VIROLOGY
89,4%-505
(1978)
3-Deazaadenosine, Inhibits Reproduction
an Inhibitor of Rous Chick
JOHN
*Laboratory tLaboratory
P. BADER,*
of Tumor of General
of Adenosylhomocysteine Hydrolase, Sarcoma Virus and Transformation of Embryo
Cells
NANCY R. BROWN,* PETER GUILIO L. CANTONIt
Virus Genetics, and Comparative
National Cancer Institute, Biochemistry, National Maryland 20014 Accepted
May
National Institute
K. CHIANG,?
Institutes of Mental
AND
of Health, and Health, Bethesda,
30, 1978
3-Deazaadenosine, a known potent inhibitor of adenosylhomocysteine hydrolase, exerts an inhibitory effect on the reproduction of Rous sarcoma virus (RSV-BH) and on the malignant transformation of chick embryo cells by this virus. The inhibitory effect is reversible. The increased capacity for glucose uptake, a biochemical characteristic of chick embryo cells transformed by RSV-BH, is inhibited by 3-deazaadenosine, and the ability of the infected cells to grow in suspension is blocked. After prolonged exposure to 3-deazaadenosine, the morphological phenotype characteristic of transformed cells largely disappeared, and transformed cells resembled noninfected cells. Deazaadenosine inhibits the reproduction of Sindbis virus, Newcastle disease virus, and vesicular stomatitis virus to a lesser degree than RSV-BH. Deazaadenosine, 0.1 n&f, has no effect on DNA or protein synthesis in cells, and only a slight effect on RNA synthesis. No incorporation of 3deaza[‘%]adenosine into cellular nucleic acids was found. Deazaadenosine produces an increase in the intracellular level of adenosylhomocysteine, with the concomitant appearance of a relatively large amount of 3-deazaadenosylhomocysteine; the ratio of intracellular adenosylmethionine to adenosylhomocysteine, or (adenosylhomocysteine + 3-deazaadenosylhomocysteine) is decreased from 150 to 19, and 1.4 respectively. It is postulated that 3. deazaadenosine inhibits virus activities by its ability to inhibit adenosylhomocvsteine hydrolase, resulting in an inhibition of methylation reaction(s) required for virus growth and replication. INTRODUCTION
5’-Deoxy-5’-isobutylthioadenosine, a synthetic analog of adenosylhomocysteine (AdoHcy), has been shown by Lederer and his colleagues to inhibit both the oncogenic transformation by chick embryo fibroblasts infected with Rous sarcoma virus and virus reproduction in culture (Robert-Gero et al., 1975). An inhibitory effect of 5’-deoxy-5’isobutylthioadenosine on polyoma virus replication in mouse embryo culture also has been demonstrated (Raies et al., 1976). These effects have been attributed to inhibition of RNA methylation, and evidence in support of this interpretation has been presented by Jacquemont and Huppert (1977) who demonstrated that under conditions which reversibly blocked multipli494 0042.6822/78/0892-0494$02.00/O Copyright 0 1978 by Academic Pres, Inc. All rights of reproduction in any form reserved.
cation of herpes simplex (type 1 virus), 5’deoxy-5’-isobutylthioadenosine inhibited the methylation of viral mRNA and, specifically, that of the 5’ cap. Chiang et al. (1977), in their survey of over 50 analogs of AdoHcy and of adenosine as potential inhibitors of AdoHcy hydrolase, have shown that 5’-deoxy-5’-isobutylthioadenosine is a good inhibitor of AdoHcy hydrolase (seealso Pierre et al., 1977). They also found that 3-deazaadenosine is nearly loo-fold more potent than 5’-deoxy-5’-isobutylthioadenosine as an inhibitor of this enzyme. It was, therefore, of interest to examine whether 3-deazaadenosine would have an effect on the growth and development of Rous sarcoma virus, or other viruses, in chick embryo cells.
