Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[CANCER RESEARCH 42. 4039-4044, 0008-5472/82/0042-OOOOS02.00 October 1982] 3-Deazaguanine: Inhibition of Initiation of Translation in L1210 Cells1 Rachel S. Rivest,2 David Irwin, and H. George Mandel3 Departments of Pharmacology ¡R.S. R. H. G. M.] and Biochemistry [D. I.], The George Washington University Medical Center, Washington. D. C. 20037 ABSTRACT In L1210 cells in culture, 3-deazaguanine (DG), a relatively new purine analog, was found to inhibit DNA and protein synthesis but not total RNA synthesis. The effect of the drug on protein synthesis was therefore further examined. Polyadenylic acid-containing RNA synthesis was not decreased by DG treatment, suggesting that the inhibition of protein synthesis was a function of an alteration in the process of translation. DG altered the polyribosome sedimentation profile in a dose-de pendent manner, increasing the numbers of monosomes and smaller polysomes and decreasing the number of larger polysomes. The nascent polypeptides in DG-treated cells were labeled with [3H]leucine, and the increased number of monosomes was not associated with a proportionate amount of [3H]leucine when compared to the polysomes. This indicated that the monosomes had not been derived directly from the breakdown of active polysomes. The shift in the polysome profile was reversed by cycloheximide, suggesting that DG inhibited the initiation of translation. This was confirmed by the demonstration of the inhibition by DG of the formation of the 43S preinitiation complex. The inhibition of the initiation of protein synthesis by DG may contribute to the antitumor actions of this new purine analog. INTRODUCTION DG" is a relatively new purine analog with interesting antitumor, antiviral, and antibacterial activity (1, 3, 7, 9, 20, 22, 23). Although most of the still limited number of studies suggested a primary effect of DG on DNA synthesis (8, 20, 23) as well as alterations in the formation of purine intermediates (26), our investigations pointed to an inhibitory effect on protein synthe sis as well (15, 16). Since 2 other growth-inhibitory purine analogs also are known to affect the synthesis of proteins (reviewed in Ref. 17), we have investigated in greater detail the inhibition of protein synthesis by DG in L1210 cells in culture. The reversal by cycloheximide of the shifts in the polyribosome sedimentation profiles from DG-treated cells suggested that DG inhibited the initiation of translation. This was confirmed by a reduction in the binding of 35S-formylated methionine tRNA ' This investigation was supported by USPHS Grants CA-02978 and CA27553 and Training Grant 5-T-32-CA-09223. awarded by the National Cancer Institute, Department of Health & Human Services. 2 National Cancer Institute Trainee (Grant 5-T-32-CA-09223). Present ad dress: Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. 06510. From a dissertation presented to the Department of Pharmacology, The Graduate School of Arts and Sciences, The George Wash ington University, in partial fulfillment of the requirements for the Ph.D. 3 To whom requests for reprints should be addressed. * The abbreviations used are: DG, 3-deazaguanine: RPMI, Roswell Park Memorial Institute Tissue Culture Medium; HBSS, Hanks' balanced salt solution; poly(A)* RNA, polyadenylic acid-containing RNA; poly(A)" RNA, polyadenylic acid-lacking RNA; TCA, trichloroacetic acid; deaza-GTP, 3-deazaguanosine 5'triphosphate. Received November 17, 1981 ; accepted July 1, 1982. OCTOBER 1982 to the ribosomal initiation complex in L1210 cells treated with DG. MATERIALS AND METHODS Materials. DG was a gift from ICN Pharmaceuticals, Irvine, Calif. Cell culture supplies (RPMI 1640, fetal calf serum, streptomycin, penicillin G, and HBSS) were received from Grand Island Biological Co., Grand Island, N. Y. RPMI 1630 without methionine was from the Media Unit, NIH, Bethesda, Md. [metf7y/-3H]Thymidine (20 Ci/mmol), [5-3H]uridine (28 Ci/mmol), [3H]adenosine (35 Ci/mmol), ['"C]uridine (50 mCi/ mmol), i_-[4,5-3H]leucine (50 Ci/mmol), Aguasol, and Omnifluor were from New England Nuclear, Boston, Mass. i_-{35S]Methionine (1200 Ci/mmol) was obtained from Amersham/Searle Corp., Arlington Heights, III. Nonidet P-40 was from Particle Data Laboratories, Elmhurst, III. Type 2 and type 3 oligodeoxythymidylic acid-cellulose was purchased from Collaborative Research, Waltham, Mass. Cell Culture. The L1210 tumor line, obtained from Dr. Kurt Kohn, National Cancer Institute, Bethesda, Md., was maintained in RPMI 1640 supplemented with 10% dialyzed fetal calf serum. The medium contained 1 x 105 units penicillin G and 0.1 g streptomycin sulfate per liter of medium. Cells were maintained in suspension at 37° in a humidified incubator with a 5% C(X>atmosphere. Measurement of Viability. Viability of L1210 cells was measured by the colony formation assay (2). Cells were exposed for 5 hr to 5, 10, 15, 20, 30, 40, 50, or 500 ¡IMDG dissolved in the medium. The cells were washed with fresh medium and diluted in RPMI 1640 containing 20% dialyzed fetal calf serum. Three ml of RPMI 1640:20% dialyzed fetal calf serum containing 0.2% Noble agar at 44° were added to L1210 cells in 2 ml of RPM11640:20% dialyzed fetal calf serum, mixed thoroughly in a 15-ml tube, placed on ice for 2 to 4 min, and then incubated upright at 