Effect of Acyclovir on the Proliferation of Human Fibroblasts and ...

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Triplicate 0.1-ml sam- ples from each culture ..... virus antigen. Each point represents triplicate sam- ... Klein, R. J., A. E. Friedman-Kien, and E. DeStefano. 1979.

ANTIMICROBIAL AGENTs AND CHEMOTHERAPY, June 1980, p. 947-953 0066-4804/80/06-0947/07$02.00/0

Vol. 17, No. 6

Effect of Acyclovir on the Proliferation of Human Fibroblasts and Peripheral Blood Mononuclear Cells MYRON J. LEVIN,* PATRICIA L. LEARY, AND ROBERT D. ARBEIT

Division of Clinical Microbiology, Sidney Farber Cancer Institute, Boston, Massachusetts 02115

The effect of acyclovir on human cells was measured. A concentration of 50 to 100 ,uM inhibited the division of fibroblasts to a variable extent, depending on the experimental design and the confluency of the monolayer. The magnitude of this effect was less than that caused by human leukocyte interferon when these antiviral agents were compared at clinically relevant concentrations. Acyclovir also inhibited thymidine incorporation by peripheral blood mononuclear cells stimulated by either phytohemagglutinin or three different herpesvirus antigens. A linear dose-response curve was observed with these cells, and their proliferation 100 was inhibited 50% by lM acyclovir. Inhibition was exerted on T-cell proliferation without apparent effect on the release of lymphokines or on monocyte function.

Acyclovir

{ACV; 9-[(2-hydroxyethoxy)- herpes simplex virus, varicella-zoster virus, or cyto-

methyl]guanine) is an analog of guanosine or

deoxyguanosine in which the C-2 and C-3 of the sugar moiety are missing. In vitro antiviral activity has been demonstrated against human herpes simplex virus types 1 and 2 and varicellazoster virus (3, 4, 6, 14). Furthermore, a favorable in vivo therapeutic ratio has been predicted for ACV because it has a selective inhibitory effect on herpesvirus-specified deoxyribonucleic acid (DNA) polymerase and because it is efficiently phosphorylated to its active form only in infected cells containing virus-specified kinases (6). In vivo efficacy has been confirmed in mice and guinea pigs infected with herpes simplex virus (7, 11, 14). Clinical trials in humans have been completed with an ophthalmic preparation (10). Although phase 1 trials of parenterally administered ACV are in progress, there has been little study of the effect of ACV on human cells (3, 4). Our report characterizes the effects of ACV on two human tissues: (i) a human fibroblast line previously used to determine the antiviral effect of ACV, and (ii) peripheral blood mononuclear (PBM) cells stimulated by mitogens and herpesvirus antigens. MATERIALS AND METHODS

Cells and reagents. A continuous line of human foreskin fibroblasts (350Q) was used at passages 17 to 22. ACV was supplied by Burroughs Welicome Co., Research Triangle Park, N.C. Human leukocyte interferon was supplied by June Dunnick of the Antiviral Substances Program of the National Institute of Allergy and Infectious Diseases, Bethesda, Md. Phytohemagglutinin was obtained from Difco Laboratories, Detroit, Mich. Antigens were prepared from uninfected tissue cultures or from cultures infected with

megalovirus by extraction with alkaline (pH 9.0) glycine-buffered saline (16). Incorporation of thymidine into proliferating fibroblasts. Fibroblasts (10k) were added to the wells of a Microtest II microtiter plate (96 flat-bottom wells per plate; Falcon Plastics, Oxnard, Calif.) in 0.05 ml of Dulbecco-modified Eagle medium supplemented with antibiotics and 10% fetal bovine serum. After 24 or 48 h, [methyl-3H]thymidine (0.25 ,ICi; 20 Ci/mmol) in 0.05 ml was added to each well. After 6 h, the wells were drained, washed with 0.2 ml of Puck saline A, incubated with 0.05 ml of trypsin-ethylenediaminetetraacetic acid for 5 min, and lysed with 0.05 ml of 0.5% sodium dodecyl sulfate. The contents of each well were harvested onto fiber glass filters with distilled water by a multiple automated sample harvester (MASH II; Microbiological Associates, Bethesda, Md.). Incorporation of radioactive thymidine into macromolecules was determined with a scintillation spectrometer. Cell growth experiments. Five milliliters of medium with 10% fetal bovine serum and 7.3 x 104 fibroblasts was added to 60-mm tissue culture dishes (Falcon Plastics). After 24 h, some plates received ACV or interferon in 0.05 ml. On successive days, triplicate plates at each inhibitor concentration were trypsinized, and the viable cells, determined by trypan blue exclusion, were counted in duplicate with a Thoma hemacytometer. In one experiment, [3H]thymidine (50 ,uCi) was added to each plate 6 h before the cells were counted. After the cells were trypsinized, a sample was removed for determining cell number, and the remaining cells were pelleted and suspended in 1 ml of cold 10% trichloroacetic acid. Incorporation of [3H]thymidine into cell DNA was determined by the Schneider procedure (15). Proliferation of PBM cells. Heparinized blood was obtained from nornal volunteers and from one patient with malignancy before chemotherapy. PBM cells, obtained by Ficoll-Hypaque density gradient