Y-DEAZAADENOSINE MATERIALS
AND
ON
GROWTH
METHODS
3-Deazaadenosine was synthesized and supplied by Dr. John A. Montgomery of Southern Research Institute, Birmingham, Ala. Cells and viruses. Chick embryo cells were prepared from lo-day-old embryos and replated at 2- and S-day intervals. Cultures were grown in Eagle’s minimal essential medium (MEM)’ supplemented with dextrose (2 g/liter final concentration), sodium pyruvate (5 mM), 10% hydrated tryptose phosphate broth (Difco), 5% fetal bovine serum, penicillin (50 units/ml), streptomycin (50 pg/ml) tylosin (50 pg/ml), and gentamycin (20 pg/ml). When effects of 3deazaadenosine or other nucleosides were being tested, tryptose phosphate broth was eliminated from the medium. Cultures were maintained in humidified, COz-atmosphere incubators. The Bryan “high titer” strain of Rous sarcoma virus (RSV-BH), a transforming virus, is mixed with Rous-associated virus (RAVI), a subgroup A nontransforming avian leukosis virus. The mutant RSV-BHTa, also called tdBElBH, transformed infected cells at 37”, but when incubated at 41”, these infected cells revert to normal phenotype (Bader, 1972a). RSV-BH-Ta stocks also contain RAVI, but the presence of this virus has no effect on the transformation process. Infection of sparsely distributed cells, and development of infected cells into foci of transformed cells, proceeded as described previously (Bader, 1972b). Growth of cells suspended in nutrient, agar also has been described (Bader, 1967). Sindbis virus, vesicular stomatitis virus, and Newcastle disease virus were propagated in chick embryo cells. For experiments with these viruses, cells were grown to confluency before infection. In examining virus reproduction, infected cultures were maintained on MEM containing 2% fetal bovine serum and antibiotics. Viruses were assayed by plaque-forming ability to ’ Abbreviations used: MEM, Eagle’s minimal essential medium; RSV-BH, Bryan “high titer” strain of Row sarcoma virus; RSV-BH-Ta, temperature-sensitive mutant of RSV-BH; FFU, focus forming units; TCA, trichloroacetic acid.
AND
DEVELOPMENT
OF
VIRIJSES
495
chick embryo cells, using a nutrient agar overlay containing MEM, 2% fetal bovine serum, antibiotics, 1% agar, and 0.004% Neutral Red. Effects of 3-deazaadenosine were measured in the same medium. Incorporation of radioactive precursors. The rates of uptake of leucine and 2-deoxyn-glucose, used as an indicator of glucose uptake, were measured after rinsing the cell cultures in Dulbecco’s phosphate-buffered saline. 2-Deoxy-n-[3H(G)]glucose (1 @.X/ml) and [l-‘4C]leucine (0.2 pCi/ml) were mixed and diluted in phosphatebuffered saline, and uptake into cells during 10 min at 37” was measured simultaneously, as described earlier (Bader et al., 1976). Incorporation of [5-3H]uridine into nucleic acids and [l-‘4C]leucine into proteins was measured by exposing cells to the mixed labeled compounds for 30 min at 37”. Cultures were then rinsed, and 5% trichloracetic acid was added. Radioactivity in both acid-soluble and acid-precipitable fractions was determined. Incorporation of [5-3H]deoxycytidine into DNA and [2-14C]uridine into RNA was determined in a similar way. Determination of intracellular levels of
AdoMet,
AdoHcy,
and 3-deaza-AdoHcy.
Levels of AdoMet, AdoHcy, and 3-deazaAdoHcy were determined by high-pressure liquid chromatography using VYDAC cation exchanger (Hoffman, 1975). Chick embryo cells were grown in a Petri dish for 7 days with 10 ml of [35S]methionine (30.1 PCi) supplemented growth medium to achieve isotopic equilibrium. Each dish contained 10 million cells. After incubation with 3-deazaadenosine, the medium was poured off. Two milliliters of fresh MEM was added to each dish and the cells were scraped off with a rubber policeman. Two dishes of cells were combined and homogenized with 0.4 ml of 50% sulfosalicylic acid in a ground-glass homogenizer. The supernatant, after centrifugation at 20,000 g for 10 min, was used for chromatographic analysis The VYDAC cation column was calibrated with radioactive standards. The radioactive fractions, 1.2 ml each, were collected and radioactivity determined by using Aquasol (New England Nuclear) as scintillator. Standard 3-deaza-AdoHcy was synthesized by purified AdoHcy hydrolase (Chiang et al., 1977).
496
BADER
ET
AL.
