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Stimulation of incorporation by triiodothyronine of thymidine-methyl-3H into hepatic DNA of the rat
ARC’HIVES
OF
BIOCHEMISTRY
AND
Stimulation
125,
BIOPHYSIL’S
of Incorporation
Thymidine-Methyl?H KAI-LIN Department
751-757
LEEI,
of Biochemistry Medicine, Tulane Received
by
into SHIH-CHIEN
and the Nutrition University School September
(lYG8)
Triiodothyronine
Hepatic
DNA
SUN,
0. NEAL
AND
and Metabolism Research of Medicine, AYew Orleans,
12, 1967;
accepted
November
of
of the
Rat
MILLER”
Laboratory,
Louisiana
Department
of
70112
14, 1967
Administration of single dose of triiodothyronine (Ta) results in hepatic cell proliferation as judged by the mitotic index and the enhanced thymidine-methyl-LH or orthophosphate-32P incorporation into DFA. Increased mitoses could be observed 24 hours after TB administration; 72 hours after the hormone injection, binuclear cells were abundant. The enhanced hepatic D?JA synthesis reached its peak 24-48 hours after Ta injection. Radioautographic techniques revealed that T1 increases 1)FiA synthesis primarily in the hepatic cells. Ta had no effect on testicular DNA synthesis. Ts-induced liver DNA synthesis was sensitive to the inhibitory effects of actinomycin D and cycloheximide. The present observations indicate that the early effects of thyroid hormone on liver protein and RNA synthesis initiates l)NA syllthesis, which in turn leads to the increased proliferation of hepatic tissue.
That thyroid hormone has a profound effect on the growt’h and development of certain vertebrates has been recognized for many years. In 1939, Sternheimer (1) studied how a single dose of thyroxine affected hepatic constituents and hepatic growth of rats, and noted changesin the size and structure of liver cells, including mitoses. Under the sameexperimental conditions, the fresh and dry weight of the liver also increased. Based on these observations, the author suggested that growth of the liver takes place after the administration of thyroid hormone, and that this growth may be directly responsible for increasing the oxygen consumption of this tissue (1). More recently, thyroid hormones have been observed to stimulate RNA synthesis and protein synthesis (2, 3), which further supports SDernheimer’s concept’. Siegel and Tobias (4) have demonstrated, by radioautographic techniques, that the nucleus of the cultured 1 Present address: Oak Ridge National Laborat,ory, Oak Ridge, Tennessee 37830. 2 Present address: Hoffmann-La Roche Inc., Nutley, New Jersey 07110; to which reprint requests should be sent. 751
human kidney epithelial cell is the primary locus that concentrates the int.racellular thyroid hormone. Recently, Tabachnick and Giorgio (5) observed that both thyroxine and triiodothyronine (Ts) have strong binding affinities for histones and that this property possibly indicates a specific histonenucleic acid complex in the nucleus as a site of thyroid hormone action. To substantiate further the effect of thyroid hormone at the nuclear level in connection with its effect on hepatic growth, the effect of T, administration on the incorporation of thymidine-methyl-3H or orthophosphate-32P into DNA was studied. The results, presented in this report, clearly demonstrate that T3 stimulates the rate of hepatic DNA synthesis and that this increased DNA synthesis is subject to inhibition by actonomycin D or cycloheximide. Radioautography revealed that the increased hepatic synthesis of DNA is located primarily in the hepatocyte. Because Ts caused no change of the rate of incorporation of thymidine-methyl-3H into the DNA of testicular tissue, the data suggest that there is an organ specificity to this response.
752
LEE, MATERIALS
AND
SUN,
METHODS
Male rats (130-160 gm) obtained from the Charles River Breeding Laboratory were used. Thyroidectomized rats, not used until 4 weeks after receipt. were given 1% calcium-gluconate in their drinking water. L-Triiodothyronine (Na, salt), a product of the Sigma Chemical Co., was given intraperitoneally in alkaline saline (6) at a dosage of 100 @g/100 gm body weight to euthryroid animals and 25 pg/lOO gm body weight to thyroidectomized rats in the volume of approximately 1 ml, depending upon the size of the animal. Actinomycin D, kindly supplied by Merck, Sharp and Dohme, or cycloheximide (California Corporation for Biochemical Research) was dissolved in saline and administered at dosages specified in the legends. Thvmidine-methyl-3H (specific activity 6 C/mmole, Schwarz BioResearch, Inc., 100 PC/ ml) was injected intraperitoneally 2 hours prior to killing the animals at a dose of 80 pC/lOO gm body weight. Carrier-free orthophosphateJ*P was obtained from New England Nuclear Corp., and was administered at a dosage of 500 rC/lOO gm body weight in the volume of about 1 ml, depending upon the size of the animal, 1 hour before the animals were killed. Extraction of DNA. Rats were killed by decapitation, and the tissues were rapidly excised. Approximately 1 gm of liver or 1 gm of testis was weighed and homogenized in 10 ml of ice-cold distilled water in a glass homogenizer tube fitted with a Teflon pestle. Perchloric acid (PCA), 0.4 N, was added to the homogenate to make the final PCA concentration 0.2 N. The precipitate was washed three times with 0.2 N PCA and then successively extracted with 15 ml of potassium acetate (1%) in ethanol, an ethanol:chloroform mixture (3:1, v/v), an ethanol:ether mixture (3:1, v/v), and finally with ether. After complete removal of the ether, the dry powder was digested in 0.4 N KOH (12 ml) at 37” for 1 hour, cooled to 0”, and mixed with 7.5 ml of 1.2 N PCA. The precipitate obtained on acidifying the alkaline digest was then washed twice with 0.2 N PCA. DNA was extracted into 10 ml of PCA (0.4 N) and separated from the residue of the RNA and lipid-free precipitate by heating for 15 minutes at 90”. It was observed later that the radioactivity of thymidine-methylJH was not incorporated into RNA or lipid; therefore, in some of the experiments, when thymidine-methyl3H was used, the DNA was extracted directly into hot PCA from the PCA-washed precipitate without removing the lipid and RNA. The pH of onehalf ml of DNA extract was adjusted to slight alkalinity with the addition of 0.04 ml of 6 N KOH; it was mixed with 10 ml of a polar scintillation fluid (7), and counted in the Packard-Tricarb
