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The synthesis of RNA by mammalian cells treated with the aminonucleoside of puromycin
424
BIOCHIMICAET BIOPHYSICAACTA
BBA 95832
T H E S Y N T H E S I S OF RNA BY MAMMALIAN CELLS T R E A T E D W I T H T H E A M I N O N U C L E O S I D E OF PUROMYCIN
J. M. TAYLOR AND C. P. STANNERS Department o] Medical Biophysics, University o[ Toronto, Toronto, Ontario (Canada)
(Received October 2nd, 1967)
SUMMARY
The aminonucleoside of puromycin will inhibit RNA synthesis in cultured hamster embryo cells with little effect on protein synthesis for several hours. In other systems, the drug-induced inhibition of RNA synthesis has been interpreted as a specific inhibition of ribosomal RNA (rRNA) synthesis with only a small effect on messenger RNA (mRNA) synthesis. In contrast, no evidence was found in this syst e m for such a selective inhibition, as the synthesis of rRNA, 'heavy nuclear' RNA, and polysomal m R N A were all inhibited to approximately equal extent b y the drug. In addition, the results are consistent with the interpretation that r R N A synthesized in the presence of the drug is open to more than normal degradation during its maturation and incorporation into ribosomes.
INTRODUCTION Until very recently, investigation of the action of the amino nucleoside of puromycin was confined to the clarification of three effects after administration in vivo: (i) its anti-trypanosomal and anti-parasitic effect, (ii) the causation of a characteristic nephrotic syndrome in rats and (iii) its greater effectiveness than the parent compound puromycin in the inhibition of mouse m a m m a r y tumors (see refs. I-3). In a recent s t u d y b y FARNHAM AND DUBIN4 it was reported that when the drug was added to L cells grown in vitro, RNA synthesis was preferentially inhibited with little effect on DNA and protein synthesis. Furthermore, these workers suggested that, relative to untreated cultures, the inhibition of RNA synthesis caused b y the aminonucleoside of puromycin (AN) involved an 85 ~o inhibition of ribosomal RNA (rRNA) and only a IO % inhibition of messenger RNA (mRNA). The purpose of this communication is to show that in hamster embryo cells growing exponentially in monolayer culture, under conditions of treatment with AN which had negligible effect on protein synthesis and yet caused an inhibition in the Abbreviations: AN, the aminonucleoside of puromycin; tRNA, transfer RNA; mRNA, messenger RNA; rRNA, ribosomal RNA. Biochim. Biophys. dcta, 155 (1968) 424-432
AMINONUCLEOSIDE ON R N A
425
SYNTHESIS
total RNA synthesis, no appreciable specificity in the inhibition could be found. That is, all species of RNA synthesized were equally inhibited. In addition, it seems t h a t the RNA that was synthesized under such conditions did not mature as did normal rRNA.
MATERIALS AND METHODS
Cells and medium Monolayer cultures of Syrian hamster embryo cells were used in their third to eighth subculture after explantation from the animal. The cultures were grown at 37 ° in glass medicine bottles containing an appropriate volume of growth medium in an atmosphere of 5 % C02-~ir (5:95, b y vol.). The cells grew with a doubling time of approx. 12 h under these conditions. Growth medium consisted of theta medium 5 (a supplemented form of EAGLE'S basal medium 6 plus IO % fetal calf serum (Flow Laboratories)).
Antibiotics and radioisotopes The aminonucleoside was purchased from the Nutritional Biochemical Corporation (Cleveland, Ohio) and the actinomycin D from Merck, Sharp and Dohme. The 14C-labelled uridine and valine were purchased from the Radiochemical Centre (Amersham, England) and the E3H]uridine from New England Nuclear (Boston, Mass.).
Measurement o~ incorporation o~ radioactive precursors into RNA and protein Cultures were grown for several generations in growth medium lacking ribonucleosides and deoxyribonucleosides and were in the exponential phase of growth at the time of the experiment. Radioactive precursor of RNA or protein: 14C-labelled uridine or valine, respectively, was added to the medium and incorporation was terminated b y immersion of the culture bottle in an ice-bath. The uridine label was considered sufficiently specific for RNA, since in a 4-h label (the longest labelling time used) less than 4 % of the activity went into DNA, as judged b y resistance to alkaline hydrolysis. The cells were removed from the glass surface b y 20 min treatment at o ° with a 1 % solution of Bacto-trypsin (Difco) in phosphate-buffered saline (see ref. 7). The cells were then centrifuged and resuspended in this buffer and then a suitable aliquot was precipitated with an equal volume of ice cold 20 % trichloroacetic acid. The precipitate was filtered and washed twice with ice cold 5 % trichloroacetic acid using a 0.45/* Millipore filter. The filter was pasted onto an aluminum planchet and counted with a Nuclear Chicago low background gas flow counter. Where two differently labelled precursors were used, the filter was placed in a vial, covered with 20 ml of scintillation fluid (4 g 2,5-bis-E2-(5-tert-butylbenzoxazolyl)]thiophene (Packard Instrument, Ilk) per 1 of toluene) and analysed with an Ansitron scintillation counter. Biochim. Biophys. Acta, 155 (1968) 424-432
420
J. M. TAYLOR, C. P. STANNERS
R N A extraction and analysis The extraction of RNA from whole cells was performed by a method very similar to that used by SCHARFF AND ROBBINSs. The method gave essentially the same results as a phenol extraction at high temperature and acid pH (see ref. io) but was more convenient and gave more reproducible data. Less than lO6 cells were supended in the extraction buffer (o.i M NaCl-o.o2 M MgC12-o.oI M Tris (pH 7.4)) at room temperature. A cytoplasmic extract containing ribosomes was prepared as described previously ° from about lO7unlabelled cells, and was added to the suspension to supply carrier RNA. The mixture was then made I ~o in sodium dodecyl sulfate to extract the RNA. For the extraction of polysomal RNA, a cytoplasmic extract from about 4" lO7 cells was prepared, as described previously 9. The extract was centrifuged on a 1530 ~o (w/w) sucrose gradient made up in the extraction buffer. The centrifugation was for 9° rain at 25 ooo rev./min in the SW 25.1 head of a Spinco Model L-2 ultracentrifuge at 4 °. The material of the polysome region was then collected and centrifuged 4.5 h at 25 ooo rev./min in the SW 25.1 head at 4 °. The pellet, which contained all of the polysome material, was suspended in I ml of the following buffer: o.I M NaCl-o.5 % sodium dodecyl sulfate-o.oo5 M Tris (pH 7.3). For both whole cell and polysomal RNA, the extracted RNA was subjected to velocity sedimentation analysis on a 15-3o % (w/w) sucrose gradient made up in the same buffer. The centrifugation was at 25 ooo rev./min in the SW 25.1 head at 19 °, and was for 7 or 11. 5 h, as indicated in the figure legends. After centrifugation the base of the tube was punctured and the contents pumped through the flow cell of a Gilford recording spectrometer which recorded the absorbance at 260 m#. The effluent from the flow cell was fractionated into 0.8 ml fractions and the acid precipitable radioactivity was measured as described above. (Before precipitation, 0.25 mg of bovine serum albumin was added to each fraction as coprecipitant.)
