OF - Cancer Research · The chicken leukocyte antiserum was obtained from a rabbit whi'ch had received 15 intravenous injections of a suspension of chicken leukocytes. As extracts

STUDIES OF GROWTH FACTORS OF EMBRYO EXTRACT

W. C. HUEPER, M.D., A. ALLEN, PFLD., M. RUSSELL, M.S., G. WOODWARD, M.S., AND M. PLATT, PH.D.

Cancer Research Laboralones, University of Pennsylvania Graduate School of Medicine, EUice McDonald, Director

The life activities of cells in Vitro in regard to growth may assume three forms: 1. Survival of cells without cell proliferation. 2. Cell multiplication without increase in protoplasmic matter,

due to the presence of substances stimulating mitosis and the absence of the proper nutritive elements in the culture medium.

3. Proliferation of cells with new formation of protoplasmic matter through utilization of nutritive substances contained in t,he culture medium under conditions which allow either (a) a restricted continuation of life and growth or (b ) an unlimited continuation of life and growth.

These variations in growth types are due to the effect of numerous endogenous (vitality, texture, type, age, quantity, synergism, and antagonism of tissues explanted) and exogenous factors of various physico-chemical nature (consistency, osmotic pressure, gas pressure, surface tension, dispersion status, chemical composi- tion of medium, position of the explant in the medium, effect of various rays, temperature, and many other factors of more or less known and unknown character) upon the explanted cells.

Among the many agents used in the culture medium for the promotion of cell multiplication and sustenance of unlimited life, embryo extract holds a preeminent and almost unique position, since it contains not only substances for the stimulation of cell division, but also nutritive material which can be assimilated by the explanted cells and used for the synthesis of new protoplasmic matter. The identification of these growth-promoting factors present in embryo extract and a study of their relations to various exogenous and endogenous factors affecting cell growth are evidently of great importance for a clearer understanding of the causes and the mechanism not only of normal growth, but also of pathological and especially blastomatous growth. Increased information on

1 Read before the American Association for Cancer Research, Philadelphia, April 1932.

74

GROWTH FACTORS O F EMBRYO EXTRACT 75

this subject should aid in improvement of existing and develop- ment of new therapeutic measures against malignant blastomas.

Numerous attempts have been made in the past to isolate and analyze the active substances of embryo extract. Various methods have been employed to this end, such as precipitation of proteins, dialysis, hydrolysis, ether extraction, adsorption, etc. (Carrel, Fischer, Baker, Ebeling and others), but the information so far gained has remained rather unsatisfactory and indefinite. No decision has been reached as to the actual character of the substances essential for the growth-promoting effect of embryo extract.

It has been maintained that these qualities are invested in or bound to the globulin fraction of the extract; that the higher protein split products, the proteoses, are the important substances; that the presence and activity of an enzyme, especially a proteolytic enzyme, are essential for the characteristic action of the extract; that the extract contains a special growth hormone; finally, that a unique physico-chemical constellation of requisite nutritive substances in the proper quantitative and qualitative relation is responsible for the growth-promoting action. In support of any of these conceptions a number of more or less well established data can be cited.

I n view of the great theoretical as well as practical importance of increasing our knowledge of the factors controlling cell growth, a renewed experimental study of these elements in embryo extract seemed to be desirable.

1. THE PROTEIN NATURE OF GROWTH SUBSTANCES IN EMBRYO EXTRACT

In view of the fact that Fischer and Carrel had shown that the active principle of embryo extract is bound to the globulin fraction, from which a separation appeared to be impossible without the simultaneous destruction of the activity, and that these authors had also found the growth-promoting action of embryo extract to be of non-species specific nature, the existence of a non-species- specific protein responsible for this quality had to be considered. The precipitin reaction seemed to offer a suitable method by which this assumption could be put to the proper test.

A chick embryo extract antiserum was prepared by semi-weekly intravenous injections of 1 C.C. of chick embryo extract into the ear vein of a rabbit. After 15 injections, the serum of this animal

76 HUEPER, ALLEN, RUSSELL, WOODWARD AND PLATT

was tested for the presence of precipitins against chick embryo extract, The antiserum was overlaid with chick embryo extract and incubated for one hour at 37" C. The appearance of a distinct turbid ring at the contact surfice of the two liquids indicated the presence of precipitins in the serum of the injected rabbit. If mouse embryo extract was used instead of the chick embryo extract, the formation of a precipitation ring was lacking. Corre- sponding results were obtained when chick embryo extract antiserum was replaced by chicken leukocyte antiserum in the tests. The chicken leukocyte antiserum was obtained from a rabbit whi'ch had received 15 intravenous injections of a suspension of chicken leukocytes. As extracts of leukocytes and bone marrow, according to Carrel, Maximow and Fischer, possess growth-promoting qualities corresponding to those of embryo extract, the results obtained with an antileukocytic serum in the reactions described above can be regarded as substantiating the outcome of the trests made with chick embryo extract antmiserum.

The plasma of the antiembryo extract rabbit was furthermore tested for the presence of non-species-specific antiproliferative antibodies by the explantation of chick bone marrow and leukocytes and mouse mammary cancer in this medium. While the chick tissues showed an inhibitory effect of the culture medium upon cell proliferation and emigration, these qualities were apparently unimpeded in the cultures of the mouse mammary cancer.

The results thus obtained with serological and biological methods seem to indicate that the active substances present in embryo extract are not of a protein nature, which gives rise to specific non-species-specific antiproliferative antibodies.

2. THE HORMONAL CHARACTER OF THE GROWTH-PROMOTING AGENT

The possible hormonal nature of the growth-promoting action of embryo extract was made the subject of another series of experiments. The recent discovery of a growth-stimulating hormone in the anterior lobe of the hypophysis and the statement of Reiter and GBbor that mitogenetic rays are exclusively produced in the heads of tadpoles suggested the examination of different parts of chick embryos for the relative amounts of growth-promoting substances.

Eight days old chick embryos were dissected into the following parts: head without eyes, eyes, body, and fluid exuding from the

GROWTH FACTORS OF EMBRYO EXTRACT 77

embryos upon dissection. The extracts obtained from these four sources were then tested for their respective growth qualities. Standardized fibroblast cultures, that is, cultures which had reached a rather uniform proliferation after ten passages, were used in the experiments. The proliferative effects obtained with these extracts, which were also tested in various dilutions (100, 50, 14, and 7 per cent), were compared with those seen in cultures which had received corresponding amounts of normal extract prepared from entire embryos. There was no evidence that extracts obtained from different parts of the embryonic body cont,ain different amounts of growth-promoting substances. The growth-activating qualities of the fluid exuding from small cuts in the skin of new-born rats or extracted from the cranial cavity through a needle have been previously reported by Martinovitch.

The results recorded are remarkable on account of the marked differences in the amounts of extract which can be pressed from the three parts of the embryo. About one third of the embryonic pulp can be recovered as embryo extract if the embryos are forced through a press and centrifuged afterwards for twenty-five minutes at 3600 revolutions. The same ratio is found if an extract is prepared from the bodies alone, while the ratio is reversed if the eyes are used, that is, two thirds of the material are liquid and one third is pulp, One ninth of the total material, however, appears as extract if the heads without eyes are crushed and centrifuged.

