Proc. Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 389-3873, September 1977

Cell Biology

Low-molecular-weight peptide inhibits RNA synthesis in human leukemic and phytohemagglutinin-stimulated leukocytes and globin mRNA transcription in differentiating Friend cells (calf thymus/complementary [3H]DNA hybridization/cell differentiation)

D. AMICI*, G. B. Rossit, L.

Ciott, G. P. MATARESEt, A. DOLEIt, L. GUGLIELMI*,

AND G. L. GIANFRANCESCHI* *Istituto di Fisiologia Generale, Universiti di Camerino, 62032 Camerino, t Sezione di Virologia, Istituto Superiore di Sanit', 00161 Roma and Cattedra di

Patologia Generale, Universiti di Camerino, 6202 Camerino; and * Istituto di Virologia, Universiti di Roma, 00185 Rome, Italy Communicated by Rita Lei-Montalcini, June 16,1977

ABSTRACT The RNA synthesis of human leukemic leukocytes and phytohemagglutinin-stimulated lymphocytes is markedly reduced by administration of a low-molecular-weight nonhistone peptide factor from calf thymus. Treatment with the factor strongly inhibits hemoglobin production and globin mRNA transcription in dimethyl sulfoxide-stimulated Friend cells without appreciably modifying the rate of cell growth. Evidence for specificity of these effects is provided by the lack of action of the factor on both growth rate and RNA synthesis of a number of nondifferentiating cell lines from various animal species. After removal of the compound, both human lymphocytes and Friend cells can be stimjK'a&ed by phytohemagglutinin and by dimethyl sulfoxide, respectively, ruling out any toxic effect. We have previously isolated, from aqueous extracts of calf thymus, an active factor, which has been partially purified by DEAE-cellulose and Dowex 50WX2 chromatography and characterized as a peptide of less than 5000 molecular weight (1). This factor causes a strong inhibition of DNA-directed (1) or chromatin-directed (2) RNA polymerase reactions in vitro. Its biological activity cannot be attributed to the presence of a nuclease or of a histone fragment. Control of chromatin and DNA transcription is apparently mediated by an ionic interaction of the peptide with the deoxyribosephosphate backbone of DNA (3) and takes place at the level of the initiation reaction (1, 2). Accordingly, the factor does not inhibit the transcription of endogenous residual RNA polymerase bound to chromatin preparations (2). The specificity of this effect is also supported by the observation of a comparatively much lower inhibition of DNA-directed DNA polymerase reaction in vitro (unpublished data). A factor endowed with similar biological properties was also isolated from calf seminiferous tubules and bull spermatozoa (2), whereas it could not be detected in calf kidney and muscles. These observations may be related to some of those described by other authors (4-7), who also isolated from thymic tissues factors capable of inhibiting cell metabolism in vivo and in vitro. The mechanism of action of these preparations, however, has not been investigated in detail. This paper describes data demonstrating that this factor specifically and reversibly reduces RNA synthesis in several animal cell systems in vitro. MATERIALS AND METHODS Preparation of Peptide Factor. The procedures used for isolation of the active peptide have been described in detail The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adiertkement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

