non-selective inhibition of transformed cell growth by a protease inhibitor

Non-Selective Inhibition of Transformed Cell Growth by a Protease InhibitorAuthor(s): Iih-Nan Chou, Paul H. Black and Richard O. RoblinSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 71, No. 5 (May, 1974), pp. 1748-1752Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/63515 .

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Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 1748-1752, May 1974

Non-Selective Inhibition of Transformed (

(contact inhibition/3T3 cells/simian virus 40 transformal

IIH-NAN CHOU, PAUL H. BLACK, AND RICHARD 0. RO:

Departmnents of Medicine and MAicrobiology and Molecular Genetics, Ha Massachusetts General Hospital, Boston, Mass. 02114

Communicated by J. D. Watson, February 11, 1974

ABSTRACT The protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) and N-tosyl-L- lysylchloromethyl ketone (TLCK) have previously been shown to selectively inhibit growth of simian virus 40- transformed cells, suggesting that proteolytic enzymes play a role in loss of cellular growth control following viral transformation. In contrast, this study shows that TPCK- mediated growth inhibition is non-selective, since the growth of both simian virus 40-transformed and untransformed 3T3 cells is similarly reduced by TPCK treatment. Under certain conditions, TPCK treatment of simian virus 40-transformed cells yields a reversible "growth plateau" condition which mimics, but is not equivalent to, contact inhibition of growth. The growth inhibitory effects of TPCK are due to inhibition of protein synthesis, since TPCK treatment resulted in a diminution of protein synthesis and since the "growth plateau" effect was also observed in cultures treated with cycloheximide.

Untransformed fibroblasts in tissue culture show a characteristic reduction in the frequency of cell division upon formation of a confluent monolayer. This phenomenon has been termed contact inhibition or density-dependent inhibition of cell growth (1, 2). In contrast, simian virus 40 (SV40)-transformed cells continue division beyond confluency and frequently exhibit multilayered cell growth.

Exposure of confluent, untransformed fibroblast monolayers to low concentrations of proteolytic enzymes can cause transient release from density-dependent inhibition of growth (3, 4). Since proteolytic enzyme treatment can thus cause the growth characteristics of untransformed cells to resemble those of transformed cells, increased production or activity of proteolytic enzymes might be responsible for the loss of density-dependent growth control which characterizes cells transformed by oncogenic viruses. In support of this hy- pothesis, Bosmann (5, 6) found increased proteolytic activity in homogenates of 3T3 cells transformed by both DNA and RNA tumor viruses. Schnebli (7) reported an increase in "protease-like" activity in DNA virus-transformed cells, and Unkeless et al. (8) and Ossowski et al. (9) recently described an enhanced fibrinolytic activity in fibroblasts transformed by either oncogenic DNA or RNA viruses. This enhanced "Fibrinolysin T" activity in transformed cell cultures apparently results from increased production or activity of a

Abbreviations: TPCK, N-tosyl-L-phenylalanylchloromethyl ketone; TLCK, N-tosyl-L-lysylchloromethyl ketone; SV40, simian virus 40; SV3T3, simian virus 40-transformed 3T3 cells; MEM X 4, Eagle's minimum essential medium containing a 4-fold concentration of vitamins and essential amino acids; FBS, fetal-bovine serum; PBS, phosphate-buffered saline; TCA, trichloroacetic acid.

17

jell Growth by a Protease Inhibitor

*ion/N-tosyl-L-phenylalanylchloromethyl ketone)

3LIN

rvard Medical School, anid Infectious Disease Unit,

cellular proteolytic enzyme (10) which subsequently cleaves serum plasminogen to plasmin (11), thus generating the fibrinolytic activity.

If proteolytic enzymes play an essential role in loss of density-dependent growth control in virus-transformed fibroblasts, then inhibition of cellular protease activity by appropriate protease inhibitors might be able to reverse the unrestrained growth of the transformed cells and cause them to reacquire density-dependent growth inhibition. Prival (12) found that the saturation density, but not the growth rate, of simian virus 40-transformed 3T3 (SV3T3) cells was reduced by treatment of cells with the chymotrypsin inhibitor, N-