947

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ANTIMICROB. AGENTS CHEMOTHER.

LEVIN, LEARY, AND ARBEIT

centrifugation (2, 16), were suspended at a concentration of 106 cells per ml in RPMI 1640 with 10% pooled AB serum. One-milliliter samples in culture tubes (no. 3033, Falcon Plastics) were stimulated with phytohemagglutinin for 3 days or with antigen for 5 to 7 days. At 6 h before harvest, duplicate cultures received 2.0 ,uCi of [methyl-3H]thymidine. Triplicate 0.1-ml samples from each culture were harvested with a multiple automated sample harvester, and incorporation of radioactivity was determined. Incorporation of L-[3,4,53H(N)]leucine (135 Ci/mmol) into PBM cells was determined in the same manner. Celi separation. Monocytes were prepared from 60 x 106 PBM cells suspended in 5 ml of RPMI 1640 containing 20% fresh autologous serum. The cells were incubated at 370C for 90 min in glass petri dishes (100 by 20 mm) prepared as described by Einstein et al. (5). Nonadherent cells were removed by repeated rinsing, and the adherent cells were released by treatment for 15 min at 370C with 100 U of acetyl trypsin (Sigma Chemical Co., St. Louis, Mo.) per ml in Hanks balanced salt solution without calcium or magnesium. The adherent cells recovered were >95% viable and were 90% monocytes as determined by staining for peroxidase and by phagocytosis of latex particles. The nonadherent cells were further purified by passage through nylon fiber columns as described by Greaves et al. (8). The column effluent consisted of >90% T lymphocytes, 95% T cells and glassadherent cells containing >90% monocytes, were prepared. ACV similarly inhibited the antigeninduced proliferation of purified lymphocytes, either alone or supplemented with monocytes (Table 5). The production of interferon in these cultures was not influenced by the presence of ACV (Table 6). Although the effect of ACV on human PBM cells was routinely tested at a single high and a single low ACV concentration, dose-response curves demonstrated some inhibition with increasing doses through the range of concentrations tested (Fig. 4). This finding was reproducible in five other dose-response curves not shown.

INHIBITION BY ACYCLOVIR OF HUMAN CELL DIVISION

VOL. 17, 1980

DISCUSSION We have confirmed the preliminary report that cultured human fibroblasts are not inhibited by ACV concentrations lower than 50 to 100MuM (4) and further observed that the threshold for inhibition of these cells by ACV may be influenced by their degree of confluence (Table 1). The effect of ACV on human fibroblast division and thymidine incorporation was compared with that caused by interferon (Fig. 2). ACV was inhibitory in the range of 40 to 60 ,uM, which is 10 to 100 times the 50% plaque reduction dose for varicella-zoster virus and herpes simplex virus, respectively, whereas interferon inhibited cell growth at 10 U/ml, which approximates the 50% plaque reduction dose for these viruses,

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although the interferon preparation and conditions used to measure the published antiviral effect were not comparable to those that we used to evaluate the anticell effect (1, 13). The effect of 10 to 50 U of interferon per ml on cell number exceeded the effect on thymidine incorporation, thereby making comparison with ACV difficult. Proliferating PBM cells were consistently inhibited by ACV. At 100 ,M, ACV depressed the response of PBM cells to phytohemagglutinin by 15 to 57% (Table 2). The antigen-specific immune response of PBM cells to herpesvirus antigens was even more readily suppressed; at 20 MuM, proliferation was reduced by 15%, and at 100 uM, it was reduced by approximately 50% (Tables 3 and 4). Inhibition was not secondary to a shift in the time course of antigen-induced

