Early events in lymphocyte transformation by phytohemagglutinin

CELLULAR

IMMUNOLOGY

14, 134-138 (1974)

Early Events in Lymphocyte III.

Inhibition

of RNA

Transformation

Synthesis

by Phytohemagglutinin

and Transformation

by Cordycepin

BEATRIZ G. T. POGO The Public Health

Research I+zstitute of the City of New York, Imorporated, New York 10016 Received

February

Nezv York,

11,1974

INTRODUCTION One of the first events in the transformation of human lymphocytes induced by phytohemagglutinin (PHA) is the stimulation of RNA synthesis (1, 2) related to an increase in the activity of DNA-dependent RNA polymerase (3). Cordycepin (3’=deoxyadenosine), an analog of adenosine, is known to inhibit messenger RNA synthesis in HeLa cells without affecting ribosomal RNA synthesis (4). It has been suggested that cordycepin inhibits more specifically the synthesis of polyadenylate sequences associated with mRNA (5) and thereby possibly blocks the transport of mRNA into the cytoplasm (6). This communication describes the effects of cordycepin on the macromolecular metabolism of human lymphocytes exposed to phytohemagglutinin (PHA) .

w

CORDYCEPIN,

pg/ml

FIG. 1. Effect of different concentrations of cordycepin on thymidine incorporation into DNA by two types of PHA-stimulated lymphocytes. Samples containing 3 X 10’ cells/ml were treated with PHA and different cordycepin concentrations for 48 hr, the 1 pCi of ‘H-thymidine (specific activity, 24 Ci/mmol) was added to the cultures. After 5 hr, the cells were processed for DNA determination and counting. 0-O and W-m, PHA-treated lymphocytes; O-O and 0-Q control lymphocytes. 134 Copyright All rights

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

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COMMUNICATIONS

PHA t

CORDYCEPIN

100

I2

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6

&

CONTROL -

0 E

TIME,

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2

.

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1:~. 2. Effect of cordycepin on uridine incorporation into RN.4 by PHrZ-stimulated lym1)hocytes. Samples containing 2 X 10” cells/ml were pretreated with PHA for 1 hr before 10 yg/ml of cordycepin were added (time 0 on the abscissa). At the times indicated, the cells were for 5 and 30 min and incubated with 4 &i/ml of ‘H-uridine (specific, activity, 28 Ci/mmol) then processed for RNA determination and coun.ing. (‘4 ) Incorporation of “H-uridine into RN:1 after 5 min; (8) incorporation after 30 min. O-0, PHA-treated lymphocytes; O--3, PHf1treated lymphocytes plus cordycepin ; n-/L, control lymphocytes.

MATERIALS

AND METHODS

Detailed descriptions of the nutrient media employed. preparation of lymphocyte cultures. and ancillary procedures have been published (2, 3 ). 1)eoxyribonucleic acid. Kh’A, and protein syntheses were determined according to previously described methods (2, 7). Likewise the preparation of nuclear fractions and assays of DNA-dependent RNA polymerases in them was tlescribed in previous papers (3 ) Cortl\;cepin was purchased from Sigma Chemical Co. RESULTS Ejject

of Cordycepin

on DNA

Synthesis

and Lplplzor~te

Transforwmtio~l

To study the effects of cordycepin on DNA synthesis and PHA-induced transformation, cultures of 3 X loo cells/ml were incubated with varying concentrations of cordycepin and 48 hr later the cells were labeled for 5 hr with “H-thymidine. harvested, and analyzed for the amount of radioactivity in their DNA. It is eyiclent in Fig. 1, summarizing data of two independent experiments, that cortl~cepin

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inhibits thymidine incorporation in PHA-treated lymphocytes in proportion to amount added in the range of S-20 pg/ml. In similar parallel experiments transformation of lymphocytes was monitored fixed and stained cells that had been harvested 72 hr after induction. Addition cordycepin, in the amounts of 10-20 pg/ml reduced the number of transformed lymphoblasts to 5% as compared with 70% present in the uninhibited cultures.