RESULTS
Inhibition of focus formation by 3-deazaadenosine. The effects of 3-deazaadenosine on development of foci of virus-transformed cells was examined. When a cell culture is exposed to an average of lessthan one infectious particle per cell, development of detectable morphological transformation requires 4 or 5 days. During this interval, the initially infected cell will go through several divisions, and many, if not all of the daughter cells, eventually change morphology, appearing as a focus of transformed cells against a background of nontransformed cells (Temin and Rubin, 1958). Transformation induced by RNA tumor viruses can be divided into two easily distinguishable phases: 1) events before, and including the synthesis of viral DNA, and 2) events following and no longer requiring the synthesis of viral DNA (Bader, 1966). Synthesis of the DNA of RSV-BH begins within the first hour after infection (Bader, 1972b), and is required for no more than 8 hr. The effects of 3-deazaadenosine on focus formation depends on the time of its addition to chick embryo cells after infection. a) Addition of 3-deazaadenosine during the first 8 hr after infection, i.e., during the time critical to DNA synthesis, had no effect on subsequent transformation. b) Addition of 3-deazaadenosine to the culture medium for longer time intervals (24 hr) immediately following infection resulted in a moderate reduction in the number of foci observable after 7 days. However, a striking inhibition of focus formation was observed if 3-deazaadenosine was added 24 hr after infection, and left in the culture for the duration of the week (Tables 1 and 2). The effective concentration, 0.1 mM, was not toxic to the cells as evidenced by their normal growth over the next 7 days. In contrast, 2’-deoxyadenosine, which is known to inhibit DNA synthesis (Maley and Maley, 1960), reduced the number of foci to one-fifth that of the control when cells were exposed to 0.1 mM of this compound during the first 9 hr after infection (Table 2). Exposure of infected cells to 2’-deoxyadenosine (0.3 n&f) during the first 24 hr resulted in a substantial decrease in the number of foci developed
TABLE
1
EFFECT OF %DEAZAADENOSINE, DEOXYADENOSINE,ANDADENOSINE FORMATIONINDUCTEDBYRSV-BW Treatment hM
Duration
2’ON Focus of treatment
0 to 24 hr
24 hr to 7 days
l.oh
1.0
0.01 0.21 -
89,4%-505
(1978)
3-Deazaadenosine, Inhibits Reproduction
an Inhibitor of Rous Chick
JOHN
*Laboratory tLaboratory
P. BADER,*
of Tumor of General
of Adenosylhomocysteine Hydrolase, Sarcoma Virus and Transformation of Embryo
Cells
NANCY R. BROWN,* PETER GUILIO L. CANTONIt
Virus Genetics, and Comparative
National Cancer Institute, Biochemistry, National Maryland 20014 Accepted
May
National Institute
K. CHIANG,?
Institutes of Mental
AND
of Health, and Health, Bethesda,
30, 1978
3-Deazaadenosine, a known potent inhibitor of adenosylhomocysteine hydrolase, exerts an inhibitory effect on the reproduction of Rous sarcoma virus (RSV-BH) and on the malignant transformation of chick embryo cells by this virus. The inhibitory effect is reversible. The increased capacity for glucose uptake, a biochemical characteristic of chick embryo cells transformed by RSV-BH, is inhibited by 3-deazaadenosine, and the ability of the infected cells to grow in suspension is blocked. After prolonged exposure to 3-deazaadenosine, the morphological phenotype characteristic of transformed cells largely disappeared, and transformed cells resembled noninfected cells. Deazaadenosine inhibits the reproduction of Sindbis virus, Newcastle disease virus, and vesicular stomatitis virus to a lesser degree than RSV-BH. Deazaadenosine, 0.1 n&f, has no effect on DNA or protein synthesis in cells, and only a slight effect on RNA synthesis. No incorporation of 3deaza[‘%]adenosine into cellular nucleic acids was found. Deazaadenosine produces an increase in the intracellular level of adenosylhomocysteine, with the concomitant appearance of a relatively large amount of 3-deazaadenosylhomocysteine; the ratio of intracellular adenosylmethionine to adenosylhomocysteine, or (adenosylhomocysteine + 3-deazaadenosylhomocysteine) is decreased from 150 to 19, and 1.4 respectively. It is postulated that 3. deazaadenosine inhibits virus activities by its ability to inhibit adenosylhomocvsteine hydrolase, resulting in an inhibition of methylation reaction(s) required for virus growth and replication. INTRODUCTION
5’-Deoxy-5’-isobutylthioadenosine, a synthetic analog of adenosylhomocysteine (AdoHcy), has been shown by Lederer and his colleagues to inhibit both the oncogenic transformation by chick embryo fibroblasts infected with Rous sarcoma virus and virus reproduction in culture (Robert-Gero et al., 1975). An inhibitory effect of 5’-deoxy-5’isobutylthioadenosine on polyoma virus replication in mouse embryo culture also has been demonstrated (Raies et al., 1976). These effects have been attributed to inhibition of RNA methylation, and evidence in support of this interpretation has been presented by Jacquemont and Huppert (1977) who demonstrated that under conditions which reversibly blocked multipli494 0042.6822/78/0892-0494$02.00/O Copyright 0 1978 by Academic Pres, Inc. All rights of reproduction in any form reserved.