These observations do not support the view that certain parts of the embryonic body are endowed with special growth-promoting qualities of possibly hormonal nature.

3. THE SIGNIFICANCE OF POLYSACCHARIDES IN EMBRYO EXTRACT In view of the recently discovered importance of polysaccharides

in certain immunological reactions, an investigation of the r61e which polysaccharides contained in the embryo extract might play as growth stimulators seemed to be indicated. The presence of glucoproteins, and especially of hexose and pentose nucleoproteins, in the protoplasm of cells is well known. The stimulating effect of the nucleoprotein decomposition products upon mitotic processes has been attested by Carrel, Fischer, and others.

The following procedure was adopted for the isolation of the polysaccharide contained in embryo extract :

The extract is acidified to about pH 4.2 by adding 0.2 C.C. of 3 per cent acetic acid for each cubic centimeter of extract, and


Gluoose

Aftor 4 hours hydrolysia with NHCl ~~f~~~ hydrolysis

5.16 per cent 22.55 per cent 34.10 per cent -

the protein precipitation is completed by the addition of absolute alcohol to make 50 per cent. After standing over night in the ice box, the mixture is centrifuged, giving a clear supernatant fluid, which is decanted. The precipitated protein, extracted twice with boiling ether to remove fats, is refluxed on a boiling water brtt,h for two hours with 30 per cent KOH. This procedure would decompose the protein and liberate the polysaccharide from it, polysaccharides being stable under these conditions. The solution is neutralized with a saturated solution of trichloracetic acid. The separation of the precipitated protein is best made by centrifuging again before filtering. The crude polysaccharide is then precipitated from the filtrate by adding absolute alcohol to make 80 per cent. A small amount of protein material is probably contained in the polysaccharide precipitate. This is purified by solution in a small amount of hot water, removal of insoluble material, followed by fractional precipitation with alcohol.

From 471 dozen eggs a total of about 900 mg. of crude polysaccharide in small quantities of varying degrees of purity was obtained.

Analyses for glucose and nitrogen have been carried out in an effort to prove that the material isolated is actually a polysaccharide. Glucose values were determined by the alkaline ferricyanide method of Hagedorn and Jensen; nitrogen determinations were made by the Kjeldahl method. Analyses on two samples were as follows:

Nitrogen

11.72 per cent 8.22 per cent

Since the glucose values by alkaline ferricyanide reduction were found considerably higher after acid hydrolysis, there is good indi- cation that the material contains a polysaccharide which is being split to glucose-like material. Similar determinations by cold acid ferricyanide reduction gave zero reducing value, which is proof that the alkaline values are due to glucose-like material. That a nitrogenous impurity (probably decomposed protein material) is present is indicated by the fact that the sample which gave the highest glucose value also contained the least nitrogen. Part of


the nitrogen, however, may be a constituent of the polysaccharide complex. A part of the polysaccharide material was carried through a further purification process by alcohol precipitation. The purest fraction of it was later used for tissue culture experiments. The material gave a negative reaction for -SH groups with the iodate titration method of Fry and Woodward.

While it was not expected that the polysaccharide as such would have a special growth-stimulating effect, it was hoped that the addition of this substance to the culture medium might result in the formation of an active polysaccharide-protein complex. It was therefore decided, following Baker's experiments with com- binations of liver ash, glutathione, and hemoglobin, to test the polysaccharide in conjunction with hemin and glutathione. The following t.issue culture experiments, using 12 standardized fibroblast cultures in each series, were performed as a test for the growth- stimulating action of polysaccharide obtained from embryo extract :

1. Equal parts of chicken plasma and Ringer solution. Equal parts of chicken plasma and Ringer solution containing

0.8 mg. polysaccharide per C.C.

2. Equal parts of chicken plasma and chick embryo extract diluted 1 : 8 with Tyrode solution.

Equal parts of chicken plasma and chick embryo extract diluted 1 : 8 with Tyrode solution containing 0.8 mg. polysaccharide per C.C.

3. Equal parts of chicken plasma and boiled embryo extract diluted 1 : 8 with Tyrode solution.

Equal parts of chicken plasma and boiled embryo extract diluted 1 : 8 with Tyrode solution containing 0.8 mg. polysaccharide per C.C.

4. Equal parts of chicken plasma and embryo extract heated to 70" C. for ten minutes and diluted 1 : 8 with Tyrode solution.

Equal parts of chicken plasma and embryo extract heated to 70" C. for ten minutes and diluted 1 : 8 with Tyrode solution containing 0.8 mg. polysaccharide per C.C.

5. Equal parts of chicken plasma and Ringer solution containing 0.8 mg. glucose, 0.4 mg. glutathione, 0.05 mg. hemin per C.C.

Equal parts of chicken plasma and Ringer solution containing 0.8 mg. polysaccharide, 0.4 mg. glutathione, 0.05 mg. hemin per C.C.

6. Equal parts of chicken plasma and Ringer solution containing 0.8 mg. glucose per C.C.


Equal parts of chicken plasma and Ringer solution containing

The amounts of polysaccharide and glucose added were calculated from the normal amounts of reducing substances, excluding glutathione present in embryo extract. All cultures were sent through two passages. The results were entirely negative. The polysaccharide did not show in any of the various types tested any growth-stimulating effect. The conclusion seems, therefore, to be justified that the polysaccharide in the form produced and used has no relation to the proliferative processes of cells grown in vitro.

In connection with these experiments on embryo extract polysaccharide, we investigated the pentose nucleotide D 96 prepared from yeast nucleic acid for growth-promoting qualities, since Jackson, Parker, Rinehart, and Taylor reported recently that this preparation has a stimulating effect upon the proliferative function of bone marrow in agranulocytic conditions. Explants of chicken bone marrow, leukocytes, and mouse mammary carcinoma were used as test materials. The nucleotide preparation was added to the embryo extract in dilutions of 1 : 10, 1 : 100, and 1 : 1000. The two higher dilutions were entirely ineffective, while the dilution 1 : 10 even produced a moderate inhibition of cell emigration. The stimulating effect of thymo-nucleic acid upon the growth of cells in witro (Carrel, Fischer, Ebeling) is therefore apparently not shared by derivatives of yeast nucleic acid. Pentose nucleotide D 96 is also unable to check the growth-inhibiting effect of antileukocytic serum upon leukocytes.

As a further outgrowth of the experiments on embryo extract polysaccharide, an examination of the stability of the carbohydrate purine complex of the thymo-nucleic acid contained in embryo extract was made with the Feulgen nucleal reaction. It was thought that the inactivation of embryo extract observed on aging might be related to decomposition processes affecting the nucleic acids which have a mitosis-stimulating effect (Fischer, Carrel, Baker). It was found that the reaction is positive in fresh extract as well as in extract which is three weeks old. The purplish color appears not only in the white precipitate of the proteins, but also in the clear supernatant fluid. Also shaking does not affect the outcome of the reaction in any way. The clear supernatant fluid, as well as the gelatinous, turbid sediment which is formed after prolonged (four to eight hours) shaking in a Kahn shaking apparatus, gives a positive reaction. Evidently no significant decomposition of the

0.8 mg. polysaccharide per C.C.


nucleoproteins in the embryo extract upon aging takes place, because the Feulgen reaction becomes negative if the hydrolysis of the nucleic acids is carried too far. Disintegration of nucleic acids is, therefore, apparently not a causative factor in the inactivation of embryo extract by aging.