RESULTS
Selection o/conditions o~ treatment with A N Our interest in AN arose from the possibility that it could selectively inhibit the synthesis of rRNA leaving m R N A synthesis relatively unaffected, as has been suggested from results in other systems 4,11. Conditions of treatment were therefore sought which would be compatible with such selectivity. The following two necessary but not sufficient criteria for selectivity were used: first, the treatment should have little effect on protein synthesis, which depends of course on mRNA; second, the treatment should depress whole cell RNA synthesis b y an amount consistent with a reasonable depression of rRNA synthesis. This amount was considered to be 50 % or greater when RNA synthesis was measured b y the incorporation of I14C~uridine for a period of I h (under such conditions, about 50 0/o of the incorporated activity appeared, by sucrose gradient velocity centrifugation analysis, to be in mature rRNA, with some of the remaining activity in rRNA precursors (unpublished observation)). A series of replicate hamster embryo cultures were incubated in the presence Biochim. Biophys. Acta, 155 (1968) 424-432
A M I N O N U C L E O S I D E ON
RNA
427
SYNTHESIS
of a range of concentrations of AN, from o to 55/zg/ml. At various times after addition of the drug, a mixture of [3H]uridine and [14C]valine was added and incubation continued in the presence of the drug for a further hour. The acid precipitable incorporation of radioactivity was taken as a measure of RNA and protein synthesis and is shown in Tables I and I I respectively. Of the treatments shown in the tables,
TABLE
I
THE EFFECT OF AMINONUCLEOSIDE
PRETREATMENT
ON R N A
SYNTHESIS
The relative incorporation of [SH]uridine per cell was measured in the last hour of the incubation period and expressed as a percentage of appropriate control. Data is a combination of 2 incorporation experiments and 3 cell growth experiments. A N concentrations of 3/,g/ml or less did not affect cell growth. At IO/,g/ml and at 55/~g/ml of A N , the cultures barely doubled their cell n u m b e r within 48 h.
A N concH. (izg/ml)
Incubation period (h)
0
o I00
I 3 IO 55
TABLE
Relative incorporation of [SH]uridine per cell
----
--
4
IO
24
I00
I00
I00
96 81 88 36
87 71 72 37
99 93 80
25
II
THE EFFECT OF AMINONUCLEOSIDE PRETREATMENT ON PROTEIN SYNTHESIS T h e r e l a t i v e i n c o r p o r a t i o n of [14C]valine p e r cell w a s m e a s u r e d in t h e l a s t h o u r of t h e i n c u b a t i o n p e r i o d a n d e x p r e s s e d a s a p e r c e n t a g e of a p p r o p r i a t e c o n t r o l . D a t a is a c o m b i n a t i o n of 2 i n c o r p o r a t i o n e x p e r i m e n t s a n d 3 cell g r o w t h e x p e r i m e n t s .
A N concH. (pg/ml)
Incubation period (h)
Relative incorporation o[ [14C]valine per cell o
4
IO
24
IOO ---
IOO IOI IOI
IOO 95 95
IO
--
105
IOI
55
--
IOO 96 98 94 75
O I 3
98
87
only 4 h at 55/~g/ml of AN satisfies both of the above criteria. It was therefore adopted for further experimentation and will be subsequently referred to as the 'standard AN pretreatment'. A more detailed study of total RNA synthesis by hamster embryo cells in the presence of AN at 55/,g/ml was carried out in order to examine the kinetics of the inhibition and the reproducibility of the effect. The results of 3 separate experiments are shown in Fig. i and demonstrate that there is some variability from experiment to experiment in the action of the drug. However, the common and important feature is that the rate of RNA synthesis experiences a rapid fall-off which is complete within 4 h, after which it levels off somewhat at about 30 o/,o of the control rate. Biochim. Biophys. Acta, 155 (1968) 4 2 4 - 4 3 2
428
J. M. TAYLOR, C. P. STANNERS
10C
\
~_
6C
g .o 4C
~=~
r~rne since
addition of drug (h)
Fig. I. The effect of AN on t h e r a t e of R N A synthesis. AN w a s added at 55/~g/ml at time zero to a series of replicate, exponential cultures and, at various t i m e s thereafter, [14C]uridine w a s added a n d the i n c u b a t i o n continued for a f u r t h e r hour. The incorporation of [14C]uridine into acid precipitable material is s h o w n as a f u n c t i o n of the time (plus 0. 5 h) of addition of [14Cluridine. The results of 3 i n d e p e n d e n t e x p e r i m e n t s are shown.
The nature o/the R N A synthesis by A N treated cells Using the standard AN pretreatment chosen above to be compatible with selective inhibition of r R N A synthesis, the whole cell RNA synthesized during a further 4 h incubation in the presence of the drug and [14Cluridine was analyzed b y the sucrose gradient velocity centrifugation technique. The absorbance and radioactivity profiles for the treated and control cultures are shown in Fig. 2. Apart from the previously seen quantitative reduction in the total radioactivity caused b y the drug treatment, an important qualitative effect can be seen in the radioactivity profile. The ratio of the activity in the 28-S and I8-S r R N A peaks was less than the approx. 2:1 ratio of the controls. Also, the radioactivity sedimenting between and ahead of the two ribosomal peaks was relatively higher than in the control. Other treatments which should favour the appearance of label in stable RNA gave similar results: RNA from cells labelled for 30 min with E14Cluridine, followed b y a 4 h 'chase' incubation in medium containing actinomycin D at IO #g/ml, or RNA from isolated cytoplasmic polysomes after continuous incubation of the cells in the presence of [14C]uridine for 2 to 8 h showed sucrose-gradient patterns similar to that of Fig. 2b. These qualitative differences in the sucrose density gradient profiles indicated that there was some specificity in the action of the drug upon the distribution of the label in the various RNA species. This specificity could have been at the level of synthesis, as has been suggested b y other workers 4,n, indicating that rRNA was selectively inhibited with respect to the other classes. The following experiments were designed to test whether this hypothesis could be applied to our system. The effect of the pretreatment with AN on the incorporation of labelled uridine into 3 different classes of RNA: rRNA, 'heavy nuclear' R N A and polysomal mRNA, was measured. First, concerning the synthesis of rRNA, the results of Fig. 2 indicate that AN detinitely inhibited the synthesis of mature rRNA. In order to test whether this inhibition was due to an inhibition of the synthesis, or to the maturation of the rRNA precursors TM or, perhaps, to both, the following experiment was performed. After the standard AN pretreatment, cultures were incubated for a further I0, 20 or 3o min in the presence of the drug and [14C]uridine. The whole cell RNA was extracted from these cultures and from control cultures not treated with Biochim. Biophys. Acta, 155 (1968) 424-432
AMINONUCLEOSIDE ON i
I
R N A SYNTHESIS i
429
i
a) C o n t r o l
a) Control
28S
3000
0.8
~~ S
I
LSO00 2000
~0000
Q,I
1BS
IOO0
5000
E
E
Bottom
0
Bottom
Top
o
Top i
'