AND
MILLER liquid scintillation counter (for counting SW). Since constant amounts of DNA extract were used to count radioactivity, no corrections were made for quenching error. For 32P radioactivity measurements, 0.25 ml of the DNS extract was applied and dried on a planchet and counted in a Nuclear Chicago gas-flow counter. DNA was determined calorimetrically with diphenylamine reagent as described by Dische (8). Preparation of tissue sections and radioautography. Specimens of liver, taken from Ts-treated and saline-treated control rats, were fixed in 10% formalin and embedded in paraffin. Sections for histological examination or radioautography were made about 54 p thick. Radioautography was carried out on fixed, unstained sections according to the dipping method of Messier and Leblond (9) with the use of Kodak NTBz emulsion. The emulsion-coated slides with tissue sections in place were first exposed for 3-4 weeks at 4” in the dark and subsequently developed with Kodak D19 developer (2 minutes), and then fixed with Kodak acid fixer (2 minutes). After being washed and dried, the tissue sections were st.ained through the emulsion with hematoxylin-eosin, and were then cleared and mounted by routine histological techniques.
RESULTS Histological examination of rat liver afte, a single injection of T, . In agreement with Sternheimer (1)) active proliferation of hepatic cells was observed after administration of a single dose of Tz , Numerous cells in different phases of mitosis were observed TABLE EFFECT OF THF, INCORPORATION HICPATIC
AND
I
ADMINISTRATION OF Ta OF THYMIDINE-MICTHYLJH
ON
THE INTO
TEXTICUL~R
DNA
OF EUTHYROID
DNA
activity
(cpm/100
RATS (;;;$a
0
1 2 3 4
No. rats
7 7 7 3 3
specific
468 973 1091 681 448
f f f f f
pg)
Testis
Liver
24 71 110 67 68
407 374 370 400
* f f f -
28” 22 14 40
a TB was given at the dosage of 100 pg/lOO gm. Control rats received alkaline saline. ThymidinemethylJH (80 pC/lOO gm) was injected 2 hours before the rats were killed. b Mean f standard error of mean.
To-STI~~~LATION TABLE
OF
THY~~IDI~~
II
EFFECT OF THE ADMINISTRATION OF Ta ON THE INCORPORATION OF THY~IDI~E-~~~THYL-~H INTO H~PATIC AND TEXTICULAR DNA OF THYROIDECTOMIZED RATS Treatmenta
so.
rats
DXA
~-.--
specific
activity
kpm/lOO
Saline Ta
05
pg)
Testis
1 i-m
263 rfr 19 881 dz 93
433 412
f 26b zk 41
* T, was given at the dosage of 25 &lOO gm. Control rats received alkaline saline. Thymidinemethyl-3H (80 #Z/100 gm) was injected 2 hours before the animals were killed. All animals were killed 24 hours after the admil~istratio~i of Ta . b Mean f standard error of mean. TABLE
III
INCORPORATION OF ORTHOPHOSPH~~I~-~~P INTO HEPATIC DNA OF EUTHYROIU THYROIDECTDMIZED AND T3-T~~,\~~~ THYROIDECTOMIZICD
RATP Animal
Treatment
2;;
Euthyroid Thyroidectomized Th~roide~tomi~ed
&$;
~~~~~~ .-
Saline Saline Ta
3 7 7
4070 1814 3123
f + *
510 283 261
"T1 , administered at the dosage of 25 pg/lOO gm, was given 24 hours before the animals were killed. OrthophosphateJzP (500 &/lo0 gm) was injected intraperitoneally 1 hour before the animals were killed. Values represent mean f standard error of mean.
in the section of livers, taken at intervals of 24,48, and 72 hours after the administration of T3 . The average mitotic index was 0.43, 1.00, and 0.30 % [four animals per group), respectively, 24,45, and 72 hours after injection of Tf . No increase in mit,otic figures was observed under identical experimental conditions in the liver of saline-treated cont$rot rats. The incidence of binuclear cells in the control livers was about 4.3%, which gradually increased to 11.6 % (the highest percentage) 3 days after administration of Tf , which indicates the end point of Tzstimulated mitosis. These observations suggest that an increase of DNA synthesis mav occur after TB-administration. .&fed of T3 on the incorporation of thymi~l~r~e-~~eth~~-3~~ and ~th.opho~hute-~z~ into
INCORPORATION
INTO
DXA
753
heputic DNA. The rate of the incorporation of thymidine or phosphate into liver DNA was dependent on the thyroid status, as indicated in Tables I-III. After administration of a single dose of T, to the euthyroid animals, the incorporation of thymidine into liver DNA was greatly enhanced. The maximum, about twofold, of the increased incorporation occurred between 24 and 48 hours after administration of T, . By 4 days after the T3 injection, the incorporation of thymidine into hepatic DNA returned to the normal rate. Thyroidectomy causes a decreased rate of incorporation of thymidine into liver DNA, and this reduced rat’e of DNA synthesis was restored to above normal level 24 hours after injection of a small single dose of T3 (Table II). In addition, it was observed that the rate of of the incorporation of orthophosphat@P into hepatic DNA was decreasedin thyroidectomized rats, and this decreased rate of the iIleorporation of orthophosphate-3zP into hepatic DNA could be greatly enhanced after the administration of T3 to thyroidectomized rats (Table III). ~~~~ioa~~to~~a~}~i~ studies of T3 ~~~~~ hepatic Dhril ~~~lthe~~~. To further evaluate the rate of DNA synthesis under the influence of T3 , radioautographic techniques were employed. The results shown in Fig. 1 clearly demonstrate that the number of labeled ceils was increased in the liver sections obtained from either euthyroid or thy~idectomized rats that had been treated with T3 . ~~~rthernlore, the increased DNA synthesis was primarily localized in the hepatocyte (Table IV) during the early stages of T3 treatment. Five times as many cells were labeled in the 4S-hour specimen of liver as compared with the control liver specimen (Table IV). However, the increased rate of DNA synthesis, as measured by the radioactivity found in DNA of whole liver homogenate, revealed less marked changes (Table I). This may be becausethe number of labeled cells is not directly propo~ional to t,he amount of DNA synthesized in the liver, and in addition, this may be also due in part to the effect of dilution caused by the cells other than hepatocytes which are lessstimulated by TI .