4. THE EFFECTS OF SHAKING AND OXYGENATION ON THE ACTIVITY

The mechanism of the auto-inactivation of embryo extract has been the subject of much speculation. It is in general stated that an extract which has been kept for two weeks in the ice box has become unsuitable for culture purposes on account of its decreased activity. Carrel and Ebeling state that this process of spontaneous inactivation can be greatly accelerated by incubation a t 37" C. Craciun observes that incubation for forty-eight hours is sufficient to destroy completely the active principle, while Shibata did not see any decrease after a period of ninety-six hours a t 37" C. He notes, however, that a marked drop in activity is noticeable after a period of nine days at 37" C.

While some authors believe that this great lability of the active substances is due to the ease with which they are oxidized by the oxygen of the air or by oxidizing systems of the tissue extract (Friedheim), others assume that this process is due to the gradual self destruction of essential enzymes or caused by a gradual denaturation of important proteins in the extract.

This change in the activity of the extract, according to Craciun, is accompanied by a shift to the acid side. Friedheim, on the contrary, has shown that a shift of the pH from normal 7.4 toward the alkaline side takes place. He adds that this shift is probably due to loss of COz and that the variations of the reaction do not explain the changes occurring in the biological activity of the extract.

Also, shaking of the extract over a prolonged period, that is four to eight hours, causes a decrease in the proliferative activity of the extract according to Carrel, Fischer, Craciun, Borger and Zenker. Carrel compares this effect on extract with the destructive action of shaking on complement. A study of the causative mechanism of the injurious effect of shaking seemed to offer an opportunity to gain some information in regard to the changes causing the normal inactivation of extract. The following four possibilities of the inactivating action of shaking were considered :

OF EMBRYO EXTRACT


(1) denaturation of the active substances in the froth formed; (2) destruction of the delicately balanced colloidal status of embryo extract; (3) enhancement of oxidation processes; (4) inactivation of important enzymes.

The effect of froth formation upon the activity of embryo extract was tested with the following method. One specimen of extract was shaken in a tube allowing sufficient space for froth formation, while a second specimen was sealed in the tube by a paraffin plug preventing thereby the production of froth. Several pyrex glass beads were placed in the tube, however, to insure the mechanical effect of shaking. The two specimens were shaken for eight hours in a Kahn shaking machine. Their activity was then tested on standardized fibroblast cultures. There was no difference in the growth-stimulating action of the two specimens, nor was there any decrease in activity compared with normal extract.

Surface tension determinationq2 performed with the ring method, on extracts in which large amounts of froth had been produced by bubbling moIecular oxygen and nitrogen through the extract for one hour, did not reveal any changes in surface active substances. The surface tension remained practically unaltered.

Normal extract. ....................... .50.80 dynes/cm. Oxygenated extract .................... .50.90 dynes/cm. Nitrogenated extract. . . . . . . . . . . . . . . . . . . .51.50 dyncs/cm.

These findings indicate that any possible changes in the activity of embryo extract after shaking are probably not caused by the inactivation of the essential factors in the froth.

Similarly, negative results were obtained in testing for effect upon activity of changes in the dispersion status of embryo extract after shaking. Again standardized fibroblast cultures were fed with the two fractions of the shaken extract, that is, the clear supernatant part and the gelatinous sediment. No differences could be discovered in cultures carried through ten passages.

Oxygenated extract, that is extract through which a fine stream of oxygen had been bubbled for one hour, was used to test for the effect of increased oxidation upon the activity of embryo extract. In this experiment two series of cultures were prepared, one of standardized fibroblast cultures and another of fresh fibroblast cultures which had not come in contact with normal extract,

Doctor Russel J. Fosbinder, of these laboratories. 2 For the determinations of surface tension on embryo extract we are indebted to


but which were explanted in the first passage in pure plasma and Tyrode. There was almost normal growth up to the tenth passage, then a general anh rapid degeneration set in, resulting in death of the cultures of the first series in the 14th passage in oxygenated extract, while the second or fresh fibroblast series died in the 7th passage in oxygenated extract.

These observations seem to support the view that increased oxidation plays a part in the asserted inactivation by shaking and that, a t least to some extent, also, the normal inactivation of embryo extract by aging is caused by oxidative processes. No injurious effect of shaking upon the growth quality of embryo extract, however, was apparent in our experiments.

The effect of shaking upon the enzymes contained in embryo extract could not be directly determined, as a t the time these experiments were done a proteolytic enzyme had not yet been isolated. An attempt was made, however, to obtain some information on the effect of shaking upon the activity of a proteolytic enzyme by submitting a freshly prepared 1 per cent pepsin solution to shaking for eight hours, to aeration for one hour, and to oxygenation for one hour. The effect of these measures upon the proteolytic activity was studied on coagulated egg albumin cylinders weighing about 0.5 gm. each. The time needed by the different enzyme fractions for the complete hydrolysis was compared with the time used for this result by normal pepsin. While this method is rather crude and will not demonstrate minor changes in the activity, the consistent absence of any difference in the times which elapsed until complete hydrolysis was obtained by the various pepsin fractions seems to support the conclusion that the measures mentioned have no marked effect upon the activity of pepsin solution. If a conclusion by analogy can be drawn from these observations, a step which appears, however, to be of doubtful value in this instance, the procedures described above should be without any effect upon the activity of the proteolytic enzymes contained in the embryo extract.

5. THE EFFECT OF HEAT UPON THE ACTIVITY OF EMBRYO EXTRACT In connection with the possible denaturation effect of shaking

upon the proteins of the embryo extract on account of froth formation, the study of another and much more intense denaturation process appeared to be suggestive. A review of the literature on the inactivating effect of heat upon embryo extract leaves the impression that heating this substance to 70" C. for ten to fifteen


minutes is sufficient to destroy completely the proliferative qualities (Carrel, Fischer). Fifteen minutes' exposure of embryo extract to a temperature of 70" C. produces, indeed, marked changes in the physical qualities of this material. It changes from a clear, serous fluid into a milky, viscous matter.

In view of these pronounced physical as well as chemical changes brought about by heat, a definite and marked effect upon the growth-promoting qualities of the extract thus treated could be expected. But standardized fibroblast cultures which received this extract grew relatively well on this material. While they lagged, in size of growth, somewhat behind the control cultures fed with normal extract, the cells appeared healthy. The strain was carried on for 29 passages. When the experiment was discon- tinued, the remaining three cultures, out of a total of twelve, were still in good condition. The result was, however, different when fresh fibroblast cultures were used which had not come in contact with normal embryo extract. These cultures did not grow well from the beginning, showed evidence of degeneration, and died after nine passages.