tosyl-L-phenylalanylchloromethyl ketone (TPCK). Goetz et al. (13) showed that the trypsin inhibitor, N-tosyl-L- lysylehloromethyl ketone (TLCK), depressed the proliferation of hamster tumor cells. In addition, Schnebli and Burger (14) reported that five protease inhibitors including TPCK and TLCK caused selective inhibition of the growth of transformed, but not untransformed, 3T3 cells. They concluded that the inhibitors blocked a "protease like activity that is required for the unrestrained growth of transformed cells." However, none of these studies evaluated the possibility that protease inhibitors might inhibit cell growth via mechanisms other than inhibition of proteolytic activity. We report here results which show that TPCK does not have a selective inhibitory effect on SV40-transformed cells and that the growth inhibitory effects of TPCK are probably due, in large part, to its inhibitory effects on cellular protein synthesis.

MATERIALS AND METHODS

Swiss 3T3 cells were originally obtained from Dr. G. Todaro and from this line a cloned SV40-transformed line was derived (15). Cells were grown in Eagle's minimal essential medium containing a 4-fold concentration of vitamins and essential amino acids (MEM X 4) and supplemented with 10% (v/v) fetal-bovine serum (FBS) and 250 units/ml penicillin plus 250 jg/ml of streptomycin.

TPCK (Sigma) was dissolved at 5 mg/ml in phosphate- buffered saline (PBS) containing 0.1 N NaOH (12), and added to MEM X 4 to give the desired concentrations. After immediate adjustment of the pH to that of MEM X 4 (7.8), the medium was quickly filtered (Nalgene, 0.45 im) and then supplemented with 10% FBS and antibiotics. TPCK solutions prepared in this way retain a minimum of 85% of their potential to inactivate chymotrypsin compared with equal concentrations of TPCK dissolved in methanol (16). Appropriate control media were prepared in exactly the

18



Proc. Nat. Acad. Sci. USA 71 (1974)

same way as the test media, except that the control media contained no protease inhibitor.

Trypsinized SV3T3 and 3T3 cells were plated at 1 to 4 X 105 and 1 to 2 X 10' cells, respectively, per 60-mm Falcon plastic tissue-culture dish. The medium was removed 20-24 hr later and immediately replaced with control or inhibitor- containing media. The media with or without inhibitor were changed daily in all experiments. Cell growth was followed by trypsinizing the cells and counting them in an automatic laser-beam cell counter. For viability tests, cell suspensions were mixed with 0.4% Trypan Blue (1:1), stained for 5-10 min, and counted in a hemocytometer.

To measure protein synthesis, a 3H-labeled L-amino-acid mixture (New England Nuclear Corp.) in PBS (500 ,Ci/ml) was added to cell cultures at 2.5 gCi/ml of medium. One hour after isotope addition, the medium was decanted and the cells were washed three times with PBS, extracted three times with 5 ml of cold 5% trichloroacetic acid (TCA), and then the cell monolayer was dissolved in 1 ml of 0.5 N NaOH. Aliquots of the sample were neutralized with 4 N HCI, precipitated with 5% TCA, and heated at 90? in a water bath for 30 min. The hot TCA precipitable materials were collected on glass-fiber filter discs (Whatman, GF/C), dried, and counted in 10 ml of toluene scintillation fluid. The radio- activity of all TCA washes was determined by counting aliquots of the TCA extracts in 10 ml of Bray's scintillation fluid (17).

For quantitative incorporation measurements, ['H]thymidine (New England Nuclear Corp., specific activity 20 Ci/mM) in PBS (100 jCi/ml) was added to the plates at a final concentration of 5 uCi/ml for 1 hr. The medium was then decanted, the cells were washed twice with 5 ml of cold Dulbecco's Tris buffer (pH 7.4) (18), extracted three times with 5 ml of cold 5% TCA, washed five times with cold 95% ethanol, and, finally, dissolved in I ml of 0.5 N NaOH. Ali- quots of NaOH-dissolved samples were precipitated with cold 5% TCA and counted as described above. For autoradiography, the cells were labeled with 5 uCi/ml of [3H]thymidine for 4 hr and processed as described previously (19). The cell lines were shown to be free from mycoplasma contamination by periodic assay of the cells using the same autoradiographic procedure (19).