elsewhere (1-3). The purified fraction designated fraction 16 (see ref. 1) was used throughout these studies. The active factor present in this fraction has a relatively high degree of purification. When 100 jg of the factor per ml are incubated with 1 mg of DNA per ml, about 20 ,ug of protein bind to DNA (3). Carboxymethyl-cellulose (Ceilex-CM, Bio-rad, Richmond, CA) chromatography of fraction 16 shows that more than 80% of it consists of active factor (data not shown). Cells. Human leukocytes were obtained from heparinized (20 units/ml) blood from normal and leukemic donors. The plasma and white cell fractions recovered by sedimentation for 3 hr at 370 were centrifuged for 5 min at 1000 X g. Pellets were resuspended in Tyrode's solution and autologous platelet-free plasma (1:1, vol/vol) supplemented with antibiotics when originating from leukemic donors. Polymorphonuclear leukocytes from normal donors were removed by an additional low-speed centrifugation (100 X g for 2 min), after which the supernatant contained about 80% small lymphocytes. Friend leukemic cells (clone 745A), obtained from C. Friend (New York), were grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum (Eurobio, Paris) and antibiotics, seeded at 105 cells per ml, and kept at 370 with no further treatment or in the presence of 1.5% (vol/vol) dimethyl sulfoxide (Me2SO) (Merck), until day 5 after seeding. Cells were counted daily with a hemocytometer in the presence of 0.05% trypan blue. Benzidine-positive cells were determined according to Orkin et al. (8); hemoglobin (Hb) contents were measured in cell lysates according to Crosby and Furth (9). Baby hamster kidney (BHK21, clone 13) cell line, obtained from P. Amati (Naples), and African green monkey kidney (CV1) cell line, obtained from F. Jensen (La Jolla, CA), were grown in minimum essential medium supplemented with 10% fetal calf serum. Mouse L929 and bovine kidney cell lines, maintained in our laboratories for several years, were grown in minimum essential medium supplemented with 8% calf serum (Flow Labs, Irvine, CA). Chinese hamster embryo fibroblasts (CHEF125), obtained from G. Olivieri (Rome), were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Preparation of RNAs and [3H]cDNA and Hybridization Assay. Total cell RNAs were extracted according to Preisler et al. (10) and stored at -20° until used. Mouse globin mRNA was extracted from reticulocytes of phenylhydrazine-treated anemic Swiss mice and purified according to Forget et al. (11). Globin mRNA (2-5 ,g) was incubated at 37° for 2 hr with avian myeloblastosis virus RNA-dependent DNA polymerase (National Institutes of Health, Bethesda, MD), [3H]dCTP (20-30 Abbreviations: PHA, phytohemagglutinin; Me2SO, dimethyl sulfoxide;

cDNA, DNA complementary to RNAs.

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I

Proc. Natl. Acad. Sci. USA 74 (1977) D

18 30A

6 B0 /

18

1220

4

12-

12

0

x E

82

0.

I*

630

2to 20 I0

4

6-

4:

16 024 6

0 246

Time, hr

FIG. 1. Effect of the peptide on RNA synthesis in human peripheral leukemic leukocytes and normal small lymphocytes. (A) Acute lymphoblastic leukemia; (B) myeloblastic leukemia; (C) chronic lymphocytic leukemia; (D) normal small lymphocytes. Incorporation

of [14C]uridine: cpm/106 cells. The sample purification, reaction mixture, and incubation were as in Materials and Methods. The leukemic leukocytes and normal small lymphocytes (after 30 min of preincubation) were incubated at a concentration of about 2 to 5 X 106 cells per ml in the presence of 12-14C]uridine (3 1ACi/ml, 60 mCi/ mmol; Radiochemical Center, Amersham), with occasional shaking under air containing 5% carbon dioxide. The purified fraction 16 (0.1 ml, corresponding to 10 ;g of protein) was added per ml of incubation mixture; controls were given 0.1 ml of saline. We chose the concentration of the factor that causes an 80-90% inhibition of the DNA and chromatin-directed transcription (1, 2); the choice was made in advance because otherwise it would have been necessary to draw too large

a

blood volume from leukemic patients to determine

a

dose/

effect ratio. The peptide and the radioactive material were added at zero time. Acid-insoluble radioactivity was determined by lysing cell aliquots with 0.5% sodium dodecyl sulfate and adding trichloroacetic acid, 10% (final concentration). Radioactivity of precipitated material was determined in a Packard Tricarb spectrometer. The data described here are the average of experiments done in duplicate. For every type of leukemia studied, the experiments were repeated with blood specimens from three donors: the results were closely comparable. 0, Control; *, fraction 16, 10 ,gg of protein per ml (final concentration). Uptake of the label was determined after a 2-min pulse. Samples were immediately diluted with ice-cold phosphate-buffered solution supplemented with unlabeled uridine 10 times more concentrated than the labeled compound. After two washes, cell aliquots (106 cells) were lysed in 0.5% sodium dodecyl sulfate and total radio-

activity

was

determined.

Ci/mmol, The Radiochemical Center, Amersham), and actinomycin D (Sigma Chem. Co.) as in ref. 11. After alkaline hydrolysis and Sephadex G-50 gel filtration, [3H]cDNA (specific activity 107 cpm/,g) was purified by alkaline sucrose density gradient centrifugation. Fractions corresponding to 9-10 S were pooled, neutralized, precipitated with ethanol, and suspended in water. [3H]cDNA (2000 cpm; 0.2 ng) was hybridized at 680 for 43 hr to different amounts of RNAs in a final volume of 15 Al with a reaction mixture described in ref. 10. The amounts of [3H]cDNA hybridized were determined by S1 nuclease (Miles, London) digestion for 40 min at 45°. A blank reaction mixture gave a value of 30 cpm. All glassware was treated with a silane and kept at 180° for 3 hr.

