The effects of protamines and histones on the nucleic acids of ascites tumor cells

Experimental

THE

Cell

Research

19, 361475

361

(1960)

EFFECTS OF PROTAMINES AND HISTONES ON THE NUCLEIC ACIDS OF ASCITES TUMOR CELLS1 F. F.

Department

of Pathology,

New

BECKER York

and

H.

GREEN

University-Bellevue N.Y., U.S.A. Received

May

Medical

Center,

New

York

City,

15, 1959

P ROTAMINES

and histones, the basic proteins of cell nuclei, have been shown to possess interesting properties with respect to a large variety of organisms. They inactivate viruses [13] and are bacteriostatic and bacteriocidal [14, 191. Their effects on animal cells have been studied largely by Fischer and his colleagues [ 11, 121 who have shown that they enter liver cells or tumor cells, and affect the respiration of these cells and their staining properties. The growth of tumors in vivo has been shown to be depressed to some extent by the injection of either protamines or histones [24, 261. The present experiments deal with the in vitro effects of protamines and histones on Krebs ascites tumor cells of the mouse. The results obtained indicate that these proteins enter the tumor cells rapidly and in large quantity, and combine with the cell nucleic acids, producing characteristic morphologic changes in both nucleus and cytoplasm. As a result presumably of their combination with cell RNA, the protamine or histone arrests the process of protein synthesis. The viability of the cells may be completely destroyed.

MATERIALS

AND

METHODS

The Krehs-2 ascites tumor was passed in mice by intraperitoneal injection of about 0.2 ml of tumor-bearing ascitic fluid. To prepare cells for experiments, 1-2 ml of ascitic fluid was placed in 40 ml of a bicarbonate buffered (pH 7.4) balanced salt solution containing glucose, 1.0 mg/ml (BSS) [lo] at 0”. The suspensionwas centrifuged at 400 RPM (International Refrigerated Centrifuge) at 0” for 5 minutes. The sedimented cells were washed twice with BSS in the sameway and resuspended to a final concentration of about 10’ per ml. Cell counts were performed by phase microscopy using a phase hemocytometer chamber. Incubations were performed in a rotary shaker-water bath at 37” under an atmosphere of 95 per cent oxygen, 5 per cent CO,. The incubation medium was BSS unlessotherwise stated. 1 Supportedby

grants

C-3249

and SF 319 from

the United

States

Public

Experimental

Health

Service.

Cell Research

19

F. F. Becker and H. Green The protamine used was a preparation of Salmine Sulfate (Nutritional Biochemicals Corporation and Ely Lilly & Company). It was dissolved in BSS to a concentration of about 10 mg/ml, dialyzed against phosphate buffered saline (pH 7.4) to remove sulfate, and then against BSS to make the final salt concentrations equal to that of the incubation medium. During the dialysis up to half the protamine nitrogen was lost to the dialysate, as determined by the Kjeldahl method. (Protamine is 30.4 per cent nitrogen [8]). The histone used was a commercial preparation from calf thymus (Nutritional Biochemicals Corporation). It was dissolved in BSS by prolonged stirring at room temperature or in the cold. Approximately half of the histone went into solution. After removal of the insoluble portion by centrifugation, the resulting histone solution was used directly, without prior dialysis. Morphological changes in the cells were observed under phase microscopy, in stained smears, and in stained sections. Cell smears were prepared by mixing a drop of cell suspension with a drop of mouse serum (to aid in fixation to the slide) smearing, and fixing the smears immediately in a mixture of equal parts 95 per cent ethyl alcohol and ether. After at least 15 minutes the smears were hydrated with alcohol solutions of decreasing concentration and after complete hydration, were stained. They were then dehydrated through alcohol and xylene and mounted. The staining techniques are described fully by Pearse [20] save for the Fast Green Stain of Alfert and Geschwind [l]. For paraffin sections, portions of cell suspension were mixed with mouse serum (4: 1); one volume of this mixture was added to two volumes of 10 per cent neutral formalin and allowed to fix for 24 hours. The cells were then centrifuged at 3000 RPM for 20 minutes and the pellets processed as cell blocks.