RESULTS

TPCK Inhibition. A comparison of the effects of TPCK on the growth of Swiss 3T3 and Swiss SV3T3 cells is shown in Fig. IA and B). The overall effect of TPCK on the growth of both cell lines is strikingly dependent upon the TPCK concentration. Thus, 50 ,ug/ml of TPCK (not shown) has no effect on the growth rate of SV3T3 cells, 100 jg/ml of TPCK decreases the growth rate of SV3T3 cells without changing the final cell density, and 200 ug/ml of TPCK causes SV3T3 cells to achieve a "growth plateau" at 4 X 105 cells per plate (Fig. 1A).

TPCK produced similar effects on the growth of untransformed 3T3 cells, in that 50 ug/ml reduced the growth rate, 100 jg/ml produced a "growth plateau" at 2 X 105 cells per plate (well below the saturation density characteristic of this line), and 150 or 200 jg/ml led to slow, progressive loss of cells from the petri dish. None of the TPCK doses used showed selective growth inhibition of SV3T3 as compared with 3T3 cells. In fact, in our cell system, 3T3 cells are more sensitive

Protease Inhibitor and Cell Growth 1749

50 40 30- A

20 -

1

10-

B o

5 -

0 2 4 6 7

DAYS IN CUL TURE

FIG. 1. Dose dependence of growth inhibition of SV3T3 and 3T3 cells by TPCK. (A) SV3T3 cells, seeded at I X 106 cells per dish (Day 0) were treated with TPCK (upward arrow) in the following concentrations: O--O, no TPCK; A--A, 100 jg/ml; O?--a-, 200 ug/ml. Parallel cultures, after growth in medium containing 200 ug/ml of TPCK for 3 days, were exposed to fresh medium without TPCK (downward arrow) and cell growth on stibsequent days was determined (D--- ). (B) 3T3 cells, seeded at 1 X 105 cells per dish (Day 0) were treated with the following concentrations of TPCK (upward arrow): 0-0, no TPCK; A--A, 50 ug/ml; EO-- O, 100 ug/ml; X--X, 150 jtg/ml; ?--*, 200 ug/ml. (The numbers on the ordinate have been multiplied by 10-5.)

to growth inhibition by TPCK than are their transformed SV3T3 counterparts.

The cell density at the time TPCK is added also influences the final cell density to which the cells grow. This is illustrated by Fig. 2, which shows the effects of 200 jg/ml of TPCK on SV3T3 cells seeded initially at 4 X 105 cells per plate and treated with TPCK 24 hr later. In this case, the SV3T3 cells reach a "growth plateau" at about 1.5 X 106 cells per plate, some four times higher than the SV3T3 cells in Fig. lA which were seeded initially at 1 X 105 cells per plate. The data in Figs. 1A and 2 also show that TPCK treatment which leads to a "growth plateau" does not kill the SV3T3 cells since, upon removal of TPCK, the cell number approximately doubles within the next 24 hr.

Inhibition of Protein Synthesis and Growth. In order for their effects on cells to be attributable solely to protease inhibition, protease inhibitors should be free from other effects on cells. However, both TPCK and TLCK at 1 X 10-4 M have previously been shown to inhibit protein synthesis in virus-infected mammalian cells (20, 21). We therefore determined whether TPCK caused inhibition of protein synthesis



1750 Cell Biology: Chou et al.

100 -

e o

114 /

- 0 4

2

1 I 1 I/ 1 1 1 1 1 0 2 4 6 8

DAYS IN CULTURE

FIG. 2. Growth "plateau" and reversibility of growth inhibition following TPCK treatment. At 28 hr after seeding (upward arrow), SV3T3 cell cultures were treated with either no TPCK (O--O) or 200 ug/ml of TPCK (A--A). After 5 days of treatment with 200 jug/ml of TPCK (downward arrow), some cultures were changed to fresh medium without TPCK, and cell growth in these "reversed" cultures was determined (0---O). The number of dead cells in the medium of SV3T3 cultures treated with 200 jtig/ml of TPCK was also determined (--un). (The numbers on the ordinate have been multiplied by 10--5.)

under our culture conditions. The results, presented in Table 1, show that TPCK at 100 and 200 jg/ml inhibits incorporation of amino acids into hot TCA-precipitable material by 60% and 70%, respectively. Thus, protein synthesis is strongly inhibited by doses of TPCK which are required to alter the growth properties of SV3T3 cells.

