EFFECT O F A RESPIRATORY ENZYME SYSTEM AND CREATINE UPON THE GROWTH O F

CELLS I N VITROl

SAUL CASPE AND GLADYS CAMERON Department of Biology, Washingtm Square College, New Pork City

ONE PLATE (SIX FIGURES)

INTRODUCTION

The reports of Doljanski ('42) on tissue and Loofbourow ('42) on yeast extracts have revived interest in cell pro- liferants. It has long been known that tissue growth and re- generation can bc promoted by various biological materials. Embryonic tissue juice, known to contain an active protein fraction and respiratory enzyme, is an outstanding example of a growth promoting agent which, since its introduction by Carrel, has been widely used in tissue culture media.

It was deemed advisable to concentrate upon the flavopro- tein enzyme (diaphorase), recently purified and isolated by Straub ( '39), and to test its activity by in vitro experiments on cells growing under controlled conditions. Diaphorase, a flavinadenine dinucleotide, is a respiratory enzyme which catalyses the oxidation of reduced coenzyme I. Oxygen will not act directly with the hydrogenated form of the coenzyme I. However, after the diaphorase accepts the hydrogen from the reduced coenzyme, oxidation will occur. The velocity of this hydrogen transfer and subsequent oxidation will depend on the concentration of enzyme and coenzyme. The turn-over number for diaphorase is 8,000. The molecular weight of this enzyme calculated from its flavin content is 70,000.

Presented before Division of Biochemistry - American Cliemical Society, Fall Meeting, 1944.

43

44 SAUL CASPE AND GLADYS CAMERON

The experiments described in this paper deal particularly with the possible function of this enzyme system as a cell pro- liferant in tissue culture. The effect of creatine in conibina- tion with this enzyme system was also studied.

METHOD

The tissues used were adult human and mouse fibroblasts obtained from lymph nodes, and embryonic chick fibroblasts from various tissues. The normal culture medium usually con- sists of fowl plasma, homologous serum and chick embryonic tissue juice which when mixed form a coagulum. Since the embryonic juice also acts as a growth promoting agent, and because of the nature of the present experiments, it was omitted from both the control and experimental media.

The medium in these experiments consisted of fowl plasma 1 part, homologous serum 3 parts and experimental solutioii 1 part. Control cultures were made for each experiment wherein Tyrode solution replaced the experimental solution. The tissues were cut into fragments from 1 to 2 mm in dia- meter and placed in the medium on coverslips or in Carrel flasks. I n some of the experiments using Carrel flasks, the experimental solution was added as the supernatant fluid.

The cultures were examined daily for the extent of the growth and the condition of the cells, the duration of the experiment being from 4 to 11 days. A total number of 187 slide cultures and 37 Carrel flasks were used. At the termina- tion of the experiments many of the slides were fixed and stained. As is well known the extent of outgrowth area of the explant varies. In order to secure adequate comparisons of experimental and control cultures an average was obtained of the radii of outgrowth from various areas. I n all the ex- perimental cultures, the greater extent of outgrowth was accompanied bp thicker growth.

PREPARATION O F THE EXPERIMENTAL SOLUTIOXS

In all the experiments reported in this paper, coenzynie I was added to the Tyrode solution containing the diaphorase. It

The concentration of coenzyme I used was 2.5 times the concentration of the diaphornse present.

FACTORS I N CELL GROWTH 45

was prepared as a sterile solution and no contaminations oc- curred in the cultures containing it. However, in the case of creatine, difficulty was encountered with bacterial and mould contamination. Filtration of the creatine solution through a Seitz and a Berkefeld filter was tried, but the solution was evidently inactivated since no difference between control and experimental cultures was obtained. Boiling the solution for 5 minutes delayed the bacterial growth but did not prevent the growth of moulds after a few days of incubation of the cul- tures. Autoclaving at 15 pounds pressure for 3 hour was finally tried. No contaminations occurred and the results re- ported with creatine are based on solutions sterilized in this manner.

RESULTS

In table 1 it is shown that a concentration equal to and greater than (up to and including) 1 part of diaphorase to 1.28 X lo5 of snl?strate i s toxic, to chick fibroblasts. This toxic

TABLE 1

Toxicrty of diapko~ase upon chi& fibroblasts.

CONCINTRATIION DIAPHORASE SUBSTRATE TOXICITY TO CBLLS

1: 2.56 x 103 1: 1.28 x 104 1: 2.56 x 104 1: 1.28 x 1oj 1: 2.56 x 10’

toxic toxic toxic toxic not toxic

or poisonous effect is made evident by inhibition of growth, excessive granulation or vacuolation of the cells followed in extreme cases by their death and disintegration.