RESULTS

The Uptake

of Protamine

and Histone

by the Tumor

Cells

Rate of uptake.-The rate of uptake of protamine by an incubating cell suspension was determined by following the disappearance of protamine from the medium. Protamine was measured calorimetrically by the Sakaguchi reaction for arginine [23], which produced a color intensity that was linear with protamine concentration over a considerable range (Text-Fig. 1). To an incubating cell suspension, protamine was added at zero time. At intervals thereafter, 2.0 ml aliquots of cell suspension were taken and centrifuged immediately at 0 degrees. To 1.0 ml aliquots of supernatant an equal volume of 10 per cent Trichloracetic Acid (TCA) was added and after standing for 15 minutes, if there was any trace of precipitated protein it was removed by centrifugation. The protamine concentration of the supernatant was then determined, The result of two such experiments is shown in Text-Fig. 2. It can be seen that at two initial concentrations (48 and 96 ,ug N/ml), protamine was removed from the medium rapidly at first, and then progressively more slowly. Experimental

Cell

Research

19

Effects of protamines

and histones on cell nucleic acids

363

Most of the protamine was taken up within the first 30 minutes, very little after the first hour. Temperature dependence of rate of uptake.-To cell suspensions incubating at 0” and 37” protamine was added to a final concentration of 76 pg protamine N per ml. The rate of disappearance of protamine from the medium was followed over a thirty minute period. It can be seen (Text-Fig. 3) that though

I 1 PROTAMINE

N

(Jg/ml)

0

30

I 60 MINUTES

I so

I

120

Text-Fig.

1. Text-Fig. 2. Text-Fig. l.-Sfandard curve for protamine determined by Sakaguchi reacfion. The extinction given by protamine is equal to 0.75 x that which would be given by free arginine equal in concentration to that contained in the protamine [8]. Histone was determined similarly, though its extinction was only 0.34 x of that of protamine at the same nitrogen concentration. Text-Fig. 2.-Removal of protamine from the medium by an incubating cell suspension. The ordinate represents the protamine concentration remaining in the medium after cells were removed by centrifugation. Cell concentration, 9.0 x loo/ml. Upper curue, initial protamine concentration, 96 lug protamine N/ml, lower curue, 48 ,ug protamine N/ml.

there was a small rate of uptake of protamine at 0”, the rate of uptake at 37” was considerably greater, indicating some degree of dependence of the rate of uptake upon metabolic reactions. Relationship between protamine uptake by the cells and protamine concentration in the medium.-Text-Fig. 4 shows the result of an experiment in which cell suspensions were incubated in the presence of different concentrations of protamine for a period of 45 minutes and then centrifuged. The quantity of protamine remaining in the supernatant was then determined, and the amount taken up by the cells calculated. At low concentrations of protamine the amount taken up was proportional to concentration, but at higher concentrations the slope of the curve was reduced. In other experiments of this Experimental

Cell

Research

19

F. F. Becker and H. Green

364

type, the uptake reached a maximum of 350-400 ,!~g of protamine S per 107 cells. The uptake of histone was followed in similar experiments but as histone is precipitated by 5 per cent TCA, the TCA treatment of the supernatants was omitted. It was found that the histone uptake by the tumor cells was very similar to that of protamine. so-

0 INITIAL

MINUTES

I 200 PROTAMINE

I I 400 600 CONCENTRATION

I 600 tpgNlml1

Text-Fig. 4. Text-Fig. 3.-Uptake of profamine by fumor cells at 37O and at OO. Cell concentration-l.2 x iO’/ml. Protamine was added at zero time to a final concentration of 76 ,ug protamine N/ml. Ordinate represents protamine concentration remaining in supernatant after centrifugation of cells. Incubation temperature, upper curue, O’, lower curue, 37”. Text-Fig. 4.-Concenfration dependence of profamine uptake. Ordinate represents amount of protamine taken up by cells of 1.0 ml of cell suspension. Abscissa, the initial protamine concentration in the medium. Cell concentration, 6.7 x loo/ml. Temperature 37”. Incubation period, 45 minutes. Text-Fig.