The TPCK-mediated inhibition of protein synthesis does not appear to be due to decreased uptake of amino acid precursors, since incorporation of a mixture of 15 different 3H-labeled amino acids into the cold TCA-soluble fraction (amino-acid pools) is slightly increased in the cells treated with TPCK at 200 jug/ml (Table 1). Although there was thus no reduction in uptake of a mixture of amino acids, we wondered whether TPCK might selectively interfere with the transport of phenylalanine, to which TPCK is structurally closely related. However, we have found, in experiments described in detail elsewhere (22), that treatment with 200 ug/ml of TPCK for 24 hr does not appreciably change the rate of ['H ]L-phenylalanine uptake by Swiss SV3T3 cells.

Concomitant with its inhibition of protein synthesis, TPCK treatment of SV3T3 cells causes a dose-dependent reduction in the level of DNA synthesis (Table 1). Cultures treated with 200 jug/ml of TPCK synthesize DNA at only half the rate of untreated cultures. In other experiments, autoradiography of cells on a "growth plateau" after TPCK treatment at 200 iug/ml for 3 days showed that about 25% of the cells in- corporated [3H]thymidine during a 4-hr labeling period. Thus, under conditions where their level of protein synthesis was depressed, an appreciable fraction of SV3T3 cells treated with 200 ug/ml of TPCK continue to synthesize DNA. In addition,


100loo -

1 50 -

-244

20

~) 3

0 2 4 6 8

DAYS IN CUL TURE

FIG. 3. Inhibition of SV3T3 cell growth by cycloheximide. At 24 hr after seeding, Swiss SV3T3 cells were treated with medium containing: 0--O, no cycloheximide; A--A, 0. jg/ml; --[n1, 0.2 ,4g/ml; ?- , 0.5 jg/ml; --U , 2 jg/ml;

--O, 10 ,g/ml of cycloheximide. (The numbers on the ordinate have been multiplied by 10-5. )

some cells probably divide successfully, since there is no net loss of cells from the petri dish during the "growth plateau" condition, yet there is a steady accumulation of dead cells in the growth medium (Fig. 2).

If a reduction in the rate of protein synthesis was responsible for the growth inhibitory effects of TPCK, we would expect other inhibitors of protein synthesis to have similar effects on the pattern of cell growth. This expectation is confirmed by the data in Fig. 3 which show that treatment with cycloheximide, a well-characterized, reversible inhibitor of polypeptide chain initiation and elongation in mammalian cells (23), also causes dose-dependent reduction of the growth of Swiss SV3T3 cells. Cycloheximide concentrations below 0.5 ug/ml reduce the growth rate, while cells treated with 0.5 ,ug/ml achieve a "growth plateau" strikingly similar to that obtained in cultures treated with TPCK. As with TPCK, concentrations of cycloheximide greater than those required to yield a "growth plateau" cause progressive loss of cells from the petri dish. In other experiments, we have shown

TABLE 1. TPCK-inhibition of protein and DNA synthesis

Amino-acid incorporation*

TCA- Thymidine precipitablet TCA-solublet incorporation*

TPCK % of % of % of jg/ml cpm~ control cpml control cpml control

0 2706 100 2730 100 353,750 100 100 1134 42 200 846 31 3175 116 174,480 49 250 647 24 - - 52,120 15 300 434 16 5165 189

* Experiments done with Swiss SV3T3 cells at 22 hr after initiation of TPCK treatment.

t Results from separate experiments. t Average cpm/106 cells from two determinations on each

sample from duplicate plates.




that the cycloheximide-induced "growth plateau" is reversible, following removal of the cycloheximide-containing medium, even after 6 days of cycloheximide treatment. In addition, cultures treated with 0.5 jg/ml of cycloheximide show a 95% inhibition of protein synthesis as compared with untreated control cultures.

TPCK-Induced Growth Plateau. The data in Figs. I and 2 show that appropriate TPCK concentrations cause a "growth plateau" condition in which there is no net increase in the number of SV3T3 cells on the petri dish. There is, however, an increase in the total number of cells in the culture, since an appreciable number of dead cells appear in the medium each day during the "growth plateau" condition (Fig. 2). The "growth plateau" condition is not due to simple toxicity, since almost all the cells which remain on the petri dish after 4-6 days of TPCK treatment are able to divide if the TPCK is removed (Figs. IA and 2). Since TPCK-treated SV3T3 cells at a plateau cell density of 1.5 X 106 cells per dish do not show extensive cell-to-cell contact and have not formed a confluent monolayer, the "growth plateau" condition does not correspond to a reacquisition of contact inhibition. Achievement of "growth plateaus" at the very low cell concentrations of 4 X 105 SV3T3 cells per dish and 2 X 105 3T3 cells per dish reinforces this conclusion.