i
i
o~
(o
b)AN-Pretreote
U 500
O.E
1500
I000 300 500
10 Bottom
20 Fro ction
30 No.
Top
Bottom
Top
Fig. 2. Sucrose gradient velocity sedimentation analysis of the total cellular R N A synthesized for 4 h in the presence of [l*C]uridine (0. 7 # C / m l a t 29 ° m C / m M ) b y c o n t r o l cultures and by cultures given the standard A N pretreatment (55/zg/ml for 4 Ix). Centrifugation was for 11.5 h a t 25 ooo r e v . / m i n to a c h i e v e m a x i m u m r e s o l u t i o n of the r R N A peaks. The circles represent the acid precipitable radioactivity in each fraction and tile solid lines the absorbance at 260 m/z due largely to a d d e d u n l a b e l l e d carrier c y t o p l a s m i c RNA. Fig. 3. Sucrose gradient velocity sedimentation analysis of whole cell R N A synthesized for IO, 20 a n d 3 ° min b y c o n t r o l cultures and by cultures given the standard A N pretreatment. Cent r i f u g a t i o n was for 7 h a t 25 ooo r e v . / m i n . T h e solid lines represent the absorbance at 260 m / , due largely to a d d e d u n l a b e l l e d carrier c y t o p l a s m i c R N A and is the same for all 3 gradients in (a) a n d (b). T h e acid precipitable radioactivity in each fraction is shown for R N A extracted f r o m c u l t u r e s e x p o s e d to [l*C]uridine (2.2/~C]ml a t 29o m C / m M ) for io min (triangles), 20 rain (closed circles) a n d 3 ° rain (open circles).
AN, and analyzed by sucrose density gradient centrifugation. The absorbance and radioactivity profiles from the gradients are shown in Fig. 3. Other workers have shown TM that the precursor of mature 28-S and I8-S rRNA is a large molecule (45 S) which matures to I8-S rRNA directly and to 28-S r R N A through an intermediate 35-S species. The radioactivity profiles in Fig. 3 for the control cultures show peaks at 45 S and 32 S approximately; it can also be seen that a small amount of maturation into mature rRNA had occurred within the 30 min. The radioactivity profiles for the treated cultures are quantitatively reduced relative to the control profiles (note the large change in the ordinate scales in Fig. 3 between control and treated profiles), and show changes in the shape somewhat similar to those seen at longer labelling times in Fig. 2. Regardless of the interpretation of the shape changes (which will be considered in DISCUSSION), the synthesis of r R N A precursors was clearly strongly depressed by drug treatment, and the depression of the synthesis of mature rRNA seen in Fig. 2 was not due solely to an inhibition of maturation. Second, in order to examine the effect of the standard AN pretreatment on the synthesis of heavy nuclear R N A 13, a sucrose gradient analysis was made of the whole Biochirn. Biophys. Acta, 155 (1968) 424-432
430
J. M. TAYLOR, C. P. STANNERS
cell R N A extracted from cells i n c u b a t e d for 3 m i n with [14C]uridine. Other workers have found t h a t after such a short i n c u b a t i o n with labelled precursor, m o s t of the labelled R N A in the cell is r a p i d l y s e d i m e n t i n g h e a v y nuclear R N A is a n d the radioa c t i v i t y profiles shown in Fig. 4 s u p p o r t the e x t r a p o l a t i o n of this finding to our system. The synthesis of h e a v y nuclear R N A was clearly depressed b y the drug t r e a t m e n t a n d b y a n a m o u n t (49 %, as estimated b y the reduction in the total acid precipitable c o u n t s / m i n s e d i m e n t i n g faster t h a n 45 S a n d in the pellet) within the range at the depression seen i n whole cell R N A synthesis over longer i n c u b a t i o n s in other experi m e n t s (Fig. I). I t is possible t h a t the p r e t r e a t m e n t with AN affected the intracellular u r i d i n e pools in such a w a y t h a t the incorporation of [14C]uridine into R N A was reduced more for short (3 min) t h a n for longer (15 m i n to I h) incubations. We consider this e x p l a n a t i o n for the observed r e d u c t i o n in labelled h e a v y nuclear R N A unlikely, however, as the rate of incorporation of [14C]uridine into total R N A was a p p r o x i m a t e l y c o n s t a n t in n o r m a l or AN pretreated cells for periods of 3 m i n up to several hours. Third, to measure the effect of the s t a n d a r d AN p r e t r e a t m e n t on the synthesis of cytoplasmic m R N A , the label in isolated polysomes from cultures i n c u b a t e d for i
1
i
160
a) C o n t r o l 0.6 28S Q4
~0 185
0.2
Bottom o
10
20
b ) A ~
30
Top
s'
80
o iC.ont to{ T ~S
0.4
,
~
-- 100
40
0.~
._c
o
18S
0.=
E 5O ~-
o~
o
10 Bottom
20 30 F r a c t i o n No.
