Action of ribonuclease on neoplastic growth

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A C T I O N OF R I B ( ) N U ( ~ L E A S E ON N E ( ) P L A S T I ( " { ; R ( ) W I ' H I. CHEMICAL ASPECTS OF NORMAl. TUM()Ut{ t;RO\VTH: T H E LANDSCH(;TZ ASCITES TUMOUI¢ by 1.. I . E 1 ) O I X * AND ,% H. R E V E l . ] .

The ('hesler Health' lCese~reh [*zstilule, [~stilute o/('r~*~ce'r Research, l?,~yal Ca~cer tb~spital, l.olzdo*~ ( l i ~ l a l l d )

With the discovery by LOEWENTHAL AND JAHN1 of the Ehrlich ascites cell carcinoma, a new type of neoplastic growth became available for biochemical studies. Following on the work of LET'I'RI~2 and KLEIN3, a large number of such turnouts has been produced and described (4, ~,~,v and others). Several authors have pointed out that these tumours are of special interest because they provide free suspensions of mammalian cells which are homogeneouss and of low chemical variability 8. One of us (L.L.) during a study of the action of ribonuclease on solid and ascites tumoursg, lo, 11,13 has been prompted to estimate the nucleic acids and the protein content of both ascitic fluid and ascites cells with regard to the growth of the latter. KLEIN4 and GOLDBERG el al. 8 had previously determined the RNA and DNA content of an ascites turnout (Ehrlich), at a fixed moment of the growth, that is, the median survival time. But results of other authors 12,1a indicated that various metabolic and chemical changes occurred during the growth of an ascites tumour. We have therefore attempted to follow the history of the nucleic acid and protein content of an ascites suspension during its growth and to correlate the characteristics of this growth with the chemical properties. Of the ascites cell tumours available, the Landschiitz offered several advantages from the cytological point of view: it has a low percentage of mitotic abnormalities 17,14 but is very similar in other respects to the Ehrlich ascites cell carcinomM 4. For this reason, we have chosen it for thv present investigation. The present paper describes experiments with this turnout in several groups of mice. The results indicate that the changes observed in the ribonucleic acid content of the cells can be correlated with changes in their growth rate. These results also suggest a possible interpretation of the effects obtained with different chemicalsa, 15,16 and particularly with ribonuclease on solid n or ascites turnouts ~° i n vivo. " Charg6 de r e c h e r c h e s du F o n d s N a t i o n a l belge de la Recherche scientiiique. Permanent address: I . a b o r a t o i r e de Morphologie a n i m a l e , l Iniversit6 libre tie Igruxelles. Belgique.

Re[ere~zces p. 426.

VOL.

18 (1955)

ACTION OF RIBONUCLEASE ON NEOPLASTIC GROWTH i

417

MATERIAL a. T u m o u r cells

T h e L a n d s c h t i t z * t u m o u r cells were p r o d u c e d b y injecting 6 week old C + s t r a i n w h i t e mice i n t r a p e r i t o n e a l l y w i t h a p p r o x i m a t e l y lO 7 cells per mouse. T h e physiological age of t h e first* a n d all t h e s u b s e q u e n t i n o c u l a w a s lO-12 days. T h e L a n d s c h i i t z t u m o u r is v e r y similar to t h e Ehrlich ascites c a r c i n o m a used b y o t h e r i n v e s t i g a t o r s 8,15 b u t h a s a n a r r o w e r d i s t r i b u t i o n of c h r o m o s o m e n u m b e r s a n d fewer m i t o t i c a b n o r m a l i t i e s 14. T h e c h r o m o s o m e m o d a l n u m b e r of t h e L a n d s c h t i t z is s l i g h t l y h i g h e r t h a n t h a t of t h e Ehrlich s t r a i n 14. b. P r o d u c t s

Ribonucleie acid (RNA) H o p k i n s a n d W i l l i a m s p r e p a r a t i o n , purified b y reprecipitation and dialysis: T h i s p r o d u c t is free of D N A c o n t a m i n a n t . Deoxyribonucleic acid (DNA) : T h y m u s D N A o b t a i n e d from calf t h y m u s , purified. No R N A c o n t a m i n a n t could be d e t e c t e d in t h e s a m p l e s used. P r o t e i n : R i b o n u c l e a s e purified b y c h r o m a t o g r a p h y TM. All inorganic a n d organic r e a g e n t s were p r o - a n a l y s i s p r o d u c t s .