DISCUSSION

Our results show that the overall effect of TPCK treatment on cell growth depends upon the cell density at the time of TPCK treatment, as well as the TPCK concentration. Cultures treated with TPCK at low initial cell densities show a more rapid inhibition of cell multiplication and a lower growth plateau cell density than those treated at higher initial cell densities (Figs. 1 and 2). Thus, meaningful comparison of the effects of TPCK treatment on different cell lines requires that the cells be treated with TPCK at approximately the same initial cell density.

When 3T3 and SV3T3 cells were seeded at the same initial cell density and treated with TPCK 24 hr later, a similar pattern of growth inhibition was observed with both cell lines. Concentrations of TPCK which are required to reduce the growth rate of SV3T3 cells are even more inhibitory to the growth of untransformed 3T3 cells (Fig. 1). Thus, 200 jg/ml of TPCK yields a "growth plateau" with SV3T3 cells, while 100 jg/ml is sufficient to cause a "growth plateau" in 3T3 cultures. The essential point is that we observed no selective effect of TPCK on the growth of the virus-transformed 3T3 cells as compared with untransformed 3T3 cells. Our results thus differ from those of Schnebli and Burger (14) who reported a selective reduction in the growth rate of SV3T3 cells treated with 10 jg/ml of TPCK. Differences in cell lines and in the manner in which TPCK-containing media were prepared might account for some of the differences in our results. However, we also observed no reduction of the growth rate of our SV3T3 cells when they were treated with TPCK dissolved in dimethylsulfoxide at concentrations up to 25 jg/ ml. At a minimum, our results show that increased sensitivity to TPCK is not an invariable consequence of transformation by SV40.

In our experiments, concentrations of TPCK which are required to reduce the growth of SV3T3 cells also depress protein synthesis. We suggest that TPCK affects cell growth

Protease Inhibitor and Cell Growth 1751

through its effect on protein synthesis, since the dose-response curve of growth inhibition in TPCK-treated cultures is exactly reproduced in cultures treated with cycloheximide, a known inhibitor of protein synthesis. The mechanism by which TPCK inhibits protein synthesis in mammalian cells is not known; it may inactivate one of the peptide chain elongation factors in protein synthesis as it does in bacteria (24, 25), and (or) it may act by inhibiting cellular RNA synthesis (26) which is required for protein synthesis. What- ever the mechanism, the growth inhibitory effects of TPCK can no longer simply be ascribed to inhibition of proteolytic activity.

Treatment with 200 yg/ml of TPCK can cause SV3T3 cells to stop increasing in net cell number at cell densities which approximate the low saturation density of untransformed 3T3 cells. However, this "growth plateau" phenomenon is not equivalent to reacquisition of contact-inhibition of cell division because (a) TPCK-treated SV3T3 cells at 1.5 X 106 cells per dish do not show extensive cell-to-cell contacts, (b) there is no marked decrease in DNA synthesis in cells on such a TPCK-induced "growth plateau'.' as there is in contact- inhibited untransformed cell monolayers, and (c) the final "growth plateau" cell density depends upon the cell density at which the cells are treated with TPCK. The TPCK-induced "growth plateau" appears to be the net result of approximately equal rates of cell multiplication and cell detach- ment from the monolayer. Thus, low "saturation" density in TPCK-treated cultures cannot be used as the sole criterion for reacquisition of contact-inhibition of cell growth.