These observations on the growth effect of heated embryo extract are in many respects comparable to those made by Carrel and Fischer on cultures fed with a proteose preparation obtained by partial hydrolysis of Witte's peptone. Those investigators, also, observed an extended growth only when the cultures had been cultured in the beginning in normal extract. While the heated embryo extract is evidently able to support cellular growth for an extended period, the explanted tissue needs the primary stimulus supplied only by normal embryo extract to carry on under less favorable conditions.

Since the proteolytic enzymes contained in the embryo extract are certainly inactivated by exposure to a temperature of 70" C. it was thought that possibly the deficiency of the heated extract might be due to this factor. A proteolytic enzyme extracted with acetic acid-glycerin from the embryo pulp, purified by absorption on kaolin followed by elution with a 1 per cent Na2HP04 solution, and representing a protease of catheptic nature, was added in a dilutio'n of 1 : 10 to the heated extract. Fresh fibroblast cultures exposed to this mixture did not show any improvement in their growth activities over the corresponding controls. While the result obtained does not support the assumption made, it has to be considered that Na2HP04 inhibits cell growth (Demuth). Any


growth-stimulating effect possibly exerted by the enzyme may thus have been neutralized by the inhibiting Na2HP04 action. Further work with enzymes eluted with non-inhibiting agents will be necessary to solve this problem in a satisfactory way.

6. THE EFFECTS OF ULTRA-VIOLET RAYS, X-RAYS, AND RADIUM RAYS UPON EMBRYO EXTRACT

In view of the growth-inhibiting action of various types of radiating energy upon cells in vivo as well as in witro, it appeared likely that corresponding effects might be produced in embryo extract, a biological material resembling in many respects the protoplasmic matter contained in the cells. The information available on this subject is scanty and contradictory. While Craciun briefly mentions that x-rays and radium rays destroy the growth-promoting qualities of embryo extract and that visible light exerts only a slightly injurious action, and while Carrel states that x-ray irradiation decreases the activity of embryo extract, Fischer asserts that x-ray irradiation of extract and plasma had no apparent effect upon the growth-activating substances contained in embryo extract. A thorough investigation of this controversial matter seemed to be indicated on account of the information such a study might yield in regard to the causative mechanism of the radiation effect upon cellular material.

( a ) Biological Eflect of Ultra-Violet Rays upon Embryo Extract Embryo extract was irradiated for eight hours in low, flat-

bottomed glass tubes covered with a quartz plate and placed in ice to prevent the complicating action of heat. The height of the liquid in the tubes did not exceed a 2 mm. level, to insure a homogeneous absorption effect. The dish containing the tubes was placed at a distance of 25 cm. from the arc of a Cooper Hewitt hori- zontal uviarc, 110 volts. This lamp gives a high ultra-violet intensity with relatively little heat. At the end of the exposure i t was noticed that the extract had acquired a peculiar odor, but no changes in color, appearance, or turbidity could be discovered.

Twelve cultures of standardized fibroblasts were halved. One series of halves was treated with ultra-violet irradiated extract, while the other series of halves received normal embryo extract. While there was no difference in the growth activity of the two series during the first ten passages, a moderate decrease in growth was observed in subsequent passages of the irradiated group. Also,

86 HUEPER, ALLEN, RUSSELL, WOODWARD AND PLAT"

the mortality rate of the irradiated extract cultures was higher, indicating a lower vitality. At the end of 23 passages two cultures with moderate growth had survived.

If fresh fibroblast cultures were exposed to the irradiated extract, a quicker and more marked toxic effect became apparent. The cultures showed an impaired growth in comparison to the controls and died after 4 passages with evidence of marked degeneration in 80 per cent of the cultures.

As it is claimed that different wavelengths may have different biological effects, embryo extract was irradiated for two to eight hours in a narrow quartz tube with monochromatic ultra-violet light selected by means of a quartz monochromator. The wavelengths 3132 A and 2535 A were selected. The wavelength 3132 A is near the wavelength 3138 A, which according to Reiter and GAbor emits the mitogenetic rays, having a growth-stimulating effect if administered in small doses and a degenerative effect if given in large doses. Rays of a wavelength below 3000 A were used because it is asserted by Pace that rays of a wavelength below 2700 8, very actively destroy enzymes and it has been stated by Kuhn, Eyer and Freudenberg that rays of a wavelength above 3000 A have almost no effect upon the destruction of insulin; that, on the other hand, rays of a wavelength between 2400 and 2725 A are well absorbed by insulin and exert a marked and rapidly destructive action upon that substance. Allen had, moreover, observed that rays of a wavelength of 2535 A have a rapid lethal effect upon Euglena, a unicellular organism. It is obvious that the absolute amount of energy absorbed by the embryo extract is lower in the monochromatic experiment than in the one first described, where rays of the entire spectrum were absorbed by the extract.

Again standardized fibroblast cultures were used as test cultures. The growth activity in the first ten passages was comparable to that seen in the normal controls. After that period degenerative changes appeared in the cultures of the two series. The cultures exposed to embryo extract irradiated with rays of 3132 A were dead after 16 passages, while those fed with extract irradiated with rays of 2535 A were dead after only 14 passages.

The results of these experiments allow the conclusion that ultra- violet irradiation of embryo extract destroys its growth-promoting qualities. The rays of shorter wavelength seem to have a somewhat more marked effect than those of longer wavelength according

GROWTH FACTORS OF EMBRfO EXTRACT 87

to the results obtained in cultures exposed to embryo extract irradiated with monochromatic ultra-violet light.

(b) Biological Efect of Radon Irradiation upon Embryo Extract Glass seeds containing radium emanation were placed a t the

bottom of a test tube containing about 2 C.C. of embryo extract. The extract received a total dose of about 320 m.c.h. As glass seeds were used, the embryo extract was exposed to p and y rays.

The action of the rays upon the proliferative activity was tested on series of standardized and fresh fibroblast cultures. There was no evidence of an inhibiting effect in the standardized series after seven passages. The set of fresh fibroblast cultures could be carried through as many as 16 passages without any definite evidence of growth inhibition. The effect of and y rays upon the active substances in embryo extract is according to these observations either very slight or entirely negligible.

(c) Biological Efect of X-ray Irradiation upon Embryo Extract Embryo extract was placed in wide test tubes and irradiated for

four hours without filtration at a target distance of 45 cm. with a machine delivering 120 k.v. a t constant potential. The total dose administered amounted to 15600 r, an enormous dose considering that the average erythema dose requires 550 r.

In these experiments, also, the test cultures were two series of Standardized and fresh fibroblast cultures, respectively. The results obtained did not differ essentially from those obtained by radon irradiation. After 12 passages both series showed a com- paratively good growth. There was no evidence of an inhibiting action of the treated extract upon the cultures in comparison with the growth activities of the control cultures.

If the results obtained with the three types of radiating energy are compared, it becomes evident that the ultra-violet rays have a definite destructive action upon the growth-promoting substances of the extract, the effect of radium emanation is doubtful, but a t any rate only very slight, and the effect of x-rays is negligible.