754
LEE,
SUN, AND MILLER
Fra. 1. Radioa~Itography of rat liver after the injection of thymidine-methyl-3H. (a) Euthyroid alkaline saline-treat,ed control animal (X288). (b) ~uthyroid rat killed 24 hours after injection of T1 (X288). (c) Euthyroid rat killed 48 hours after injection of Ta (X288). (d) Thyroidectomy control rat (X288). (e) Thyroidectomy rat killed 24 hours after injection of Ts (X288).
E$ect of Ts on the incorporation of thymi~~n~-3~ethyl-3~into ~e~~~~~r DNA. Thyroid hormone increasesoxygen consumption, prot&n synthesis, and the level of mitochondrial
ar-glycerophosphate dehydrogenase (MGPDH) only in certain organs but not in others (10-12). The testis is an example of a tissue that is not responsive to 113treatment,
Ta-STIMULATION
OF
TABLE EFFECT
OF THE
OF T3
11.9 43.1 60.3
0 24 48
per
INTO
cells/1000
RAT
cells
(hours) Hepatocytes
a Mean group).
f
f f f
standard
Kupffer
0.4 3.6 5.5 error
24.7 26.5 32.9 of mean
cells
f f f
2.9” 2.2 1.1
(four
animals
and it w-as used in these experiments as an internal reference tissue. The results shown in Tables I and II demonstrate that T3 had no significant effect on testicular DNA synthesis. These results were expected because of the observations that oxygen consumption, protein synthesis, and the enzyme level of M-GPDH in the testis are not altered by differences in thyroid function (10-12). E$ect of inhibitors on T&nducad hepatic DNA synthesis. Considerable evidence (13, 14) indicates that an increased rate of synthesis of RNA and protein is an important event that precedes the entrance of cells into DNA synthesis. Consequently, the effect of actinomycin D and cyclohexamide on the TB-induced hepatic DNA synthesis was investigated. The result’s, presented in TABLE EFFECT
OF
D AND
ACTINOMYCIN
CYCLOHEXIMIDE
METHYI~H Treatmenta
Expt.
I
II
INTO
Inhibitor
None None Actinomycin Actinomycin None Noue Cycloheximide Cycloheximide
l-2
I> D
+ + + + -
INTO
7.55
DNA
Table V, demonstrate that actinomycin D (10 pg/lOO gm) slightly reduced the incorporation of thymidine-methyl-3H into liver DNA, but it completely prevented the Ts-induced hepatic DNA synthesis. The amount of actinomycin D used in the present study was considerably less than the amount used to produce maximum inhibition of RNA synthesis. Nevertheless, Chiga et al. (15) observed that actinomycin D at the dose of 13 pg/lOO gm body weight caused the nucleolar partition and delayed hepatic regeneration. These investigators suggested that this specific action of actinomycin D may result from its impairing RNA synthesis in the nucleolus. In addition, a low dosage of actinomycin D (0.S pg/lOO gm) could inhibit RNA synthesis in crypt cells of the epithelial lining of the jejunum of mice, which in t,urn blocks cells passing from G1 to the S-phase of the cell cycle (16). Cycloheximide, which has been shown to inhibit protein synthesis by blocking the transfer of amino acids from aminoacyl transfer RNA to the growing peptide (17), also blocked the Ts-induced liver DKA synthesis (Table V). Under identical experimental conditions, both ant)ibiotics were without effect on DNA synt’hesis in the testis (Table V). The present observations are consistent with the requirement of both RNA and protein synthesis before T, induces hepatic DNA synthesis.
THE
KUPFFER CELLS OF BY RADIOAUTOGRAPHY Labeled
Time
ON
OF THYMIDINE-METHYL-3H
HEPAT~CYTES >YND LIVER AS DETERMINED
[NCORPORATION
IV
ADMINISTRATION
INCORPORATION
THYMDINE
V ON
HEPATIC
THE
~~~~~~~~~~
DNA
DNA
No. rats
5 5 4 5 3 3 5 3
INCORPORATION
OF THYMINE-
OF RUTS specific
activity
(cpm/lOO
Liver
485 f 69.7 1066 It 50.5 320 f 39.7 312 f 19.7 451 920 291 288
f f f f
pg) Testis
22.3 52.3 39.6 25.7
329 349 311 325
f f f zb
27.3b 32.4 28.3 26.7
360 386 353 331
f f f f
58.4 37.0 51.6 48.1
n Tt was given (100 @g/100 gm) at zero time. Actinomycin D (10 pg/lOO gm) or cycloheximide (100 pg/lOO pm), in the volume of about 1 ml, depending upon the size of the animal, was administered intraperitoneally with the Ta . Thymidine-methyl-3H (8Opc/lOO gm) was injected 2 hours before the animals were killed. All rats were killed 24 hours after T, injection. b Mean f standard error of mean.
