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Liver regeneration in the hypophysectomized rat: Influence of corticosteroids on the size distribution and synthesis of ribosomal components
ARCHIVES
OF
Liver
BIOCHEYISTRY
AND
Regeneration
in the
Corticosteroids Synthesis THOMAS McGill,
University
Cancer
126,
BIOPHYSICS
Research University
69-75
(1968)
Hypophysectomized on the
Size
of Ribosomal
E. WEBB
AND
Unit, McInfyre of Manitoba, Received
Distribution
September
Influence
of
and
Components’
LEOKARD
Medical Winnipeg,
Rat:
R. WOZNEY
Sciences B&ding, Montreal, Manitoba, Canada
&uebec;
and
the
18, 1967
The increased incorporation of label from erotic-G-1°C acid into ribosomal RNA and changes in the size distribution of ribosomal components, normally occurring during t,he early phases of liver regeneration in the intact rat, are also observed in the regenerating liver of the hypophysectomized rat. Neither parameter in the resting and regenerating liver was adversely affected by the administration of pharmacological doses of corticosteroids to the int,act rat. However, identical treatment of the hypophysectomized rat resulted in a marked inhibition of the incorporation of label from erotic-6-l% acid into ribosomal RNA and partial breakdown of the polyribosomes in the regenerating liver within 20 hours of partial hepatectomy. The phenomena appear to be peculiar to proliferating tissue, since pharmacological doses of hydrocortisone neither caused polyribosome breakdown nor inhibited erotic-6-l% acid incorporation iu the resting liver of the hypophgsectomized rat.
The rate of RSA and prot,ein biosynthesis in the liver of the adult rat can be influenced by nutritional or hormonal shifts and by partial hepact,ectomy. Increased RYA or protein synthesis is observed in the liver of the irlt& rat after the administ~ration of growth hormone (1, 2) or cortico&eroids (3, 4). Conversely, protein and RNA synthesis is decreased in the liver of the hypophysectomized rat (5, 6). After the surgical removal of approximately two-thirds of the liver of the rat, numerous biochemical and qklogical changes occur in the cells of the remaining lobe, including (see 7) an early irlcrease in RKA and protein synbhesis. Although hormones do not’ appear to be
obligatory for liver regeneration (8), there is evidence that they can drastically modify the process. The rate of growth of the regenerating liver remnant is slower in the hypophysectomized rat (9). Hemingway and Cater found that hypophysectomy delays the peak of the first wave of mitosis and decreases the maximum mitotic rat’e normally observed in rats (10). These investigators also report,ed that the administration of cortisone (80 mg/kg twice daily) caused cyt,oplasmic and nuclear degeneration in the regenerating liver of hypophysectomized rats; no mit’osis was observed between 27 and 60 hours after partial hepatectomy. This phenomenon is of interest since corticosteroids are sometimes effective in suppressing the growth of neoplastjic tissue (11). The present investigation represents an attempt to identify the early biochemical events responsible for the inhibit,ion of tissue growth and proliferation by pharmacological doses of corticosteroids. Since early cffect,s of t)he hormone must be opera-
1 A port ion of this work was sltbmitted by L. l(. Wozney in partial fulfillment for the B.S. in Medicine degree, Vlliversity of Manitoba. We are grntefkll to 1)r. I’. G. Scholefield for suggestions during the preparation of the manuscript. This work was sLIpported by grants from the Medical Research Colmcil of Canada (MA 1949) and the National Callcer Instit Ilte of Canada. 69
WEBB
AND
tive at either the transcriptional or translational levels of protein synthesis, and since the polyribosomes are influenced by changes at either level, the effect of pharmacological doses of hydrocortisone on the labeling of ribosomal RNA and the polyribosome size distribution forms the basis of the present study. Hydrocortisone had a marked inhibitory effect on the incorporation of label from erotic-6-14C acid into ribosomal RNA and caused polyribosome breakdown in the early stages of liver regeneration in the hypophysectomized rat; neither RNA synthesis nor the integrity of the polyribosomes was adversely affected in the regenerating liver of intact rats, nor in the resting liver from hypophysectomized or intact rats.2 MATERIALS
AND
METHODS
Experimental animals. Male Spraque Dawley rats @O&250 gm) were maintained on a Purina chow diet with water ad libitum. Lighting was controlled from 6 AM to 6 PM. Hypophysectomies were performed by the parapharyngeal approach: partial hepatectomies were routinely performed at 4 PM by the method of Higgins and Anderson (12). Hypophysectomized rats were used from 10 to 30 days after the operation; t,hey were maintained on Purina chow, and drinking water that contained 2.57, glucose. The survival of the hypophysectomized, partially hepatectomixed rats was significantly improved by intubating 5 ml of 5% glucose immediately after partial hepatectomy, and at S-hour intervals therafter. All rats were fasted in wire bottom cages from 3 PM; the hypophysectomized rats received 2.5y0 glucose during the fasting period. Hydrocortisone was usually administered in two doses, one immediately after partial hepatectomy and a second dose 8 hours later. Carrier-free orotic-6-14C acid (2-10 mC/mmole) was injected i.p. (10 rC/250 gm) at 8 AM the following day, and the livers were removed at 12 noon. The livers of rats not subjected to partial hepatectomy were perfused before removal with cold 0.25 M sucrose via the portal vein. hemisuccinate Chemicals. Hydrocortisone (Mann Research Laboratories), hydrocortisone sodium succinate (Solucortef, Upjohn Co.), and prednisolone (Nutritional Biochemicals) were employed in these studies. Sodium deoxycholate was purchased from Mann Research Laboratories. 2 The terms “intact rats” and “resting refer to rats not subjected to hypophysectomy to nonregenerating liver, respectively.
