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EVIDENCE FOR THE^ STEROID-INDUCED ACCUMULATION OF
TYROSINE-AMINOTRANSFERASE MESSENGER^ RNA IN
THE ABSENCE OF PROTEIN SYNTHESIS
BY BEVERLY PETERKOFSKY* AND GORDON Mv1. ToMKINS LABORATORY OF MOLECULAR BIOLOGY, NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, BETHESDA, MARYLAND Communicated by Marshall Nirenberg, February 20, 1968
We have been studying the mechanism of TAT1 induction by glucocorticoid hormones in an established line of tissue culture cells (HTC cells).2 This system allows us to perform experiments which would be technically difficult or impossi- ble using intact animals. Although no gross changes in RNA synthesis were observed after treating these tissue culture cells with steroid, it was concluded on the basis of inhibitor studies that the synthesis and accumulation of enzyme- specific mRNA is required for induction. The present experiments were designed to determine whether steroid hormone could stimulate the appearance of TAT mRNA in the absence of protein syn- thesis. Several mechanisms of induction could be proposed in which protein synthesis would be required for mRNA accumulation. For example, the initial effect of the hormone could be to increase the rate of translation of the^ TAT mRNA itself which could secondarily lead to its accumulation; or^ TAT^ induc- tion might be the consequence of the prior induction of other enzymes which^ then produce the "true inducer" of TAT.^ The^ experiments^ described^ in^ this paper, however, show that protein synthesis is not^ needed^ for^ the^ accumulation^ of^ TAT- specific RNA resulting from treatment of^ HTC cells with steroid.^ We therefore conclude that the hormone acts directly either^ to^ increase^ mRNA^ synthesis^ or^ to decrease its degradation resulting in^ an^ increase in^ its^ concentration^ in^ the^ cell.
Materials and Methods.-Chemicals: Dexamethasone phosphate, sodium salt, a syn- thetic glucocorticoid, was obtained through the courtesy of the Merck Chemical Company. Actinomycin D and cycloheximide were obtained from the Cancer Chemotherapy Section, National Cancer Institute. Cell growth and incubation conditions: HTC cells, an established line derived from Morris rat hepatoma 7288c, were grown in Swim's medium as described previously. Cells were detached from the surface of the culture bottle as described previously and suspended in "induction medium" S774 which also contained penicillin, 100 units/ml; neomycin, 25 ,tg/ml; and 0.5% bovine serum. The cell concentration was approximately
106 cells/ml. Experiments were carried out in two steps: a preincubation period of^ 1.5-
2.5 hr followed by washing of the cells and then reincubation in fresh medium. For the preincubation, 10-ml portions of cell suspension were transferred to sterile 50-ml^ Erlen- meyer flasks and incubated in a Dubnoff metabolic shaker under 95% air-5% C02; inducer and inhibitors were added as described in the legends to the figures. The cultures were then transferred to sterile 45-ml conical centrifuge tubes with tightly closed screw caps and the tubes centrifuged for 4 min at 700 X g at room temperature. The super- natant medium was poured off and the cells were resuspended in fresh S77-medium equili- brated at 37°. The tubes were again centrifuged and the cells resuspended in^10 ml S77-medium at 37°. Then 2-ml portions were removed from each culture for TAT assays, and the remainder of the culture was transferred to clean sterile flasks,^ containing various reagents to be tested, for reincubation as described above. At various^ intervals, additional samples were removed for TAT assays. For enzyme assay, the cells were iso- 222
VOL. 60, 1968 BIOCHEMISTRY: PETERKOFSKY AND TOMKINS
lated by centrifugation, washed with buffer, and sonicated as described previously.4 The TAT activity was measured by the method of Diamondstone5 and protein by the method of Lowry et al.6 Specific activity is expressed as milliunits/mg of cell protein. The in- corporation of radioactive uridine into RNA and radioactive valine into protein, and the uptake of radioactive actinomycin D into the cells were measured according to methods described previously.