756
LEE,
SUN, AND MILLER
DISCUSSION
The reported data confirm and extend the early observations which showed that an active hepatic cell pro~eration takes place after a single injection of thyroid hormone (1). The incorporation of DNA precursor into liver DNA provides a more reliable method for studying this effect, because an increase of the mitotic index may not necessarily represent active cell proliferation but rather a mitotic arrest, such as that produced by colchicin, which also increasesthe mitotic index. The data, shown in Tables I-III, indicate that T3 administration increases DNA replication of cells in the liver. Radioautographic studies of T&reated livers that were in the rapid phase of DNA synthesis revealed that this effect occurs primarily in the parenchymal cells (Table IV). The dosage of thyroid hormone used was much higher than the amount of thyroid hormone produced under physiological conditions; therefore, two questions should be considered. First, does the increased DNA synt,hesis under the described conditions represent a true physiolo~cal action of thyroid hormone; and second, does the administration of large dosesof Ts increase the production of endogenous pituitary growth hormone which in turn causesthe increased DNA4 synthesis. The following seemsto support the view that the data reported represent a physiological rather than a pharmacological response to T3 administration. Thyroid hormone appears to st~ulate both oxygen consumption and protein synthesis in the liver but not in the testis. The hepatic DNA synthesis, but not testicuIar DNA synthesis, responded to TS stimulation (Tables I and II). Furthermore, the incorporation of thymidine or phosphate into liver DNA of the thyroidectomized rat was less than the incorporation observed in the liver DNA of euthyroid rat (Tables I-III). This decreased liver DNA synthesis in the thyroidectomized rat could be rapidly re&ored to normal after TQ administration (Tables II and III), thus indicating that the action of thyroid hormone on DNA synthesis is a true physiological action. The laek of effect of growth hormone in stimulating liver DNA synthesis (18) suggeststhat the
TV-increased hepatic DNA synthesis is not mediated through the action of growth hormone. It is not possible at the present time to explain the actual mechanism by which thyroid hormone stimulates hepatic DNA synthesis. Since we studied the inco~oration of radioactive precursors of DNA into total liver DNA, the observed incorporation could result from either directly stimulating the synthesis of DNA by the hormone or the alteration in permeability, transport, or pool-size of the precursor(s) by the hormone. Nevertheless, the enhanced incorporation of either thymidine or orthophosphate into liver DNA along with the finding that thyroid hormone induces the hepatic proliferation supports the idea that thyroid hormone directly stimulates hepatic DNA synthesis. In addition, the inhibitory effects of actinlmycin D and cycloheximide on the T3-increased thymidine incorporation indicate that normal protein and RNA synthesis are required for the stimulation. Hormonal control of genetic expression (19) has been considers as one of the important functions of hormones. Although considerable evidence indicates that the nucleus may be the site of thyroid hormone action (4, 5), the enhanced hepatic DNA synthesis caused by TZ administration is unlikely to be a primary action of thyroid hormone for the following reasons: (1) there is a relatively long delay of 2448 hours before the induced DNA synthesis reaches its peak (Tables I and IV), and @) the increased RNA and protein synthesis take place in the liver during this lag period after T3 administration (2, 20) and before DNA synthesis begins. It can be speculated that thyroid hormone regulates the expression of specific regions of the genome and produces the early anabolic effects such as protein synthesis and RNA synthesis, which in turn initiates DNA synthesis. The inhibiting effects of actinomycin D and cycloheximide on the TB-induced DNA synthesis (Table IV) are consistent with such a concept. If this proves to be correct, then the enhanced DNA synthesis caused by T3 administ,ration can be considered as a final phase of the sequential events of thyroid
T,-STIMULATION
OF
hormone anabolic effects in the this increased DNA synthesis cells may be the earliest effect hormone on the proliferation tissue.
THYJIMIIDINE
liver, but of hepatic of thyroid of hepatic
ACKNOWLEDGMENTS This study was supported by grant TIGM-64% 06 from the United States Public Health Service. The authors thank Mrs. Marta N. Gutierrez for her preparation of tissue sections. REFERENCES 1. STERNHEIMER, R., Endocrinology 26, 899 (1939). 2. TATA, J. R., in “hlechanisms of Hormone Action” (P. Karlson, ed.), p. 173. Academic Press, New York (1965). 3. SOKOLOFF, L., ANDKSUFMAN,~., J.Biol.Chem. 236, 795 (1961). 4. SIEGEL, E., AND TOBIAS, C. A., Science 163, 763 (1966). 5. TABACHNICK, AM., AND GIORGIO, N. A., JR., Nature 212, 1610 (1966). 6. PITTMAN, C. A., AND BARKER, S. B., Am. J. Physiol. 197,127l (1959). 7. BRAY, G. A., Anal. Biochem. 1,279 (1960).