liver” and
WOZNEY The deoxycholate (6.25%) and all sucrose solutions used in t,he preparation of density gradients and homogenizing medium contained TKM buffer (5 mM Tris, 25 mM KCl, and 5 mM MgC12, pH 7.5). Preparation and analysis of ribosomal components. Certain of the procedures outlined below have been described in detail elsewhere (13). Livers from rats fasted overnight to reduce liver glycogen were homogenized in 0.25 M sucrose (1 gm/2 ml). Cell breakage was estimated to be 85 90% by microscopic examination. The postmitochondrial supernatant fluid, prepared at 10,OOOg for 10 minutes, was treated with 1.25% deoxycholate, and then aliquots were layered over exponential (10-40%) sucrose gradients. The gradients were centrifuged in an SW 25.1 rotor of a Beckman centrifuge at 69,000g for 4.5 hours. After centrifugation, the effluent flowing from the bottom of the tube was passed through a flow cell, and the optical density of the effluent at 260 rnr was recorded automatically. With the passage of each 1 ml of effluent through the flow cell, a mark was recorded on the optical density tracing by an event marker which was activated by a drop counter. The resolution of the various ribosomal components in the density gradient was improved by first diluting the postmitochondrial supernatant fluid with 1 volume of buffer before layering aliquots over the gradients. Ferritin corrections were applied to t,he monomer peak of each size distribution pattern by analyzing duplicate gradients at 320 rnp (14). Polyribosomes were prepared by centrifuging the deoxycholate-treated postmitochondrial supernatant fraction through 2 M sucrose (15). The pellets were resuspended in water and aliquots were precipitated twice with perchloric acid. The acid-insoluble material from one pellet was dissolved in hyamine hydroxide and then transferred to vials containing 15 ml of liquid scintillant. The vials were counted in a Packard liquid scintillation counter at a tifficiency of approximately 80%. The concent,ratinn of RNA in a second aliquot was determined by the orcinol reaction (16). In a few inst,ances the concentration of RNA was calculated from the optical density at 260 rnp by using the relation 1 OD unit = 50 pg RNA. RESULTS
Within 6 hours of the surgical removal of two-thirds of the liver of the rat, there is a detectable increase in the rate of incorporation of labeled precursors into cytoplasmic RNA in the regenerating liver remnant (17, 18). In the present investigation a comparative study was made of the 4-hour incorpora-
CORTICOSTEROIDS
AND
tion of label from erotic-6J4C acid into ribosomal RNA of resting and regenerating liver of the intact and hypophysectomized rat and the effect of hydrocortisone on t,his process. In the following experiments bhe intact or hgpophysect,omized rats were part.ially hepnt,ectomized at 4 PM. Where applicable, hydrocort,isone, because of it’s rapid clearance from the animal, was given in t\vo doses, one at 4 PM and one at’ 11 P.M. Orotic-G-14C acid n-as injected at S AM the followiug day, and t,he livers were processed 4 hours later. This procedure reduced the possibility of variat,ions in labeling due to circadian rhythm. The result,s of preliminary experiments ut8ilizing large doses of hydrocortisone showed t,hat there was a significant inhibit,ion of the incorporation of label from erotic-6-14C acid into the regenerating liver of the hypophysectomized rat. Figure 1 shows a dose-response curve in which the percentage iuhibition of the incorporat,ion of label into the RNA of the polyribosomes from the regenerating liver of the hypophysectomized rat is plotted as a function of the size of the administered dose of hydrocortisone sodium succinate (Solucortef). Each rat, received the indicat,ed dose of hydrocortisone at partial hepatectomy and 7 hours later. Approximately 50 % inhibition is obtained at 20 mg/kg/dose and approximately 95-100% inhibition at’ 250 mg/kg/ dose. Subsequent experiments were designed to determine whether the inhibitory effect of hydrocortiswe was peculiar to the regenerating liver system in the hypophysectomized rat. Large doses of hydrocortisone (250 mg/kg/dose) were used to ensure that near optimal effects would be obtained and t,o thereby eliminate overlap due to individual variations in the response of t.he animals to partial hepat,ectomy or to the hydrocortisone. The result,s are summarized in Table I. The data obtained from Expts. I and II indicat,e that hydrocortisone had little effect on t,he incorporat,ion of label from erotic-614C acid int,o ribosomal RNA of the resting liver of the intact rat over a 4 hour labeling period. Predictably, t.he incorporat,ion of
LIVER
REGENERATIO?S
71
FIG. 1. Percentage inhibition of the incorporation of label from erotic-6-W acid into ribosomal RI\;A in the regenerating liver of the hypophysectomized rat as a fnnction of the size of the dose of hydrocortisone sodium succinate. The indicated doses were administered at partial hepatect,omy and 8 hours later. label into the 20-hour regenerating liver of the intact rat (Expt. III) was fivefold greater than in the resting liver. Furthermore, the specific activity of ribosomal RNA in the regenerating liver \vas approximately sixfold greater in the hydrocortisone-treated intact rats (Expt. IV) than in the control rats (Expt. II). The administration of two doses of hydrocortisone to the hypophysectomized rat resulted in a twofold increase in the incorporation of label from erotic-6-14C acid into the resting liver (Expts. V and VI). When three doses (a total of 7FiO mg/kg) If-ere administered to the hypophysectomized rat, there was a threefold increase in the incorporation of label from erotic-6-14C acid int,o ribosomal RNA of the resting liver (Expt. VII). The results of Expt. VIII shorn that the specific activity of ribosomal RNA in the 20-hour regenerating liver of the hypophysectomized rat was 4.3-fold greater than the unoperated control rats (Expt. V). This suggests that the response of the liver of the hypophysectomixed rat to the stimulus of partial hepat’ectomy is only slightly less t’han in the intact animal. However, the administration of hydrocortisone (Expt. IX) or an equivalent concentration of prednisolone (Expt,. X) reduced the incorporation of label from orotic-6-X4C
72
WEBB
AND TABLE
INFLUENCE
Expt.