METHODS a. C h e m i c a l

T h e ascitic fluid w a s w i t h d r a w n with a h y p o d e r m i c syringe, fitted with a No. 12 needle, from an a n i m a l killed b y neck dislocation. T h i s w i t h d r a w a l w a s m a d e as q u i c k l y as possible after killing t h e a n i m a l a n d a given v o l u m e of fluid w a s i m m e d i a t e l y c e n t r i f u g e d at 3,500 r.p.m, for 3 rain. T h e s u p e r n a t a n t was s e p a r a t e d a n d stored. B o t h cells a n d s u p e r n a t a n t were c o n s e c u t i v e l y t r e a t e d according to SCHNEIDER TMa n d different fractions were o b t a i n e d : acid-soluble, trichloro-acetic acid (TCA) e x t r a c t (DNA a n d R N A ) a n d proteins. T h e acid-soluble was a n a l y s e d b y t h e U.V. s p e c t r o p h o t o m e t r i c m e t h o d , u s i n g t h e difference E200-E300 as a m e a s u r e of t h e free nucleotide content. N u m e r o u s t e s t s h a v e p r o v e d t h a t t h e r e s u l t s so o b t a i n e d were identical w i t h t h o s e o b t a i n e d b y t h e orcinol colorimetric m e t h o d TM. T h e T C A e x t r a c t w a s a n a l y s e d according to SCHNEIDER: for R N A b y u s i n g t h e modification of LUSENA~°; D N A w a s e s t i m a t e d b y t h e u s u a l t e c h n i q u e TM. T h e p r o t e i n s were d e t e r m i n e d by t h e application of MEHL'S m e t h o d 21, after digestion of t h e T C A residue b y N K O H . b. Cytological

A s m a l l p a r t of t h e ascitic fluid w i t h d r a w n w a s fixed a n d s t a i n e d b y dilution w i t h a b o u t t w o p a r t s of a solution of orceine in 60 % acetic acid. E s t i m a t e s of t h e m i t o t i c indices (totals of cells in prophase, m e t a p h a s e , a n a p h a s e a n d t e l o p h a s e p e r iooo cells), a n d of p r o p o r t i o n s of polyploid cells a n d blood cells in t h e total population, were all m a d e from t h e s e samples.

RESULTS

a. C h e m i c a l results

The first three graphs (Figs. I, 2 and 3) show the variation in concentrations (per unit volume) of diffelent components in a typical group of experiments. The points represent means of duplicate determinations made on a mixture of the pooled fluids of one or several animals (according to the available quantity of fluid). The graphs also show the mortality curve of the group used. This curve is expressed in terms of percentage of numbers of surviving mice, plotted against time. It can be seen from these graphs that the DNA content per unit volume remains approximately constant throughout the growth period. Similarly KLEIN had observed, in the case of Ehrlich ascites ~3 that the concentration of cells per unit volume * T h i s s t r a i n h a s b e e n p r o v i d e d b y Dr. KLEIN, to w h o m t h e a u t h o r s are v e r y grateful.

Re/erences p. 426.

4I~

L. LEDOUX, 8. H. RF.VF.LL

VOI.. 1 8

(r(~55!

was constant during the growth. These graphs also demonstrate that the amounts of RNA, free nucleI DNA ~ I N/;I.ACE LL 0 ,~, FN ) otides and protein in the external fluid increaoe slightly during growth, although being always 8 0 0 0 comparatively small. The viscosity of this fluid also increases greatly, o which is probably due to the accumulation of a component or O 0 0 O O components, not studied during 0 0 0 0 0 0 this investigation. 5 Table I sums up the observed I 0 0 mean contents of both ascites fluid 4 and ascites cells at different mo. . _ . % " 0 " ~ ments of the growth. The concen- ? II II II trations are expressed in mg per 2 unit volume. ,A A ~ n ~'~ ,5 A L% z~ A A & A instead of expressing the conA centrations per unit volume, it is more useful to express them per r~M~ (oQys) amount of DNA, thus obtaining a Fig. I. Intracellular content in different nucleotidepicture of their relative proporcontaining substances at various times of g r o w t h of tions in the cells. The validity of a L a n d s c h u t z ascites t u m o r (concentrations per unit this procedure will he discussed volume of fluid). below. Figs. 4, 5 and 0 represent the variation of the ratio RNA/DNA, free nucleotides/DNA, protein/DNA respectively, during tumour growth. These results have been obtained from five groups of 5o animals, each group representing a new transfer of inoculum.