TABLE 4. Effect of ACV on the proliferative response of PBM cells to varicella-zoster virus antigen [3H]thymidine incorporation' Donora

Day of harvest

5 6 7

a No ACV 9,192 ± 792 15,077 ± 909 13,751 ± 117

8,079 ± 483 12,056 ± 5,191 12,724 ± 932

2a

5 6 7

5,377 ± 383 11,417 ± 106 15,600 ± 424

2b

5 6 7

3

ACV (20 MM)d

(0.88)"

ACV (100 ,1M)e

(0.80) (0.92)

5,133 ± 142 9,196 ± 518 7,688 ± 324

(0.55) (0.61) (0.55)

4,407 ± 218 10,785 ± 263 13,415 ± 800

(0.82) (0.94) (0.84)

3,069 ± 134 7,108 ± 207 9,033 ± 162

(0.57) (0.62) (0.57)

23,536 ± 75 47,642 ± 373 43,818 ± 480

15,426 ± 1,116 43,373 ± 1,584 NDI

(0.65) (0.91)

13,812 ± 42 24,128 ± 277 27,721 ± 797

(0.59) (0.51) (0.63)

5

31,999 ± 1,052

22,917 ± 318

(0.72)

17,962 ± 555

(0.56)

4

5

13,209 ± 147

9,143 ± 481

(0.69)

9,047 ± 313

(0.68)

5

5 6 7

3,257 ± 267 6,940 ± 278 13,069 ± 1,471

2,713 ± 132 5,664 ± 303 8,642 ± 1,737

(0.83) (0.81) (0.65)

1,999 ± 97 4,081 ± 267 7,404 ± 748

(0.60) (0.58) (0.55)

6

5

31,999 ± 1,052

(0.72)

17,962 ± 555

(0.56)

1

22,917 ± 318

5 (0.59) (0.76) (0.58) 6 (0.87) 7 (0.58) (0.80) a Donors 1 to 3 are the same as shown in Table 2; donors 2a and 2b are studies on separate days on patient

Mean

2.

Day after addition of antigen and ACV on which [3H]thymidine was added. minute per filter with standard error; represents triplicate determinations on duplicate tubes at each ACV concentration. Incorporation in the presence of mock (uninfected tissue culture) antigen was a mean of 183 and was less than 312 for all patients. d Proliferative response in the presence of 20 ,uM ACV differed from the control value with P < 0.005 or smaller (t-test) except for donor 1, day 5 (P < 0.025) and days 6 and 7 (P < 0.05), and donor 5, days 5 and 7 (P < 0.01). e Proliferative response in the presence of 100,uM ACV differed from the control value with P < 0.001. f Numbers within parentheses represent fractional values compared with control tubes without ACV: [3H]thymidine incorporation at 20 or 100,uM ACV/[3H]thymidine incorporation with no ACV. 5 ND, Not done. '

C Mean counts per

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LEVIN, LEARY, AND ARBEIT

ANTIMICROB. AGENTS CHEMOTHER.

proliferation (Fig. 3) and could be demonstrated on a purified population of T lymphocytes (Table 5). These cells are the class of lymphocytes which proliferate in response to a specific antigen, whereas glass-adherent cells act as accessory cells to augment lymphocyte proliferation. ACV inhibited the purified lymphocytes to the same degree as the Ficoll-Hypaque-prepared PBM cells from which these subsets were defined (Table 5). Antigen-stimulated proliferation was optimal in cultures of purified lymphocytes supplemented with monocytes. ACV also inhibited the response of these cultures, but sometimes to a lesser degree than the other cell populations studied. These results support the hypothesis that ACV suppresses the in vitro resporse of PBM celLs by inhibiting the proliferation of antigen- or mitogen-stimulated T lymphocytes. Herpesvirus antigens also elicit interferon from PBM cells (12). ACV, even at 100lM, had