I II III IV V VI VII VIII IX X
OF HYDROCORTISONE
SUrgerya
None None Hepx. Hepx. Hypox. Hypox. Hypox. Hypox.-Hepx. Hypox.-Hepx. Hypox.-Hepx.
ON THE ACID INTO
WOZNEY I
INCORPORATION RIBOSOMAL
Corticosteroid”
OF LABEL
No. of rats
None Hydrocortisone None Hydrocortisone None Hydrocortisone Hydrocortisoneb None Hydrocortisone Prednisolone
FROM
OROTIC-~-~~C
RNA
2 2 6 4 7 3 2 7 10 1
Specific cpm/,ug RNAC
27 24 134 145 26 60 82 115 10
3
(25-28) (22-26) (106-149) (124-180) (1440) (49-72) (79-85) (104-130) (2-20)
activity R&t& 100
90 504 548 96 227 304 415 37 13
a Hepx. = partial hepatectomy 20 hours before removal of the liver. Hypox. = lo- to 20-day hypophysectomized rat. b Two doses of 250 mg/kg each at 0 and 8 hours after partial hepatectomy, except for Expt. VII where three doses were given at 0,4, and 8 hours. c Orotic-6-i% acid (1OpC) injected 4 hours before killing. The average specific activity is followed by the range of values. d The normal rat was arbitrarily assigned a value of 100.
acid into ribosomal RNA in the regenerating liver of the hypophysectomized rat by at least 90 %. Collectively, the data suggest that the inhibitory effect of hydrocortisone is restricted to the regenerating liver of the hypophysectomized rat under the conditions employed in the present investigation. Further support for this conclusion was obtained from an analysis of the size distribution profiles of ribosomal components in the postmitochondrial supernatant fraction of the liver or the regenerating liver remnant from intact or hypophysectomized rats, before and after the administration of pharmacological doses of hydrocortisone. The results of an earlier study (19) established that the monomer concentration of the regenerating rat liver decreased shortly after partial hepatectomy and that there is an apparent shift in the mean polyribosome size distribution to the heavier species. Figure 2 shows the size distribution pattern of the ribosomal components in the postmitochondrial supernatant fraction of the 20-hour regenerating liver from the intact rat. The peak of ribosomal monomers, centered at fraction 21, has been corrected in this and subsequent figures for the absorp-
30.6 g 2 : ?? o’4 E 2 0 g 0.2
FIG. 2. Size distribution profile of ribosomal components from the 20-hour regenerating liver of an intact control rat (-) and from the 20-hour regenerating liver of an intact rat that received two doses of 250 mg hydrocortisone/kg (. .). Top of the gradient is to the right.
tion of ferritin components which sediment in this region (14). The height of this peak is about 40% of that of the corresponding peak in profiles of the resting liver of the
CORTICOSTEROIDS
AND
LIVER
73
REGENERATION
r
0
5
20 FRACTlPON
25
N{MBER
FIG. 3. Size distribution profile of ribosomal components from the resting liver of a 20.day hypophysectomized control rat (-) and a hypophysectomized rat that received three doses of 250 mg hydrocortisone/kg (. ..).
FIG. 4. Size distribution profile of ribosomal components from the 20-hour regenerating liver of a 20-day hypophysectomized control rat (-) and a hypophysectomized rat that received two doses of 250 mg of hydrocortisone/kg (. .).
normal rat (unpublished results). The administration of two doses of hydrocortisone (250 mg/kg/dose) caused no significant change in the size distribution profile (Fig. 2). Similarly, Fig. 3 shows the size distribution pattern of ribosomal components in the resting liver from t’he 20.day hypophysectomized rat and from an 11-day hypophysectomized rat, treated with three doses of hydrocortisone at 12,16, and 20 hours before processing the liver. The results suggest that up to 750 mg hydrocortisone/kg has no significant effect on the size distribution. Ferritin corrections, amounting to over 60% of the observed height of the monomer peak, were applied to both patterns; since the corrections were very large and since the ferritin and ribosomal monomer peaks did not exactly coincide, t,he corrected heights shown are only approximate. The biochemical significance of the excessive concentrations of ferritin in the liver of t’he hypophysectomized rat has not been established. Figure 4 shows the size distribution pattern of ribosomal components in the 20-hour regenerating liver from a 20-day hypophysectomized rat, as well as the pattern of the 30-hour regenerating liver of a hypophy-
sectomized rat which received two doses of hydrocortisone. Again, large ferritin corrections, amounting to 100 %, have been applied so that the heights of the monomer peaks are only approximate. However, a comparison of the control patterns in Figs. 3 and 4 suggests that there is a decrease in the monomer concentration in the regenerating liver of a hypophysectomized rat. Significantly, although two or even three doses of hydra: cortisone had little or no effect on the pat’tern in the resting liver of the hypophysectomized rat, two doses caused extensive breakdown of the polyribosomes to monomers and dimers and a shift of the mean polyribosome size distribution to the lighter species in t,he regenerating liver of the hypophysectomized rat’. DISCUSSION
The results of the present investigation show that pharmacological doses of hydrocortisone cause an inhibition of IZISA synt’hesis and polyribosome breakdown in the regenerating liver of the hypophysectomized rat’, but not, in the regenerating liver of t,he intact rat. This suggests that it is not the ratio of the size of the administ)ered dose to
74
WEBB
AND