RESULTS AND DISCUSSION RNA synthesis of human leukemic leukocytes and of

hytohemagglutinin-stimulated small lymphocytes from

healthy humans

a much higher RNA synthesis than normal small lymphocytes (12), thereby providing a suitable system for studying the possible regulatory activity of the peptide factor in living cells. Fig. 1 shows kinetics data of RNA synthesis after treatment with the factor of human leukocytes obtained from patients with acute lymphoblastic, myeloblastic, and chronic lymphocytic leukemias, as well as of human small lymphocytes from healthy subjects. [14C]Uri-

Human leukemic leukocytes show

dine was continuously present in the incubation mixture. Leukemic patients had not yet been treated with any antimitotic drug. RNA synthesis was notably decreased in leukemic cells. It is also apparent that the factor-induced inhibition was much more marked whenever RNA synthesis was more pronounced, which is reminiscent of a similar correlation observed in the case of the inhibition of DNA- and chromatin-directed transcription (1, 2). Uptake of labeled uridine after 1- to 2-min pulses was comparable in the presence and in the absence of the factor, the addition of which, therefore, did not impair uptake of the label. RNA synthesis of normal small lymphocytes, although unaffected by the addition of the factor, was too low to represent a suitable control. In order to investigate the action

of the factor on nonleukemic lymphocytes actively synthesizing RNA, we tested the factor on phytohemagglutinin (PHA)stimulated human small lymphocytes. The data shown in Fig. 2 indicate. that the factor (10 jug/ml) causes a 50% reduction of RNA synthesis up to the 20th hr of incubation in conditions where PHA, the peptide, and the labeled uridine were continuously present. This effect progressively diminishes at later intervals. The addition of 20 ,gg of the factor per ml causes at the 20th hr the almost complete abolishment of PHA-induced stimulation of RNA synthesis (data not shown). Furthermore, data from pulse experiments done at the 18th (inset A) and 46th (inset B) hr of incubation also show that the factor inhibits RNA synthesis only at the early interval tested. Data shown in inset C indicate that a 30% reduction of RNA synthesis is also observed when the peptide is added to cultures of PHA-stimulated lymphocytes 17 hr after seeding. [3H]Uridine was added 1 hr thereafter. Specific activities of total cell RNAs extracted from cells treated with PHA and with PHA plus peptide 2 hr after labeling time were 2506 + 250 (SEM) cpm/Aug of RNA for PHA-stimulated cells and 1750 + 185 (SEM) for cells treated with PHA plus peptide. The reduction of RNA synthesis, as measured by incorporation of [3H]uridine into trichloroacetic acid-precipitable material, is already visible shortly after addition of the labeled precursor (inset D). Also, in this system the uptake of labeled uridine was indistinguishable regardless of the presence of the factor. In the PHA-stimulated system DNA synthesis does not yet occur at the 20th hr and is detectable only from the 36th hr onward (13, 14). Treatment with the factor does not appreciably affect DNA synthesis at the 46th hr of incubation. When tested at the 18th hr, no culture showed any thymidine incorporation, confirming published evidence (data not shown). The fact that RNA synthesis was reduced in human leukemic leukocytes treated with the factor is in agreement with the reduction of RNA synthesis observed in PHA-stimulated lymphocytes. The former, in fact, undergo a much higher RNA synthesis than do controls, similar to what the latter do in response to PHA stimulation. Leukemic cells may synthesize more RNA than normal cells because of the lack of factor(s) controlling cell metabolism and the ratios between active and inactive genes. This would be in keeping with data of Sawada et al. (15), indicating that chromatin of leukemic cells possesses a higher template activity than chromatin from normal cells. Similarly, also in PHA-stimulated lymphocytes, RNA synthesis increases and previously repressed genetic loci appear to be activated (13). The data of Fig. 2 (left and insets A, C, and D) indicate that the inhibition of RNA synthesis caused by the factor is independent of the timing of the addition of the peptide. This apparently rules out the possibility that the factor may compete with PHA for some common receptor(s) and may act, therefore, as an antimitogenic

stimulus.

Cell Biology: Amici et al.