no detectable effect on the ability of PBM cells

to produce interferon (Table 6). Thus, although ACV inhibited proliferation, it was not generally toxic to lymphocytes since lymphokine production, as assessed by interferon, was not appreciably impaired. The metabolism of ACV in herpesvirus-infected fibroblasts proceeds through phosphorylation by the action of a virus-specified thymidine kinase before synthesis of the triphosphate, which is the active inhibitor of viral DNA polymerase (6). It has been postulated that uninfected human fibroblasts are protected from ACV because they phosphorylate ACV poorly and because cellular DNA polymerase has a low affinity for ACV triphosphate. However, our observations indicate that the metabolism of ACV may differ in rapidly dividing fibroblasts and clearly is different in proliferating human PBM cells. It is unlikely, though possible, that PBM cells contain a unique polymerase which is sen-

TABLE 5. Effect of ACV on the proliferative response of PBM cell subsets to herpesvirus antigens [3H]thymidine incorporationb with following antigen added: Subsete

VariceRla-zoster virus

Herpes simplex virus

Cytomegalovirus

No ACV

ACV (20 AM)c

ACV (100 uM)d

No ACV

ACV (20 AM)c

ACV (100 pM)

No ACV

ACV (20 uM)

ACV (100puM)

Ficoll-Hypaque

3,766

Lymphocyte plus monocyte'

5,572

3,814 5,420

2,090 4,392

31,999 34,414

22,917 28,424

17,962 19,603

20,218 24,826

18,699 22,922

11,663 16,487

Lymdphocytef

1,935 1,581 1,098 17,945 13,089 8,793 3,882 3,558 1,275 'Subsets consisted of PBM cells from the interface of a Ficoll-Hypaque gradient, nylon column-purified lymphocytes, or a mixture of purified lymphocytes with glass-adherent cells. b Mean counts per minute per filter, triplicate determinations on duplicate tubes at each ACV concentration. The standard deviation for these determinations was always less than 10% of the values shown. Incorporation in the presence of mock antigen wag 119 for Ficoll-Hypaque cells, 517 for lymphocytes plus monocytes, and 57 for lymphocytes alone. 'The proliferative response at 20 ,uM ACV differed from the control value with P < 0.01 or smaller except when FicollkHypaque or lymphocyte-plus-monocyte cultures were stimulated with varicella-zoster virus antigen (t-test). d The proliferative response in the presence of 100,uM ACV differed from the control value with P < 0.001 in all cases. e Percent inhibition of this subset by 100 ,uM ACV was the same as that for other subsets with varicella-zoster virus antigen and was less than that of other subsets with herpes simplex virus and cytomegalovirus antigens (P < 0.001; paired t-test). f Percent inhibition of this subset by 20 or 100MuM ACV was equal to or greater than the inhibition seen with the other subsets.

TABLE 6. Effect of ACV on the production of interferon by PBM cells exposed to herpesvirus antigens Interferon production (IU/ml of supernatant) induced by following antigen:

Herpes simplex virus

Subseta

Ficoli-Hypaque

Varicella-zoster virus

No ACV

ACV (20 pM)

ACV (100 pM)

No ACV

ACV (20 pM)

ACV (100 pM)

75

75

150 37 150

200 49 123

150 30 75

150 49 75

37 Lymphocyte plus monocyte 24 75 Lymphocyte 100 a Same subsets as described in Table 5, footnote a.

'

INHIBITION BY ACYCLOVIR OF HUMAN CELL DIVISION

VOL. 17, 1980 20

;N

1C

"C~

2.

18

3.

6

14

12

4.

0( O 10 20

40

60

80

100

Acyclovir (QM) FIG. 4. Dose-response curve of the effect of ACV on the proliferation of PBM cells. Cells from an immune donor were stimulated with varicella-zoster virus antigen. Each point represents triplicate samples from duplicate cultures harvested on day 5 after addition of antigen. The standard deviations are shown.