( d ) The Effect of the Rays upon Certain Chemical and Physical Properties of Extract

The discrepancy observed between the biological action of the three types of radiating energy seemed to indicate that corresponding differences in the quantity and quality of the physical and


chemical changes produced by the rays in the substrate could be demonstrated. A thorough study of this matter seemed to be justified, as the effect produced by these rays in protoplasmic material is still a subject of lively controversy.

Some authors believe that x-rays and radium rays produce a denaturation of the cell proteins (Freundlich, Dessauer-point heat effect, Hardy, Spiegel-Adolf and others). Other investigators assert that the rays have primarily an effect upon the oxidation-reduction mechanism (Waterman, Loew-Beer and Reiss, Gottschalk and Nonnenbruch, etc.), while Heeren and Pansdorf consider an effect upon the dispersion of the colloidal protein solutions and secondarily upon the cell ferments as the main factor involved in the cellular action of these rays. But Spiegel-Adolf remarks that denaturation of proteins does not occur in well buffered protein solutions and that, in spite of the presence of changes in odor and color, definite chemical changes have not yet been demonstrated, although changes in the absorption spectra of the irradiated protein solutions, showing increased absorption, have been noted.

Our investigation of the physico-chemical effects of the ultra- violet rays, radium rays and x-rays was extended to a study of the following factors : glutathione content ; activity of cathepsin; absorption of ultra-violet light; denaturation of proteins; surface tension.

Glutathione Content: The determination of the glutathione content seemed to be important in view of the numerous claims which have recently been made in regard to the significance of this substance for the regulation of the oxidation-reduction potential in the cell, the activation of several enzymes such as cathepsin, amylase, and arginase, the detoxication of heavy metals, the stimulation of cell multiplication and differentiation, and the production of malignant tumors. It has been shown, moreover, by various authors that sulphydryl compounds are sensitive to radiating energy. Lieben and Molnar have demonstrated that cysteine is oxidized under the influence of visible as well as ultra- violet light. Shoji made a similar observation on the cysteine of the lens after ultra-violet irradiation. Coldwater reported a drop in the glutathione content of Planarians after exposure to 2000 R and correlated this decrease with an inhibition of the rate of division of these organisms. Also, Hammett asserts that the oxidation of -SH groups by radium rays is responsible for the growth-


1 hour 4 hours 8 hours

GSH Total GSH Total GSH Total mg. % mg. % mg. % mg. % mg. % mg. %

14.9 - 3.1 20.0 - - - - - - 0 14.7

- _ _ _ ~ ~ ~ ~ -

1 6 - - - - - 0 3.5 - - -

inhibiting action of the rays. He states that the rays produce an oxidation of the -SH groups to suboxidized sulphur compounds which impair the multiplication process of cells.

The total glutathione and the -SH fraction present in embryo extract were determined before and after irradiation with ultra- violet rays, x-rays, and radium emanation in glass seeds. The doses given and results obtained are recorded on the following charts. For the purpose of comparison, identical determinations were made on extract oxygenated for one hour.

TABLE I Changes in Glutathione of Embryo Extract by Ultra-violet Light

- -

7.03 -

Extract No.

33.5 - 25.8 35.8 29 38 32 37

1 . 2. 3. 4.

PH

- 7.03

L

Ex- tract NO.

- 3. 4. -

Original content

GSH Total mg. % mg. '%

29 38 32 37

---

Original content

PH

GSH * I Total mg. % mg. %

Amt. of treat.

8 hr. = 31,200r 4 hr. = 15,600 r

GSH Total GsH mg. % mg. % Am'. treat. mg. % mg. %

25 38 323 m.c.h. 25 37 33 40 255 m.c.h. 21 , 32

-- --

I Glutathione found after irradiating for

* GSH denotes glutathione in the reduced form.

TABLE IT Changes in Glutathione of Embryo Extract by X-rays and Radium

Glutathione found after treatment with

Radium I X-rays

The tables show that ultra-violet rays cause both oxidation and destruction of the glutathione in embryo extract. This is observed to a different extent in different extracts, probably dependent upon the pH, as was observed in pure solutions of glutathione. The glutathione of embryo extract is much less stable when exposed to ultra-violet rays than when present in pure solution, as could be demonstrated in separate investigations by one of us (Woodward).

Toward x-rays, the glutathione of embryo extract behaves the

90 HUEPER, ALLEN, RUSSELL, WOODWARD AND PLATY!

Original content

GSH I Total PH

mg. % w. %

same as in pure solution. There is no destruction, but possibly slight oxidation, so slight that i t may be due to an experimental error.

Radium causes partial oxidation of the glutathione in embryo extract, and slight destruction. This is as observed in pure solution of glutathione.

Toward molecular oxygen, the glutathione of embryo extract is much more stable than when in pure solution of the same pH. This stability varies in different extracts. In one extract it was not

Glutathione found after oxygenating one hour

GSH 1 Total nw. % mg. %

TABLE I11 Changes in Glutathione of Embryo Extract by Molecular Oxygen

mg. ______

O * 0 6 9

I” 8.

%

0 0

13 16

6.87 6.95 6.98 6.97

36 49 44 57 31 53 31 53 47 56 41 57 55 64 46 66

* In this single case there was an increase of 8 mg. in both the reduced and total glutathione, which we have explained as an error due to introduction of some impurity. There was no oxidation.

possible to produce any oxidation. This stability toward molecular oxygen is also apparent when extract is analyzed for its glutathione content after standing for days or weeks. There is a gradual oxidation of the SH to the SS form, but it is very slow. The stability is perhaps explained by the presence of hydrogen-donators in the extract which reduce the glutathione with a speed about equal to the oxidation. Hopkins has observed this to be the case in liver tissue pulp. Thus he states: “The rate of disappearance of -SH, as observed, is the resultant of associated reduction and oxidation. The agencies responsible for the former become grad- ually exhausted in excised tissue, and the observed velocity of oxidation then increases.”

As it has been claimed by some investigators that the destruction of the growth-stimulating -SH groups by irradiating energy and the resulting production of growth-inhibiting suboxidized sulphur (SO-, SOz- and SOa-) groups are responsible for the arrest of the mitotic processes in irradiated tissues, the following experiment was devised to test the correctness of this assertion.


In duplicate experiments two series of standardized fibroblast cultures were explanted in embryo extract irradiated with ultra- violet rays for eight hours. When the first evidence of degenerative processes appeared in the two series, one of them was put on an embryo extract diet to which glutathione was added in amounts corresponding to those destroyed by the irradiation. The series was carried on for about 6 passages without any stimulating effect upon growth whatever. The cultures in both series were dead at the same time. As the possibility had to be considered that the addition of glutathione to the irradiated extract had been too late to stimulate a culture already in the state of degeneration, two additional series were set up, the one of which received straight irradiated extract, while the other received irradiated extract plus glutathione. The results were identical. No stimulating effect upon cell mitosis by the glutathione added was observed in 23 passages.