INCORPORATION
INTO
NA
c ,- Id i
8. L)ISCHE, z., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), lrol. 1. Academic Press, New York (1955). 9. MESSIER, B., AND LEBLOND, C. P., Proc. Sot. Exptl. Biol. Med. 96, 7 (1957). 10. GORDON, E. S., AND HEMING, A. E., Endocrinology 34,353 (1944). 11. MICHELS, R., CASON, J., AND SOI
OF
BIOCHEMISTRY
AND
Stimulation
125,
BIOPHYSIL’S
of Incorporation
Thymidine-Methyl?H KAI-LIN Department
751-757
LEEI,
of Biochemistry Medicine, Tulane Received
by
into SHIH-CHIEN
and the Nutrition University School September
(lYG8)
Triiodothyronine
Hepatic
DNA
SUN,
0. NEAL
AND
and Metabolism Research of Medicine, AYew Orleans,
12, 1967;
accepted
November
of
of the
Rat
MILLER”
Laboratory,
Louisiana
Department
of
70112
14, 1967
Administration of single dose of triiodothyronine (Ta) results in hepatic cell proliferation as judged by the mitotic index and the enhanced thymidine-methyl-LH or orthophosphate-32P incorporation into DFA. Increased mitoses could be observed 24 hours after TB administration; 72 hours after the hormone injection, binuclear cells were abundant. The enhanced hepatic D?JA synthesis reached its peak 24-48 hours after Ta injection. Radioautographic techniques revealed that T1 increases 1)FiA synthesis primarily in the hepatic cells. Ta had no effect on testicular DNA synthesis. Ts-induced liver DNA synthesis was sensitive to the inhibitory effects of actinomycin D and cycloheximide. The present observations indicate that the early effects of thyroid hormone on liver protein and RNA synthesis initiates l)NA syllthesis, which in turn leads to the increased proliferation of hepatic tissue.
That thyroid hormone has a profound effect on the growt’h and development of certain vertebrates has been recognized for many years. In 1939, Sternheimer (1) studied how a single dose of thyroxine affected hepatic constituents and hepatic growth of rats, and noted changesin the size and structure of liver cells, including mitoses. Under the sameexperimental conditions, the fresh and dry weight of the liver also increased. Based on these observations, the author suggested that growth of the liver takes place after the administration of thyroid hormone, and that this growth may be directly responsible for increasing the oxygen consumption of this tissue (1). More recently, thyroid hormones have been observed to stimulate RNA synthesis and protein synthesis (2, 3), which further supports SDernheimer’s concept’. Siegel and Tobias (4) have demonstrated, by radioautographic techniques, that the nucleus of the cultured 1 Present address: Oak Ridge National Laborat,ory, Oak Ridge, Tennessee 37830. 2 Present address: Hoffmann-La Roche Inc., Nutley, New Jersey 07110; to which reprint requests should be sent. 751
human kidney epithelial cell is the primary locus that concentrates the int.racellular thyroid hormone. Recently, Tabachnick and Giorgio (5) observed that both thyroxine and triiodothyronine (Ts) have strong binding affinities for histones and that this property possibly indicates a specific histonenucleic acid complex in the nucleus as a site of thyroid hormone action. To substantiate further the effect of thyroid hormone at the nuclear level in connection with its effect on hepatic growth, the effect of T, administration on the incorporation of thymidine-methyl-3H or orthophosphate-32P into DNA was studied. The results, presented in this report, clearly demonstrate that T3 stimulates the rate of hepatic DNA synthesis and that this increased DNA synthesis is subject to inhibition by actonomycin D or cycloheximide. Radioautography revealed that the increased hepatic synthesis of DNA is located primarily in the hepatocyte. Because Ts caused no change of the rate of incorporation of thymidine-methyl-3H into the DNA of testicular tissue, the data suggest that there is an organ specificity to this response.
752
LEE, MATERIALS
AND
SUN,
METHODS
Male rats (130-160 gm) obtained from the Charles River Breeding Laboratory were used. Thyroidectomized rats, not used until 4 weeks after receipt. were given 1% calcium-gluconate in their drinking water. L-Triiodothyronine (Na, salt), a product of the Sigma Chemical Co., was given intraperitoneally in alkaline saline (6) at a dosage of 100 @g/100 gm body weight to euthryroid animals and 25 pg/lOO gm body weight to thyroidectomized rats in the volume of approximately 1 ml, depending upon the size of the animal. Actinomycin D, kindly supplied by Merck, Sharp and Dohme, or cycloheximide (California Corporation for Biochemical Research) was dissolved in saline and administered at dosages specified in the legends. Thvmidine-methyl-3H (specific activity 6 C/mmole, Schwarz BioResearch, Inc., 100 PC/ ml) was injected intraperitoneally 2 hours prior to killing the animals at a dose of 80 pC/lOO gm body weight. Carrier-free orthophosphateJ*P was obtained from New England Nuclear Corp., and was administered at a dosage of 500 rC/lOO gm body weight in the volume of about 1 ml, depending upon the size of the animal, 1 hour before the animals were killed. Extraction of DNA. Rats were killed by decapitation, and the tissues were rapidly excised. Approximately 1 gm of liver or 1 gm of testis was weighed and homogenized in 10 ml of ice-cold distilled water in a glass homogenizer tube fitted with a Teflon pestle. Perchloric acid (PCA), 0.4 N, was added to the homogenate to make the final PCA concentration 0.2 N. The precipitate was washed three times with 0.2 N PCA and then successively extracted with 15 ml of potassium acetate (1%) in ethanol, an ethanol:chloroform mixture (3:1, v/v), an ethanol:ether mixture (3:1, v/v), and finally with ether. After complete removal of the ether, the dry powder was digested in 0.4 N KOH (12 ml) at 37” for 1 hour, cooled to 0”, and mixed with 7.5 ml of 1.2 N PCA. The precipitate obtained on acidifying the alkaline digest was then washed twice with 0.2 N PCA. DNA was extracted into 10 ml of PCA (0.4 N) and separated from the residue of the RNA and lipid-free precipitate by heating for 15 minutes at 90”. It was observed later that the radioactivity of thymidine-methylJH was not incorporated into RNA or lipid; therefore, in some of the experiments, when thymidine-methyl3H was used, the DNA was extracted directly into hot PCA from the PCA-washed precipitate without removing the lipid and RNA. The pH of onehalf ml of DNA extract was adjusted to slight alkalinity with the addition of 0.04 ml of 6 N KOH; it was mixed with 10 ml of a polar scintillation fluid (7), and counted in the Packard-Tricarb
AND
MILLER liquid scintillation counter (for counting SW). Since constant amounts of DNA extract were used to count radioactivity, no corrections were made for quenching error. For 32P radioactivity measurements, 0.25 ml of the DNS extract was applied and dried on a planchet and counted in a Nuclear Chicago gas-flow counter. DNA was determined calorimetrically with diphenylamine reagent as described by Dische (8). Preparation of tissue sections and radioautography. Specimens of liver, taken from Ts-treated and saline-treated control rats, were fixed in 10% formalin and embedded in paraffin. Sections for histological examination or radioautography were made about 54 p thick. Radioautography was carried out on fixed, unstained sections according to the dipping method of Messier and Leblond (9) with the use of Kodak NTBz emulsion. The emulsion-coated slides with tissue sections in place were first exposed for 3-4 weeks at 4” in the dark and subsequently developed with Kodak D19 developer (2 minutes), and then fixed with Kodak acid fixer (2 minutes). After being washed and dried, the tissue sections were st.ained through the emulsion with hematoxylin-eosin, and were then cleared and mounted by routine histological techniques.