i

°

TABLE i Mean c o n t e n t (mg)/ml ascitic fluid T[DI6 (!'ll.q'l'* 1~Y~gt~]

H - o.25

o.25

U.50

0.50--o.75

0.75-f.OU

1 . 0 0 - I .Su

a. Intracel. RNA DNA F.N Protein No. of samples

7-75 2.46 1.48 44.4 (14)

7.55 2.90 1.08 52.0 (34)

5 .ro 2.58 1.47 46-5 (27)

4.95 2.75 1.65 49.5 (32 )

4 .26 2.79 1.54 49-3 (29)

b. Extracel. RNA F.N Protein No. of sample

0.34 0.3; 1o.2 (5)

0.37 0.35 tl. 3 (io)

o.35 0.38 r3.o (12)

0.40 0.38 [4.2 (I l)

0-43 0.40 L6.1 (to)

* Median survival time unit: the period between t u m o u r i m p l a n t a t i o n and the trine when - o °' :o of the animals were dead.

.')

The points represent means of duplicates obtained from the pooled fluid of two different mice. tfe/ere~wes p. 426.

i

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u n i t v o l u m e of fluid).

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The abscissae represent time expressed in units of m e d i a n s u r v i v a l time (51S'1) ~ This unit is nsed because the MS'I" has varied from i o - L # d a y s (meau aNA/o~A I r.5 - o.8) although the sam~. ~tnlotlnt of inocllhlnl has beell use(1 in several groui)s. It can be d e d u c e d from these results t h a t the RNA/1)NA r a t i o •I " - . . " ." decreases by 5 o " . during turnout oo ~ • • growth while n o n e of the o t h e r • ,, . , - •'-~..._ " I[ o| ratios observed change a p p r e c i a b l y . • :".'"'~ :,. " . 1 ]-~v using a U.V. Inicrophot~> " ." . - • L ". • graphic m e t h o d , which will be des:F.-4 • . - :... cribed in detail elsewhere, R. ,[. : ":': . KIN(; 22 of this I n s t i t u t e has followed the same p h e n o m e n o n in living cells. Briefly, the m e t h o d consists in measuring the U.V. c y t o p l a s m i c absorpMST UNITS tion, at e57 m#, of a large n u m b e r of g ,.o t:5 i n d i v i d u a l cells taken at r a u d o m lqg. 4- Changes in intracellular RNA/DNA ratios from t h e suspensions used in the during the growth of the ascites tumor. Time in chemical analysis. A v e r y good M.S.T. units (el. text).

"" ]: :""

PlROT./DNA a g r e e m e n t was found between the 3•results o b t a i n e d b y the two methods, I t should he p o i n t e d out t h a t there was a r e g u l a r a d j u s t m e n t of 0 .'.: t . .| o'o. •. tile cellular c o n t e n t b y t r a n s f e r to a • ~ • e 00 • o e fresh a n i m a l : t h e values of the R N A / 2oee • oe • e • D N A r a t i o in different inocula could • Q eIm • : • $ v a r y greatly, b u t after a v e r y short • coo • • ~eo p e r i o d in tile new host, t h e y all e • . : :.:. . . : ". reached t h e same level. Table I I (columns I a n d 2) shows t h e observed Ic~ results. The occurrence of such a d j u s t m e n t agrees with t h e o b s e r v a t i o n b y KLEIN t h a t t h e m o r t a l i t y curve deMST UNITS pends only on t h e n u m b e r of ceils 4 J.o i m p l a n t e d a n d is i n d e p e n d e n t of their Fig. 5- Intracellular Protem/DN A ratios during the p h y s i o l o g i c a l age~,% I t can also be growth of the tumor. s e e n (Table I I , column 3) t h a t the

d i f f e r e n c e s in t h e i n o c u l a h a v e no "nfluence on t h e s u r v i v a l t i m e (MST).

* Determined from a probit/log time curve. Re/ere**ces p. 4-~6.