The outcome of these experiments agrees very well with observations made some time ago on the effect of copper upon the growth of cells in vitro. It was observed at-that time that the growth toxic effect of copper (electrolytically powdered copper and CuC12) could be partially overcome by the addition of hemin and cysteine or embryo extract to the culture medium. As it is known that -SH groups combine with and detoxicate copper (Voegtlin, Myrbaeck), the growth-stimulating action of cysteine in this instance was an indirect one, due to the removal of an inhibitor (copper). A similar growth-inhibiting mechanism is apparently present in growth inhibition by lead. It is apparently the direct effect of the heavy metal upon the cell and not the binding of the -SH groups which produces the inhibition of mitosis. The correctness of this conception is borne out by the action of arsenic, which is also bound by -SH groups (Voegtlin), which, however, stimulates growth.

It is also highly improbable that the presence of suboxidized sulphur groups in the tissue would inhibit cell growth, since it can justifiably be assumed that at the decomposition of the glutathione by ultra-violet rays, not inconsiderable amounts of suboxidized sulphur are formed. No inhibitory effect of these substances became apparent in our experiments with ultra-violet irradiated embryo extract.

A growth-inhibiting effect of x-rays through the mechanism of -SH destruction is also highly improbable, in view of the fact


that even a dose amounting to almost thirty times an erythema dose fails to produce any appreciable changes in the SH content of embryo extract.

Also, the relatively moderate oxidative and destructive effect of radium emanation upon the -SH glutathione is scarcely sufficient to account for the growth-inhibiting action of this agent. The occurrence of osteogenic sarcomas on the basis of chronic radium poisoning, a condition in which the cancer-producing tissues are constantly exposed to a heavy bombardment with p and y rays, contradicts the assumption that -SU groups are the only growth- and cancer-producing agents and suboxidized sulphur groups the only growth-inhibiting ones. There exists abundant additional evidence which refutes such a conception, as, for instance, the production of cutaneous cancer by prolonged ultra-violet treatment (Putschar and Holtz, Herlitz, Jundell and Wahlgren, Abrikosoff and Wail, Findlay), a measure which is certainly not suited to increase the -SH content of the exposed epithelial cells. Also, the production of arsenic cancer does not support the -SH etiology of malignant growth. Labes even suggested the use of small doses of arsenic to neutralize -SH hyperfunction. The identification of the Rous agent with a -SH action is without any proof; in fact, Yaoi and Nakahara found that the Rous tumor contains little glutathione. Witte’s peptone stimulates cell growth without containing any demonstrable -SH groups. Morgulis and Green using various -SH preparations did not see any stimulation of the regenerative processes in experiments with the worm, Podarlce obscura. Similar results were obtained by Gaunt with water snakes. The tissue culture experiments of Baker on this subject are quite inconclusive, since the control series was kept in a medium deficient in the proper nutritive substances. Moreover, almost no stimulation to mitosis was seen if glutathione was added alone. The stimulative effect of insulin medication on the healing of skin wounds can not be considered as a support of the -SH theory, because insulin is inactivated by cysteine (Du Vigneaud et al.) and numerous observations on the cancer-inhibiting action of insulin therapy are available (Munzner and Rupp, Stuehlern, v. Witzleben, Mori and Nakamura, Cioff ari and Piccaluga). Voegtlin, moreover, showed that the mitosis-stimulating effect of glutathione on ameba is an indirect one, through stimulation of differentiation. Guder- natsch observed that cystine may stimulate differentiation and growth, but that arginine stimulates growth and inhibits differenti-


ation in tadpoles. Cancer is not simply a manifestation of increased cell multiplication; it is characterized, also, by inhibited differentiation (Zakrzewski), which would indicate that arginine rather than glutathione is involved in the cancerization process. Gilroy did not see any cancer stimulating effect by cystine and cysteine medication, while arginine accelerated the growth of transplantable mouse tumors. The frequency of liver metastases and the high incidence of mitoses are not necessarily due to the high glutathione content of the liver, but may be attributed, possibly with more justification, to the high glycogen content of that organ and the ready utilization of the lactic acid produced by the malignant cells in glycogen synthesis by the liver.

Nor is the conception of an exclusive control of the cellular multiplication process by the -SH-SOS equilibrium (Reimann) supported by the fact that the optimal growth of tissue cultures is observed in a medium having a pH 7.4 to 7.8 (Fischer). This represents a range in which the SH groups are relatively labile, because the reaction favors the spontaneous oxidation of -SH to -S-S-. It remains to be seen, if the sudden, temporary increase in -SH content of sea-urchin eggs shortly before mitosis has any causative connection with the division process or is only the result of an intracellular change in reaction of the protoplasm on occasion of the multiplication process (Chatton, Lwoff and Rapkine).

It is moreover still quite doubtful whether malignant tumors contain actually more glutathione than normal tissues, or whether possible increases may only represent an index of their cellularity (Kennaway and Hieger), since glutathione is also increased in granulation tissue. While Binet and Magrou, Delbet and Franicevic, Voegtlin, and Lecloux, Vivario, and Firket record an increased glutathione content in various malignant tumors examined, Bierich and Kalle found only a normal content, and Heinlein, Holmes, and others report subnormal values. Similar contradictory statements are made in regard to the glutathione content of the blood in cancerous conditions.

The status of glutathione in the tissues is also bound to the activity of the enzymes, especially the cathepsin, for which it acts as an activator. Also, in this capacity glutathione is indirectly linked to the proliferative activity. Waldschmidt-Leitz has maintained that the excessive production of lactic acid by the cancer cells produces an acid reaction favoring the stability of the -SH

6


form of glutathione, which in turn stimulates and activates the proteolytic cathepsin. Krebs has stated that the proteolytic activity of tumor tissue is high but within normal limits, while Kleinmann and Werr report that there are neither qualitative nor quantitative differences in the proteolytic activity of normal and malignant tissues. The significance of the activity of a proteolytic enzyme in embryo extract rests upon the claims of Carrel, Fischer, Borger and Zenker, who point out that the production of proper protein decomposition substances is necessary for the growth- promoting quality of the extract and that the inactivation of the extract by heat, shaking, or oxidation suggests the presence of a proteolytic enzyme. Fischer, however, raises an objection to an enzyme action, as he notes that the medium has not the proper reaction of pH 4-5 to insure an active enzyme (Willstaetter). But it is known that the presence of electrolytes and proteins in the enzyme solution may shift the optimum pH considerably (Northrop, Willstaetter, Grassmann).

Cathepsin: For the demonstration of the presence of a proteolytic enzyme of catheptic nature, a glycerin-acetic acid extract was prepared from embryo pulp according to the method of Rona and Kleinmann. This crude extract was purified by adsorption on kaolin, followed by elution with 1 per cent Na2HP04 solution. The purified preparation, which gives a faint turbidity yith 20 per cent sulphosalicylic acid, was tested for its proteolytic qualities against gelatine, according to the method of Kleinmann and Werr. The turbidity readings were made with a colorimeter which, according to these two authors, gives more reliable results than a nephelometer, if gelatine is used as a substrate. The extract was found to be active and gave the following results, expressed in per- centage of digested gelatine :

I. Activated with H2S water and kept at 37" C. after 30 minutes. .................................... .2O per cent after 60 minutes. .................................... .33 per cent after 90 minutes. .................................... .33 per cent

after 30 minutes. ..................................... 9 per cent after60 minutes ...................................... 13 per cent after 90 minutes. .................................... .33 per cent

11. Not activated with H1.S water and kept at 37" C.

The extract was diluted for the experiment with Tyrode in a ratio 1 : 10. The activity of a watery and purified extract was somewhat weaker.