RESULTS Histological examination of rat liver afte, a single injection of T, . In agreement with Sternheimer (1)) active proliferation of hepatic cells was observed after administration of a single dose of Tz , Numerous cells in different phases of mitosis were observed TABLE EFFECT OF THF, INCORPORATION HICPATIC
AND
I
ADMINISTRATION OF Ta OF THYMIDINE-MICTHYLJH
ON
THE INTO
TEXTICUL~R
DNA
OF EUTHYROID
DNA
activity
(cpm/100
RATS (;;;$a
0
1 2 3 4
No. rats
7 7 7 3 3
specific
468 973 1091 681 448
f f f f f
pg)
Testis
Liver
24 71 110 67 68
407 374 370 400
* f f f -
28” 22 14 40
a TB was given at the dosage of 100 pg/lOO gm. Control rats received alkaline saline. ThymidinemethylJH (80 pC/lOO gm) was injected 2 hours before the rats were killed. b Mean f standard error of mean.
To-STI~~~LATION TABLE
OF
THY~~IDI~~
II
EFFECT OF THE ADMINISTRATION OF Ta ON THE INCORPORATION OF THY~IDI~E-~~~THYL-~H INTO H~PATIC AND TEXTICULAR DNA OF THYROIDECTOMIZED RATS Treatmenta
so.
rats
DXA
~-.--
specific
activity
kpm/lOO
Saline Ta
05
pg)
Testis
1 i-m
263 rfr 19 881 dz 93
433 412
f 26b zk 41
* T, was given at the dosage of 25 &lOO gm. Control rats received alkaline saline. Thymidinemethyl-3H (80 #Z/100 gm) was injected 2 hours before the animals were killed. All animals were killed 24 hours after the admil~istratio~i of Ta . b Mean f standard error of mean. TABLE
III
INCORPORATION OF ORTHOPHOSPH~~I~-~~P INTO HEPATIC DNA OF EUTHYROIU THYROIDECTDMIZED AND T3-T~~,\~~~ THYROIDECTOMIZICD
RATP Animal
Treatment
2;;
Euthyroid Thyroidectomized Th~roide~tomi~ed
&$;
~~~~~~ .-
Saline Saline Ta
3 7 7
4070 1814 3123
f + *
510 283 261
"T1 , administered at the dosage of 25 pg/lOO gm, was given 24 hours before the animals were killed. OrthophosphateJzP (500 &/lo0 gm) was injected intraperitoneally 1 hour before the animals were killed. Values represent mean f standard error of mean.
in the section of livers, taken at intervals of 24,48, and 72 hours after the administration of T3 . The average mitotic index was 0.43, 1.00, and 0.30 % [four animals per group), respectively, 24,45, and 72 hours after injection of Tf . No increase in mit,otic figures was observed under identical experimental conditions in the liver of saline-treated cont$rot rats. The incidence of binuclear cells in the control livers was about 4.3%, which gradually increased to 11.6 % (the highest percentage) 3 days after administration of Tf , which indicates the end point of Tzstimulated mitosis. These observations suggest that an increase of DNA synthesis mav occur after TB-administration. .&fed of T3 on the incorporation of thymi~l~r~e-~~eth~~-3~~ and ~th.opho~hute-~z~ into
INCORPORATION
INTO
DXA
753
heputic DNA. The rate of the incorporation of thymidine or phosphate into liver DNA was dependent on the thyroid status, as indicated in Tables I-III. After administration of a single dose of T, to the euthyroid animals, the incorporation of thymidine into liver DNA was greatly enhanced. The maximum, about twofold, of the increased incorporation occurred between 24 and 48 hours after administration of T, . By 4 days after the T3 injection, the incorporation of thymidine into hepatic DNA returned to the normal rate. Thyroidectomy causes a decreased rate of incorporation of thymidine into liver DNA, and this reduced rat’e of DNA synthesis was restored to above normal level 24 hours after injection of a small single dose of T3 (Table II). In addition, it was observed that the rate of of the incorporation of orthophosphat@P into hepatic DNA was decreasedin thyroidectomized rats, and this decreased rate of the iIleorporation of orthophosphate-3zP into hepatic DNA could be greatly enhanced after the administration of T3 to thyroidectomized rats (Table III). ~~~~ioa~~to~~a~}~i~ studies of T3 ~~~~~ hepatic Dhril ~~~lthe~~~. To further evaluate the rate of DNA synthesis under the influence of T3 , radioautographic techniques were employed. The results shown in Fig. 1 clearly demonstrate that the number of labeled ceils was increased in the liver sections obtained from either euthyroid or thy~idectomized rats that had been treated with T3 . ~~~rthernlore, the increased DNA synthesis was primarily localized in the hepatocyte (Table IV) during the early stages of T3 treatment. Five times as many cells were labeled in the 4S-hour specimen of liver as compared with the control liver specimen (Table IV). However, the increased rate of DNA synthesis, as measured by the radioactivity found in DNA of whole liver homogenate, revealed less marked changes (Table I). This may be becausethe number of labeled cells is not directly propo~ional to t,he amount of DNA synthesized in the liver, and in addition, this may be also due in part to the effect of dilution caused by the cells other than hepatocytes which are lessstimulated by TI .