sitive to a product of ACV. It is more likely that the stimulated lymphocyte either is more permeable to ACV, contains new phosphorylating enzymes, or lacks enzymes which degrade ACV products. These alternatives are currently being examined by measuring intracellular concentrations of ACV and ACV nucleotides. The therapeutic index of systemically administered ACV for human infection is unknown, although herpes simplex virus and varicella-zoster virus are inhibited in vitro by ACV at concentrations which have been readily achieved in phase 1 trials (Burroughs Weilcome Co., personal communication). The observation that ACV inhibits proliferation of PBM cells emphasizes the need during clinical trials to monitor rapidly dividing tissues, such as bone marrow and gastrointestinal epithelium, for evidence of toxicity. ACKNOWLEDGMENTS This work was supported by Public Health Service grants CA-19589-04 from the National Cancer Institute and Al07061-03 from the National Institute of Allergy and Infectious Diseases and by funds provided by Burroughs Wellcome Co. and the Louis and Sidelle Bruckner Memorial Fund. We thank Barbara WilLhams for providing technical support for this work.

1.

LITERATURE CITED Armstrong, R. W., and T. C. Merigan. 1971. Varicellazoster virus: interferon production and comparative in-

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

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terferon sensitivity in human cell cultures. J. Gen. Virol. 12:53-54. Boyum, A. 1968. Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. Suppl. 97 21: 51-76. Crumpacker, C. S., L. E. Schnipper, J. A. Zaia, and M. J. Levin. 1979. Growth inhibition by acycloguanosine of herpesviruses isolated from human infections. Antimicrob. Agents Chemother. 15;642-645. De Clercq, E., J. Descamps, P. De Somer, P. J. Barr, A. S. Jones, and R. T. Walker, 1979. (E)-5-(2-Bromovinyl)-2'-deoxyuridine: a potent and selective antiherpes agent. Proc. Natl. Acad. Sci. U.S.A. 76M29472951. Einstein, P., E. E. Schneeberger, and IL It. Colton. 1976. Synthesis of the second component of complement by long term primary cultures of human monocytes. J. Exp. Med. 143:114-126. Elion, G. B., P. A. Furman, J. A. Fyfe, P. deMlran4a, L Beauchamp, and H. J. Schaeffer. 1977. Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl)guanine. Proc. Natl. Acad. Sci. U.S.A. 74:57165720. Field, H. J., S. E. Bell, G. B. Elion, A. A. Nash, and P. Wildy. 1979. Effect of acycloguanosine treatment on acute and latent herpes simplex infections in mice. Antimicrob. Agents Chemother. 16;554-561. Greaves, M. F., G. Janossy, and P. Curtis. 1976. Purification of human T lymphocytes using nylon fiber columns, p. 217-229. In B. P. Bloom and J. R. David (ed.), In vitro methods in cell-mediated and tumor immunity. Academic Press, Inc., New York. Havell, E. A., and J. Vil6ek. 1972. Production of hightitered interferon in cultures of human diploid cells. Antimicrob. Agents Chemother. 2:476-484. Jones, B. R., D. J. Coster, P. N. Fison, G. M. Thompson, L. M. Cobo, and M. G. Falcon. 1979. Efficacy of

acycloguanosine (Wellcome 248U) against herpes-simplex comeal ulcers. Lancet i:243-247. Klein, R. J., A. E. Friedman-Kien, and E. DeStefano. 1979. Latent herpes simplex virus infections in sensory ganglia of hairless mice prevented by acycloguanosine. Antimicrob. Agents Chemother. 15:723-729. Levi, M. J., KI Parkman, M. N. Oxman, J. M. Rappeport, M. Simpson, and P. L Leary. 1978. Proliferative and interferon responses by peripheral blood mononuclear cells after bone marrow transplantation in humans. Infect. Immun. 20:678-684. Rasmussen, L, and L. B. Farley. 1975. Inhibition of Herpesvirus homuzis replication by human interferon. Infect. Immun. 12:104-108. Schaeffer, H. J., L Beauchamp, P. deMiranda, G. B. Elion, D. J. Bauer, and P. Collins. 1978. 9-(2-Hydroxyethoxymethyl)guanine activity against viruses of the herpes group. Nature (London) 272:583-585. Schneider, W. C. 1945. Phosphorus compounds in animal tissues. I. Extraction and estimation of desoxypentose nucleic aci4 and of pentose nucleic acid. J. Biol. Chem. 161:293-303. Zaia, J. A., P. L. Leary, and M. J. Levin. 1978. Specificity of the blastogenic response of human mononuclear cells to herpesvirus antigens. Infect. Imamun. 20:646651.

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