cation of herpes simplex (type 1 virus), 5’deoxy-5’-isobutylthioadenosine inhibited the methylation of viral mRNA and, specifically, that of the 5’ cap. Chiang et al. (1977), in their survey of over 50 analogs of AdoHcy and of adenosine as potential inhibitors of AdoHcy hydrolase, have shown that 5’-deoxy-5’-isobutylthioadenosine is a good inhibitor of AdoHcy hydrolase (seealso Pierre et al., 1977). They also found that 3-deazaadenosine is nearly loo-fold more potent than 5’-deoxy-5’-isobutylthioadenosine as an inhibitor of this enzyme. It was, therefore, of interest to examine whether 3-deazaadenosine would have an effect on the growth and development of Rous sarcoma virus, or other viruses, in chick embryo cells.
Y-DEAZAADENOSINE MATERIALS
AND
ON
GROWTH
METHODS
3-Deazaadenosine was synthesized and supplied by Dr. John A. Montgomery of Southern Research Institute, Birmingham, Ala. Cells and viruses. Chick embryo cells were prepared from lo-day-old embryos and replated at 2- and S-day intervals. Cultures were grown in Eagle’s minimal essential medium (MEM)’ supplemented with dextrose (2 g/liter final concentration), sodium pyruvate (5 mM), 10% hydrated tryptose phosphate broth (Difco), 5% fetal bovine serum, penicillin (50 units/ml), streptomycin (50 pg/ml) tylosin (50 pg/ml), and gentamycin (20 pg/ml). When effects of 3deazaadenosine or other nucleosides were being tested, tryptose phosphate broth was eliminated from the medium. Cultures were maintained in humidified, COz-atmosphere incubators. The Bryan “high titer” strain of Rous sarcoma virus (RSV-BH), a transforming virus, is mixed with Rous-associated virus (RAVI), a subgroup A nontransforming avian leukosis virus. The mutant RSV-BHTa, also called tdBElBH, transformed infected cells at 37”, but when incubated at 41”, these infected cells revert to normal phenotype (Bader, 1972a). RSV-BH-Ta stocks also contain RAVI, but the presence of this virus has no effect on the transformation process. Infection of sparsely distributed cells, and development of infected cells into foci of transformed cells, proceeded as described previously (Bader, 1972b). Growth of cells suspended in nutrient, agar also has been described (Bader, 1967). Sindbis virus, vesicular stomatitis virus, and Newcastle disease virus were propagated in chick embryo cells. For experiments with these viruses, cells were grown to confluency before infection. In examining virus reproduction, infected cultures were maintained on MEM containing 2% fetal bovine serum and antibiotics. Viruses were assayed by plaque-forming ability to ’ Abbreviations used: MEM, Eagle’s minimal essential medium; RSV-BH, Bryan “high titer” strain of Row sarcoma virus; RSV-BH-Ta, temperature-sensitive mutant of RSV-BH; FFU, focus forming units; TCA, trichloroacetic acid.