754
LEE,
SUN, AND MILLER
Fra. 1. Radioa~Itography of rat liver after the injection of thymidine-methyl-3H. (a) Euthyroid alkaline saline-treat,ed control animal (X288). (b) ~uthyroid rat killed 24 hours after injection of T1 (X288). (c) Euthyroid rat killed 48 hours after injection of Ta (X288). (d) Thyroidectomy control rat (X288). (e) Thyroidectomy rat killed 24 hours after injection of Ts (X288).
E$ect of Ts on the incorporation of thymi~~n~-3~ethyl-3~into ~e~~~~~r DNA. Thyroid hormone increasesoxygen consumption, prot&n synthesis, and the level of mitochondrial
ar-glycerophosphate dehydrogenase (MGPDH) only in certain organs but not in others (10-12). The testis is an example of a tissue that is not responsive to 113treatment,
Ta-STIMULATION
OF
TABLE EFFECT
OF THE
OF T3
11.9 43.1 60.3
0 24 48
per
INTO
cells/1000
RAT
cells
(hours) Hepatocytes
a Mean group).
f
f f f
standard
Kupffer
0.4 3.6 5.5 error
24.7 26.5 32.9 of mean
cells
f f f
2.9” 2.2 1.1
(four
animals
and it w-as used in these experiments as an internal reference tissue. The results shown in Tables I and II demonstrate that T3 had no significant effect on testicular DNA synthesis. These results were expected because of the observations that oxygen consumption, protein synthesis, and the enzyme level of M-GPDH in the testis are not altered by differences in thyroid function (10-12). E$ect of inhibitors on T&nducad hepatic DNA synthesis. Considerable evidence (13, 14) indicates that an increased rate of synthesis of RNA and protein is an important event that precedes the entrance of cells into DNA synthesis. Consequently, the effect of actinomycin D and cyclohexamide on the TB-induced hepatic DNA synthesis was investigated. The result’s, presented in TABLE EFFECT
OF
D AND
ACTINOMYCIN
CYCLOHEXIMIDE
METHYI~H Treatmenta
Expt.
I
II
INTO
Inhibitor
None None Actinomycin Actinomycin None Noue Cycloheximide Cycloheximide
l-2
I> D
+ + + + -
INTO
7.55
DNA
Table V, demonstrate that actinomycin D (10 pg/lOO gm) slightly reduced the incorporation of thymidine-methyl-3H into liver DNA, but it completely prevented the Ts-induced hepatic DNA synthesis. The amount of actinomycin D used in the present study was considerably less than the amount used to produce maximum inhibition of RNA synthesis. Nevertheless, Chiga et al. (15) observed that actinomycin D at the dose of 13 pg/lOO gm body weight caused the nucleolar partition and delayed hepatic regeneration. These investigators suggested that this specific action of actinomycin D may result from its impairing RNA synthesis in the nucleolus. In addition, a low dosage of actinomycin D (0.S pg/lOO gm) could inhibit RNA synthesis in crypt cells of the epithelial lining of the jejunum of mice, which in t,urn blocks cells passing from G1 to the S-phase of the cell cycle (16). Cycloheximide, which has been shown to inhibit protein synthesis by blocking the transfer of amino acids from aminoacyl transfer RNA to the growing peptide (17), also blocked the Ts-induced liver DKA synthesis (Table V). Under identical experimental conditions, both ant)ibiotics were without effect on DNA synt’hesis in the testis (Table V). The present observations are consistent with the requirement of both RNA and protein synthesis before T, induces hepatic DNA synthesis.
THE
KUPFFER CELLS OF BY RADIOAUTOGRAPHY Labeled
Time
ON
OF THYMIDINE-METHYL-3H
HEPAT~CYTES >YND LIVER AS DETERMINED
[NCORPORATION
IV
ADMINISTRATION
INCORPORATION
THYMDINE
V ON
HEPATIC
THE
~~~~~~~~~~
DNA
DNA
No. rats
5 5 4 5 3 3 5 3
INCORPORATION
OF THYMINE-
OF RUTS specific
activity
(cpm/lOO
Liver
485 f 69.7 1066 It 50.5 320 f 39.7 312 f 19.7 451 920 291 288
f f f f
pg) Testis
22.3 52.3 39.6 25.7
329 349 311 325
f f f zb
27.3b 32.4 28.3 26.7
360 386 353 331
f f f f
58.4 37.0 51.6 48.1
n Tt was given (100 @g/100 gm) at zero time. Actinomycin D (10 pg/lOO gm) or cycloheximide (100 pg/lOO pm), in the volume of about 1 ml, depending upon the size of the animal, was administered intraperitoneally with the Ta . Thymidine-methyl-3H (8Opc/lOO gm) was injected 2 hours before the animals were killed. All rats were killed 24 hours after T, injection. b Mean f standard error of mean.