Results.-The role (^) of protein synthesis during initial steroid action: (^) The pur- pose of these experiments was to see whether the inducer stimulated the (^) accumu- lation of TAT mRNA in the absence of protein synthesis. (^) Cycloheximide was used to inhibit protein synthesis. Several concentrations were tested to (^) deter- mine their effect on the rate of C14-L-valine incorporation into protein; at 0.025 (^) Amole/ml there was an inhibition of (^95) per cent, and in the (^) present exper-
iments, a concentration of 0.10lsmole/ml, which produced an inhibition of about
97 per cent, was used routinely. As described under Materials and Methods, the following general procedure was used in these experiments: Cells were (^) preincubated with Dex in the (^) presence of cycloheximide, then both hormone and inhibitor were removed (^) by washing the cells, and finally the cells were reincubated in the absence of either steroid (^) or inhibitor. Preincubation and washing by themselves do not affect TAT (^) syn- thesis, as shown by the results illustrated in Figure 1, curve 1. In this (^) case,
E (^30)
20 E
10
40, A
- (^) Wash Wash I -2 -I^0 2 3 4 5 -2^ -I^0 HOURS
2 3 4 5
FIG. 1.-Level of TAT activity after preincubation of HTC cells with or without various additions. Cells were (^) preincubated for 1.5 hr, washed with medium, and reincubated as described in the Materials and Methods section.
(A) Curve 1 O---@ Curve 2 0 0 (B) Curve 1 *---- Curve2A 2 Curve S A---A Curve 4 0 o
Preincubation No addition No (^) addition Preincubation CH (^) + Dex CH CH CH (^) + Dex
Reincubation No addition Dex Reincubation No addition Dex No addition Dex
223
l-T
VOL. 60, (^1968) BIOCHEMISTRY: PETERKOFSKY AND TOMKINS
FIG. 2.-The effect of actinomycin D added (^) during preincubation on (^) subsequent 20 induction of TAT. Cells were preincubated for (^) 2.5 hr with 5 -/ cycloheximide and steroid with or without (^) / actinomycin which was added 15 (^) min prior X to the other additions. _
Preincubation (^) Reincubation - -O Act (^) D, CH, Dex No addition E A ush -@ @CH, Dex No addition o F^ I , O-O (^) CH, Dex (^) Act D -3 -2 (^) -I 0 1 2 3 4 5 6 HOURS
HTC (^) cells are exposed to actinomycin, the antibiotic cannot be removed by the washing (^) procedure, as shown by several experiments (not illustrated) using HI (^) -actinomycin D. (^) Furthermore, actinomycin-treated washed cells do not show steroid-dependent TAT^ induction. It must be assumed, then, that actinomycin remained in the cells during the reincubation period in the (^) experiments described above (Fig. 2). (^) Consequently, it was (^) necessary to determine whether it was this residual actinomycin which inhibited the (^) burst of TAT (^) activity. This was accomplished by first (^) preincubating cells with Dex and (^) cycloheximide and then reincubating them with 0.125 (^) ,gg/ml actinomycin D (^) (Fig. 2, open circles). Obvi- ously, the antibiotic, when present during the reincubation (^) period, did not in- hibit the rise in TAT activity. Therefore, the inhibitory effect of (^) actinomycin D on TAT induction in these experiments depended specifically on its (^) action during the preincubation period. For this reason, we conclude that the (^) steroid acts in the preincubation period, when protein (^) synthesis is (^) inhibited, to (^) cause the accumulation of RNA required for the induced synthesis of TAT. Inhibition of increase in TAT synthesis by readdition of cycloheximide: The increase in TAT activity observed after preincubation with steroid and (^) cyclo- heximide appears to result from new protein synthesis, since the rise could be prevented if inhibitory concentrations of cycloheximide were replaced after
washing, as illustrated in Figure 3 (closed circles). The smaller increase in enzyme
activity observed when actinomycin D was present during preincubation was also inhibited by cycloheximide (Fig. 3, triangles). Discussion.-The results of these experiments suggest that the steroid hormone brings about an increase in the amount of TAT mRNA in HTC cells without the obligatory prior synthesis of either TAT itself or any other protein. This
conclusion is based on the following observations: (1) induction of TAT oc-
curred in^ the absence of the steroid if HTC cells had been preincubated with
hormone in^ the presence of cycloheximide and (2) if actinomycin had been added
during preincubation, the induction was markedly inhibited. After preincuba-
tion with inducer and cycloheximide, further transcription was not required for
the increase in TAT activity in the present experiments, as evidenced by the fact
that (^) actinomycin did not inhibit (^) enzyme synthesis if (^) the antibiotic was added
only after removal^ of^ steroid and^ cycloheximide. Needless to say, these experi-
ments do not show whether the Dex molecule itself is the true inducer (i.e., inter-
acts directly in the processes of RNA synthesis or degradation). For the moment,
225
BIOCHEMISTRY: PETERKOFSKY AND TOMKINS (^) PROC. N. A. S.
