Dissociation of androgen-induced enzyme synthesis and amino acid incorporation in mouse kidney by actinomycin D

Dec. 1964

S T E R 0 I D S

777

DISSOCIATION OF ANDROGEN-INDUCED ENZYME SYNTHESIS AND AMINO ACID INCORPORATION IN MOUSE KIDNEY BY ACTINOMYCIN D (I) Edward H. Frieden (2), Annabel A. Harper, Fulton Chin, and William H. Fishman From The Arthur G. Rotch Laboratory, The Boston Dispensary and The Department of Pathology (Oncology), Tufts University School of Medicine, Boston. Received October 13, 1964 The renotrophic response of a number of strains of mice to the injection of testosterone, its esters, and other androgens is accompanied by increase in the total activity of a number of enzymes.

Early eleva-

tions in specific enzyme activity have been noted, however, in the case only of D-amino acid oxidase (3) and ~-glucuronidase (4). In the latter instance, the view of a de novo synthesis is supported (5) by the results of 8glucuronidase-protein labeling experiments in animals undergoing prolonged androgenic stimulation. Moreover, only 24 to 48 hours after the initial dose of testosterone propionate, one can observe (6) a transitory increase in the rate of incorporation of several 14C-amino acids into kidney proteins. Both processes (G-glucuronidase synthesis and renal protein production) were considered to have the same endocrinologic significance. In this paper, it will be shown that although actinomycin D inhibits the testosterone propionateinduced increase in B-glucuronidase completely and in D-amino acid oxidase significantly, it fails to affect the extent of amino acid incorporation.

It

778

S T E R 0 I D S

4:6

has also been observed that inhibition of testosterone propionate-induced enzyme changes occurs only if administration of the antibiotic is begun before the first steroid injection.

Even prolonged

treatment with actinomycin has little or no effect if it is commenced one or two days later. Experimental A Cloudman mice (7) of both sexes were used. Testosterone propionate (1.O mg in 0.1 ml sesame oil) was injected intramuscularly daily in 2- or 3- day experiments, and every other day in experiments lasting four days or longer. Actinomycin D (8) was dissolved in buffered saline and injected intraperitoneally once daily at the dosage indicated in the legends to figures. At the end of the experiment, the animals were killed by a blow on the head and the kidneys were removed and weighed. For incorporation studies the kidneys {one from each animal) were slicsd and the slices incubated for 2 hours at 38.5 in Krebs-Ringer phosphate containing the appropriate labeled amino acid (9). After incubation, radioactive protein was isolated and counted as previously described (6). Kidneys used for enzyme determinations were homogenized in buffer. G-Glucuronidase was determined in the homogenate according to the method of Fishman (10); D-amino acid oxidase was measured by the method of Corrigan, Wellner and Meister (ll) using D (-) allo-hydroxyproline as the substrate, and acid phosphatase as described by Stolbach et. al. (12). Figure 1 shows the effect of actinomycin D upon changes in the rate of leucine incorporation and kidney ~-glucuronidase content observed 24 hours after the second of two daily injections of testosterone propionate. In this experiment, actinomycin (200 ~gm/Kg.) was given for four days, beginning two days before the first testosterone propionate injection. Under these conditions, actinomycin diminished the rate of leucine incorporation by approximately 25% in otherwise untreated animals, but did not significantly alter the stimulatory effect of testosterone propionate (31% vs 39%). On the other hand, actinomycin-

Dec. 1 9 6 4

S T E R 0 I D S

779

250 LEUCINE INCORPORATION

ONIDASE

200

I--lCONTROLS

~

~IR,2

~ A C " [ + 11?

xlrng.

ACI,4 x 200,u.gm/~gI

Figure 1 Effects of Actinomycin D upon testosterone propionate-induced increase~4in the rate of incorporation of leucine-1-" C into mouse kidney protein in vitro (left), and in mouse kidney G-glucuronidase (right). Ordinate: per cent of control value (unshaded bars). For each experiment, the number of animals used and the standard errors are indicated.

treated animals failed to exhibit the pronounced increase in ~-glucuronidase concentration characteristic of testosterone propionate-treated mice (14% vs 170%). It may also be noted that actinomycin alone, even after four days, does not reduce the enzyme content of the kidneys. With regard to D-amino acid oxidase, because significant increases in its response to testosterone propionate administration occur slowly, the period of exposure to androgen was increased to three days; actinomycin injections were begun one day earlier. The kidneys of animals receiving testosterone propionate for three days contained 69% more D-amino acid oxidase than did those of untreated animals; in mice subjected to the same stimulus whilst simultaneously receiving actinomycin, the increase, referred to actinomycin-

780

S T E R O I D S

treated controls, was only 23% (Figure 2). The fact that the effect of actinomycin upon D-amino acid oxidase is less complete than its effect on ~-glucuronidase reflects, perhaps, the shorter interval between the initiation of actinomycin and testosterone dosage, respectively. In any event, it seems clear that the stimulation of D-amino acid oxidase activity, like that of ~-glucuronidase, is inhibited by prior administration of actinomycin.