Morphologic

3.

Evidence of the Entrance Cells, and of their Interaction

of Protamine and Histone with Cell Nucleic Acids

into the

Phase microscopy.-Untreated ascitic tumor cells examined under phase microscopy have a clearly delineated cellular margin but very indistinct nuclear boundary. Refractile inclusions and vacuoles are visible in the cyto-

Fig. l.-Untreated ascific tumor cells. The cells reveal sharp margins and indistinct nuclei. Occasional cytoplasmic granules are noted. Phase x 690. Fig. 2.-Cells freafed wifh profamine (75 pg N/ml). The cytoplasm is swollen, but cell outline is still clear. The nuclei appear discrete, and cytoplasmic granules are clearly visible. Phase x 500. Fig. 3.-Cells treated with profamine (175 pg N/ml). Some cells are of the typical granular type (G), others are granular but possess in addition a rim of pale cytoplasm characteristic of the swollen type (GS). Phase x 535. Experimental

Cell Research

19

Effects of profamines

and hisfones on cell nucleic acids

Experimental

Cell Research

365

19

F. F. Becker and H. Green

366

plasm (Fig. I). Two types of alteration were produced by the addition of either protamine or histone (50-300 fig N/ml) to the medium. In one type the cells revealed severe cytoplasmic swelling, the swollen cytoplasm apwhile the nucleus became more discrete. pearing pale and homogeneous, Cytoplasmic granules sometimes became clearly demarcated (Fig. 2). In a second more frequently observed type of change the cells appeared densely granular and darker than normal and the nucleus, though distinguishable, was partially obscured by this general granularity. Swelling of the cell was also apparent though there was sometimes only a thin rim of paler cytoplasm (Fig. 3). High concentrations of protamine usually produced these granular cells rather than swollen cells. This type of granular change has been shown to occur in protamine treated, isolated liver cell nuclei by Philpot and Stanier [22]. The effects induced by protamine began to appear within 5 minutes after its addition when the final concentration was 300 ,ug N/ml. By 10 minutes roughly half of the cells and by 15 minutes all the cells showed visible changes. Clumping was occasionally encountered but chiefly at higher concentrations of protamine or after prolonged incubation. The changes described above were also induced by histone, but not by equal concentrations (by weight) of arginine or of lysine. If serum protein was present in the incubation medium, the effects of a given protamine concentration were reduced, presumably due to binding of a portion of the protamine to the serum protein. A fairly short period of exposure to protamine was sufficient to induce irreversibly the changes observed by phase microscopy. To a washed cell suspension (6 X 106 cells/ml), protamine was added to a concentration of 100 ,ug N/ml. After 5 minutes incubation, at which time none of the cells had yet developed visible changes, the suspension was centrifuged, and the cells reincubated in protamine-free BSS. After 20 minutes incubation, 80-90 per cent of the cells showed typical protamine induced changes. Control cells carried through the two incubations and the centrifugations appeared perfectly normal. Staining reactions---These were carried out either on sections prepared from formalin fixed cell blocks, or on smears fixed with alcohol-ether or with formalin, and the results were substantially identical in the two cases. Cells treated with protamine or histone in the range 50-300 pg N/ml showed loss of affinity for basic stains by both nucleus and cytoplasm. The staining intensity of the cytoplasm with hematoxylin or toluidine blue and of the considerably enlarged nucleus by hematoxylin or methyl green was sharply Experimental

Cell Research

19

Effects of protamines

and histones on cell nucleic acids

Fig. 4.--Unfreafed fumor cells. Both nuclei and cytoplasm stain (Cell section) x 580. Fig. 5.-Cells freufed luifh protomine (75 pg N/ml). Cytoplasm intensity of staining greatly reduced in both. The mean nuclear Elliptical, cytoplasmic bodies with intense staining qualities are toxylin, x 615. Fig. B.--Cells treated with profamine (75 pg N/ml). Bodies more plate Fig. 5. (Cell smear), Papanicolau Stain, x 320.