the size of the regenerating remnant but rather the lack of the hypophysis which is the determining factor. Furthermore, since up to three doses did not cause deleterious effects in the resting liver of the hypophysectomized rat, the inhibitory effect of hydrocortisone appears to be restricted to regenerating liver and possibly to proliferating tissue in general. Although the sodium salt of hydrocortisone hemisuccinate was administered at the rate of 250 mg/kg/ dose (equivalent to 170 mg/kg of cortisone), equivalent results could probably have been obtained with lower doses and most certainly by using lower doses of less soluble derivatives or more frequent injections. As in the intact rat, the monomer concentration appears to decrease to some extent in the regenerating liver of the hypophysectomized rat. Although the results of preliminary experiments designed to prevent the inhibitory effect of hydrocortisone by simultaneous administration of porcine growth hormone were equivocal, it seems probable that the level of circulating growth hormone is important for the effect. Growth hormone is reported (10) to prevent the nuclear and cytoplasmic degeneration in regenerating liver caused by the administration of hydrocortisone to the hypophysectomized rat. In other relevant studies, low doses of corticosteroids inhibited the growth of hypopituitary patients (20), and DNA synthesis in regenerating mouse liver (21). The inhibition of tumor growth in experimental animals and the remission of leukemia in man by administration of pharmacological doses of hydrocortisone is well documented (11, 22). The molecular basis for the inhibitory action of hydrocortisone on proliferating tissue has not been determined. Since the present results indicate that RNA synthesis is almost completely inhibited and polyribosome breakdown occurs within 20 hours of partial hepatectomy, the lesion must precede the period when DNA synthesis is maximal (18-24 hours) and the period when both DNA synthesis and mitosis are prominent (see 23). Thus the lesion occurs before or during the period of accelerated RNA synthesis and is independent of both DNA
WOZNEY
synthesis and mitosis. Polyribosome breakdown may be related to inhibited messenger RNA synthesis. However, the fact that the mean size distribution of the residual polyribosomes has shifted to the lighter species would suggest that direct or indirect effects also occur at the translational level. The observation (24) that the ability of ribonucleoprotein particles from rat liver to catalyze the incorporation of leucine-1% into protein increases with the removal of endogenous corticosteroids by adrenalectomy, and is decreased by pretreatment of the adrenalectomized rat by hydrocortisone, is in agreement with the present data. The dimers observed in Fig. 4 are typical of situations where in viva polyribosome breakdown occurs as a result of interference with messenger RNA production or the attachment of the ribosomes to the messenger RNA (18). It should be noted also that in those instances where the administration of hydrocortisone resulted in an increased RNA synthesis, there was no apparent increase in the concentration of the polyribosomes in the postmitochondrial supernatant fraction. This can be explained by the observation (7) that a detectable increase occurs in the incorporation of labeled precursors into RNA within 6 hours after partial hepatectomy, but no significant net increase in RNA is observed until 24 hours after partial hepatectomy. In view of the report (21) that DNA synthesis is negligible in the 2-day regenerating liver of the intact mouse treated with high doses of hydrocortisone acetate, while RNA synthesis was not significantly affected, the inhibition of DNA synthesis may be possible at low doses of corticosteroid, i.e., without the extensive decreases in RNA synthesis obtained in the present study. As in the actinomycin-treated rats, it seems unlikely that the parenchymal cells in the regenerating liver of the hypophysectomized, hydrocortisone-treated rat would remain viable for any extended period of time. Further studies will be necessary to determine the relative sensitivity of the cells at various stages of regeneration and at various stages of cellular differentiation (25) during the regenerative process.
CORTICOSTEROIDS
AND
REFERENCES 1.
LEE,
N.
ology 2. TALWAR, AND
(1962). 3. KENNEY, D. L., 4. GBRREN, KINS,
5. KORNER, 6. ULRICH, Biol. 7. BUCHER, (1963). 8. HARKNESS,
D., BND WILLIIMS, R. H., ,%docrin61, 451 (1962). G. P., p.4~~~1, N. C., SARIN, G. S., TOLANI, A. J., Biochem. J. 62, 173 F. T., WICKS, W. D., AND GREENMSN, J. Cell. Comp. Physiol. 66, 125 (1966). L. D., HOWELL, R. R., SND TOMG. M., J. Mol. Biol. 9, 100 (1964). A., Biochem. J. 73, 61 (1959). F., TSRVER, H., .4ND LI, C. H., J. Chem. 209, 117 (1954). N. R. L., Intern. Rev. Cytol. 16, 245 R.
D.,
Brit.
Med.
Bull.
13,
87
(1957). 9. DOLJANSKI, F., AND NOVOGROTZKI, M., Lab. Invest. 8, 989 (1959). 10. HEMINGWAY, J. T., AND CATER, D. B., Nature 182, 1065 (1958). 11. DORFMAN, R. I. (Ed.), in “Methods in Hormone Research,” Vol. 4, pp. 165-192. Academic Press, New York (1965). 12. HIGGINS, G. M., END ANDERSON, R. M., Arch. Pathol. 12, 186 (1931).
LIVER
REGENERATION
73
T. E., Biochem. Biophys. Acta 138, 307 13. WEBB, (1967). 14. WILSON, S. H., .~ND HOAGLAND, RI. B., Proc. Natl. Acad. Sci. U.S. 64, 600 (1965). 15. WEBB, T. E., BLOBEL, G., POTTER, V. R., AND MORRIS, H. P., Cancer Res. 26, 1219 (1965). 16. CERIOTTI, G., J. Biol. Chem. 214, 59 (1955). 17. HECHT, L., AND POTTER, V. R., Cancer Res. 18, 186 (1958). 18. SCHNEIDER, J. H., AND POT’FER, V. IX., Cancer Res. 17, 701 (1957). 19. WEBB, T. E., BLOBEL, G., AND POTTER, V. R., Cancer Res. 26, 253 (1966). 20. SOYKA, L. F., AND CRAWFORD, J. D., J. CZin. Endocrinol. Metab. 26, 469 (1965). 21. ROBERTS, K. B., FLOREY, H. W., .IXD JOKLIK, W. K., Quart. J. Physiol. 37, 239 (1952). 22. POLLI, E., ERIDINI, S., AND BIAYL’CHESSI, M., in “Hormonal Steroids” (L. Martini and A. Pecile,eds.), Trol. 2, p.351. Academic Press, New York (1965). R. E., VAN LSNCKER, J., .YND POTTER, 23. BELTZ, V. R., Cancer Res. 17, 688 (1957). 24. DEVENUTO, F., AND LANGE, R. J. G., Proc. Sot. Exptl. BioZ. Med. 124, 793 (1967). 25. LANE, B. P., AND BECKER, F. F., Am. J. Pathol. 60, 435 (1967).