Top
0 ~ottom
10
20 Fraction
30 No.
40 Top
Fig. 4. Sucrose gradient velocity sedimentation analysis of whole cell RNA synthesized for 3 min by control cultures and by cultures given the standard AN pretreatment. Centrifugation was for 7 h at 25 ooo rev./min. The solid lines represent absorbance at 260 m/z due largely to added unlabelled carrier cytoplasmic RNA. The circles represent the acid precipitable radioactivity in each fraction from RNA extracted from cultures exposed to [x4C]uridine (o.75/,C/ml at 325 mC/ mM) for 3 min. Fig. 5. Sucrose gradient velocity sedimentation analysis of polysomal RNA from control cultures and cultures given the standard AN pretreatment after exposure of the cultures to [14C]uridine (0.4/2C/ml at 324 mC/mM) for 15 mill. Centrifugation was for 7 h at 25 ooo rev./min. Polysomal RNA was obtained as outlined in MATERIALSAND METHODS,The solid line represents absorbance at 26o m#. The open and closed circles represent acid precipitable radioactivity in each fraction for control and AN pretreated cultures, respectively. Biochim. Biophys. Acta, 155 (1968) 424-432
AMINONUCLEOSIDE ON R N A SYNTHESIS
431
15 rain with [14C]uridine was analyzed by sucrose-density-gradient analysis. Since the radioactivity profiles shown in Fig. 5 are heterodisperse with no peaks at the position of I8-S and 28-S rRNA or at the 4-S transport RNA (tRNA) and if polysomes do indeed contain only mRNA, tRNA and rRNA, then this heterogeneous RNA must be mRNA. Its synthesis can be seen to be depressed in the drug treated cells. The extent of this depression (52 %) was the same as the depression in the whole cell RNA synthesis in 15 rain (53 %) observed in this particular experiment. Thus the synthesis of rRNA, heavy nuclear RNA, and polysomal mRNA all appear to be inhibited to approximately the same extent by the drug. DISCUSSION
The above results establish the fact that under suitable conditions AN can depress the synthesis of RNA in cultured hamster embryo cells with little effect on protein synthesis. Similar results have been obtained b y FARNHAM AND DUBIN4 for mouse L cells and b y STUDZINSKIAND ELLEM11 for human HeLa cells. The fact that protein synthesis is unaffected for relatively long times (4 to IO h) under such conditions indicates that the inhibition of RNA synthesis does not cause the cell to become greatly depleted in the mRNA required for the majority of protein synthesis. This result can be interpreted in one of two ways: either the cells can maintain protein synthesis for some time with the mRNA present prior to the drug treatment or they continue suitable mRNA synthesis in the presence of the drug. In accordance with the second interpretation, both FARNHAM AND DUBIN and STUDZINSKI AND ELLEM have suggested that the drug selectively inhibits the synthesis of rRNA, leaving mRNA synthesis relatively unaffected. No evidence for selective inhibition of RNA synthesis was found, however, in our study of the synthesis of ribosomal RNA precursors, heavy nuclear RNA and polysomal mRNA, since each class of RNA was inhibited to approximately the same extent. We have no conclusive evidence that the synthesis of polysomal mRNA, as detected by early incorporation of E14C~uridine into cytoplasmic polysomes, is actually representative of all of the cellular mRNA. Since the polysomes account for about 9 ° % of the total protein synthesis in hamster embryo cells 14, however, this interpretation seems likely. Our evidence thus strongly favours the interpretation that the inhibition of RNA synthesis by AN in our system and under our conditions of treatment, at least, is not selective for any particular class of RNA and that protein synthesis continues unabated by virtue of the stability of pre-existing mRNA. Work in our system (unpublished) with actinomycin D supports this latter view: protein synthesis is only slightly affected for several hours after addition of this drug to cultures at concentrations which severely depress the synthesis of cell RNA. The distribution of RNA synthesized over relatively long time periods in the presence of AN, as detected b y sucrose density gradient analysis (see Fig. 2) showed a qualitative difference from the control distribution: the ratio of the radioactivity incorporated into 28-S and into I8-S rRNA was less than the 2 : I ratio of the controls and the radioactivity sedimenting between and ahead of the two ribosomal peaks was relatively higher than in the controls. This finding has been observed in other systems and has been interpreted as evidence for a selective inhibition of rRNA synthesis 4. Our failure to detect such selective inhibition leads to the alternative suggesBiochim. Biophys. Acta, 155 (1968) 424-432
432
J. M. TAYLOR, C. P. STANNERS
tion that the drug also affects the maturation of the rRNA precursors in such a way t h a t they are left open to more than normal degradation. The proposed selective degradation would tend to broaden the peaks of the synthesized r R N A seen in a sucrose gradient giving relatively more material between and ahead of these peaks, and, if the more complex maturation to 28-S r R N A 12 is more affected than the maturation to I8-S rRNA, to raise the height of the I8-S r R N A peak relative to the 28-S r R N A peak, as observed (see Figs. 2 and 3). A further aspect ot the experiment shown in Fig. 3 is also consistent with this proposal. The change in sedimentation distribution of the labelled RNA in the AN treated cultures as the labelling time increased from io to 30 min was different from that of the control, thus indicating something abnormal about the r R N A maturation. In s u m m a r y then, our chosen pretreatment with AN did not give selective inhibition of RNA synthesis. This result is inconsistent with the previously mentioned interpretation of the drug's action b y FARNHAM AND DUBIN*. STUDZINSKI AND ELLEMu conceded both selective inhibition of r R N A synthesis and selective degradation of rRNA as consistent with their data. The inconsistency between our results and those of others could be attributed to the use of the different cell systems or to differences in the dose and time of treatment with the drug. For example, it is possible that in this system a longer exposure to a slightly lower concentration of AN m a y have given a selective effect. This type of treatment with AN was avoided here as the longer exposures to the drug required for pretreatment plus the time needed for labelling studies resulted in a detachment of the cells from the glass surface. Though AN has not proven useful in our system as a means of labelling m R N A selectively, it could be used as a mild non-selective in hibitor of cellular RNA synthesis. ACKNOWLEDGEMENTS
This work was supported b y the National Cancer Institute of Canada and by grant MA-I877 from the Medical Research Council of Canada. One of us (J.M.T.) acknowledges the assistance of an award from the Commonwealth Scholarship and Fellowship Plan.
REFERENCES i M, NAKAMURA AND S. JONSSON, Arch. Biochem. Biophys., 66 (1957) 183. 2 S. FRENK, I. ANTONOWICZ, J. M. CRAIG AND J. METCOFF, Proc. Soc. Exptl. Biol. Med., 89 (1955) 424 . 3 S. G. F. WILSON, D. B, HACKEL, S. HORWOOD, G. NASH AND N. HEYMANN, Pediatrics, 21 (1958 ) 963. 4 A. E. FARNHAM AND O. T. DUBIN, J. Mol. Biol., 14 (1965) 55" 5 R. W. READER, M. A. Thesis, U n i v e r s i t y of Toronto, Canada, 1966, p - A I - 3. 6 H. EAGLE, Science, 13o (1959) 423 • 7 R. DULBECCO AND M. VOGT, J. Exptl. Med., 99 (1954) 167. 8 M. D . SCHARFF AND E . ROBBINS, Nature, 208 (1965) 464 . 9 C. P. STANNERS, Biochem. Biophys. Res. Commun., 24 (1966) 758. IO K . SCHERRER AND J . E . DARNELL, Biochem. Biophys. Res. Commun., 7 (1962) 486. I I G. P . STUDZlNSKI AND Z . A . O. ELLEM, J. Cell. Biol., 29 (1966) 411. 12 S. PENMAN, I. SMITH AND E. HOLTZMAN, Science, 154 (1966) 786. 13 J. R. WARNER, R. SOEIRO, H. C. BIRNBOIM, M. GIRARD AND J. E. DARNELL, J. Mol. Biol., 19 (1966) 349" 14 C. P. STANNERS, Biophys..]'., in the press.