I n tables 2 and 3 are reported several series of tests com- paring the minimum and maximum proliferation effects ob- tained with creatine and with diaphorase. It is shown that 50 and 100 mg % of creatine increase the growth. A concentra- tion of diaphorase 1: 2.56 x lo6 also produces an increase, while a concentration of 1: 1.28 X lo6 increases the growth

46 S A U L CASPE AND GLADYS CAMERON

still more. The greatest amount of growth occurred when the creatine and diapliorase were combined. I n these cultures the growth of those containing diaphorase at a concentration of 1: 2.56 >( loF was actually greater than when the diaphorase was present in the higher concentration, viz. 1: 1.28 x lo6.

TABLE 2

The effect of diaplkorase and creatine on chick fibroblasts (heart) .

ACTIVATING AGEST

Creatine

Creatine

Diaphorase with coenzyme I

Diaphorase with coenzynie I and creatine

Diaphorase with coenzyme I

NUMBER OF EXPERIXENTS CONCENTTUTIO N

100 nig % 11

50 1ng % ~ 7

50 nig % ~

6 I: 2..56 X 10'

Diaphorase with coenzyme I and creatine

I 1: 2.56 x 10' ~

I

50 1ng 7%

4

RATIO OF INDUCED OUTGROWTH AREA TO

CONTRQL AREA

16: 1

6: 1

16: 1 ---____ _.

20: 1

7: 1

30: 1

TABLE 3

The effect of creutiqie and diapkorase on chick mesonephros.

ACTIVATING AGENT I NUI\IBER RATIO OF INDUCED

EXPERIMENTS ~ OUTGROWTH AREA To COXCENTRATION CONTROL AREA

~

Crea tine I 50 nig % 3 2: 1 I

- _____ . __ - Embryonic juice 5 70 by V O ~ . 2 8 : 1

I-:;;-- ---

Diaphorase 1: 2.56 X l o E 6 with coenzyme I

, Diaphorase 1: 2.36 x 10" 3 ' 16: 1

with coeiizynie I and creatine 50 ing 7%

FACTORS IX CELL G R O W T H 47

This indicates that the effect of the two agents combined is greater than their expected additive effects.

These results are illustrated by photomicrographs of fixed and stained representative cultures of chick fibroblasts. Fig- ures 1 and 3 are controls in plasma and Tyrode solution only, no growth promoting substance being present. The explant shows black in the photograph and a certain extent of out- growth is seen beyond it.

Figure 2 is of a culture containing twice the amount of creatine than that present in the culture of figure 4, the amount of growth being commensurate with the concentration.

Figure 5 shows a culture with diaphorase and coenzyme in the medium. Note the extensive thin outgrowth with wander- ing cells (macrophages) showing beyond the periphery par- ticularly at the upper right corner of the figure.

Figure 6 is of a culture containing 50 mg % creatine added to diaphorase and coenzyme. This medium produced a maxi- mum proliferation Both in extent and thickness of the out growth.

A number of experiments, comprising one-fifth of the work, were also done upon adult mouse and adult human fibroblasts. In every case the results were the same as those with the more extensive experiments on chick fibroblasts.

When diaphorase was present alone or with creatine, the number of wandering cells in the outgrowth was abnormally large. I n cultures containing fragmented segments of kidney tubules there was a n enhancement of cellular proliferation a t the broken ends. I n all the treated cultures the outgrowth mas thicker as well as greater in extent, and the cells were moi'e derisely packed.

DISCUSSION

Considering the weight of the diaphorase molecule, its pro- nounced activity at such low concentrations (shown in tables 2 and 3) attests to its physiological importance. Doljanski's work was concerned with the effects of extracts of a variety of tissues. He reported a stimulating effect from extracts of,

48 SAUL CASPE AND GLADYS CAMERON

among others, brain, heart, liver and testes on the growth of fibroblasts without specifying the activating components. Since these tissues, brain, heart, liver and testes are known to contain large amounts of creatine, it is reasonable to snp- pose that a t least one of the activating agents in his extracts of these tissues was creatine. Loofbourow, Webb and Abram- owitz ( '42), in their studies on the effect of products from in- jured yeast cells upon yeast growth, found evidence that a respiratory mechanism was responsible. However, they were unable to identify the presence of any specific coenzyme in their product. Our results on animal cells are in line with their findings but go further in demonstrating the action of a co- enzyme with diaphorase and establishes a link between the role of the respiratory enzyme system in glycolysis and amino acid oxidation and its translation physiologically into cell proliferation.