OF
Liver
BIOCHEYISTRY
AND
Regeneration
in the
Corticosteroids Synthesis THOMAS McGill,
University
Cancer
126,
BIOPHYSICS
Research University
69-75
(1968)
Hypophysectomized on the
Size
of Ribosomal
E. WEBB
AND
Unit, McInfyre of Manitoba, Received
Distribution
September
Influence
of
and
Components’
LEOKARD
Medical Winnipeg,
Rat:
R. WOZNEY
Sciences B&ding, Montreal, Manitoba, Canada
&uebec;
and
the
18, 1967
The increased incorporation of label from erotic-G-1°C acid into ribosomal RNA and changes in the size distribution of ribosomal components, normally occurring during t,he early phases of liver regeneration in the intact rat, are also observed in the regenerating liver of the hypophysectomized rat. Neither parameter in the resting and regenerating liver was adversely affected by the administration of pharmacological doses of corticosteroids to the int,act rat. However, identical treatment of the hypophysectomized rat resulted in a marked inhibition of the incorporation of label from erotic-6-l% acid into ribosomal RNA and partial breakdown of the polyribosomes in the regenerating liver within 20 hours of partial hepatectomy. The phenomena appear to be peculiar to proliferating tissue, since pharmacological doses of hydrocortisone neither caused polyribosome breakdown nor inhibited erotic-6-l% acid incorporation iu the resting liver of the hypophgsectomized rat.
The rate of RSA and prot,ein biosynthesis in the liver of the adult rat can be influenced by nutritional or hormonal shifts and by partial hepact,ectomy. Increased RYA or protein synthesis is observed in the liver of the irlt& rat after the administ~ration of growth hormone (1, 2) or cortico&eroids (3, 4). Conversely, protein and RNA synthesis is decreased in the liver of the hypophysectomized rat (5, 6). After the surgical removal of approximately two-thirds of the liver of the rat, numerous biochemical and qklogical changes occur in the cells of the remaining lobe, including (see 7) an early irlcrease in RKA and protein synbhesis. Although hormones do not’ appear to be
obligatory for liver regeneration (8), there is evidence that they can drastically modify the process. The rate of growth of the regenerating liver remnant is slower in the hypophysectomized rat (9). Hemingway and Cater found that hypophysectomy delays the peak of the first wave of mitosis and decreases the maximum mitotic rat’e normally observed in rats (10). These investigators also report,ed that the administration of cortisone (80 mg/kg twice daily) caused cyt,oplasmic and nuclear degeneration in the regenerating liver of hypophysectomized rats; no mit’osis was observed between 27 and 60 hours after partial hepatectomy. This phenomenon is of interest since corticosteroids are sometimes effective in suppressing the growth of neoplastjic tissue (11). The present investigation represents an attempt to identify the early biochemical events responsible for the inhibit,ion of tissue growth and proliferation by pharmacological doses of corticosteroids. Since early cffect,s of t)he hormone must be opera-
1 A port ion of this work was sltbmitted by L. l(. Wozney in partial fulfillment for the B.S. in Medicine degree, Vlliversity of Manitoba. We are grntefkll to 1)r. I’. G. Scholefield for suggestions during the preparation of the manuscript. This work was sLIpported by grants from the Medical Research Colmcil of Canada (MA 1949) and the National Callcer Instit Ilte of Canada. 69
WEBB
AND
tive at either the transcriptional or translational levels of protein synthesis, and since the polyribosomes are influenced by changes at either level, the effect of pharmacological doses of hydrocortisone on the labeling of ribosomal RNA and the polyribosome size distribution forms the basis of the present study. Hydrocortisone had a marked inhibitory effect on the incorporation of label from erotic-6-14C acid into ribosomal RNA and caused polyribosome breakdown in the early stages of liver regeneration in the hypophysectomized rat; neither RNA synthesis nor the integrity of the polyribosomes was adversely affected in the regenerating liver of intact rats, nor in the resting liver from hypophysectomized or intact rats.2 MATERIALS
AND
METHODS
Experimental animals. Male Spraque Dawley rats @O&250 gm) were maintained on a Purina chow diet with water ad libitum. Lighting was controlled from 6 AM to 6 PM. Hypophysectomies were performed by the parapharyngeal approach: partial hepatectomies were routinely performed at 4 PM by the method of Higgins and Anderson (12). Hypophysectomized rats were used from 10 to 30 days after the operation; t,hey were maintained on Purina chow, and drinking water that contained 2.57, glucose. The survival of the hypophysectomized, partially hepatectomixed rats was significantly improved by intubating 5 ml of 5% glucose immediately after partial hepatectomy, and at S-hour intervals therafter. All rats were fasted in wire bottom cages from 3 PM; the hypophysectomized rats received 2.5y0 glucose during the fasting period. Hydrocortisone was usually administered in two doses, one immediately after partial hepatectomy and a second dose 8 hours later. Carrier-free orotic-6-14C acid (2-10 mC/mmole) was injected i.p. (10 rC/250 gm) at 8 AM the following day, and the livers were removed at 12 noon. The livers of rats not subjected to partial hepatectomy were perfused before removal with cold 0.25 M sucrose via the portal vein. hemisuccinate Chemicals. Hydrocortisone (Mann Research Laboratories), hydrocortisone sodium succinate (Solucortef, Upjohn Co.), and prednisolone (Nutritional Biochemicals) were employed in these studies. Sodium deoxycholate was purchased from Mann Research Laboratories. 2 The terms “intact rats” and “resting refer to rats not subjected to hypophysectomy to nonregenerating liver, respectively.