the in of cell

Efect of Cordycepin on RNA and Protein Synthesis Rates of RNA synthesis were measured by uptake of SH-uridine into RNA. According to Cooper (S), the RNA of lymphocytes labeled during a 5-min pulse sediments in a sucrose density gradient as polydisperse material of heterogeneous molecular weight, while the RNA labeled during a 30-min pulse sediments in the position of ribosomes or ribosomal precursors; furthermore, this 30-min labeled RNA is selectively inhibited (by 90%) by low concentrations of actinomycin D (0.05 pg/ml). To distinguish synthesis of the two types of RNA after cells had been pretreated with PHA for 1 hr, incorporation of either 5- or 30-min duration was used in the presence or absence of cordycepin. At the times indicated in Fig. 2, 3H-uridine was added to the cultures and the incorporation of label into an RNA product was determined. The rates of synthesis after labeling in pulses of 5 min are shown in Fig. 2A, and those after 30-min pulses in Fig. 2B, from which it became apparent that within 1 hr after addition of cordycepin the drug inhibited more profoundly synthesis of the rapidly labeled RNA fraction. This is especially evident during the initial 2 hr after induction. Later cordycepin blocked formation of the “30-min RNA”, as shown in Fig. 2B. Thirty-minute pulses of SH-leucine were used to measure synthesis of protein at various times following PHA induction in the presence and absence of cordycepin. The results, summarized in Fig. 3, showed as expected that protein synthesis in-

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WA t CORDYCEPlN

3,000

E /,b

P \ E 2 1,000

I-------

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4

6

24

TIME, HOURS

FIG. 3. Effect of cordycepin on amino acid incorporation into proteins by PHA-stimulated lymphocytes. Samples ccntaining 3 X 108 cells/ml were pretreated with PHA for 1 hr before 10 pg/ml of cordycepin were added (time 0 on’ the abscissa). At the times indicated the cells were transferred to a leucineless medium containing 2% fetal calf serum and 1 &/ml of Lleucine-4,5-‘H (specific activity, 36 Cijnmol) and incubated for 30 min. The cells were washed with phosphate-buffered saline, precipitated with 0.5 N perchloric acid (PCA) in the cold, washed with 0.5 N PCA, 95% alcohol, and alcohol-ether (3:l). The precipitates were resuspended in 0.1 N NaOH for protein determinsation and scintillation counting.

SHORT

TAPLE RNA

POLYMERASK

1

ACTIVITIES IN ISOLATED NUCIXI WITH OR WITHOUT CORDYCEPIN

(nmol UhlPJH/mg Time after addition of rordycepin b-1

Conditions of incubation

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COMMUNICATIONS

DNA

FKOM PH.I-TRKATED AND CONTROL CELLS

CELLS

1NCTT11.41.1~I)

PER 10 min)a

Control cell5

I’HA-treated CCllS

PHA-treated ceils and cordyccpin

~

Mg2f M n2+ M g2+ M n2+ Mg2+ TV1 n2+ hlg2+ XIII”?

1.0 2.0

2.2 2.5

1.5 3.0 2.5 4.0 .1.8 4.4 6.9 0.0

1.2 3.0 2.0 3.1 3.5 4.9 5.0 5.0

a Experimental conditions similar to those for Figs. 2 and 3. .4t times indicated, 1.5 X 10’ cells/ml were washed with phosphate-buffered saline, and then resuspended in 1 ml of 0.01 J/ tris-HCI buffer (Schwarz/Mann, Orangeburg, NY), pH 7.8, with 1 mM MgClz (or hlnCl2) and 10 m&’ KCI. Swelling of the cells was allowed to proceed for 10 min at 4°C. Triton >< 100 was thrll added at a linal concentration of 0.5yc, and the cells were disrupted by 10 strokes of a Dounre homogenizer and centrifuged at 800 g for 3 min. The pellet was washed with the same solution plus 0.17; sodium deoxycholate. The nuclear fraction was then resuspended in 0.25 ml of a mixture containing 0.3 M sucrose in 0.01 M tris-HCI buffer, pH 8.0, 4 mM L’IgClr (or 1.8 mM MnCle), 0.06 dl NaCI, 30 mAJ 2-@-mercaptoethanol, 0.1 pm01 of adenosine triphosphate (ATI’), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and 0.03 pmol of “H-labeled uridine triphosphate (UTP) (specific activity, 20 &i/pmol). The reaction was allowed to proceed for 10 min at 37’C and then stopped by adding 5 ml of lOojo trichloroacetic acid (TCA) with 0.05 M sodium pyrophosphate. The precipitates were collected in Millipore filters, washed twice lvith 10yc TCA, and the radioactivity was determined.