Numerous investigations have been made on the effect of the


various types of irradiation upon the activity of enzymes, but the results obtained are quite contradictory. Maubert, Jaloustre, Lemay and Guilbert reported the inactivation of liver catalase by x-rays, the effect depending upon the dose and time of exposure Richards, on the other hand, found x-rays quite ineffective against rennin, pepsin, and papain. He even saw an acceleration of the enzyme action after weak doses of x-rays. Packard claims that radium radiation activates autolytic enzymes in cells which bring about a degeneration of the complex proteids. Gayer, on the contrary, observed a retardation of enzyme action by radium. Henri and Mayer reported that invertin, emulsin, and trypsin lose their activity when exposed to radium. Neuberg and Wohlge- muth noticed an acceleration of the autolytic processes after radium irradiation, and Neuberg added that autolytic enzymes of cancer cells are not as susceptible to radium as enzymes from normal tissues, because there is a much more rapid autolysis of cancer tissue than of normal tissue after irradiation. X-rays he considered less active in this respect. Loewenthal and Wohlge- muth reported that radium emanation accelerates the diastatic enzyme of the blood, liver, saliva, and pancreas. Euler saw an activation of pepsin and trypsin after radium emanation irradiation. Richards stated that only weak x-rays are comparable to radium irradiation. He mentioned that an exposure of four minutes to x-rays increases the activity of pepsin, five minutes exposure is ineffective, while a thirty minutes’ exposure inhibits the enzyme action. Similar results were obtained by him with Taka diastase. Lockmann, Thies and Wichern used x-rays without effect upon catalase, as did Euler, who tested the catalase activity in the cell, because he thought that this enzyme might be more sensitive in the cell.

Schwarz and Friedrich, using higher doses of x-rays, saw an immediate drop in the activity of the catalase followed by a gradual recovery and finally by an increased activity. Wels, who re- peated this experiment, could substantiate only the decreasing phase of the x-ray action, but in no instance saw a recovery. He stated that catalases from other sources than the liver are not definitely affected by x-rays, while serum lipase and pancreatin are not affected by x-rays. He noted, furthermore, that the x-ray effect upon enzymes depends upon environmental factors which may or may not react with a dispersity decrease upon the x-rays. The proteins may protect the enzyme against the ray effect. Also,


the pH of the substrate is important, as it controls the colloidal stability. According to Wels, very active enzymes are more sensitive than less active ones. Mueller and Jochmann observed that x-rays have no effect upon leukocytic proteases.

The effects of ultra-violet rays upon enzymes have been studied by Takuma, who reports a decrease in the activity of liver lipase proportional to the dose and exposure. Wels, Batelli and Stern, Euler, Wentig and Stecke, Morgulis, Pincussen, and Baker and Nanavutty, saw an inactivation of catalase by ultra-violet irradiation. This effect was the same in living and dead tissue. Hussey and Thompson inactivated pepsin, Pincussen amylase, serum lipase and esterase and Pace trypsin by exposure to ultra-violet rays. Finally it may be mentioned that the Rous agent can be inactivated with ultra-violet rays, requiring a dose below that necessary for the inactivation of most of the enzymes and above that needed for the killing of bacteria and the common viruses (Murphy; Sturm, Gates and Murphy). This observation is important, because Murphy believes that the Rous agent and the growth-promoting factor of embryo extract are related and similar in nature.

On account of the contradictory statements, some preliminary experiments were conducted in which the effect of the different rays upon the activity of pepsin was tested.

In the first experiment 10 C.C. of a 1 per cent pepsin solution were exposed to an ultra-violet lamp for four hours, the container of the pepsin solution being kept in ice water during this period. Five C.C. of a n/lO normal HC1 solution were added, and a cylinder of coagulated egg albumin measuring 2 mm. x 10 mm., weighing about 0.5 gm., was put into the mixture, which was placed in the incubator with the proper controls. After six hours of incubation, the albumin cylinders in the controls were entirely digested, while about haif of the cylinder was left in the irradiated pepsin solution. A marked decrease in activity due to ultra-violet irradiation was thereby established. To obtain more accurate results, the edestin method of Fuld and Levison was used. The pepsin solution was again irradiated with ultra-violet rays. The results of this experiment showed that the enzyme number of the control was 80, while the enzyme number of the irradiated pepsin had dropped to 20. The hydrolytic activity of the pepsin had been reduced four times by ultra-violet irradiation. In a third series of experiments, in which the edestin method was again used, one part of the pepsin solution was irradiated for four hours with ultra-violet rays, another


portion for four hours with x-rays, and another portion was oxygenated for one hour. The normal enzyme number in this experiment was 36, the oxygenated pepsin had an enzyme number of 30, the x-rayed of 26, and the ultra-violet irradiated of 10. The ultra- violet irradiation, according to these findings, had the strongest effect upon the activity of pepsin, decreasing it by about 75 per cent. The addition of cysteine to the pepsin solution before or after irradiation did not change the results appreciably.

The glycerin-acetic acid extract of embryo pulp was then sub- jected to the same type of treatment, that is, four hours' ultra- violet or x-ray irradiation. The irradiated enzyme was activated with H2S water and the hydrolysis of gelatine determined with the colorimeter, after two hours incubation at 37" C.

Normal embryo extract cathepsin ............................ .55 per cent X-rayed embryo extract cathepsin ........................... .33 per cent Ultra-violet irradiated embryo extract cathepsin . . . . . . . . . . . . . . . .20 per cent

Embryo extract was then tested for its proteolytic qualities after irradiation with ultra-violet rays, x-rays, and radium emanation. The extract was diluted for this purpose with Tyrode solution in a ratio 1 : 11. The results were as follows after two hours incubation at 37" C.

Normal embryo extract. ............................ .33 per cent X-rayed embryo extract. ........................... .23 per cent Radon irradiated extract. ........................... .23 per cent Ultra-violet irradiated extract, ...................... .18 per cent

Also, in these experiments it is evident that the ultra-violet rays have the most injurious effect upon the activity of the cathepsin contained in embryo extract. A certain parallelism between the effect of the different rays updn the glutathione content and the cathepsin activity is remarkable. But this can not be due to an inactivation of the cathepsin on account of the destruction of the glutathione, since the cathepsin was always activated in the digestion experiments by the addition of H2S water.

In the consideration of the action of radiating energy upon enzymes, thought should also be given to the possibility that rays may have not only a purely destructive effect upon enzymes, but also a mutating effect, resulting in changes of the type of substances and chemical group attacked and also in the character of degrada- tion substances produced. Experiments on this phase of the problem will be undertaken in the near future.