X

Proc. Natl. Acad. Sci. USA 74 (1977)

3871

1 2 U 1 2 E T Time, hr Timehrr a B 46--)48 hr D 18 hr o ~~~~~~~~~~~~~ 3 4 -2502 -1

C1o

Time,

E

0 c

1

024 6

2

Time, hr

-JI 7 20 32 48 Time, hr

Time, min

FIG. 2. Effect of the peptide on RNA synthesis of human peripheral small lymphocytes stimulated by PHA (Calbiochem; 15 /g/ml of culture). Incorporation of [2-14C]uridine (3 gCi/ml; 60 mCi/mmol): cpm/106 cells. The preparation of samples of small lymphocytes, the reaction mixture, and the incubation were as in Materials and Methods. The peptide, PHA, and radioactive material were added at zero time. The data are the average of eight experiments made in triplicate: cpm are mean ± SEM. Trichloroacetic acid-insoluble radioactivity was determined as in Fig. 1. 0, PHA-stimulated cells; *, PHA-stimulated cells in the presence of fraction 16, 10 ,ug of protein per ml (final concentration); 0, unstimulated control cells. (Insets A and B) Cells were exposed to [14C]uridine at the indicated times (18th and 46th hr) and labeled for 2 hr. Other conditions for incubation were as described before. (Insets C and D) Cells were exposed to [3H]uridine (50 1ACi/ml, 60 Ci/mmol) at the 18th hr after stimulation with PHA, and were labeled either for 2 hr (C) or for 6 min (D). The factor was added 1 hr prior to the label. Uptake of the label was determined as for Fig. 1.

Erythroid differentiation of Friend leukemic cells Growth of Friend cells in the presence of Me2SO (16) and several other compounds results in a marked shift of the large majority of these cells towards the erythroid pathway, with production of Hb and globin mRNA. The stimulation of globin gene transcription by Me2SO is apparently dependent upon the occurrence of one to two cell cycles prior to the synthesis of globin mRNA, although this does not seem to be a prerequisite for other inducers (data reviewed in ref. 17). In view of these data, we chose in a pilot experiment the appropriate doses and timing of peptide administration that would be least active on cell growth, so that Me2SO-stimulated Friend cells could double even more than twice in the presence of the factor. Fig. 3 A and B shows that the administration on day 1 of the peptide to cultures of untreated and of Me2SO-stimulated

Friend cells resulted in a slight inhibition of cell growth. It is apparent, though, that, even with the highest dose of the factor, the Me2SO-treated cell cultures attained a density of 1.7 X 106 cells per ml, by far fulfilling the cell-cycle requirements and making toxic effect rather unlikely. Under these conditions, administration of the peptide resulted in a reduction of the amount of Hb detected on days 4 and 5 after Me2SO administration to 10% of the original value. The percent of benzidine-positive cells determined on the same cell preparations decreased accordingly (Fig. 3C). More detailed dose-response and kinetics data will be published elsewhere (Rossi et al., unpublished data). Hb production was similarly depressed, even when the peptide was added on day 2. By hybridizing total cell RNA extracted on day 5 from untreated Friend cells, Me2SO-stimulated cells, or-Me2SO-stimulated cells given the peptide on day 1 with 3H-labeled complementary globin DNA, amounts of globin mRNA present in

3X 1

a

1 X1

0

I-E

0. C.

C-) C ._;

c

oa

1x A 3 Days

1.0) Days

0

3

4

Days

5

FIG. 3. Kinetics of growth and Hb synthesis in untreated and Me2SO-stimulated Friend cells given the peptide factor on day 1 after seeding

(arrow). Data shown are the average viable cell counts from triplicate samples. (A) Growth curves of untreated cells (0); of cells given the peptide, 7 ,g of protein per ml, (final concentration) (-). (B) Growth curves of Me2SO-stimulated cells (-); of Me2SO-stimulated cells given the factor, 5 ,ug of protein per ml (A) or 7 ug of protein per ml (-) (final concentration). (C) Percentages of benzidine-positive cells in the same Friend cell preparations shown in (B). Numbers in parentheses represent Hb amounts (,Ug/107 cells).

3872

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au 9R

Proc. Nati. Acad. Sci. USA 74 (1977)

Cell Biology: Amici et al. 'O N .