distinctly

with

36’7

hematoxylin.

and nuclei are swollen and the diameter is increased by 30 %. present. (Cell section), hemanumerous Experimenfcd

and smaller

than

Cell Research

in 19

F. F. Becker and H. Green

368

reduced (Figs. 4-6). The same was true for the Feulgen reaction of the nucleus, and the pyronin reaction of the cytoplasm. The staining intensity of cytoplasm with eosin was, on the other hand, somewhat increased. In addition to these changes there appeared in the cytoplasm clearly demarcated bodies of elliptical or crescentic shape with sharp borders, frequently appearing compressed against the cell membrane (Figs. 4-6). They varied in size from the limit of visibility to about 9 micra in length, and in number up to about 5 per cell, higher concentrations of protamine tending to produce bodies in large number and of smaller size. Their frequency also varied considerably, and sometimes unaccountably from one experiment to another. In some experiments 100 per cent of the cells showed bodies, and in others as little as 10 per cent. These bodies stained intensely with the basic dyes hematoxylin and toluidine blue, and frequently were the only parts of the cell which did so. They were also strongly stained by pyronin, but not by the Feulgen reaction, or by methyl green. They did not take up the fat stain oil Red 0 and were negative to the periodic acid-Schiff reaction. On the basis of these reactions it was concluded that the bodies contained RNA. They also stained with the dye Fast Green at pH 8.0, which is specific for protamines and histone [l]. Tumor cells untreated with protamine took up none of this dye whatever.1 The effect of ribonuclease2 on the staining reactions of the bodies was examined by treatment of the hydrated sections with the enzyme for varying periods of time and then staining with toluidine blue. When sections of untreated tumor cells were exposed to ribonuclease (0.5 mg/ml in water) for 3 hours at 37X, the RNA was completely removed from the cytoplasm, as judged by its failure to then stain with the basic stain. Similar exposure of protamine treated cells failed to significantly reduce the staining ability of the bodies. When sections of protamine treated cells were incubated with the enzyme for periods of 15 hours or more the bodies did lose their ability to bind basic stains. However, this treatment of control tumor cells not only removed cytoplasmic staining properties but significantly reduced the ability of the nucleus to take up basic stain. Even incubation in distilled water for this period of time reduced the nuclear staining intensity, though not to the same degree as incubation in the enzyme solution. It seems, therefore, that no firm conclusions can be drawn as to the specificity of the enzyme effect on the cytoplasmic bodies. 1 If a smear of untreated then stains with Fast Green * Three times recrystallized, Experimental

Cell

Research

tumor cells is extracted with hot TCA, the cells own nuclear histone (1). obtained from the Worthington Corporation, Freehold, New Jersey. 19

Effects of profamines Metabolic

Effects

and hisfones on cell nucleic acids of Protamine

on the Tumor

369

Cells

Oxygen consumption.-Cells were washed in the usual manner but were suspended finally in a phosphate-buffered (pH 7.4) balanced salt solution [25] containing a glucose concentration of 2.0 mg/ml. The oxygen consumption of the cells was determined manometrically by the direct method of Warburg [25], in the presence and in the absence of protamine. It can be seen (Text-Fig. 5) that the addition of protamine to a final concentration of 300 ,ug N/ml had no effect on the oxygen consumption of the cell suspension over at least a 40 minute period.

Text-Fig.

5.

Text-Fig.

6.

Text-Fig. 5.-Effect of protamine on the oxygen consumption of fhe tumor cells. Flask contents3.0 ml of cell suspension (1.0 x lO’/ml) in phosphate buffered balanced salt solution pH 7.4 [25). Side arm contained 0.3 ml protamine solution (3.3 mg N/ml). At time = 10 minutes, protamine was tipped in from the arm of the experimental flask, and an equal volume of buffer from side arm of control flask. Gas phase, air. Final pH 7.1. 0, cells in absence of protamine. 0, Cells in presence of protamine (300 pg N/ml). Text-Fig. 6.-Effect of protamine on the rate of anaerobic glycolysis by tumor cells. Flask contents, 3.0 ml cell suspension (1.1 x 10’ cells/ml) in BSS. Side arm contained 0.3 ml protamine (3.3 mg N/ml) in BSS. At time-10 minutes, protamine was tipped in from the side arm of the experimental flask and an equal volume of BSS from side arm of control flask. Gas phase, 95 per cent N,, 5 per cent CO*. Final pH in all flasks, 7.3. 0, cells in absence of protamine. 0, cells in presence of protamine (300 pg N/ml).