Biochim. Biophys. Acta, 155 (1968) 424-432
BIOCHIMICAET BIOPHYSICAACTA
BBA 95832
T H E S Y N T H E S I S OF RNA BY MAMMALIAN CELLS T R E A T E D W I T H T H E A M I N O N U C L E O S I D E OF PUROMYCIN
J. M. TAYLOR AND C. P. STANNERS Department o] Medical Biophysics, University o[ Toronto, Toronto, Ontario (Canada)
(Received October 2nd, 1967)
SUMMARY
The aminonucleoside of puromycin will inhibit RNA synthesis in cultured hamster embryo cells with little effect on protein synthesis for several hours. In other systems, the drug-induced inhibition of RNA synthesis has been interpreted as a specific inhibition of ribosomal RNA (rRNA) synthesis with only a small effect on messenger RNA (mRNA) synthesis. In contrast, no evidence was found in this syst e m for such a selective inhibition, as the synthesis of rRNA, 'heavy nuclear' RNA, and polysomal m R N A were all inhibited to approximately equal extent b y the drug. In addition, the results are consistent with the interpretation that r R N A synthesized in the presence of the drug is open to more than normal degradation during its maturation and incorporation into ribosomes.
INTRODUCTION Until very recently, investigation of the action of the amino nucleoside of puromycin was confined to the clarification of three effects after administration in vivo: (i) its anti-trypanosomal and anti-parasitic effect, (ii) the causation of a characteristic nephrotic syndrome in rats and (iii) its greater effectiveness than the parent compound puromycin in the inhibition of mouse m a m m a r y tumors (see refs. I-3). In a recent s t u d y b y FARNHAM AND DUBIN4 it was reported that when the drug was added to L cells grown in vitro, RNA synthesis was preferentially inhibited with little effect on DNA and protein synthesis. Furthermore, these workers suggested that, relative to untreated cultures, the inhibition of RNA synthesis caused b y the aminonucleoside of puromycin (AN) involved an 85 ~o inhibition of ribosomal RNA (rRNA) and only a IO % inhibition of messenger RNA (mRNA). The purpose of this communication is to show that in hamster embryo cells growing exponentially in monolayer culture, under conditions of treatment with AN which had negligible effect on protein synthesis and yet caused an inhibition in the Abbreviations: AN, the aminonucleoside of puromycin; tRNA, transfer RNA; mRNA, messenger RNA; rRNA, ribosomal RNA. Biochim. Biophys. dcta, 155 (1968) 424-432
AMINONUCLEOSIDE ON R N A
425
SYNTHESIS
total RNA synthesis, no appreciable specificity in the inhibition could be found. That is, all species of RNA synthesized were equally inhibited. In addition, it seems t h a t the RNA that was synthesized under such conditions did not mature as did normal rRNA.
MATERIALS AND METHODS
Cells and medium Monolayer cultures of Syrian hamster embryo cells were used in their third to eighth subculture after explantation from the animal. The cultures were grown at 37 ° in glass medicine bottles containing an appropriate volume of growth medium in an atmosphere of 5 % C02-~ir (5:95, b y vol.). The cells grew with a doubling time of approx. 12 h under these conditions. Growth medium consisted of theta medium 5 (a supplemented form of EAGLE'S basal medium 6 plus IO % fetal calf serum (Flow Laboratories)).
Antibiotics and radioisotopes The aminonucleoside was purchased from the Nutritional Biochemical Corporation (Cleveland, Ohio) and the actinomycin D from Merck, Sharp and Dohme. The 14C-labelled uridine and valine were purchased from the Radiochemical Centre (Amersham, England) and the E3H]uridine from New England Nuclear (Boston, Mass.).
Measurement o~ incorporation o~ radioactive precursors into RNA and protein Cultures were grown for several generations in growth medium lacking ribonucleosides and deoxyribonucleosides and were in the exponential phase of growth at the time of the experiment. Radioactive precursor of RNA or protein: 14C-labelled uridine or valine, respectively, was added to the medium and incorporation was terminated b y immersion of the culture bottle in an ice-bath. The uridine label was considered sufficiently specific for RNA, since in a 4-h label (the longest labelling time used) less than 4 % of the activity went into DNA, as judged b y resistance to alkaline hydrolysis. The cells were removed from the glass surface b y 20 min treatment at o ° with a 1 % solution of Bacto-trypsin (Difco) in phosphate-buffered saline (see ref. 7). The cells were then centrifuged and resuspended in this buffer and then a suitable aliquot was precipitated with an equal volume of ice cold 20 % trichloroacetic acid. The precipitate was filtered and washed twice with ice cold 5 % trichloroacetic acid using a 0.45/* Millipore filter. The filter was pasted onto an aluminum planchet and counted with a Nuclear Chicago low background gas flow counter. Where two differently labelled precursors were used, the filter was placed in a vial, covered with 20 ml of scintillation fluid (4 g 2,5-bis-E2-(5-tert-butylbenzoxazolyl)]thiophene (Packard Instrument, Ilk) per 1 of toluene) and analysed with an Ansitron scintillation counter. Biochim. Biophys. Acta, 155 (1968) 424-432
420
J. M. TAYLOR, C. P. STANNERS
R N A extraction and analysis The extraction of RNA from whole cells was performed by a method very similar to that used by SCHARFF AND ROBBINSs. The method gave essentially the same results as a phenol extraction at high temperature and acid pH (see ref. io) but was more convenient and gave more reproducible data. Less than lO6 cells were supended in the extraction buffer (o.i M NaCl-o.o2 M MgC12-o.oI M Tris (pH 7.4)) at room temperature. A cytoplasmic extract containing ribosomes was prepared as described previously ° from about lO7unlabelled cells, and was added to the suspension to supply carrier RNA. The mixture was then made I ~o in sodium dodecyl sulfate to extract the RNA. For the extraction of polysomal RNA, a cytoplasmic extract from about 4" lO7 cells was prepared, as described previously 9. The extract was centrifuged on a 1530 ~o (w/w) sucrose gradient made up in the extraction buffer. The centrifugation was for 9° rain at 25 ooo rev./min in the SW 25.1 head of a Spinco Model L-2 ultracentrifuge at 4 °. The material of the polysome region was then collected and centrifuged 4.5 h at 25 ooo rev./min in the SW 25.1 head at 4 °. The pellet, which contained all of the polysome material, was suspended in I ml of the following buffer: o.I M NaCl-o.5 % sodium dodecyl sulfate-o.oo5 M Tris (pH 7.3). For both whole cell and polysomal RNA, the extracted RNA was subjected to velocity sedimentation analysis on a 15-3o % (w/w) sucrose gradient made up in the same buffer. The centrifugation was at 25 ooo rev./min in the SW 25.1 head at 19 °, and was for 7 or 11. 5 h, as indicated in the figure legends. After centrifugation the base of the tube was punctured and the contents pumped through the flow cell of a Gilford recording spectrometer which recorded the absorbance at 260 m#. The effluent from the flow cell was fractionated into 0.8 ml fractions and the acid precipitable radioactivity was measured as described above. (Before precipitation, 0.25 mg of bovine serum albumin was added to each fraction as coprecipitant.)