_

.0

z a

0

wU

3.0 2 4 6 8 10 12 2.0 0.0 0.5 1.0 1 /Total cell RNA, pg Total cell RNA, ug FIG. 4. Effect of the peptide on transcription of globin mRNA

in Me2SO-stimulated Friend cells: [3H]cDNA.RNA hybridization curves. RNA extractions and hybridization assays were done as described in Materials and Methods. On day 3, Friend cells treated with Me2SO and with Me2SO plus factor were labeled for 18 hr with [3H~uridine (1 ,Ci/ml; 42 Ci/mmol). Aliquots (5 X 105 cells) were lysed with 0.5% sodium dodecyl sulfate (final concentration) and trichloroacetic acid-precipitable radioactivity was determined. Incorporation of [3H]uridine was: Friend cells treated with Me2SO, 3.2 X 105 cpm; treated with Me2SO plus factor, 3.5 X 105 cpm. (A) Total cell RNAs from untreated cells (0); from Me2SO-stimulated cells (0); from Me2SO-stimulated cells given, on day 1, 5Mgg (A) or 7Mug (E) of fraction 16 per ml. (B) Double reciprocal plots of data from (A), except for total cell RNA from untreated cells, which were omitted.

these cell preparations were measured. From previous data (18) we knew that 0.10 ng of pure globin mRNA was needed to obtain a 50% hybridization value with [3H]cDNA. This 50% saturation value was used, in turn, to measure the amounts of globin mRNA per ,g of total cell RNA present in the cell populations tested. Fig. 4A shows saturation curves of total cell RNAs from the above conditions. By measuring the 50% saturation points (Fig. 4B) the various populations of Friend cells tested were found to contain the following amounts of globin mRNA (ng/,ug of total cell RNA): Me2SO-stimulated Friend cells, 0.50; cells treated with Me2SO plus active factor (5 ,ug/ml), 0.17; cells treated with Me2SO plus active factor (7 ,ug/ml), 0.10. It is apparent, therefore, that the peptide factor inhibits the transcription of globin genes induced by Me2SO in a dosedependent way, whereas the overall RNA synthesis appears to be unaffected (see legend to Fig. 4). In order to provide evidence for the specificity of the preceding observations and to obtain an internal negative control, we studied growth curves and [3H]uridine incorporation into trichloroacetic acid-precipitable material in several established tissue culture lines originating from different animal species and exposed to 10 ,ug of the factor per ml. Treatment with the peptide does not at all affect these parameters in BHK21 cell line or in murine (L-929), chinese hamster (CHEF12), simian (CV1), and bovine (MDBK) cell lines (data omitted for brevity). To conclusively prove that the treatment with the peptide was not toxic, human small lymphocytes given 10 or 20 ,ug of the peptide per ml for 8 hr were either stimulated with PHA and labeled with [14C]uridine (Fig. 5A) or were thoroughly washed, then stimulated with PHA and labeled (Fig. SB). A dose-dependent inhibition of PHA-stimulated RNA synthesis up to 20th hr is demonstrated in Fig. 5A. The data in Fig. 5B, in contrast, show that already at the 5th hr after removal of the factor, PHA-stimulated RNA synthesis of lymphocytes that had been treated with various doses of the peptide is indistinguishable from controls. Similar results were obtained in the Friend system. Friend

O

5

10

0 20 Time, hr

FIG. 5. Lack of toxicity of peptide treatment after removal of the compound. Human peripheral small lymphocytes, prepared and incubated as in Materials and Methods, were treated with 10 or 20 Mg of protein per ml of the peptide. After 8 hr of incubation, cells were either (A) stimulated with PHA and labeled (as in Fig. 2) or (B) thoroughly washed to remove the factor and then stimulated with PHA and labeled. Incorporation of [2-14C]uridine into trichloroacetic acid-precipitable material was assessed at the indicated time intervals. Untreated PHA-stimulated cells (0); PHA-stimulated cells given 10 (N) and 20 (A) Mg of protein per ml of the peptide, final concentrations; untreated unstimulated cells (0).