D-AMINO-ACID OXIDASE

20(3

15£

I0£

50

I

Ic°NT~Ls

~A~,4~2oo/~,~/k~

W/~IP.,3,,,,~ ~Ac"r..T.P. Figure 2 Effects of Actinomycin D upon testosterone propionate-induced stimulation of D-amino acid oxidase in mouse kidney. Details as in Figure 1. Figure 3 shows the r e s ~ t s of i n c o r p o r a t i ~ experiments with glycine-1- C and arginine-U- C as the labeled amino acids. In these experiments, the testosterone propionate-induced increments in the absence of actinomycin-D were 35% and 33%, respectively; the corresponding figures for actinomycin-treated animals were 49% and 42%. As with leucine, the administration of actinomycin alone caused a diminution of about 25% in the rates of incorporation of both amino acids. In some of these experiments, renal acid phosphatase activity was also measured. This enzyme, which is unaffected by androgen administration, also showed no change in response to actinomycin.

4:6

Dec. 1 9 6 4

S T E R 0 I D S

•

' GLYCINE

7B1

ARGININE

~ i

ACI, 4 X 200/.Cgm./kO.

F"---'IACI:f T. R

Figure 3 Effects of Actinomycin D upon testosterg~e p r o p i o n a t e - i n d u ~ d stimulation of glycine-l-~-C and arginine-1C incorporation into mouse kidney protein in vitro. Five to six animals were included in each group. Other details as in Figure 1. In all of the experiments described thus far, actinomycin injections were begun one or two days before the first injection of testosterone propionate. In view of the apparent inability of the antibiotic to reduce the level of either enzyme below the control levels, whether or not testosterone propionate was given simultaneously, it seemed of interest to see what effect actinomycin would have if the sequence was reversed, i.e., if testosterone propionate was injected first and actinomycin administration was delayed until after some increase in enzyme concentration had occurred. Accordingly, such an experiment was performed. Its design and the results appear in Figure 4. The results indicated that even prolonged administration of actinomycin failed to prevent the increase of either enzyme in response to androgen provided the administration of the latter is started first. To verify this point, the experiment shown in Figure 5 was performed.

S T E R 0 I D S

782

4:6

J

D-AMINO ACID OXiDASE

iiiiii

!!iii iii!i !~ili iiili

~O

ii!i !!i!i

20

~!iii i o 0 DAYS ON T.P

.

2

4

8

'

Figure 4 Effects of delayed Actinomycin D administration upon testosterone propionate-induced stimulation of kidney enzyme activity. Testosterone propionate (1.0 mg.) was injected on alternate days, beginning on day O. Actinomycin D, 200 ~gm/kg, was given daily, beginning on day 2. Animals were sacrificed in groups of 6 (2 in one instance) at 2 day intervals, beginning on day 4, and the kidneys were assayed for ~-glucuronidase and D-amino acid oxidase (shaded bars). The solid lines were obtained by using animals undergoing the same schedule of testosterone propionate injections, but receiving no Actinomycin D~ Lefit ordinate: Units/gm. G-glucuronidase (xlO-°); Right ordinate: Colorimeter readings in the D-amino acid oxidase assay. The results (Fig. 5) indicate that actinomycin D has comparatively little effect upon the increase in kidney G-glucuronidase activity due to androgen administration if initiation of actinomycin treatment is begun either simultaneously with or subsequent to the first injection of androgen. A significant inhibition appears, however, if actinomycin is started before testosterone propionate. (The fact that inhibition of ~-glucuronidase synthesis is relatively less complete in this experiment compared to the experiment depicted in Figure l, ,may be attributed, in part at least, to the lower dose of actinomycin.)

Dec. 1 9 6 4

S T E R O I D S

783

i _

[

!

+

E

DAYS ON ACTINOMFYCN, 50 p,gm./Ko.

I

+oi =

J

t+

. i

+

I

i,,

,,

,,

o w T.P.

autP's

Figure 5 Influence of Actinomycin D upon B-glucuronidase response to testosterone propionate. Testosterone propionate (1 mg.) was injected on day l, and again on day 3. Actinomycin (150 ~gm/kg. daily) was started on day O, l, or 2. All animals were sacrificed on day 5 for G-glucuronidase determinations. Details as in Figure 1. Discussion Williams-Ashman,

et al. (!3) have summarized

the evidence which indicates vivo can increase

that testosterone

the incorporation

of amino

acids into prostatic protein by regulating centration of template RNAs nucleoprotein.

the con-

associated with ribo-

Such a process should presumably

be actinomycin-sensitive; our knowledge,

in

however,

there are, to

no published data concerning

extent to which androgen-dependent

the

protein synthe-

sis in prostate or other reproductive

tract tissue

is inhibited by actinomycin. Our data indicate

that the effects of testos-

784

S T E R O I D S

4:6

terone propionate,

and presumably of other androgens,

upon protein synthesis in the mouse kidney can be separated into components distinguishable

by their

sensitivity to actinomycin D. It is possible that the two components represent qualitatively different androgen-stimulable mechanisms

for protein synthesis.

hand, it is also possible reflect the differential

On the other

that our observations sensitivity of various

template RNA-synthesizing

reactions

to actinomycin

or a difference in the half-life of the corresponding mRNAs.