756
LEE,
SUN, AND MILLER
DISCUSSION
The reported data confirm and extend the early observations which showed that an active hepatic cell pro~eration takes place after a single injection of thyroid hormone (1). The incorporation of DNA precursor into liver DNA provides a more reliable method for studying this effect, because an increase of the mitotic index may not necessarily represent active cell proliferation but rather a mitotic arrest, such as that produced by colchicin, which also increasesthe mitotic index. The data, shown in Tables I-III, indicate that T3 administration increases DNA replication of cells in the liver. Radioautographic studies of T&reated livers that were in the rapid phase of DNA synthesis revealed that this effect occurs primarily in the parenchymal cells (Table IV). The dosage of thyroid hormone used was much higher than the amount of thyroid hormone produced under physiological conditions; therefore, two questions should be considered. First, does the increased DNA synt,hesis under the described conditions represent a true physiolo~cal action of thyroid hormone; and second, does the administration of large dosesof Ts increase the production of endogenous pituitary growth hormone which in turn causesthe increased DNA4 synthesis. The following seemsto support the view that the data reported represent a physiological rather than a pharmacological response to T3 administration. Thyroid hormone appears to st~ulate both oxygen consumption and protein synthesis in the liver but not in the testis. The hepatic DNA synthesis, but not testicuIar DNA synthesis, responded to TS stimulation (Tables I and II). Furthermore, the incorporation of thymidine or phosphate into liver DNA of the thyroidectomized rat was less than the incorporation observed in the liver DNA of euthyroid rat (Tables I-III). This decreased liver DNA synthesis in the thyroidectomized rat could be rapidly re&ored to normal after TQ administration (Tables II and III), thus indicating that the action of thyroid hormone on DNA synthesis is a true physiological action. The laek of effect of growth hormone in stimulating liver DNA synthesis (18) suggeststhat the
TV-increased hepatic DNA synthesis is not mediated through the action of growth hormone. It is not possible at the present time to explain the actual mechanism by which thyroid hormone stimulates hepatic DNA synthesis. Since we studied the inco~oration of radioactive precursors of DNA into total liver DNA, the observed incorporation could result from either directly stimulating the synthesis of DNA by the hormone or the alteration in permeability, transport, or pool-size of the precursor(s) by the hormone. Nevertheless, the enhanced incorporation of either thymidine or orthophosphate into liver DNA along with the finding that thyroid hormone induces the hepatic proliferation supports the idea that thyroid hormone directly stimulates hepatic DNA synthesis. In addition, the inhibitory effects of actinlmycin D and cycloheximide on the T3-increased thymidine incorporation indicate that normal protein and RNA synthesis are required for the stimulation. Hormonal control of genetic expression (19) has been considers as one of the important functions of hormones. Although considerable evidence indicates that the nucleus may be the site of thyroid hormone action (4, 5), the enhanced hepatic DNA synthesis caused by TZ administration is unlikely to be a primary action of thyroid hormone for the following reasons: (1) there is a relatively long delay of 2448 hours before the induced DNA synthesis reaches its peak (Tables I and IV), and @) the increased RNA and protein synthesis take place in the liver during this lag period after T3 administration (2, 20) and before DNA synthesis begins. It can be speculated that thyroid hormone regulates the expression of specific regions of the genome and produces the early anabolic effects such as protein synthesis and RNA synthesis, which in turn initiates DNA synthesis. The inhibiting effects of actinomycin D and cycloheximide on the TB-induced DNA synthesis (Table IV) are consistent with such a concept. If this proves to be correct, then the enhanced DNA synthesis caused by T3 administ,ration can be considered as a final phase of the sequential events of thyroid
T,-STIMULATION
OF
hormone anabolic effects in the this increased DNA synthesis cells may be the earliest effect hormone on the proliferation tissue.
THYJIMIIDINE
liver, but of hepatic of thyroid of hepatic
ACKNOWLEDGMENTS This study was supported by grant TIGM-64% 06 from the United States Public Health Service. The authors thank Mrs. Marta N. Gutierrez for her preparation of tissue sections. REFERENCES 1. STERNHEIMER, R., Endocrinology 26, 899 (1939). 2. TATA, J. R., in “hlechanisms of Hormone Action” (P. Karlson, ed.), p. 173. Academic Press, New York (1965). 3. SOKOLOFF, L., ANDKSUFMAN,~., J.Biol.Chem. 236, 795 (1961). 4. SIEGEL, E., AND TOBIAS, C. A., Science 163, 763 (1966). 5. TABACHNICK, AM., AND GIORGIO, N. A., JR., Nature 212, 1610 (1966). 6. PITTMAN, C. A., AND BARKER, S. B., Am. J. Physiol. 197,127l (1959). 7. BRAY, G. A., Anal. Biochem. 1,279 (1960).
INCORPORATION
INTO
NA
c ,- Id i
8. L)ISCHE, z., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), lrol. 1. Academic Press, New York (1955). 9. MESSIER, B., AND LEBLOND, C. P., Proc. Sot. Exptl. Biol. Med. 96, 7 (1957). 10. GORDON, E. S., AND HEMING, A. E., Endocrinology 34,353 (1944). 11. MICHELS, R., CASON, J., AND SOI