cells treated for 48 hr with the factor, thoroughly washed, and reseeded at 105 cells per ml, were fully inducible by Me2SO to synthesize Hb (data not shown). The data obtained with Friend cells provide direct evidence of a major reduction in transcription of globin mRNA under conditions of virtually unaffected cell growth. Since Friend cells as well as all other cell lines tested grow, it is certainly unlikely that the active factor affects ribosomal RNA synthesis to any significant extent. By inference, one is tempted to surmise that it is mRNA synthesis which is impaired, as shown by the hybridization experiments. The observed reduction of RNA synthesis in human lymphocytes and globin mRNA transcription in Me2SO-stimulated Friend cells is of potentially great interest because it was observed only in cells engaged in some kind of differentiation (Friend cells) or at least specifically stimulated to an increased RNA production (PHA-stimulated lymphocytes). This may explain the lack of effect of the peptide on growing "nondifferentiating" cell lines from several species. As a working hypothesis, one could suggest that the peptide may be operative only under conditions where the synthesis of RNAs (or of mRNAs) is enhanced by some factors. At this time we have no information concerning the mechanism of action of the peptide factor in the cell systems studied. Since neither Hb synthesis in Friend cells nor blastic transformation of PHA-stimulated lymphocytes constitutes unequivocal bona fide examples of cell differentiation, it remains to be seen whether the peptide effects can be interpreted in terms of regulation of cell differentiation. On the basis of the available evidence, one may be simply dealing with a quantitative control of transcription or mRNA processing for genes already activated by independent mechanisms. We are indebted to Prof. G. Sprovieri, Clinical Laboratory, Umberto 10 Regional Hospital, Ancona, Italy, for kindly providing leukemic blood samples and to Drs. C. Friend, L. Luzzatto, and E. Calef for reading this manuscript. The generous gift of avian myeloblastosis virus RNA-dependent DNA polymerase by Dr. J. Beard, Life Sciences, Inc., St. Petersburg, FL, through Dr. 1. Gruber, Office of Resources and Logistics of the Virus Cancer Special Program, National Cancer In-

Cell Biology: Amici et al.

stitute, Bethesda, MD is gratefully acknowledged. This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CNR), Progetto Finalizzato Virus (76.00686.84 and 76.00690.84), and CT. 76.01151.04, Rome, NATO (no. 1152) and Fondazione Rusconi.

1. Gianfranceschi, G. L., Amici, D. & Guglielmi, L. (1975) Biochim. Biophys. Acta 414,9-19. 2. Gianfranceschi, G. L., Amici, D. & Guglielmi, L. (1976) Nature 262,622-623. 3. Gianfranceschi, G. L., Amici, D. & Guglielmi, L. (1976) Mol. Biol. Rep. 3, 55-62. 4. Szent-Gyorgyi, A., Hegyeli, A. & McLaughlin, J. A. (1962) Proc. NatI. Acad. Sci. USA 48, 1439-1443. 5. Goldstein, A. L., Banerjee, S. & White, A. (1967) Proc. Natl. Acad. Sci. USA 57,821-828.

6. Milcu, S. M. & Potop, L. (1973) in Thymic Hormones, ed. Luckey, T. D. (Urban & Schwarzenberg, Munich), pp. 97-133. 7. Trainin, N. (1974) Physiol. Rev. 54, 272-316.

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8. Orkin, S. H., Harosi, F. D. & Leder, P. (1975) Proc. Nati. Acad. Sci. USA 72,98-102. 9. Crosby, W. H. & Furth, F. W. (1956) Blood 11, 380-385. 10. Preisler, H. D., Housman, D., Scher, W. & Friend, C. (1973) Proc. Natl. Acad. Sci. USA 70,2956-2959. 11. Forget, B. G., Marotta, C. A., Weisman, S. M., Verna, I. M., McCaffrey, R. P. & Baltimore, D. (1974) Ann. N.Y. Acad. Sci. 241,290-309. 12. Cline, M. J. & MacKenzie, M. R. (1967) Nature 214,496-497. 13. Pogo, B. G. T., Allfrey, V. G. & Mirsky, A. E. (1966) Proc. Natl. Acad. Sci. USA 55,805-812. 14. Cooper, H. L. (1966) J. Biol. Chem. 243,34-43. 15. Sawada, H., Gilmore, V. H. & Saunders, G. F. (1973) Cancer Res. 33,428-434. 16. Friend, C., Scher, W., Holland, J. G. & Sato, I. (1971) Proc. Natl. Acad. Sci. USA 68,378-382. i7. Friend, C. (1977) Harvey Lect., in press. 18. Rossi, G. B., Dolei, A., Cioe, L., Benedetto, A., Matarese, G. P. & Belardelli, F. (1977) Proc. Natl. Acad. Sci. USA 74, 20362040.