Anaerobic glycolysis.-The effect of protamine on the ability of cells to produce acid in the absence of oxygen was also determined manometrically. Washed cell suspensions were incubated in BSS (containing for this experiExperimental

Cell Research

19

370

F. F. Becker and H. Green

ment 2.0 mg/ml of glucose), under a gas phase of 5 per cent CO,, 95 per cent N, in Warburg vessels, and CO, production from the bicarhonatc in the medium was followed (Text-Fig. 6). It can be seen that for lo-15 minutes after the addition of protamine there was no change in the rate of CO, evolution. However, thereafter the rate declined somewhat faster than that of the control cells, and by 40 minutes after the addition of the protamine, the total CO, evolved was about 30 per cent less than by the control cells. Incorporation of amino acids into cell protein.-The effect of protamine on the ability of an incubating cell suspension to incorporate 14C-labelled isnleucine was determined as follows. To an incubating cell suspension the labelled amino acid was added at zero time (Text-Fig. 7). At time = 15 minutes, the cell suspension was divided equally into 2 flasks. To one was added protamine, such that the final concentration was 300 pg protamine N/ml. To the control was added an equal volume of BSS. It can bc seen that the addition of protamine produced immediate and complete cessation of isoleucine incorporation into cell protein. Similar experiments were performed with nL-14C-lysine, and L-~~Carginine, and in each case 300 ,ug/ml of protamine N produced total and virtually immediate arrest of amino acid incorporation. At protamine concentrations lower than this, the suppression was not complete. Histone at a concentration of 100 ,ug N/ml was effective in suppressing amino acid incorporation. In these experiments on oxygen consumption, anaerobic glycolysis and amino acid incorporation, all treated cells showed, 15 minutes after the addition of protamine, the typical changes visible by phase microscopy.

E$ect of Protamine

Exposure in Vitro on the Ability to Grow in Vivo

of the Cells

Incubation of cells with concentrations of protamine of the same order of magnitude as used in the experiments described above retards or completely prevents the growth of the tumor when the cells are reinjected into mice. Two experiments of this kind are illustrated in Text-Figs. 8 and 9. A washed cell suspension was incubated in vitro with protamine at a concentration of 300 ,ug N/ml, for one hour. At the end of this time examination of the cells by phase microscopy showed no undamaged cells. Aliquots of the suspension were then injected into each of a group of mice, and aliquots containing the same number of cells incubated in the absence of protamine were injected Experimental

Cell

Research

19

Effects of profamines as controls into a second group the mice receiving control cells of the growing tumor, and all protamine treated cells gained growth over a 3 week period. tumor by this time.

and histones on cell nucleic acids

371

of mice. It can be seen from Text-Fig. 8 that began to gain weight within 2 days as a result died within 10 days. The mice injected with a small amount of weight due to normal They showed no gross autopsy evidence of

i!li! 0

Fig.6

I?

0

.

.

2 ECTIOY

MINUTES

Text-Fig.

7.

Text-Fig.

8.

4

6

Text-Fig.

6

IO

I2

9.