Although we have not investigated growth inhibition by TLCK in detail, we have previously reported (22) our in- ability to demonstrate a selective growth inhibitory effect of TLCK on the final cell density of SV3T3 cells. However, in our previous experiments (22), Balb SV3T3 cells were seeded and treated with TLCK at higher cell densities than the Balb 3T3 cells. In more recent experiments, treatment of Swiss SV3T3 cell cultures seeded at I X 105 cells per 60-mm dish with medium containing 25 ug/ml of TLCK led to a short-lived growth plateau at 3 X 105 cells per 60-mm dish. Again, this TLCK-induced growth plateau does not correspond to reacquisition of contact inhibition as the cells are not in extensive contact with one another at this low cell density. Treatment of Swiss 3T3 cell cultures with 25 jug/ml of TLCK produced a slight (<25%) reduction in both the growth rate and final cell density. Thus, we have not observed as striking a selective effect of TLCK on SV3T3 cells as that reported by Schnebli and Burger (14). In addition, TLCK has been shown to inhibit protein synthesis in poliovirus- infected HeLa cells (21) and RNA synthesis in lymphocytes (26) in the concentration range (1 X 10-4 M) previously. reported (14) to selectively inhibit the growth of SV40- transformed cells. Thus, TLCK may also cause growth inhibition via inhibition of macromolecular synthesis rather than by inhibiting cellular proteases.

Our results with TPCK illustrate the many difficulties involved in using protease inhibitors like TPCK and TLCK in cell cultures to investigate the role of proteolytic enzymes in virus-induced cell transformation. Protease inhibitors without adverse side effects on cell metabolism and which are stable under cell culture conditions are required if this ap- proach is to be fruitful.



1752 Cell Biology: Chou et al.

This research was supported by Grants VC-31 from the Ameri- ican Cancer Society and CA-10126 from the National Institutes of Health. I.N.C. holds a Postdoctoral Fellowship from the National Cancer Institute (1-F02-CA54314) and R.O.R. is a Faculty Research Associate (PRA-75) of the American Cancer Society.

1. Todaro, G. J., Lazar, G. K. & Green, H. (1965) J. Cell. Comp. Physiol. 66, 325-334.

2. Stoker, M. P. G. & Rubin, H. (1967) Nature 215, 171-172. 3. Burger, M. M. (1970) Nature 227, 170-171. 4. Sefton, B. M. & Rubin, H. (1970) Nature 227, 843-845. 5. Bosmann, H. B. (1969) Exp. Cell Res. 54, 217-221. 6. Bosmann, H. B. (1971) Biochim. Biophys. Acta 264, 339-343. 7. Schnebli, H. P. (1972) Schweiz. Med. Wochenschr. 102,

1194-1197. 8. Unkeless, J. C., Tobia, A., Ossowski, L., Quigley, J. P.,

Rifkin, D. B. & Reich, E. (1973) J. Exp. Med. 137, 85-111. 9. Ossowski, L., Unkeless, J. C., Tobia, A., Quigley, J. P.,

Rifkin, D. B. & Reich, E. (1973) J. Exp. Med. 137, 112-126. 10. Unkeless, J., Tobia, A., Ossowski, L., Quigley, J., Rifkin,

D. & Reich, E. (1973) Fed. Proc. 32, 851. 11. Quigley, J. & Unkeless, J. (1973) Fed. Proc. 32, 851. 12. Prival, J. T. (1971) Ph.D. Thesis, Massachusetts Institute

of Technology, pp. 120-148.


13. Goetz, I. E., Weinstein, C. & Roberts, E. (1972) Cancer Res. 32, 2469-2474.

14. Schnebli, H. P. & Burger, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 3825-3827.

15. Black, P. H. (1966) Virology 28, 760-763. 16. Shaw, E. (1970) Physiol. Rev. 50, 244-296. 17. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 18. Dulbecco, R. & Vogt, M. (1962) Virology 16, 41-51. 19. Culp, L. A. & Black, P. H. (1972) J. Virol. 9, 611-620. 20. Pfefferkorn, E. R. & Boyle, M. K. (1972) J. Virol. 9, 187-

188. 21. Summers, D. F., Shaw, E. N., Stewart, M. L. & Maizel,

J. V. (1972) J. Virol. 10, 880-884. 22. Chou, I. N., Black, P. H. & Roblin, R. 0. (1974) in Control

of Proliferation in Animal Cells, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, N.Y.), in press.

23. Pestka, S. (1971) Annu. Rev. Microbiol. 25, 488-562. 24. Jonak, J., Sedlacek, J. & Rychlik, I. (1973) Biochim. Bio-

phys. Acta 294, 322-328. 25. Richman, N. & Bodley, J. W. (1973) J. Biol. Chem. 248,

381-383. 26. Darzynkiewicz, Z. & Arnason, B. G. W. (1974) Exp. Cell.

Res., in press.



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