RESULTS
Selection o/conditions o~ treatment with A N Our interest in AN arose from the possibility that it could selectively inhibit the synthesis of rRNA leaving m R N A synthesis relatively unaffected, as has been suggested from results in other systems 4,11. Conditions of treatment were therefore sought which would be compatible with such selectivity. The following two necessary but not sufficient criteria for selectivity were used: first, the treatment should have little effect on protein synthesis, which depends of course on mRNA; second, the treatment should depress whole cell RNA synthesis b y an amount consistent with a reasonable depression of rRNA synthesis. This amount was considered to be 50 % or greater when RNA synthesis was measured b y the incorporation of I14C~uridine for a period of I h (under such conditions, about 50 0/o of the incorporated activity appeared, by sucrose gradient velocity centrifugation analysis, to be in mature rRNA, with some of the remaining activity in rRNA precursors (unpublished observation)). A series of replicate hamster embryo cultures were incubated in the presence Biochim. Biophys. Acta, 155 (1968) 424-432
A M I N O N U C L E O S I D E ON
RNA
427
SYNTHESIS
of a range of concentrations of AN, from o to 55/zg/ml. At various times after addition of the drug, a mixture of [3H]uridine and [14C]valine was added and incubation continued in the presence of the drug for a further hour. The acid precipitable incorporation of radioactivity was taken as a measure of RNA and protein synthesis and is shown in Tables I and I I respectively. Of the treatments shown in the tables,
TABLE
I
THE EFFECT OF AMINONUCLEOSIDE
PRETREATMENT
ON R N A
SYNTHESIS
The relative incorporation of [SH]uridine per cell was measured in the last hour of the incubation period and expressed as a percentage of appropriate control. Data is a combination of 2 incorporation experiments and 3 cell growth experiments. A N concentrations of 3/,g/ml or less did not affect cell growth. At IO/,g/ml and at 55/~g/ml of A N , the cultures barely doubled their cell n u m b e r within 48 h.
A N concH. (izg/ml)
Incubation period (h)
0
o I00
I 3 IO 55
TABLE
Relative incorporation of [SH]uridine per cell
----
--
4
IO
24
I00
I00
I00
96 81 88 36
87 71 72 37
99 93 80
25
II
THE EFFECT OF AMINONUCLEOSIDE PRETREATMENT ON PROTEIN SYNTHESIS T h e r e l a t i v e i n c o r p o r a t i o n of [14C]valine p e r cell w a s m e a s u r e d in t h e l a s t h o u r of t h e i n c u b a t i o n p e r i o d a n d e x p r e s s e d a s a p e r c e n t a g e of a p p r o p r i a t e c o n t r o l . D a t a is a c o m b i n a t i o n of 2 i n c o r p o r a t i o n e x p e r i m e n t s a n d 3 cell g r o w t h e x p e r i m e n t s .
A N concH. (pg/ml)
Incubation period (h)
Relative incorporation o[ [14C]valine per cell o
4
IO
24
IOO ---
IOO IOI IOI
IOO 95 95
IO
--
105
IOI
55
--
IOO 96 98 94 75
O I 3
98
87
only 4 h at 55/~g/ml of AN satisfies both of the above criteria. It was therefore adopted for further experimentation and will be subsequently referred to as the 'standard AN pretreatment'. A more detailed study of total RNA synthesis by hamster embryo cells in the presence of AN at 55/,g/ml was carried out in order to examine the kinetics of the inhibition and the reproducibility of the effect. The results of 3 separate experiments are shown in Fig. i and demonstrate that there is some variability from experiment to experiment in the action of the drug. However, the common and important feature is that the rate of RNA synthesis experiences a rapid fall-off which is complete within 4 h, after which it levels off somewhat at about 30 o/,o of the control rate. Biochim. Biophys. Acta, 155 (1968) 4 2 4 - 4 3 2
428
J. M. TAYLOR, C. P. STANNERS
10C
\
~_
6C
g .o 4C
~=~
r~rne since
addition of drug (h)
Fig. I. The effect of AN on t h e r a t e of R N A synthesis. AN w a s added at 55/~g/ml at time zero to a series of replicate, exponential cultures and, at various t i m e s thereafter, [14C]uridine w a s added a n d the i n c u b a t i o n continued for a f u r t h e r hour. The incorporation of [14C]uridine into acid precipitable material is s h o w n as a f u n c t i o n of the time (plus 0. 5 h) of addition of [14Cluridine. The results of 3 i n d e p e n d e n t e x p e r i m e n t s are shown.
The nature o/the R N A synthesis by A N treated cells Using the standard AN pretreatment chosen above to be compatible with selective inhibition of r R N A synthesis, the whole cell RNA synthesized during a further 4 h incubation in the presence of the drug and [14Cluridine was analyzed b y the sucrose gradient velocity centrifugation technique. The absorbance and radioactivity profiles for the treated and control cultures are shown in Fig. 2. Apart from the previously seen quantitative reduction in the total radioactivity caused b y the drug treatment, an important qualitative effect can be seen in the radioactivity profile. The ratio of the activity in the 28-S and I8-S r R N A peaks was less than the approx. 2:1 ratio of the controls. Also, the radioactivity sedimenting between and ahead of the two ribosomal peaks was relatively higher than in the control. Other treatments which should favour the appearance of label in stable RNA gave similar results: RNA from cells labelled for 30 min with E14Cluridine, followed b y a 4 h 'chase' incubation in medium containing actinomycin D at IO #g/ml, or RNA from isolated cytoplasmic polysomes after continuous incubation of the cells in the presence of [14C]uridine for 2 to 8 h showed sucrose-gradient patterns similar to that of Fig. 2b. These qualitative differences in the sucrose density gradient profiles indicated that there was some specificity in the action of the drug upon the distribution of the label in the various RNA species. This specificity could have been at the level of synthesis, as has been suggested b y other workers 4,n, indicating that rRNA was selectively inhibited with respect to the other classes. The following experiments were designed to test whether this hypothesis could be applied to our system. The effect of the pretreatment with AN on the incorporation of labelled uridine into 3 different classes of RNA: rRNA, 'heavy nuclear' R N A and polysomal mRNA, was measured. First, concerning the synthesis of rRNA, the results of Fig. 2 indicate that AN detinitely inhibited the synthesis of mature rRNA. In order to test whether this inhibition was due to an inhibition of the synthesis, or to the maturation of the rRNA precursors TM or, perhaps, to both, the following experiment was performed. After the standard AN pretreatment, cultures were incubated for a further I0, 20 or 3o min in the presence of the drug and [14C]uridine. The whole cell RNA was extracted from these cultures and from control cultures not treated with Biochim. Biophys. Acta, 155 (1968) 424-432
AMINONUCLEOSIDE ON i
I
R N A SYNTHESIS i
429
i
a) C o n t r o l
a) Control
28S
3000
0.8
~~ S
I
LSO00 2000
~0000
Q,I
1BS
IOO0
5000
E
E
Bottom
0
Bottom
Top
o
Top i
'