liver” and
WOZNEY The deoxycholate (6.25%) and all sucrose solutions used in t,he preparation of density gradients and homogenizing medium contained TKM buffer (5 mM Tris, 25 mM KCl, and 5 mM MgC12, pH 7.5). Preparation and analysis of ribosomal components. Certain of the procedures outlined below have been described in detail elsewhere (13). Livers from rats fasted overnight to reduce liver glycogen were homogenized in 0.25 M sucrose (1 gm/2 ml). Cell breakage was estimated to be 85 90% by microscopic examination. The postmitochondrial supernatant fluid, prepared at 10,OOOg for 10 minutes, was treated with 1.25% deoxycholate, and then aliquots were layered over exponential (10-40%) sucrose gradients. The gradients were centrifuged in an SW 25.1 rotor of a Beckman centrifuge at 69,000g for 4.5 hours. After centrifugation, the effluent flowing from the bottom of the tube was passed through a flow cell, and the optical density of the effluent at 260 rnr was recorded automatically. With the passage of each 1 ml of effluent through the flow cell, a mark was recorded on the optical density tracing by an event marker which was activated by a drop counter. The resolution of the various ribosomal components in the density gradient was improved by first diluting the postmitochondrial supernatant fluid with 1 volume of buffer before layering aliquots over the gradients. Ferritin corrections were applied to t,he monomer peak of each size distribution pattern by analyzing duplicate gradients at 320 rnp (14). Polyribosomes were prepared by centrifuging the deoxycholate-treated postmitochondrial supernatant fraction through 2 M sucrose (15). The pellets were resuspended in water and aliquots were precipitated twice with perchloric acid. The acid-insoluble material from one pellet was dissolved in hyamine hydroxide and then transferred to vials containing 15 ml of liquid scintillant. The vials were counted in a Packard liquid scintillation counter at a tifficiency of approximately 80%. The concent,ratinn of RNA in a second aliquot was determined by the orcinol reaction (16). In a few inst,ances the concentration of RNA was calculated from the optical density at 260 rnp by using the relation 1 OD unit = 50 pg RNA. RESULTS
Within 6 hours of the surgical removal of two-thirds of the liver of the rat, there is a detectable increase in the rate of incorporation of labeled precursors into cytoplasmic RNA in the regenerating liver remnant (17, 18). In the present investigation a comparative study was made of the 4-hour incorpora-
CORTICOSTEROIDS
AND
tion of label from erotic-6J4C acid into ribosomal RNA of resting and regenerating liver of the intact and hypophysectomized rat and the effect of hydrocortisone on t,his process. In the following experiments bhe intact or hgpophysect,omized rats were part.ially hepnt,ectomized at 4 PM. Where applicable, hydrocort,isone, because of it’s rapid clearance from the animal, was given in t\vo doses, one at 4 PM and one at’ 11 P.M. Orotic-G-14C acid n-as injected at S AM the followiug day, and t,he livers were processed 4 hours later. This procedure reduced the possibility of variat,ions in labeling due to circadian rhythm. The result,s of preliminary experiments ut8ilizing large doses of hydrocortisone showed t,hat there was a significant inhibit,ion of the incorporation of label from erotic-6-14C acid into the regenerating liver of the hypophysectomized rat. Figure 1 shows a dose-response curve in which the percentage iuhibition of the incorporat,ion of label into the RNA of the polyribosomes from the regenerating liver of the hypophysectomized rat is plotted as a function of the size of the administered dose of hydrocortisone sodium succinate (Solucortef). Each rat, received the indicat,ed dose of hydrocortisone at partial hepatectomy and 7 hours later. Approximately 50 % inhibition is obtained at 20 mg/kg/dose and approximately 95-100% inhibition at’ 250 mg/kg/ dose. Subsequent experiments were designed to determine whether the inhibitory effect of hydrocortiswe was peculiar to the regenerating liver system in the hypophysectomized rat. Large doses of hydrocortisone (250 mg/kg/dose) were used to ensure that near optimal effects would be obtained and t,o thereby eliminate overlap due to individual variations in the response of t.he animals to partial hepat,ectomy or to the hydrocortisone. The result,s are summarized in Table I. The data obtained from Expts. I and II indicat,e that hydrocortisone had little effect on t,he incorporat,ion of label from erotic-614C acid int,o ribosomal RNA of the resting liver of the intact rat over a 4 hour labeling period. Predictably, t.he incorporat,ion of
LIVER
REGENERATIO?S
71
FIG. 1. Percentage inhibition of the incorporation of label from erotic-6-W acid into ribosomal RI\;A in the regenerating liver of the hypophysectomized rat as a fnnction of the size of the dose of hydrocortisone sodium succinate. The indicated doses were administered at partial hepatect,omy and 8 hours later. label into the 20-hour regenerating liver of the intact rat (Expt. III) was fivefold greater than in the resting liver. Furthermore, the specific activity of ribosomal RNA in the regenerating liver \vas approximately sixfold greater in the hydrocortisone-treated intact rats (Expt. IV) than in the control rats (Expt. II). The administration of two doses of hydrocortisone to the hypophysectomized rat resulted in a twofold increase in the incorporation of label from erotic-6-14C acid into the resting liver (Expts. V and VI). When three doses (a total of 7FiO mg/kg) If-ere administered to the hypophysectomized rat, there was a threefold increase in the incorporation of label from erotic-6-14C acid int,o ribosomal RNA of the resting liver (Expt. VII). The results of Expt. VIII shorn that the specific activity of ribosomal RNA in the 20-hour regenerating liver of the hypophysectomized rat was 4.3-fold greater than the unoperated control rats (Expt. V). This suggests that the response of the liver of the hypophysectomixed rat to the stimulus of partial hepat’ectomy is only slightly less t’han in the intact animal. However, the administration of hydrocortisone (Expt. IX) or an equivalent concentration of prednisolone (Expt,. X) reduced the incorporation of label from orotic-6-X4C
72
WEBB
AND TABLE
INFLUENCE
Expt.