AND
DEVELOPMENT
OF
VIRIJSES
495
chick embryo cells, using a nutrient agar overlay containing MEM, 2% fetal bovine serum, antibiotics, 1% agar, and 0.004% Neutral Red. Effects of 3-deazaadenosine were measured in the same medium. Incorporation of radioactive precursors. The rates of uptake of leucine and 2-deoxyn-glucose, used as an indicator of glucose uptake, were measured after rinsing the cell cultures in Dulbecco’s phosphate-buffered saline. 2-Deoxy-n-[3H(G)]glucose (1 @.X/ml) and [l-‘4C]leucine (0.2 pCi/ml) were mixed and diluted in phosphatebuffered saline, and uptake into cells during 10 min at 37” was measured simultaneously, as described earlier (Bader et al., 1976). Incorporation of [5-3H]uridine into nucleic acids and [l-‘4C]leucine into proteins was measured by exposing cells to the mixed labeled compounds for 30 min at 37”. Cultures were then rinsed, and 5% trichloracetic acid was added. Radioactivity in both acid-soluble and acid-precipitable fractions was determined. Incorporation of [5-3H]deoxycytidine into DNA and [2-14C]uridine into RNA was determined in a similar way. Determination of intracellular levels of
AdoMet,
AdoHcy,
and 3-deaza-AdoHcy.
Levels of AdoMet, AdoHcy, and 3-deazaAdoHcy were determined by high-pressure liquid chromatography using VYDAC cation exchanger (Hoffman, 1975). Chick embryo cells were grown in a Petri dish for 7 days with 10 ml of [35S]methionine (30.1 PCi) supplemented growth medium to achieve isotopic equilibrium. Each dish contained 10 million cells. After incubation with 3-deazaadenosine, the medium was poured off. Two milliliters of fresh MEM was added to each dish and the cells were scraped off with a rubber policeman. Two dishes of cells were combined and homogenized with 0.4 ml of 50% sulfosalicylic acid in a ground-glass homogenizer. The supernatant, after centrifugation at 20,000 g for 10 min, was used for chromatographic analysis The VYDAC cation column was calibrated with radioactive standards. The radioactive fractions, 1.2 ml each, were collected and radioactivity determined by using Aquasol (New England Nuclear) as scintillator. Standard 3-deaza-AdoHcy was synthesized by purified AdoHcy hydrolase (Chiang et al., 1977).
496
BADER
ET
AL.
RESULTS
Inhibition of focus formation by 3-deazaadenosine. The effects of 3-deazaadenosine on development of foci of virus-transformed cells was examined. When a cell culture is exposed to an average of lessthan one infectious particle per cell, development of detectable morphological transformation requires 4 or 5 days. During this interval, the initially infected cell will go through several divisions, and many, if not all of the daughter cells, eventually change morphology, appearing as a focus of transformed cells against a background of nontransformed cells (Temin and Rubin, 1958). Transformation induced by RNA tumor viruses can be divided into two easily distinguishable phases: 1) events before, and including the synthesis of viral DNA, and 2) events following and no longer requiring the synthesis of viral DNA (Bader, 1966). Synthesis of the DNA of RSV-BH begins within the first hour after infection (Bader, 1972b), and is required for no more than 8 hr. The effects of 3-deazaadenosine on focus formation depends on the time of its addition to chick embryo cells after infection. a) Addition of 3-deazaadenosine during the first 8 hr after infection, i.e., during the time critical to DNA synthesis, had no effect on subsequent transformation. b) Addition of 3-deazaadenosine to the culture medium for longer time intervals (24 hr) immediately following infection resulted in a moderate reduction in the number of foci observable after 7 days. However, a striking inhibition of focus formation was observed if 3-deazaadenosine was added 24 hr after infection, and left in the culture for the duration of the week (Tables 1 and 2). The effective concentration, 0.1 mM, was not toxic to the cells as evidenced by their normal growth over the next 7 days. In contrast, 2’-deoxyadenosine, which is known to inhibit DNA synthesis (Maley and Maley, 1960), reduced the number of foci to one-fifth that of the control when cells were exposed to 0.1 mM of this compound during the first 9 hr after infection (Table 2). Exposure of infected cells to 2’-deoxyadenosine (0.3 n&f) during the first 24 hr resulted in a substantial decrease in the number of foci developed
TABLE
1
EFFECT OF %DEAZAADENOSINE, DEOXYADENOSINE,ANDADENOSINE FORMATIONINDUCTEDBYRSV-BW Treatment hM
Duration
2’ON Focus of treatment
0 to 24 hr
24 hr to 7 days
l.oh
1.0
0.01 0.21 -
