cell lines (4). These investigators also observed that TNF enhances growth in some non-transformed diploid human cell lines.
Others have also shown that TNF
stimulates normal fibroblast division (5). In addition TNF has a large number of other biological actions including being a prime mediator of the metabolic effects of sepsis, especially hyperlipidemia (1-3,6,7). Recently studies have demonstrated that TNF regulates hepatic function. Perlmutter, et al, and Darlington, et al, have shown that TNF stimulates the production of acute phase proteins by hepatoma cells (8,9). Our laboratory has demonstrated that TNF stimulates beth hepatic fatty acid and cholesterol synthesis in intact animals (i0). Durir~ these studies we observed that TNF 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
576
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
administration produces a 21% increase in liver mass which could not be accounted for by an increase in either lipid accumulation or glycogen storage. In this manuscript we demonstrate that TNF administration stimulates DNA synthesis in the liver of intact rats. MATERIAIS AND METHODS Materials: [3H-Methyl]thymidine (78.8 Ci/ mmole) was purchased from New England Nuclear. Male Sprague-Dawley rats were purchased from Simonsen animal vendors. Aphidicolin and calf thymus DNA Type I were purchased from Sigma Chemical Company. 7Human tumor necrosis factor alpha (TNF) with a specific activity of 5 x i0 units/mg was provided by Dr. Shepard of Genentech, Inc. Methods: On the day prior to study animals were injected intravenously with TNF in 0.9% saline or saline alone. Animals were fasted after injection. The next day the rats were injected intraperitoneally with 50 uCi of [ H]thymid~_e. One hour later the rats were killed and the liver excised and weighed. [ H]thymidine incorporation into DNA and total DNA was determined according to the method of Schneider (Ii). Briefly, a 250 mg portion of liver was homogenized in 1 ml of H_O after which 5 ml of cold 10% TCA was added. Ten minutes later the material was centrifuged at 2,000 g at 4° for 5 rain. The supernatant was discarded and the precipitant material washed 2 times with 2 ml of 5% cold TCA followed by centrifugation 6 The precipitated material was then suspended in 2 ml, 5% TCA, heated at 90 C in a water bath for 15 rain. and centrifuged. An aliquod of the supernatant containing the hydrolyzed DNA was counted by liquid scintillation. Total DNA was then determined by incubating 1 ml of the supernatant with 2 ml diphenylamine reagent (i g diphenylamine ~lus i00 ml glacial acetic acid plus 2.75 ml concentrated sulfuric acid) at i00 C for 15 min. After cooling, the absorbance was determined at 600 nm. . ~ polymerase alpha activity was determined by measuring the incorporation of [ H]thymidine into DNA in liver slices incubated in the presence or absence of aphidicolin, a specific inhibitor of DNA polymerase alpha (12,13). Twentyfour hours after TNF administration the animals were killed and 150 mg of liver slices were incubated in a 1 ml Krebs phosphate buffer, pH 7.4 with or w i ~ o u t 25 ug/ml aphidicolin. The mixture was preincubated for 5 m ~ u t e s at 37 C in a shaking water bath followed by the addition of 20 uCi of [~H]thymidine with further incubation for 30 minutes and incorporation determined as above. As a positive control, 60-70% hepatectomy was carried out in another set of animals 24 hours prior to study. Values are mean + S.E.M. Statistical significance was determined by using a two-tailed Student' s t-test. RESULTS The time course of effect of the administration of TNF (25 ug/200 gm rat) on the incorporation of tritiated thymidine into ENA in the liver of intact animals is shown in Figure i.
A significant increase in [3H]thymidine incor-
poration is first seen 20 hours following TNF administration. increase in E~A synthesis is observed at 24 hours. returns to baseline.
The maximal
By 30 hours DNA synthesis
TNF administration did not alter [3H]thymidine incorpo-
ration into DNA in the heart or small intestine, indicating that the TNF 577
Vol. 153, No. 2, 1988
=~
~,
(b
25-
/I\
20-
0,.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
z
o_ 2o
o:o
15-
~
~o-
>-
5-
0
o
--A/ E
18
2'4
0
3'o
0
Hours
[
I
I
I
I
I0
20
30
40
50
TNF (/zg)
Q
Fig. 1. Time co~rse of the effect of TNF on ~ synthesis in the liver. At ~.e times indicated after TNF administration, animals were injected with H-thymidine, One hour later incorporation of thymidine into ~ was determined. Closed circles - TNF animals (N=5 for each point). Open circles - control animals (N=5 for each point), *p <. 05. FiG. 2. The effect of various doses of TNF on ~ synthesis. Animals were administered the indicated dose of TNF and 24 hours later the incorporation of thymidine into E~A determined. TNF animals (N=5 for each point), 0 point
(N=20).
induced increase in E~A synthesis in the liver is not a generalized nonspecific phenc~enon. Figure 2 illustrates the dose response curve for the stimulation of [3H]thymidine incorporation into DNA in the liver of intact animals 24 hours following TNF.
The lowest dose of TNF that significantly increases DNA
synthesis in the liver is i0 ug/200 g rat (1.7 fold increase).
The maximal
increase occurs with the administration of 25 ug/200 g rat (4.8 fold increase).
Higher doses of TNF (50 ug/200 g rat) resulted in only a 2.5 fold
increase in DNA synthesis.
These results thus indicate that relatively small
quantities of TNF stimulate hepatic DNA synthesis in vivo. E~A polymerase alpha is associated with replication of DNA whereas DNA polymerase beta plays a role in repair of E~A (14,15).
DNA polymerase alpha
can be specifically inhibited by aphidicolin and, thus, incorporation of [3H]thymidine into E~A in the presence or absence of aphidicolin can be used to quantitate the activity of DNA polymerase alpha and by ialolication, DNA replication (12,13).