Text-Fig. 7.-Effect of profamine on fhe incorporation of L isoleucine info cellular protein. To a cell suspension (8.0 x 10’ cells/ml) at 37” uniformly “C labelled L isoleucine was added to a final concentration of 0.26 mg/ml. (Time = 0.) At time = 15 minutest the cell suspension was divided, and at time = 17 minutes (indicated by arrow) protamine was added to one flask. 1.0 ml aliquots of cell suspension were taken at intervals and placed in 20 ml of 5 per cent TCA at 80°C. After 10 minute incubation, the suspension was chilled and poured through a cellulose membrane filter.’ The precipitated protein was washed on the filter by the addition of 20 ml of cold 5 per cent TCA, and air dried. The filters were then glued to copper planchets and heated in a forced draft oven at 70” for one hour. They were counted with a thin end window GeigerMiiller counter. Ordinate represents counts per minute in the protein. 0, Cells in presence of protamine. 0, Cells in absence of protamine. Text-Figs. 8 and 9.-Effect of incubation of cells with protamine in vitro on cell viability in vivo. Washed cell suspensions were incubated for one hour at 37” in the presence and in the absence of protamine. Aliquots of each cell suspension were then injected intraperitoneally into 6 mice. The mice were weighed daily, weight gain reflecting quantity of ascitic tumor fluid in the animal. 0, Mice receiving cells incubated in presence of protamine. 0, Mice receiving cells incubated in absence of protamine. t, indicates death of last surviving mouse of each group. Ordinate represents mean weight gain of the surviving mice. Fig. 8. Protamine concentration 300 pg N/ml. Cell concentration during incubation 1.2 x lO’/ml. Each mouse received 0.30 ml cell suspension (3.6 x 10’ cells). Fig. 9. Protamine concentration 250~~ N/ml. Cell concentration during incubation 2.0 x lO’/ml. Each mouse received 0.30 ml cell suspension (6.0 x 10’ cells). 1 Obtained from Millipore

Filter Corporation,

Watertown,

Mass. Experimental

Cell Research

19

372

F. F. Becker and H. Green

The same experiment performed at a slightly lower concentration of protamine (250 pg Protamine N/ml) left an occasional normal looking cell after one hour incubation in vitro. When these cells were injected into mice (Text-Fig. 9) the weight gain of the mice was delayed by about 3 days as compared with the controls. Since the generation time of the cells in the mouse can be as short as 14 hours, the observed delay in weight gain of 3 days is enough for 5 generations. A surviving fraction of cells of 3 per cent would therefore be consistent with this delay. DISCUSSION

The experiments reported here indicate that ascites tumor cells took LIP protamine and histone very rapidly from the medium and in large quantity. The maximum amount of protamine which could be held by 107 cells was at least 350 pugof protamine N (or 1.2 mg of protamine base). To compare this quantity with the quantity of macromolecular substances in the tumor cell, it should be noted that the same number of cells contains about 3.0 mg of protein, 185 lug of DNA, and about 350 ,IJ~ of RNA. It is evident therefore that the cells can take up an amount of protamine greater than their contents of RNA and DNA combined, and not much less than their total protein content. Apparently the cell membrane does not constitute a very strong barrier to the penetration of the positively charged molecules of protamine (molecular weight 4000 [8]) and histone (molecular weight 11,000 or higher [3]). The accumulation of these substances within the cells to such remarkably high concentrations probably depends on their ability to combine with intracellular constituents and the evidence suggests that the ribonucleic acid and desoxyribonucleic acid of the cell are the principal substances involved. No attempt was made to measure breakdown. of protamine within the cells, but it seems unlikely that more than a minute fraction of the large quantity taken up could have broken down. The presence of the protamine or histone within both nucleus and cytoplasm of the cells was evident from changes observed under phase microscopy, or more characteristically, in stained smears or sections. There was evident a much reduced staining intensity of nucleus and cytoplasm with basic stains or stains specific for RNA and DNA. In addition, there appeared in the cytoplasm typical bodies which stained with basic stains, with stains specific for RNA, and with stains specific for protamine. These properties of the bodies suggest that they are precipitates of the cell RNA with the protamine or histone. The fact that the RNA type of staining behavior of the Erperimenfal