I II III IV V VI VII VIII IX X
OF HYDROCORTISONE
SUrgerya
None None Hepx. Hepx. Hypox. Hypox. Hypox. Hypox.-Hepx. Hypox.-Hepx. Hypox.-Hepx.
ON THE ACID INTO
WOZNEY I
INCORPORATION RIBOSOMAL
Corticosteroid”
OF LABEL
No. of rats
None Hydrocortisone None Hydrocortisone None Hydrocortisone Hydrocortisoneb None Hydrocortisone Prednisolone
FROM
OROTIC-~-~~C
RNA
2 2 6 4 7 3 2 7 10 1
Specific cpm/,ug RNAC
27 24 134 145 26 60 82 115 10
3
(25-28) (22-26) (106-149) (124-180) (1440) (49-72) (79-85) (104-130) (2-20)
activity R&t& 100
90 504 548 96 227 304 415 37 13
a Hepx. = partial hepatectomy 20 hours before removal of the liver. Hypox. = lo- to 20-day hypophysectomized rat. b Two doses of 250 mg/kg each at 0 and 8 hours after partial hepatectomy, except for Expt. VII where three doses were given at 0,4, and 8 hours. c Orotic-6-i% acid (1OpC) injected 4 hours before killing. The average specific activity is followed by the range of values. d The normal rat was arbitrarily assigned a value of 100.
acid into ribosomal RNA in the regenerating liver of the hypophysectomized rat by at least 90 %. Collectively, the data suggest that the inhibitory effect of hydrocortisone is restricted to the regenerating liver of the hypophysectomized rat under the conditions employed in the present investigation. Further support for this conclusion was obtained from an analysis of the size distribution profiles of ribosomal components in the postmitochondrial supernatant fraction of the liver or the regenerating liver remnant from intact or hypophysectomized rats, before and after the administration of pharmacological doses of hydrocortisone. The results of an earlier study (19) established that the monomer concentration of the regenerating rat liver decreased shortly after partial hepatectomy and that there is an apparent shift in the mean polyribosome size distribution to the heavier species. Figure 2 shows the size distribution pattern of the ribosomal components in the postmitochondrial supernatant fraction of the 20-hour regenerating liver from the intact rat. The peak of ribosomal monomers, centered at fraction 21, has been corrected in this and subsequent figures for the absorp-
30.6 g 2 : ?? o’4 E 2 0 g 0.2
FIG. 2. Size distribution profile of ribosomal components from the 20-hour regenerating liver of an intact control rat (-) and from the 20-hour regenerating liver of an intact rat that received two doses of 250 mg hydrocortisone/kg (. .). Top of the gradient is to the right.
tion of ferritin components which sediment in this region (14). The height of this peak is about 40% of that of the corresponding peak in profiles of the resting liver of the
CORTICOSTEROIDS
AND
LIVER
73
REGENERATION
r
0
5
20 FRACTlPON
25
N{MBER
FIG. 3. Size distribution profile of ribosomal components from the resting liver of a 20.day hypophysectomized control rat (-) and a hypophysectomized rat that received three doses of 250 mg hydrocortisone/kg (. ..).
FIG. 4. Size distribution profile of ribosomal components from the 20-hour regenerating liver of a 20-day hypophysectomized control rat (-) and a hypophysectomized rat that received two doses of 250 mg of hydrocortisone/kg (. .).
normal rat (unpublished results). The administration of two doses of hydrocortisone (250 mg/kg/dose) caused no significant change in the size distribution profile (Fig. 2). Similarly, Fig. 3 shows the size distribution pattern of ribosomal components in the resting liver from t’he 20.day hypophysectomized rat and from an 11-day hypophysectomized rat, treated with three doses of hydrocortisone at 12,16, and 20 hours before processing the liver. The results suggest that up to 750 mg hydrocortisone/kg has no significant effect on the size distribution. Ferritin corrections, amounting to over 60% of the observed height of the monomer peak, were applied to both patterns; since the corrections were very large and since the ferritin and ribosomal monomer peaks did not exactly coincide, t,he corrected heights shown are only approximate. The biochemical significance of the excessive concentrations of ferritin in the liver of t’he hypophysectomized rat has not been established. Figure 4 shows the size distribution pattern of ribosomal components in the 20-hour regenerating liver from a 20-day hypophysectomized rat, as well as the pattern of the 30-hour regenerating liver of a hypophy-
sectomized rat which received two doses of hydrocortisone. Again, large ferritin corrections, amounting to 100 %, have been applied so that the heights of the monomer peaks are only approximate. However, a comparison of the control patterns in Figs. 3 and 4 suggests that there is a decrease in the monomer concentration in the regenerating liver of a hypophysectomized rat. Significantly, although two or even three doses of hydra: cortisone had little or no effect on the pat’tern in the resting liver of the hypophysectomized rat, two doses caused extensive breakdown of the polyribosomes to monomers and dimers and a shift of the mean polyribosome size distribution to the lighter species in t,he regenerating liver of the hypophysectomized rat’. DISCUSSION
The results of the present investigation show that pharmacological doses of hydrocortisone cause an inhibition of IZISA synt’hesis and polyribosome breakdown in the regenerating liver of the hypophysectomized rat’, but not, in the regenerating liver of t,he intact rat. This suggests that it is not the ratio of the size of the administ)ered dose to
74
WEBB
AND