10 O-I^ ~^ ~~~~~~~~~~~~I
(^9) _ * (^) FIG. 3.-The inhibition of induction after
_ preincubation^ with^ steroid and^ cycloheximide
by readdition^ of^ cycloheximide during the uj 7 I _ second^ incubation.^ Actinomycin was^ added^15 min prior to other additions made during the 6 2.5hr^ preincubation. Additions were made _ r (^) as follows:
5 _/,>_Preincubation 2 Reincubation I- 4 _^ |^ /^ _I CH,^ Dex^ CH O-O (^) CH, Dex No addition A---A (^) Act D, CH, Dex CH iHo>8 A* (^) iA ActD, CH, Dex No addition
*-0-_ CH, Dex CH, act D
Wash * ~ (^) ~ - CH, Dex Act D
-3 -2 - 0 2 3 4 HOURS
it is an open question whether the added steroid, one of its metabolites, or any
metabolic consequence of its presence is the immediate cause for mRNA accumu- lation. In certain respects, the induction of TAT by steroid hormones in HTC cells is
similar to the induction of #-galactosidase in E. coli. Pardee and Prestidge'
first showed that the induction of (^) f3-galactosidase in E. coli occurred even if the inducer was removed from cells after a few minutes' exposure, but before enzyme
synthesis started. Nakada and Magasanik" went on to show that during these
first few minutes, the fl-galactosidase-mRNA concentration was increased by the
inducer, whether or not protein synthesis occurred. A somewhat analogous situation occurs when larvae of the insect Chironomus
tentans are treated with the insect-molting hormone, ecdysone. This hormone
stimulates the incorporation of radioactive uridine into RNA at specific sites in
the salivary gland chromosomes of the insect and also causes the enlargement or "puffing" of these same loci in^ a definite sequence. When protein synthesis is inhibited by puromycin, the^ ecdysone-induced puffing of the loci affected earli-
est still occurs, although the^ hormone can no longer affect those sites which show
puffing later in the sequence.'2 Although the gene products of these loci have
not been identified, the fact that^ ecdysone is also a steroid hormone and that
RNA accumulation at^ specific sites^ occurs shortly after exposure of larvae to the
hormone suggests that its^ action^ may be^ similar^ to^ that of Dex in^ HTC cells. In some other (^) instances, the (^) accumulation of mRNA appears to depend upon
prior protein synthesis. During induction of^ the^ lac^ system of E. coli, it^ has
been shown that although the mRNA for ,-galactosidase appears when protein
synthesis is^ inhibited, mRNA for another^ enzyme coded^ for in^ the same operon,
thiogalactoside transacetylase, does^ not.'3^ As mentioned^ above, in^ Chironomus
tentans, those chromosomal loci which (^) normally exhibit (^) puffing late in the se-
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(^228) BIOCHEMISTRY: PETERKOFSKY AND TOMKINS PROC. N. A. S.
I (^) Even though TAT induction was significantly inhibited by the actinomycin present (^) during preincubation, in several experiments, the inhibition was never complete. The other experi- ments on the effect of actinomycin D on uridine incorporation into RNA mentioned in the text indicate that RNA synthesis should have been completely abolished under these conditions by the time the steroid and cycloheximide were added. The cause of this incomplete inhibition of induction, therefore, is not apparent and is currently under investigation. 10Pardee, A.^ B., and L.^ S. Prestidge, Biochim. Biophys. Acta, 49, 77 (1961). "Nakada, D., and B. Magasanik, J. Mol. Biol., 8, 105 (1964). 1" Clever, U., Science, 146, 794 (^) (1964). (^13) Alpers, D., and G. M. (^) Tomkins, J. Biol. Chem., 241, 4434 (1966). '4Sussman, M., Federation Proc., 26, 77 (1967).
RLeive, L., and V. Kollin, J. Mol. Biol., 24, 247 (1967).
(^16) Tomkins, G. (^) M., E. B. (^) Thompson, S. Hayashi, T. Gelehrter, D. Granner, and B. Peter- kofsky, in^ Cold^ Spring Harbor Symposia on Quantitative Biology, vol. 31 (1966), p. 349.