Published investigation has shown that phosphocreatine is low in the resting muscle of scorbutic guinea pigs arid of pig- eons with polyneuritis ( '29) ; that it is low in human muscular dystrophies ( '38) ; and in experimentally induced rabbit mus- cular dystrophies ( '39). Sure ( '42) notes that in thiamine deficiency, there are excretions of creatine in the urine. It is apparent that a relationship exists between the creatine body l e ~ e l and vitamin B,. Kenyon ('42) has shown a rela- tionship between the weight of a eunuchoid correlated with the retention by the body of orally ingested creatine. He con- jectures that in this case the reduction of creatinuria suggests a non-genital site (possibly muscle) of new tissue deposit. These cited studies and others constitute indirect evidence that creatine is a growth stimulant. In a previous study ('44), it was reported that creatine stimulates the growth of chick embryo fibroblasts and skin, ra t and mouse epithelium from liver, kidney, bladder and adult human connective tissue in tissue culture. Baker and Carrel ('26) observed little or no stimulation of cell growth in tissue culture by the dialyzable and ultrafilterable components of embryonic juice. When our creatine solutions were filtered as described under prepara-

FSCTOBS I N CELL G R O W T H 49

tion, these filtrates also were inactive. Creatiiie iiiaiiifested activity as a cell proliferant only when its unfiltered solution TWS introdneed into the medium.

Since creatine is a simple amino acid possessing a stable configuration mliich is not degraded in the body, its value per se in developmental growth is of importance. It is known to be stored or present in a great number aiid variety of tissues where developmental growth is accelerated, but its many physiological activities have not been as yet fully investigated.

We are greatly indebted to Prof. Robert Chambers for his interested advice and cooperation.

8UMlfARY

1. The respiratory enzyme, diaphorase (plus coenzyme) a t a coiicentratioii of 1: 2.56 X lo6 in tissue culture, elicited cell-proliferation response which increased with an increase iii coiicentratioii of tlie eiixyme. Diapliorase is not injurious to the cells up to a concentration of 1: 2.56 X lo5 in tissue culture.

2. Creatine, a t a concentration of 50 mg 7. in tissue cul- ture, also gave evidence of a stimulating action which was defiiiitely increased by an increase in concentration of the ereatine.

Diaphorase (plus coenzyme) and creatine combined a t the above concentrations in tissue culture elicited a pro- nounced and enhanced cell proliferation response beyond that of their expected additive effects.

3.

LITERATURE CITED

E ~ K E R , L. E., -4XD A . CARREL 1926 Effect of the aiiiiiio acids and dialyzable constituents of eiiibryoiiic tissue juice on the growth of fiborblasts. J. Esp. Med., vol. 54, pp. 397-407. 1944 The role of creatine in cell growth iu ri tro aiitl its use in wound healing. J. Lab. aad Cliii. Med., vol. 29, pp. 483-485.

1942 The effect of extracts of lieterologous adult tissue on cell growth in \-itro aiid their use in \round healing. Nature, vol. 1-50, p. 23.

CASPE, S.

DoL~assk- I , L., R. s. HOFFIIAN 9 N D E. TENENBSUJI

GOETTSCH, M., I. LONSTEIN AND J. J. HUTCHINSOS 1939 hIuscle phosphorus in nutritional muscular dystrophy in rabbits. J. Biol. Cheiii., rol. 198, pp. 9-21.

KENYON, A. T. 1912 Sex Hormones. PI). 12-13. The Jaques Cattell Press. I i ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ T , J. R. 1912 Further observatioiis regarding the iiiecliaiiisni of

the production and release of proliferation promoting factors by injured cells. Biocliciii. J., rol. 36, pp. 631--638.

LOOFBOWROW, J. R., A. 14. WEBB A N D R. I<. ABRAJIOWITZ 1942 Relation of aemtioii to activity of proliferation - 1)roniotiiig factors from injured cells. Nature, vol. 149, p. 272.

Poliferation - promoting intercellular hormones. Biochem. J., vol. 36, pp. 613-518.

PALIADIN, A., ASD S. EPELBAUM 1929 Ujber deli kreatinpliospliorsauregelialt in deli weissen uiid roteii Muskelii bei experiineiitellen skorbut uiid bei Polyneuritis. 2. Bioclieiii., vol. 204, pp. 140-149.

1938 Cheiiiical composition of voluntary muscle in muscle disease. J. Clin. Investigation, vol. 1 7 , pp. 3ii-383.

Iiifluciice of thiamine, riboflavin, 11)-ridoxine and paiitotheiiic acid deficiencies on iiitrogeu metaboljsm. J. Sutritioii, vol.

LOOBBOUROW, J. R., A. M. WEBB, D. G. LOOFBOK-ROW AND R. r<. ABRAXOTVITZ 1942

HEINHOLD, J. G., AKD G. R. KINGSLEY

SURE, B., AND Z. W. FORD 1942

24, pp. 403-126.

FACTORS 1s CELL GROTTTII SAW, CASPE A N D G L A D Y S C A X E I W S

ESPLISATIOX OF FIGURES

1. Contiol 2. 100 ing % cieatiiie 3. Control 4. 50 iiig yo creatiiie 5 . Diapliorase aiid coeiiz-iiie 6. Diapliorase ant1 coenzyiile plus 30 mg % creatiilt

51