Spectrographic Studies: An attempt was then made to correlate

98 HUEPER, ALLEN, RUSSELL, WOODWARD AND PLA'M'

the effects of the different radiations upon the extract with changes in the absorption by the quartz ~pectrograph.~ A Hilger quartz E 2 spectrograph with a hydrogen continuous light source and East- man 33 plate were used. The spectrograms were analyzed in the Kruss densitometer. While the decreasing sensitivity of the plates used prevented absolute values from being obtained, the curves still have a comparable value.

The densitometer curve of a freshly prepared glutathione solution containing 50 mg. glutathione per 100 C.C. shows practically

GRAPH I. SPECTROQRAPHIC ABSORPTION CURVES OF GLUTATHIONE water normal glutathione (50 mg. in 100 C.C. of water buffered to pH 7.0)

---

* . . . . . . . glutathione irradiated with ultra-violet rays The marked shift in the absorption band of the glutathione solution upon irradiation

toward the visible part of the spectrum indicates radical changes of the glutathione molecules.

no absorption. A solution which is'several days old shows a slight increase in absorption, while a solution irradiated for four hours with ultra-violet light shows a markedly increased absorption toward the visible light. This increase in absorption is due possibly to the formation of colloidal sulphur from the irradiated glutathione.

The densitometer curves of normal embryo extract and embryo extract diluted forty times before irradiation with ultra-violet rays are almost identical. The effect of x-ray irradiation upon the absorption curve is also negligible, while markedly increased absorption is present if the embryo extract is irradiated with ultra-

SThe spectrographic work wm done in the laboratories of the Bartol Research Foundation, with kind permission of Doctor W. F. G. Swann, Director.


violet rays and radium emanation before it is diluted 40 times, the absorption after radium emanation treatment surpassing in this instance even that produced by the ultra-violet ray effect. A dilution of the extract was necessary because investigations on concentrated extract showed such a homogeneous and extensive absorption that the irradiation effects were covered.

Also, the irradiation of a glycerin-acetic acid extract with the three ray sources showed the increase in absorption produced by the rays. The radon effect is the most marked, then that pro-

r

Wavelength 22 23 25 27 30 35 40 50 60

GRAPH 11. SPECTROQRAPHIC ABSORPTION CURVES OF EMBRYO EXTRACT normal extract

.-.- irradiated with ultra-violet before dilution 1: 40 . . . . . . . . irradiated with ultra-violet after dilution 1: 40 --- irradiated with x-rays -. .- irradiated with radon

The numbers on the abscissa indicate A units in hundreds The shifts in the absorption band toward the visible part of the spectrum i n the

embryo extract exposed to the various types of rays indicate that intramolecular re- arrangements in some of the substances contained in the extract have been produced by the rays.

duced by x-rays, and finally that resulting from ultra-violet irradiation.

The absorption curves show that the radium emanation effect is outstanding in the embryo extract and the enzyme. The ultra- violet rays have in all three experiments a definite absorption- increasing effect, while the action of the x-rays is somewhat ir- regular, being almost zero in the embryo extract, but surpassing the ultra-violet effect in the enzyme. Our present knowledge in this matter does not allow a decision as to the causes of these variations. Differences in the amount of absorbed energy may


possibly be responsible for these inconsistent observations, also variations in specific absorption may play a rble.

Ultramicroscopic examinations of the various types of embryo extracts in the course of the experiments reported above, using the cardioid as well as the Spierer condenser, gave results too dis- crepant for any reliable conclusions to be drawn.

.I g \ * I I I

I !2 I

).

i WavdenEth 22 23 25 27 30 35 40 60 60

GRAPH I1 1. SPECTROQRAPIIIC ABSORPTION CURVES OF ENZYME normal enzyme - . . . - irradiated with ultra-violet

--- irradiated with x-rays -. .- irradiated with radon The numbers on the abscissa indicate A units in hundreds. The shifts in the absorption of the enzyme irradiated with the three types of rays

indicate that intramolecular changes in some of the substances contained in the enzyme solution have taken place.

Determinations4 of the surface tension of the extract after irradiation with ultra-violet rays and radium emanation did not reveal any changes in this factor. The ring method was used.

Normnl embryo extract. ....................... .60.80 dynes@. Ultra-violet irradiated .......................... .49.30 dyneslcm. Radon irradiated. ............................. .50.10 dynes/cm.

The minor variations recorded are within the range of experimental error.

CONCLUSIONS 1. Tests with an antiembryo extract serum indicate that the

non-species specific growth-promoting substances contained in the

Doctor Russel J. Fosbinder, of these laboratories. 4 For the determinations of surface tension on embryo extract we are indebted to


embryo extract are apparently not protein, capable of producing specific non-species specific antiproliferative antibodies.

2. Extracts made from different portions of chick embryos have approximately the same growth-promoting power.

3. Polysaccharides isolated from embryo extract have no growth-stimulating qualities.

4. Embryo extract gives a positive Feulgen nucleal reaction. This result is not changed by aging, shaking, etc., attesting to the stability of the nucleic acid complex responsible for this reaction.

5. Oxygenation, nitrogenation, ultra-violet irradiation, and radon irradiation do not change materially the surface tension of embryo extract.

6. Prolonged shaking of embryo extract does not affect seriously the activity of embryo extract.

7. Any possible inactivating effect caused by shaking is probably due to an increase in the oxidative processes and is not caused by denaturation of substances in the froth formed or by inactivation of enzymes or by change of the dispersion status.

8. Oxygenated extract has a markedly decreased activity. 9. Heating of extract to 70" C. for fifteen minutes does not make

extract unsuitable for the cultivation of cultures which have been grown for some time in a medium containing normal extract. The heated extract is, however, unsuitable for the cultivation of fresh cultures.

10. Irradiation of embryo extract with ultra-violet rays decreases its growth-promoting power.

11. Irradiation of embryo extract with x-rays or radium emanation does not markedly change the growth-stimulating qualities.

12. Ultra-violet rays oxidize and destroy considerable portions of the glutathione contained in embryo extract.

13. X-rays cause only a slight oxidation and radium emanation a moderate oxidation and slight destruction of glutathione in embryo extract.

14. There is no evidence that the -SH group has a selective mitosis-stimulating effect and that suboxidized sulphur is a growth inhibitor, according to our experiments.

15. A proteolytic enzyme of catheptic nature was prepared from embryo pulp.

16. Ultra-violet rays have the most injurious effect upon the activity of this enzyme.

17. Spectrograms have been taken of normal embryo extract


and embryo extract irradiated with ultra-violet rays, x-rays and radium.

18. The preeminent effect of ultra-violet rays upon the growth qualities of embryo extract, upon its glutathione content and the activity of its proteolytic enzyme, seems to indicate that u l h - violet rays have a more marked injurious effect upon the cell than x-rays or radium.

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OF - Cancer Research · The chicken leukocyte antiserum was obtained from a rabbit whi'ch had received 15 intravenous injections of a suspension of chicken leukocytes. As extracts