i
i
o~
(o
b)AN-Pretreote
U 500
O.E
1500
I000 300 500
10 Bottom
20 Fro ction
30 No.
Top
Bottom
Top
Fig. 2. Sucrose gradient velocity sedimentation analysis of the total cellular R N A synthesized for 4 h in the presence of [l*C]uridine (0. 7 # C / m l a t 29 ° m C / m M ) b y c o n t r o l cultures and by cultures given the standard A N pretreatment (55/zg/ml for 4 Ix). Centrifugation was for 11.5 h a t 25 ooo r e v . / m i n to a c h i e v e m a x i m u m r e s o l u t i o n of the r R N A peaks. The circles represent the acid precipitable radioactivity in each fraction and tile solid lines the absorbance at 260 m/z due largely to a d d e d u n l a b e l l e d carrier c y t o p l a s m i c RNA. Fig. 3. Sucrose gradient velocity sedimentation analysis of whole cell R N A synthesized for IO, 20 a n d 3 ° min b y c o n t r o l cultures and by cultures given the standard A N pretreatment. Cent r i f u g a t i o n was for 7 h a t 25 ooo r e v . / m i n . T h e solid lines represent the absorbance at 260 m / , due largely to a d d e d u n l a b e l l e d carrier c y t o p l a s m i c R N A and is the same for all 3 gradients in (a) a n d (b). T h e acid precipitable radioactivity in each fraction is shown for R N A extracted f r o m c u l t u r e s e x p o s e d to [l*C]uridine (2.2/~C]ml a t 29o m C / m M ) for io min (triangles), 20 rain (closed circles) a n d 3 ° rain (open circles).
AN, and analyzed by sucrose density gradient centrifugation. The absorbance and radioactivity profiles from the gradients are shown in Fig. 3. Other workers have shown TM that the precursor of mature 28-S and I8-S rRNA is a large molecule (45 S) which matures to I8-S rRNA directly and to 28-S r R N A through an intermediate 35-S species. The radioactivity profiles in Fig. 3 for the control cultures show peaks at 45 S and 32 S approximately; it can also be seen that a small amount of maturation into mature rRNA had occurred within the 30 min. The radioactivity profiles for the treated cultures are quantitatively reduced relative to the control profiles (note the large change in the ordinate scales in Fig. 3 between control and treated profiles), and show changes in the shape somewhat similar to those seen at longer labelling times in Fig. 2. Regardless of the interpretation of the shape changes (which will be considered in DISCUSSION), the synthesis of r R N A precursors was clearly strongly depressed by drug treatment, and the depression of the synthesis of mature rRNA seen in Fig. 2 was not due solely to an inhibition of maturation. Second, in order to examine the effect of the standard AN pretreatment on the synthesis of heavy nuclear R N A 13, a sucrose gradient analysis was made of the whole Biochirn. Biophys. Acta, 155 (1968) 424-432
430
J. M. TAYLOR, C. P. STANNERS
cell R N A extracted from cells i n c u b a t e d for 3 m i n with [14C]uridine. Other workers have found t h a t after such a short i n c u b a t i o n with labelled precursor, m o s t of the labelled R N A in the cell is r a p i d l y s e d i m e n t i n g h e a v y nuclear R N A is a n d the radioa c t i v i t y profiles shown in Fig. 4 s u p p o r t the e x t r a p o l a t i o n of this finding to our system. The synthesis of h e a v y nuclear R N A was clearly depressed b y the drug t r e a t m e n t a n d b y a n a m o u n t (49 %, as estimated b y the reduction in the total acid precipitable c o u n t s / m i n s e d i m e n t i n g faster t h a n 45 S a n d in the pellet) within the range at the depression seen i n whole cell R N A synthesis over longer i n c u b a t i o n s in other experi m e n t s (Fig. I). I t is possible t h a t the p r e t r e a t m e n t with AN affected the intracellular u r i d i n e pools in such a w a y t h a t the incorporation of [14C]uridine into R N A was reduced more for short (3 min) t h a n for longer (15 m i n to I h) incubations. We consider this e x p l a n a t i o n for the observed r e d u c t i o n in labelled h e a v y nuclear R N A unlikely, however, as the rate of incorporation of [14C]uridine into total R N A was a p p r o x i m a t e l y c o n s t a n t in n o r m a l or AN pretreated cells for periods of 3 m i n up to several hours. Third, to measure the effect of the s t a n d a r d AN p r e t r e a t m e n t on the synthesis of cytoplasmic m R N A , the label in isolated polysomes from cultures i n c u b a t e d for i
1
i
160
a) C o n t r o l 0.6 28S Q4
~0 185
0.2
Bottom o
10
20
b ) A ~
30
Top
s'
80
o iC.ont to{ T ~S
0.4
,
~
-- 100
40
0.~
._c
o
18S
0.=
E 5O ~-
o~
o
10 Bottom
20 30 F r a c t i o n No.