Cell

Research

19

Effects of profamines

and histones on cell nucleic acids

373

bodies was not readily eliminated by RNAse may indicate decreased susceptibility of the RNA to the enzyme action when it is in the form of a precipitate with the protamine. The origin of the RNA which appears in the bodies has not been elucidated in these experiments. According to Hoagland et al. [ 161, in the similar Ehrlich asdites tumor cells, 10 per cent of the cytoplasmic RNA is soluble. Presumably this would be true also of the Krebs ascites cells. This RNA would be free to move about in the cytoplasm and to form localized aggregates with protamine. The rest of the Ehrlich ascites cell cytoplasmic RNA is in the form of sedimentable particles (ribosomes) of the endoplasmic reticulum and of the cytoplasmic matrix [16]. Whether these would be free to move about within the cell, and aggregate with protamine is unknown, though it has been stated that such particles from other cell types can be agglutinated by protamine when they are removed from the cells [21]. The presence of a high concentration of protamine tends to produce larger numbers of bodies of smaller size or may even prevent their appearance altogether. The presence of an excess of protamine may limit aggregate size in a manner similar to the effect of antigen excess on antigen-antibody precipitates. In model experiments involving precipitation of soluble yeast RNA with protamine, such precipitates were found to be slightly soluble in the presence of protamine excess. A similar effect has been reported by Lewis et al. [18]. The effects of moderate protamine concentrations on cellular respiration were very small. The ability of the cells to consume oxygen was not affected at all, and the ability to produce acid in the absence of oxygen was only slightly affected, and then only some time after the addition of protamine. In a study of the effect of protamine on a variety of cell types, Fischer [l l] concluded that protamine sulfate was taken up in large quantities by the cells and altered their staining reactions, though in a manner different from what was observed in our experiments. He also found that cellular respiration could be depressed by protamine, but only in concentrations higher than those employed here. At a protamine concentration of 300 ,ug N/ml Fischer found, as we did, virtually no effect on the oxygen consumption of tumor cells [12]. On the other hand the experiments reported here show virtually immediate arrest of incorporation of labelled amino acids into cell proteins, upon the addition of that concentration of protamine. This metabolic result might be expected from an intracellular combination of protamine with RNA, in view of what is known about the functions of RNA in protein synthesis [B, 151, and is reported to occur in the onion root tip by Brachet Experimental

Cell

Research

19

F. F. Becker and H. Green

374

[5, 61. It seems most likely therefore, that respiratory effects of protamine are not the primary effects, or at least not those which occur at the lowest protamine concentrations. Though the tendency of protamine to produce some clumping may be taken as evidence of effects on the cell membrane, protamine treated cells did not, with rare exception, appear physically disrupted. In addition, there was no appreciable loss from the cells of nucleic acids or proteins, insofar as could be judged from determination of light absorption at 260 and 250 m,u of the medium after incubation. Though the osmotic effects of protamine inside the cells may be responsible for the swelling changes observed by microscopy, it seems doubtful that they play an important role in the other effects observed. It appears unlikely also that the mechanism of the protamine effect is related to direct interaction between the protamine and amino acids, since the uptakes of both neutral and basic amino acids were equally affected. In experiments on calf-thymus nuclei, Allfrey et al. [2], showed that protamine had virtually no effect on the incorporation of labelled amino acids into nuclear protein. This observation would be consistent with the present experiments if the nuclear rate of amino acid incorporation accounted for only a very small part of that of the whole Krebs ascites tumor cell, or if the behavior of thymus nuclei and ascites tumor nuclei were basically different in this respect. The reinjection of cells treated with protamine into mice shows that after one hour incubation in vitro with protamine in high enough concentration (300 ,ug protamine N/ml) cells were no longer viable. Zbarskii and Perevozhchikova [26] have shown that the injection of tumor bearing mice with histones slowed the rate of growth of a transplanted tumor. They postulated that histone enters the nucleus of the tumor cells and exerts a rate controlling effect on cell duplication. It would seem likely from the present experiments that the effects of histone, like those of protamine, could be lethal to the tumor cells and the lethality might as likely be due to cytoplasmic as to nuclear effects. Protamines and histones were the only basic proteins studied in the present work. However, it is quite likely that other basic proteins would be capable of produc,ing similar effects; their ability to do so might depend on their molecular size and degree of basicity. Such biological effects as have been reported for lysine polypeptides on viruses and bacteria [7, 17 1, ribonuclease on the onion root tip [4] and lysozyme on the Rous sarcoma [9] probably depend, as in the present experiments, on the ability of the basic macromolecules to combine with acidic cellular macromolecules. Experimental