The incorporation of thymidine into DNA that is not
inhibited by aphidicolin is representative of DNA polymerase beta and garm~ activity.
To validate this assay we studied animals who had undergone a 578
60
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
12 /
I0
D
O3
__J
B c ~5 E ~
8
--
4
O
=~
6
E 2
c mf total
,iii
c tnf c tnf alpha beta & gamma DNA polymerase
Fiq. 3. Effect of TNF on E~A polymerase alpha, beta, and garana. Animals were injected with 25 ug TNF. Twenty-four hours later the incorporation of Hthymidine into E~A was determined in liver slices incubated in the presence or absence of 25 ug/ml of aphidicolin. Controls (N=7), TNF animals (N=6) 60-70% hepatectomy 24 hours prior to study.
Incorporation of [3H]thymidine
into DNA was, as expected, markedly increased in the hepatectomized animals (control (N=7) 1.03 + .09 vs. hepatectamy (N=3) 34.7 + 4.33 pmoles incorporated/ug DNA, p <.001).
Moreover, DNA polymerase alpha activity was 40 fold in-
creased in hepatectomized animals (control 0.853 + .091 vs. hepatectomy 34.1 + 4.31 pmoles/incorporated/ug DNA, p <.001).
In contrast, the activity of DNA
polymerase beta plus gar~a was only slightly increased (control 0.176 _+ .020 vs. hepatectomy 0.593 + .070 pmoles/incorporated/ug DNA, p <.001).
These
results indicate, as expected, that partial hepatectomy leads to a marked increase in hepatic DNA replication. Figure 3 presents the effect of TNF administration on [3H]thymidine incorporation in liver slices mediated by DNA polymerase alpha and DNA polymerase beta plus gamma.
In agreement with our in vivo observations, total thymidine
incorporation into ~
is i n c h e d
9 fold in animals administered TNF.
Of
more iai0ortance, DNA polymerase alpha activity is increased 10.5 fold in the TNF treated animals whereas the activity of beta plus gamma DNA polymerase is increased only 1.6 fold.
These results indicate that the increased incorpora-
tion of [3H]thymidine into E~A in animals administered TNF is due almost entirely to an increase in DNA replication. 579
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION Studies have demonstrated that hormones can stimulate [3H]thymidine incorporation into DNA in the liver.
For exani01e, triiodothyronine (I00 ug/160 g
rat) leads to a 7 fold increase in the incorporation of thymidine into DNA in vivo (16). similarly EGF (34 ug/200 g rat) results in 3 fold increase in hepatic thymidine incorporation (17). In addition, glucagon, insulin and vasopressin have all been reported to enhance hepatic DNA synthesis (17,18). In the present manuscript we report the first evidence that the cytokine TNF can stimulate DNA synthesis in the liver. As illustrated in Figure 2, the administration of small doses of TNF (I0 ug/200 g rat) can stimulate hepatic DNA synthesis.
The dose of TNF that
increases DNA synthesis is considerably less than the dose that half maximally increases plasma triglycerides, a well described biological effect of TNF (7,10). TNF.
A maximal increase in DNA synthesis (4.8 fold) is observed with 25 ug
This dose is approximately one-quarter of that shown to produce tumor
necrosis in vivo (19). The increase in the incorporation of thymidine into I~A following TNF could result from injury to ENA followed by DNA repair, or alternatively might represent a stimulation of de novo DNA replication.
Obviously the signifi-
cance of the TNF effect could be quite different depending on which process was responsible.
Our study also presents evidence indicating that this
stimulation is due to an increase in ENA polymerase alpha activity.
DNA
polymerase alpha activity is associated with the replication of DNA and is increased 10.5 fold in the TNF animals.
In contrast, the activity of DNA
polymerase beta, which is believed to play a role in the repair of DNA and I~A polymerase gaam~, is increased only 1.6 fold in the animals administered TNF. These results indicate that the increased incorporation of thymidine into DNA in the liver of TNF treated animals is aecounted for by an increase in DNA replication.
Furthermore, the ability of TNF to stimulate hepatic DNA repli-
cation is not a generalized non-specific phenomenon, as 3H-thymidine incorporation was not increased in the heart and small intestine. 580
Vol. 153, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Of interest is that the time course of the stimulation of E~A synthesis following TNF (Fig. i) is very similar to that observed following hepatic resection (20).
In this light, Cornell has demonstrated that endotoxin can
stimulate thymidine incorporation into hepatic DNA (21). Endotoxin stimulates the release of TNF by macrophages and it is therefore possible that the endotoxin effect could be mediated by TNF (1-3). More significantly, Cornell has also demonstrated that the inhibition of the usual increase in circulating endotoxin concentrations that occurs following hepatic resection prevents the characteristic increase in hepatic DNA synthesis that occurs in response to liver resection (22). His observations suggest that endotoxin (and possibly TNF) may play a role in mediating liver regeneration following hepatectomy. The mechanism by which TNF increases hepatic DNA synthesis and the specific cell types stimulated have not yet been elucidated.
Whether TNF acts directly
or via inducing the release of other cytokines (such as interleukin-i or interferon-beta2) remains to be determined (23-26). cytokines will also affect hepatic DNA synthesis.
It is possible that other In conclusion, the present
study provides the first evidence that the cytokine TNF stimulates DNA replication in the liver.
ACKNOWLEDG~NTS: We thank J. Patton, H.M. Shepard, M.D. Siperstein, and M. Joe for their support. This work was supported by grants from the Veterans Administration and the NIH (AM-37102, CA 15979). Dr. Feingold and Dr. Grunfeld are the recipients of clinical Investigator Awards from the V.A.
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