the size of the regenerating remnant but rather the lack of the hypophysis which is the determining factor. Furthermore, since up to three doses did not cause deleterious effects in the resting liver of the hypophysectomized rat, the inhibitory effect of hydrocortisone appears to be restricted to regenerating liver and possibly to proliferating tissue in general. Although the sodium salt of hydrocortisone hemisuccinate was administered at the rate of 250 mg/kg/ dose (equivalent to 170 mg/kg of cortisone), equivalent results could probably have been obtained with lower doses and most certainly by using lower doses of less soluble derivatives or more frequent injections. As in the intact rat, the monomer concentration appears to decrease to some extent in the regenerating liver of the hypophysectomized rat. Although the results of preliminary experiments designed to prevent the inhibitory effect of hydrocortisone by simultaneous administration of porcine growth hormone were equivocal, it seems probable that the level of circulating growth hormone is important for the effect. Growth hormone is reported (10) to prevent the nuclear and cytoplasmic degeneration in regenerating liver caused by the administration of hydrocortisone to the hypophysectomized rat. In other relevant studies, low doses of corticosteroids inhibited the growth of hypopituitary patients (20), and DNA synthesis in regenerating mouse liver (21). The inhibition of tumor growth in experimental animals and the remission of leukemia in man by administration of pharmacological doses of hydrocortisone is well documented (11, 22). The molecular basis for the inhibitory action of hydrocortisone on proliferating tissue has not been determined. Since the present results indicate that RNA synthesis is almost completely inhibited and polyribosome breakdown occurs within 20 hours of partial hepatectomy, the lesion must precede the period when DNA synthesis is maximal (18-24 hours) and the period when both DNA synthesis and mitosis are prominent (see 23). Thus the lesion occurs before or during the period of accelerated RNA synthesis and is independent of both DNA
WOZNEY
synthesis and mitosis. Polyribosome breakdown may be related to inhibited messenger RNA synthesis. However, the fact that the mean size distribution of the residual polyribosomes has shifted to the lighter species would suggest that direct or indirect effects also occur at the translational level. The observation (24) that the ability of ribonucleoprotein particles from rat liver to catalyze the incorporation of leucine-1% into protein increases with the removal of endogenous corticosteroids by adrenalectomy, and is decreased by pretreatment of the adrenalectomized rat by hydrocortisone, is in agreement with the present data. The dimers observed in Fig. 4 are typical of situations where in viva polyribosome breakdown occurs as a result of interference with messenger RNA production or the attachment of the ribosomes to the messenger RNA (18). It should be noted also that in those instances where the administration of hydrocortisone resulted in an increased RNA synthesis, there was no apparent increase in the concentration of the polyribosomes in the postmitochondrial supernatant fraction. This can be explained by the observation (7) that a detectable increase occurs in the incorporation of labeled precursors into RNA within 6 hours after partial hepatectomy, but no significant net increase in RNA is observed until 24 hours after partial hepatectomy. In view of the report (21) that DNA synthesis is negligible in the 2-day regenerating liver of the intact mouse treated with high doses of hydrocortisone acetate, while RNA synthesis was not significantly affected, the inhibition of DNA synthesis may be possible at low doses of corticosteroid, i.e., without the extensive decreases in RNA synthesis obtained in the present study. As in the actinomycin-treated rats, it seems unlikely that the parenchymal cells in the regenerating liver of the hypophysectomized, hydrocortisone-treated rat would remain viable for any extended period of time. Further studies will be necessary to determine the relative sensitivity of the cells at various stages of regeneration and at various stages of cellular differentiation (25) during the regenerative process.
CORTICOSTEROIDS
AND
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(1962). 3. KENNEY, D. L., 4. GBRREN, KINS,
5. KORNER, 6. ULRICH, Biol. 7. BUCHER, (1963). 8. HARKNESS,
D., BND WILLIIMS, R. H., ,%docrin61, 451 (1962). G. P., p.4~~~1, N. C., SARIN, G. S., TOLANI, A. J., Biochem. J. 62, 173 F. T., WICKS, W. D., AND GREENMSN, J. Cell. Comp. Physiol. 66, 125 (1966). L. D., HOWELL, R. R., SND TOMG. M., J. Mol. Biol. 9, 100 (1964). A., Biochem. J. 73, 61 (1959). F., TSRVER, H., .4ND LI, C. H., J. Chem. 209, 117 (1954). N. R. L., Intern. Rev. Cytol. 16, 245 R.
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T. E., Biochem. Biophys. Acta 138, 307 13. WEBB, (1967). 14. WILSON, S. H., .~ND HOAGLAND, RI. B., Proc. Natl. Acad. Sci. U.S. 64, 600 (1965). 15. WEBB, T. E., BLOBEL, G., POTTER, V. R., AND MORRIS, H. P., Cancer Res. 26, 1219 (1965). 16. CERIOTTI, G., J. Biol. Chem. 214, 59 (1955). 17. HECHT, L., AND POTTER, V. R., Cancer Res. 18, 186 (1958). 18. SCHNEIDER, J. H., AND POT’FER, V. IX., Cancer Res. 17, 701 (1957). 19. WEBB, T. E., BLOBEL, G., AND POTTER, V. R., Cancer Res. 26, 253 (1966). 20. SOYKA, L. F., AND CRAWFORD, J. D., J. CZin. Endocrinol. Metab. 26, 469 (1965). 21. ROBERTS, K. B., FLOREY, H. W., .IXD JOKLIK, W. K., Quart. J. Physiol. 37, 239 (1952). 22. POLLI, E., ERIDINI, S., AND BIAYL’CHESSI, M., in “Hormonal Steroids” (L. Martini and A. Pecile,eds.), Trol. 2, p.351. Academic Press, New York (1965). R. E., VAN LSNCKER, J., .YND POTTER, 23. BELTZ, V. R., Cancer Res. 17, 688 (1957). 24. DEVENUTO, F., AND LANGE, R. J. G., Proc. Sot. Exptl. BioZ. Med. 124, 793 (1967). 25. LANE, B. P., AND BECKER, F. F., Am. J. Pathol. 60, 435 (1967).