A choice between these (and pos-

sibly other) hypotheses must await the completion of additional experiments the quantitative

more carefully defining

relationships

involved.

Studies

of the effects of actinomycin upon isotope labeling of specific

tissue proteins would also be helpful.

One of the most interesting

findings of

these experiments was that the effect of actinomycin D upon the ~-glucuronidase

response

to tes-

tosterone propionate was greatly reduced if the latter is given first.

If, as current views

suggest, actinomycin D acts as a specific inhibitor of DNA-primed RNA synthesis,

its inability to

interrupt the stimulation of enzyme synthesis once the latter has been set in motion by androgen suggests metabolic

that the latter also acts at the same locus, i.e., that actinomycin D and tes-

tosterone are "competitive"

at the cell nucleus.

That a similar phenomenon may be involved in enzyme induction by other steroid hormones is suggested by the observations

of Greengard,

et al.

of the effects of actinomycin D on the neonatal increase in rat liver tyrosine-~ keto glutaric transaminase

(14).

As shown by Sereni,

the increase in enzyme concentration,

et al. (15),

which begins

two hours after birth, is adrenal dependent.

Dec. 1 9 6 4

S T E R 0 I D S

Greengard, et al. have shown that if actinomycin is given one hour after birth, the subsequent rise in liver transaminase is completely inhibited; in contrast, if administration of the antibiotic is delayed until the third hour, the subsequent rise in enzyme concentration is only partially inhibited.

Summary Actinomycin D, 200 ~gm/Kg/da, when given to mice, completely inhibits the increase in ~glucuronidase or D-amino acid oxidase concentrations in the kidneys which normally follows the injection of testosterone propionate.

In contrast, the

androgen-dependent increase in the rate of amino acid incorporation into kidney protein in vitro, is not significantly affected. Inhibition of ~-glucuronidase synthesis by actinomycin D occurs only if administration of the drug is begun before the first androgen injection.

If testosterone propionate is given first,

even prolonged administration of actinomycin D is ineffective. REFERENCES

.

Supported by research grants (A-1891 and FR05176 to E. H. Frieden) from the National Institutes of Health, US Public Health Service and by research grant (CA-07538, contract SA43-PH-3090 and Cancer Award, CA-K6-18453) from the National Institutes of Health, US Public Health Service and by Program Grm]t P-106, P107 of the American Cancer Society, Inc. to W. H. Fishman.

.

Present address: Department of Chemistry, Kent State University, Kent, Ohio.

.

Kochakian, C.D., Hill~ J., and Aonuma, S. Endocrinology, 72, 354 (1963).

785

786

.

S T E R 0 I D S

Plaut, A.G., and Fishman, W.H., J. Cell. Biol., 16, 253 (1963).

.

Pettengill, 0.S., and Fishman, W.H., Exp. Cell Research, 28, 248 (1962).

.

Frieden, E.H., and Harper, A.A., Endocrinology, 7_22, 465 (19o3). '

.

Obtained from Jackson Laboratories, Bar Harbor, Maine.

.

Obtained through the generosity of Merck, Sharp and Dohme, West Point, Pennsylvania.

.

lO.

DL~ucine-l-14C, 0.4 ~C/ml, 0.73 mM; glyci~1 -± C, 0.2 ~C/ml, 0.37 mM; L(+)-arginine-U -~ C, 0.2 ~C/ml, 0.37 mM. Fishman, W.H., in Methods of Hormone Research (R. Dorfman, Ed.) Vol. 4, Chapter lO, 1964, Academic Press, Inc., New York.

ll.

Corrigan, J.J., Wellner, D., and Meister, A. Biochimica et Biophysica Acta, 73, 50 (1963).

12.

Stolbach, L., Nisselbaum, J.S., and Fishman, W.H., Am. J. Clin. Path., 29, 379 (1958).

13.

14.

15.

Williams-Ashman, H.G., Liao, S., Hancock, R.L., Jurkowitz, L., and Silverman, D.A., Recent Progress in Hormone Research, 20, 292 (1964). Greengard, 0., Smith, M.A., and Acs, G., J. Biol. Chem., 238, 1548 (1963). Sereni, F., Kenney~ F~T., and Kretchner, N., J. Biol. Chem., 234, b09 (1959).

4:6