RNA

c o n t e n t at t h e m e d i a n

v o L 18 (]955)

ACTIONOF RII;ONUCLEASEON N-EOPL:\STIC C,ROWTH I

421

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Fig. 6. Intracellular free nucleotides/DNA ratios during the growth of the tumor. TABLE II (RNA/DNA) * Inoculum

4-23 1.87 1,55 1.34 1,29

A/ter o.z unit M S T

2.94 2.54 2.96 2.82 2.50

Alter s.o unit M S T

1.45 1,34 1,55 1,55 1.5o

* Means of duplicate determinations made on 4 different ascitic fluids.

b. Cytological study Results obtained by previous workers with different ascites tumours 23, 24 indicated that during the growth of the tumour the mitotic index decreases, the mean cell generation time increases and the rate of growth decreases continuously. I n the present work mitotic index was measured in two or three a n i m a l s every d a y (taken from a group of a n i m a l s i m p l a n t e d together) b y the e x a m i n a t i o n of IOOO t u m o u r cells from each, a n d some estimates were also made of the proportions of these mitoses which were aberrant, of the proportion of t u m o u r cells which were polyploid a n d of the relative n u m b e r s of blood cells in the ascites population. Mitotic index. I n the m e a s u r e m e n t of mitotic i n d e x all prophases, metaphases, anaphases a n d telophases were c o u n t e d (each telophase being c o u n t e d as one cell). The i n d e x of the t u r n o u t cell p o p u l a t i o n used for i n o c u l a t i n g all the animals (itself a m i x t u r e of the t u r n o u t s from two animals) was 3.6%. Each a n i m a l received a b o u t IO~ cells. Table I I I shows the results obtained. A variable n u m b e r of the mitoses scored appeared to be a b n o r m a l in v a r i o u s ways (e.g. m e t a p h a s e degeneration, m u l t i p o l a r anaphase). The n u m b e r s observed r a n g e d from o to 40% of the t o t a l mitotic c o u n t (average 13.5 %), b u t this v a r i a t i o n

Re/erences p. 426.

422

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s e e m e d not to be correlated with age of the t u m o n r or any of the other parameters measured. H o w e v e r , a close s t u d y was not m a d e of this side of the problem and these figures should be regarded as approximate. Fig. 7 expresses the relationship existing between the R N A / D N A ratio and the actual m i t o t i c index of the population. As seen from this graph, a v e r y good correlation is obtained. The equation of the regression line is y = 1.97 -t- ° . I 8 9 (x - - 3.56) where x indicated the percentage of mitosis and y the R N A / D N A ratio. The t w o Re/erences

p. 426.

VOL. 1 8

(1955)

ACTION OF RIBONUCLEASE ON NEOPLASTIC GROWTH I

423

exterior lines indicate the limits of probable error (P = o.o5). The correlation coefficient thus is very significant. Polyploidy. Polyploidy was estimated in two ways. Resting cells which were obviously highly polyploid (as judged by the size or number of their nuclei) were scored separately: their numbers ranged from I to 31 per thousand (average lO.6). No consistent trend was apparent in this variation. In addition, mitoses which were obviously above the modal chromosome number of 4 6'4 , 25were also separately scored: these varied from o to 5 per thousand (average: I.I), apparently at random. There was thus no detectable tendency for the proportion of polyploid cells to change during the development of the tumour (Fig. 8).

%

00. o

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g F i g . 7- R e l a t i o n

between

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Blood cells. The proportion of blood cells in the ascites population was highest at I day after inoculation but declined rapidly to an average figure of below lO%. Fig. 8 also shows the variation in percentage of tumour cells during the growth of the tumour. The results obtained here agree perfectly with those described by other authors 23. Bearing in mind that the RNA content of non-tumour cells is much lower than that of the tumour cells, one can conclude that the values of RNA/DNA observed during the first period of the growth are underestimated. DISCUSSION

The study of the variations of the ratios: RNA/DNA, free nucleotide/DNA and protein/DNA throughout the growth of the Landschiitz ascites tumour reveals that RNA is the only one of these constituents to vary during the ageing of the tumour. The decrease observed amounts to 5° % of the initial value and reaches its end point at the median survival time (MSF). Re/erences p. 426.