Cell Research

19

Effects of protamines

and histones on cell nucleic acids

375

SUMMARY

When Krebs ascites tumor cells are exposed to protamine or histone, these basic proteins are rapidly incorporated into the cells. At fairly high medium concentrations of these substances the cells may finally take up more protamine than their normal content of DNA and RNA combined. This protamine combines with the DNA of the cell nucleus, and with cytoplasmic RNA forming precipitated bodies in the cytoplasm. By their staining reactions these bodies were shown to contain RNA and protamine. The exposure of the cells to protamine or histone was followed by abrupt cessation of incorporation of amino acids into cell protein, though only relatively minor effects on cellular respiration were produced by the same protamine concentrations. The viability of the cells was destroyed. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26.

M. and GESCHWIND, I. I., Proc. Nat!. Acad. Sci. US 39, 991 (1953). V. G., MIRSKY, A. E. and OSAWA, S., J. Gen. Physiol. 40, 451 (1957). B., KOLB, J. J. and TOENNIES, G., Arch. Biochem. Biophys. 58, 144 (1955). J., Nature 174, 876 (1954). __ Biochim. et Biophys. Acta 19, 583 (1956). __ Biochemical Cytology. New York Academic Press, 1957. BURGER, W. C. and STAHMANN, M. A., J. Biol. Chem. 193,13 (1951). CALLANAN, N. J., CARROLL, W. R. and MITCHELL, E. R:, J. ‘Biol.’ Chem. 229, 279 (1957). CASELLI, P. and SCHUMACHER, H., Boll. Ocul. 34, 513 (1955). EAGLE, H., Science 122, 501 (1955). FISCHER, H. and BRANDIS, H., Naturwissenschaften 41, 533 (1954). FISCHER, H., KREUZER, L. and ARGENTON, H., Thrombose and Emboli, p. 136. I. Internationale Tagung, Basel, 1954. FISCHER, H. and WAGNER, L., Nafurwissenschaften 41, 532 (1954). HIRSCII, J. G., J. Ezptl. Med. 108, 925 (1958). HOAGLAND, M. B., Symposium No. VIII at IVth International Congress of Biochemistry, Vienna, Sept. 1958. In press. HOAGLAND, M. B., STEPHENSON, M. L., SCOTT, J. F., HECHT, L. I. and ZAMECNIK, P. C., J. Biol. Chem. 231, 241 (1958). KATCHALSKI, E., BIEKOWSKI-SLOMNITZKI, L. and VOLCANI, B. E., Biochem. J. 55, 671 (1953). LEWIS, G. T., BLOOM, W. L. and SMART, C. W., Cancer Research 9, 103 (1949). MILLER, B. F., ABRAMS, R., DORFMAN, A. and KLEIN, M., Science 96, 428 (1942). PEARSE, A. G. E., Histochemistry, Theoretical and Applied. Little Brown Br Co., Boston, 1953. PETERMANN, M. L., 2’excr.s Reps. Biol. Med. 12, 921 (1954). PHILPOT, J. ST. L. and STAINER, J. E., Biochem. J. 63, 214 (1956). SAKAGUCHI, S., J. Biochem. (Japan) 37, 231 (1950). STEDMAN, E., STEDMAN, E. and PETTIGREW, F. W., Biochem. J. 38, XXXI (1944). UMBREIT. W. W.. BURRIS. R. H. and STAUFFER. J. F., Manometric Techniaues in Tissue Metabolism.’ Minneapolis, Burgess, 1957. ZBARSKII, I. B. and PEREVOZHCHIKOVA, K. A., Byu!l. Ekspll. Biol. i. Med. (USSR) 38, 61 ALFERT, ALLFREY, BAKAY, BRACHET,

(1954).

25 - 603702

Experimental

Ccl1 Research

19