crease in lymphocytes stimulated by PHA (2, 9, 10) and cordycepin had no effect on protein synthesis during the initial 4 hr. Exposure to the drug for longer periods resulted in a gradual reduction of synthesis until the level reached by 24 hr was the same as that recorded with control lymphocytes. Efect

of Covdycepin

on the Nuclear

DNA-Depelzdent

RNA

Polymerascs

The activity of DNA-dependent RNA polymerases was assayed in nuclei isolated from cells exposed to varying periods to PHA with or without cordycepin. The data summarized in Table 1 indicated that in the absence of the analog the anticipated increase in RNA polymerase activity due to PHA induction (3) was found. Addition of cordycepin failed to influence the elevation of polymerase activities during the first 6 hr of treatment. However, after 24 hr of treatment there was a small decrease in both the Mg”+- and Mn’+-dependent activities. DISCUSSIOfl The data presented herein show that cordycepin suppresses transformation most probably by inhibiting synthesis of RKA. One of the earliest events associated with transformation of human lymphocytes is the stimulation of RNA synthesis,

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evident within 1 hr after exposure to PHA (1, 2). The analog inhibits the synthesis of the rapidly labeled RNA first, and later the ribosomal RNA. Since inhibition of protein synthesis by cordycepin develops much later, it may be assumed that the primary effect is on the synthesis of RNA. Unlike actinomycin D, cordycepin did not affect the stimulation of RNA polymerase induced by PHA (3), although some decrease in the activities was found after several hours. Suppression of RNA synthesis is obviously not related to the level of the Mg2+- and Mn2+-dependent polymerase activities, both of which increase as much as in the cordycepin untreated cells, at least during the initial 6 hr of the experiment. This result should be compared with observations made on the same system using rifampicin which specifically blocks the increase in RNA polymerase activity (7). It has been suggested that in HeLa cells cordycepin interferes selectively with mRNA biosynthesis or its transport from the nucleus into the cytoplasm, perhaps by blocking the formation of polyadenylate sequences covalently linked to mRNA. Under any of these circumstances the effect of cordycepin would be the incorporation of this analog into the RNA molecule and, therefore, genesis of incomplete chains of RNA. The data suggest that cordycepin inhibits lymphocyte transformation by interfering with early RNA synthesis without affecting the enhancement in the activity of DNA-dependent RNA polymerases. It also provides indirect evidence that PHA stimulation results in an increase of mRNA. ACKNOWLEDGMENTS The author is indebted to Dr. Dales for advice and criticism and to J. R. Katz for her capable technical assistance. Aided by Grant No. E-623 from the American Cancer Society.

REFERENCES 1. Rubin, A. D., and Cooper, H. L., Proc. Nat. Acad. Sci. U.S.A. 54, 469, 1965. 2. Pogo, B. G. T., Allfrey, V. G., and Mirsky, A. E., Proc. Nat. Acad. Sci. U.S.A. 55, 805, 1966. 3. Pogo, B. G. T., J. Cell. Biol. 53, 635, 1972. 4. Penman. S.. Rosbash. M., and Penman, M., Proc. Nat. Acad. Sci. U.S.A. 67, 1878, 1970. 5. Adesnick, k., Salditt; M.; Thomas, W.; and Darnell, J. E., J. Mol. Biol. 71, 21, 1972. 6. Darnell, J. E., Phillipson, L., Wall, R., and Adesnick, M., Science 174, 507, 1971. 7. Pogo, B. G. T., J. Cell. Biol. 55, 515, 1972. 8. Cooper, H. L., J. Biol. Chem. 243, 34, 1968. 9. Bach, F., and Hirschhorn, K., Exp. Cell. Res. 32, 592, 1%3. 10. Levy, R., and Rosenberg, S. A., Cell, Immunol. 7, 92, 1973.