424

L.I.t,2I)()UX, S. H. I{EVEI.I

X()I. I 8 (I(}55

Of course, the use of the r a t i o R N A / D N A as a measure of tlw ratio RNA.cclI m i g h t be criticised : the r a t i o could decrease by increase of the I)NA content, l.:nde~these conditions, however, the protein or free nucleotide c o n t e n t s]totlld iner
VOL. 18 (1955)

ACTION OF RIBONUCLEASE ON NEOPLASTIC GROWTH I

425

I t has also b e e n o b s e r v e d in t h e c o u r s e of o u r i n v e s t i g a t i o n s t h a t cells of l o w R N A a n d slow g r o w t h r a t e c o u l d be r e j u v e n a t e d a g a i n a n d a g a i n b y t r a n s p l a n t i n g t h e m i n t o fresh a n i m a l s w h e n b o t h R N A c o n t e n t a n d g r o w t h r a t e i n c r e a s e again. T h e a d j u s t m e n t b y t h e h o s t of t h e c e l l u l a r c o n t e n t of t h e i m p l a n t e d t u m o u r cells a g r e e s w i t h t h e r e s u l t s of KLEIN et al. 8 w h o o b s e r v e d t h a t w h a t e v e r t h e a m o u n t of i m p l a n t e d cells, t h e nucleic a c i d c o n t e n t was c o n s t a n t at t h e m e d i a n s u r v i v a l t i m e a n d t h a t v a r i a t i o n in p h y s i o l o g i c a l age of i n o c u l a h a d no d i s c e r n i b l e effect on t h e p r o l i f e r a t i o n of E h r l i c h ascites 23. On t h e o t h e r h a n d , this a u t h o r has s h o w n t h a t b y d e c r e a s i n g c o n s i d e r a b l y t h e R N A c o n t e n t of t h e cells (by s t o r a g e at 4 ° C15), t h e i n f e c t i v i t y of t h e i n o c u l u m was c o n s i d e r a b l y d e c r e a s e d . I t s e e m s t h e r e f o r e t h a t an R N A t h r e s h o l d for this a d j u s t m e n t p r o c e s s exists. I n c o n s e q u e n c e , if one c o u l d b y c h e m i c a l m e a n s d e c r e a s e sufficiently t h e c o n t e n t of n o r m a l R N A b e l o w its c r i t i c a l limit, one m i g h t e x p e c t to m o d i f y g r e a t l y t h e r a t e of t h e n e o p l a s t i c g r o w t h . I n fact, t h i s has b e e n f o u n d in t h e case of ascites t u m o u r s t r e a t e d b y a c r i d i n e s 3, w h e r e t h e s l o w i n g d o w n of t h e t u m o u r is a c c o m p a n i e d b y a d r o p in t h e R N A c o n t e n t (the D N A c o n t e n t r e m a i n i n g u n c h a n g e d ) . T h i s also s e e m i n g l y occurs in e i t h e r ascites or solid t u m o u r s t r e a t e d b y r i b o n u cleaselO, 11, t h e n u c l e o l y t i c e n z y m e d i r e c t l y c o n c e r n e d w i t h R N A m e t a b o l i s m . T h e a u t h o r s wish to t h a n k P r o f e s s o r s A. HADDOW, F. BERGEL, P. C. KOLLER AND J. BRACHET for c r i t i c i s m s a n d suggestions. SUMMARY During the normal growth of an ascites tumour (Landschfitz) the intracellular concentration of the ribonucleic acids (RNA) decreases by about 5 ° %. At the same time, the amounts of protein, free nucleotides and deoxyribonucleic acids (DNA) do not vary appreciably. The depletion of cellular RNA is accompanied by a corresponding decrease of the mitotic index and by a general slowing down of the tumour growth. This depletion is not due to the disappearance, from the ascitic fluid, of RNA precursors. It is reversible by transplanting the tumour into a fresh animal. There is presumably a relation between these facts and the chemotherapeutic action of certain anti-tnmour agents. R~:SUM~: Au cours de la croissance normale d'une tumeur d'ascites (Landschtitz), la concentration intracellulaire des acides ribonucMiques d6cruit de 5o % environ. Pendant ce temps, la teneur en prot6ines, en "nucl6otides libres", et en ADN ne varie pas sensiblement. L'appauvrissement en ARN cellulaire est accompagn6 d'une diminution correspondante de l'index mitotique et d'une ralentissement g6n6ral de la croissance tumorale. Cet appauvrissement n'est pas dfi /~ la disparition dans le milieu nutritif (plasma ascitique) des pr6curseurs de I'ARN. I1 est r6versible si on transplante la tumeur dans un organisme sain. I1 existe vraisemblablement une relation entre ces faits et l'action ch6motherapeutique de certains agents anticanc6reux. ZUSAMMENFASSUNG "vV~.hrend des normalen Wachstums einer Aszit-Geschwulst (Landschiitz) sinkt die intrazellulare RibonukleinsAurekonzentration um etwa 5o%. Gleichzeitig kann keine bemerkbare VerAnderung des Gehaltes an Proteinen, freien Nukleotiden und DNA beobachtet werden. Das Verschwinden des zellularen RNA wird yon einem entsprechenden Herabsinken des mitotischen Index und einer allgemeinen Verlangsamung des Geschwulstwachstumes begleitet. Der RNASchwund wird nicht durch das Verschwinden der RNA-Vorg/~nger aus der Aszitischen Flfissigkeit verursacht. Der Schwund kann durch Umpflanzung der Geschwulst in ein neues Tier rtickgAngig gemacht werden. Es darf angenommen werden, dass zwischen diesen Tatsachen und der chemotherapeutischen Wirkung yon bestimmten geschwulstheilenden Mitteln eine Beziehung besteht. Re/erences p. 426.