Top
0 ~ottom
10
20 Fraction
30 No.
40 Top
Fig. 4. Sucrose gradient velocity sedimentation analysis of whole cell RNA synthesized for 3 min by control cultures and by cultures given the standard AN pretreatment. Centrifugation was for 7 h at 25 ooo rev./min. The solid lines represent absorbance at 260 m/z due largely to added unlabelled carrier cytoplasmic RNA. The circles represent the acid precipitable radioactivity in each fraction from RNA extracted from cultures exposed to [x4C]uridine (o.75/,C/ml at 325 mC/ mM) for 3 min. Fig. 5. Sucrose gradient velocity sedimentation analysis of polysomal RNA from control cultures and cultures given the standard AN pretreatment after exposure of the cultures to [14C]uridine (0.4/2C/ml at 324 mC/mM) for 15 mill. Centrifugation was for 7 h at 25 ooo rev./min. Polysomal RNA was obtained as outlined in MATERIALSAND METHODS,The solid line represents absorbance at 26o m#. The open and closed circles represent acid precipitable radioactivity in each fraction for control and AN pretreated cultures, respectively. Biochim. Biophys. Acta, 155 (1968) 424-432
AMINONUCLEOSIDE ON R N A SYNTHESIS
431
15 rain with [14C]uridine was analyzed by sucrose-density-gradient analysis. Since the radioactivity profiles shown in Fig. 5 are heterodisperse with no peaks at the position of I8-S and 28-S rRNA or at the 4-S transport RNA (tRNA) and if polysomes do indeed contain only mRNA, tRNA and rRNA, then this heterogeneous RNA must be mRNA. Its synthesis can be seen to be depressed in the drug treated cells. The extent of this depression (52 %) was the same as the depression in the whole cell RNA synthesis in 15 rain (53 %) observed in this particular experiment. Thus the synthesis of rRNA, heavy nuclear RNA, and polysomal mRNA all appear to be inhibited to approximately the same extent by the drug. DISCUSSION
The above results establish the fact that under suitable conditions AN can depress the synthesis of RNA in cultured hamster embryo cells with little effect on protein synthesis. Similar results have been obtained b y FARNHAM AND DUBIN4 for mouse L cells and b y STUDZINSKIAND ELLEM11 for human HeLa cells. The fact that protein synthesis is unaffected for relatively long times (4 to IO h) under such conditions indicates that the inhibition of RNA synthesis does not cause the cell to become greatly depleted in the mRNA required for the majority of protein synthesis. This result can be interpreted in one of two ways: either the cells can maintain protein synthesis for some time with the mRNA present prior to the drug treatment or they continue suitable mRNA synthesis in the presence of the drug. In accordance with the second interpretation, both FARNHAM AND DUBIN and STUDZINSKI AND ELLEM have suggested that the drug selectively inhibits the synthesis of rRNA, leaving mRNA synthesis relatively unaffected. No evidence for selective inhibition of RNA synthesis was found, however, in our study of the synthesis of ribosomal RNA precursors, heavy nuclear RNA and polysomal mRNA, since each class of RNA was inhibited to approximately the same extent. We have no conclusive evidence that the synthesis of polysomal mRNA, as detected by early incorporation of E14C~uridine into cytoplasmic polysomes, is actually representative of all of the cellular mRNA. Since the polysomes account for about 9 ° % of the total protein synthesis in hamster embryo cells 14, however, this interpretation seems likely. Our evidence thus strongly favours the interpretation that the inhibition of RNA synthesis by AN in our system and under our conditions of treatment, at least, is not selective for any particular class of RNA and that protein synthesis continues unabated by virtue of the stability of pre-existing mRNA. Work in our system (unpublished) with actinomycin D supports this latter view: protein synthesis is only slightly affected for several hours after addition of this drug to cultures at concentrations which severely depress the synthesis of cell RNA. The distribution of RNA synthesized over relatively long time periods in the presence of AN, as detected b y sucrose density gradient analysis (see Fig. 2) showed a qualitative difference from the control distribution: the ratio of the radioactivity incorporated into 28-S and into I8-S rRNA was less than the 2 : I ratio of the controls and the radioactivity sedimenting between and ahead of the two ribosomal peaks was relatively higher than in the controls. This finding has been observed in other systems and has been interpreted as evidence for a selective inhibition of rRNA synthesis 4. Our failure to detect such selective inhibition leads to the alternative suggesBiochim. Biophys. Acta, 155 (1968) 424-432
432
J. M. TAYLOR, C. P. STANNERS
tion that the drug also affects the maturation of the rRNA precursors in such a way t h a t they are left open to more than normal degradation. The proposed selective degradation would tend to broaden the peaks of the synthesized r R N A seen in a sucrose gradient giving relatively more material between and ahead of these peaks, and, if the more complex maturation to 28-S r R N A 12 is more affected than the maturation to I8-S rRNA, to raise the height of the I8-S r R N A peak relative to the 28-S r R N A peak, as observed (see Figs. 2 and 3). A further aspect ot the experiment shown in Fig. 3 is also consistent with this proposal. The change in sedimentation distribution of the labelled RNA in the AN treated cultures as the labelling time increased from io to 30 min was different from that of the control, thus indicating something abnormal about the r R N A maturation. In s u m m a r y then, our chosen pretreatment with AN did not give selective inhibition of RNA synthesis. This result is inconsistent with the previously mentioned interpretation of the drug's action b y FARNHAM AND DUBIN*. STUDZINSKI AND ELLEMu conceded both selective inhibition of r R N A synthesis and selective degradation of rRNA as consistent with their data. The inconsistency between our results and those of others could be attributed to the use of the different cell systems or to differences in the dose and time of treatment with the drug. For example, it is possible that in this system a longer exposure to a slightly lower concentration of AN m a y have given a selective effect. This type of treatment with AN was avoided here as the longer exposures to the drug required for pretreatment plus the time needed for labelling studies resulted in a detachment of the cells from the glass surface. Though AN has not proven useful in our system as a means of labelling m R N A selectively, it could be used as a mild non-selective in hibitor of cellular RNA synthesis. ACKNOWLEDGEMENTS
This work was supported b y the National Cancer Institute of Canada and by grant MA-I877 from the Medical Research Council of Canada. One of us (J.M.T.) acknowledges the assistance of an award from the Commonwealth Scholarship and Fellowship Plan.
REFERENCES i M, NAKAMURA AND S. JONSSON, Arch. Biochem. Biophys., 66 (1957) 183. 2 S. FRENK, I. ANTONOWICZ, J. M. CRAIG AND J. METCOFF, Proc. Soc. Exptl. Biol. Med., 89 (1955) 424 . 3 S. G. F. WILSON, D. B, HACKEL, S. HORWOOD, G. NASH AND N. HEYMANN, Pediatrics, 21 (1958 ) 963. 4 A. E. FARNHAM AND O. T. DUBIN, J. Mol. Biol., 14 (1965) 55" 5 R. W. READER, M. A. Thesis, U n i v e r s i t y of Toronto, Canada, 1966, p - A I - 3. 6 H. EAGLE, Science, 13o (1959) 423 • 7 R. DULBECCO AND M. VOGT, J. Exptl. Med., 99 (1954) 167. 8 M. D . SCHARFF AND E . ROBBINS, Nature, 208 (1965) 464 . 9 C. P. STANNERS, Biochem. Biophys. Res. Commun., 24 (1966) 758. IO K . SCHERRER AND J . E . DARNELL, Biochem. Biophys. Res. Commun., 7 (1962) 486. I I G. P . STUDZlNSKI AND Z . A . O. ELLEM, J. Cell. Biol., 29 (1966) 411. 12 S. PENMAN, I. SMITH AND E. HOLTZMAN, Science, 154 (1966) 786. 13 J. R. WARNER, R. SOEIRO, H. C. BIRNBOIM, M. GIRARD AND J. E. DARNELL, J. Mol. Biol., 19 (1966) 349" 14 C. P. STANNERS, Biophys..]'., in the press.
Biochim. Biophys. Acta, 155 (1968) 424-432