420

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2 M. l.ETTR12, Z. pll3'siol. ('he~l~.. 2()8 (tq41) 50. 3 G. KLEIN, ('a~zcer, 3 (195°) 1°52. a G. KLEIN, Expll. Cell Rc:*ar(h, 2 (1951) 51,S. 5 H . (;OLDIE AND X]. l). ]?f(l.lN, ( ' a m ' e r Research, i1 ( i 0 5 1 ) 736 A. ]~EVAN AND T. J. HAUS;CHKA, ]qeredilas, 38 (1952) 18. 7 G. I(LEIN AND E. b~Ll':IX, (airier Research, I l (1951) 400. s L. (;OLDBERG, E. KLEIN AND (i. KLEIN, t'2xptl. Cell leeseareh, I (1()5o) 543 t 9 L. I~EDOUX AN> E. B..xl.Lt~s, ILrperie./ia, Jo (1954) 5 ° o . l0 L. IA~2DOUX, Nalure, 175 (1055) 258. H L. I.EDOUX, Nature (in p r e s s ) . 12 ].. I.Er}OUX, Bi*~ehim. Bi.phv.;. Acta (in p r e s s ) . ~a G. A. I.E PACE, ( ' a . ( e r Re.;earch, 13 (I953) I 7 8 . 14 j . I h N T J O ANI) .\. I.J..vax, I.ztmls U m v . . l r s s k r . , 15 (1954) 5 ° . is E . KLEIN, N. B. KURNJCK aND G. KLEIN, F.Xptl. ('ell. Research, I (195 o) I I , 7. 16 If. E. SKIPPER, ('aH~er l~t'd('al(h, 13 ( r 9 5 3 ) 545. 17 13. C. KOLLER, p e r s o n a l c o m m u n i c a t i o n . is N . I. SCHNEH)ER, ,/. t~ol. (7hem., ~{,i ( I 9 4 5 ) -'93, lu C. N . W . H I R s , J . MOORE AND W . H . STEIN, ./. 13iol. ('he.~., - o o ( t 9 5 3 ) 4~3 • 20 C. V. I.USEXA, ( ' a . . . / . ( ' h e m , -'9 ( t O 5 / ) Io7. 21 j . W . MEHL, ./. l~i.[. ( ' & m . , 157 (1945) 173. 22 i . J . I~[ING, p e r s o n a l 1"Oll1131tli/ic{ltioi1. ea G. KLEIN AXD 1.. REVESZ, ./. Nail. Ca*zcer l~sl., 14 U 0 5 3 ) -'29 24 H. M. PATT AND AT. tL. ]{I..\('KEORD, Proc. NOC. F.xpII. Biol. Meg., 83 (1053) 52o. 2s K. BAY~EX'rREF, Z. N a / u r / o r s c h . , to (1952) 726 E. KLEIN, Expll. ('ell Research, 8 ( t 9 5 5 ) ISS. 27 j . BRACHET, li'~.:3'mrd~)gi~t, IO (I94~) S7. 28 ],~. JEENER, Nature, t03 (I(~4(}) 837. 2!1 j . lgRACH15T AND R . JEENF-R, ti~eymolog, i~*, I 3 (t 944) I 0 ( " 30 T. CASPERSSON, .qymp,~sia .'~'(m. F.xpll. l:tiol., 2 (t 947) I 27aI j . t3RACHET, (The*~/ica/ et,d)ryotogy, l n t e r s c i e n c e , N e w Y o r k , 1945. :*2 j . B~ACUET, /.e Rdle des acides m*cldiques darts la vie de' la cellule el de l'embrwm, D e s o e r , LiE,g e .

t~52. R e c e i v e d M a y 2nd, I 9 5 5