37°.The colonies were counted visually 10 to 12 days later. Measurement of Macromolecular Synthesis. The effect of DG on DNA, RNA, and protein synthesis was determined by the method of Mandel ef al. (11). The L1210 cells were exposed to 5, 50, or 500 fiM DG for 5 hr and then incubated with either [3H]thymidine, [3H]uridine, or [3H]leucine at 1 /iCi/ml. An equal volume of ice-cold 0.9% NaCI solution was added to each sample to stop the reaction after 60 min, and the samples were put on ice. The cell suspensions were filtered directly on Whatman GF/C filters wetted with 0.9% NaCI solution and washed with 5 volumes of 0.9% NaCI solution, 10 volumes of 0.2 N perchloric acid, and 5 volumes of 0.9% NaCI solution. All of the filtering solutions were ice cold. The filters were dried and counted in 0.4% Omnifluortoluene. To determine the effect of DG on poly(A)+ and poly(Ar RNA synthe sis, L1210 cells were exposed to 50 JJ.MDG for 5 hr and labeled during the last hr of drug treatment with either [3H]adenosine (10 /iCi/ml) or [14C]uridine (0.1 juCi/ml). The cells were chilled rapidly with ice-cold HBSS, centrifuged, and lysed with gentle vortexing in isotonic buffer (150 mw NaCI:10 mw Tris HCI, pH 7.4:10 mw MgCI2) containing 0.5% Nonidet P-40, a nonionic detergent. The crude nuclei were pelleted by centrifugation at 1000 x g for 5 min, and the supernatant was centri fuged at 10,000 x g for 15 min to obtain a postmitochondrial super natant. The supernatant was diluted in 10 volumes of 0.1 M NaCI:0.2% sodium dodecyl sulfate: 10 ITIM Tris, pH 9.0:10 ITIM EDTA and then extracted several times with an equal volume of phenol and of chlorofornrisoamyl alcohol (96:4). The last extraction was done with chloro- 4039 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. R. S. Rivest et al. form:isoamyl alcohol (96:4), and the RNA in the resultant aqueous phase was precipitated with 2 volumes of 95% ethanol overnight at -20°. was added with mixing, followed by 0.11 volume of 40% formaldehyde neutralized to pH 7 on the day of use. The solution was held on ice for a minimum of 30 min and was then layered over 9-ml preformed CsCI The pellet of RNA obtained after ethanol precipitation was resuspended in binding buffer (0.5 M NaCI:0.01 M Tris HCI, pH 7.5:0.5% sodium dodecyl sulfate) and applied to an oligodeoxythymidylic acid- gradients, 1.35 to 1.60 g/ml. The gradients were centrifuged for 40 hr at 27,000 rpm at 4°. Fractions were collected on ice, and 1 drop of cellulose column [0.25 g each of type 2 and type 3 oligodeoxythymi dylic acid-cellulose] equilibrated with binding buffer. After elution with 15 ml binding buffer to collect the poly(A)~ RNA, the poly(A)+ RNA was eluted with 15 ml of eluting buffer (0.01 M Tris HCI, pH 7.5:0.5% sodium dodecyl sulfate). Five M NaCI was added to the poly(A)* RNA 0.5% bovine serum albumin was added to each fraction as a carrier, followed by 5 volumes of cold 20% TCA. The precipitate was collected on Whatman GF/C filters and washed with 10% TCA, 5% TCA, water, and 95% ethanol. The filters were dried, and radioactivity was mea sured in 0.4% Omnifluortoluene. fraction to make a final concentration of 0.1 M NaCI. Two volumes of 95% ethanol were added, the RNA was precipitated overnight at —20°, RESULTS and the precipitate was collected by centrifugation at 10,000 x g for 20 min at -20°. The pellets were resuspended in 0.1 M NaCI:0.2% sodium dodecyl sulfate:! 0 mM Tris, pH 9.0:10 mM EDTA, and the aliquots were measured for radioactivity using Aquasol as the scintil lation cocktail. Polyribosome Sedimentation Profile Analysis. L1210 cells were exposed to 10, 30, 50, 100, or 500 ,UMDG for 5 hr. The cells were chilled rapidly with ice-cold HBSS, and the postmitochondrial super natant, isolated as described above, was layered over 35-ml 15 to 40% linear sucrose gradients made in lysing buffer without the Nonidet P- Viability and Macromolecular Synthesis. After treatment with 50 or 500 /ÕMDG for 5 hr, the viability of L1210 cells decreased to 25% of control. At such concentrations, DG did not inhibit [3H]uridine incorporation into nucleic acids, whereas [3H]thymidine and [3H]leucine incorporation was decreased to near 50% of control values (Chart 1). Although total RNA synthesis was not depressed by DG, the possibility of a de crease in mRNA synthesis, which could explain the inhibition of protein synthesis, needed to be excluded. Since this fraction 40. The gradients, centrifugea for 2.5 hr at 26,000 rpm in a SW 27 represents only a small proportion of RNA, such an effect might rotor, were pumped through a flow cell, and the absorbance at 260 nm have been masked in the previous studies. However, DG was was recorded continuously. shown to produce no significant effect on the synthesis of To rule out the possibility that the shift in the polysome profile was poly(A)+ RNA (mRNA), as measured by the incorporation of due to fragmentation of mRNA, L1210 cells were exposed to 50 /¿M [3H]adenosine (Table 1). Since measurement of the incorpo DG for 5 hr, and during the last 3 min of the incubation, the cells were ration of this precursor into poly(A)+ RNA actually measured pulse labeled with [3H]leucine (20 juCi/ml) to label the nascent polypeptide chains on the polysomes. The cells were chilled rapidly with HBSS, and the polysomes were displayed on sucrose gradients as described above. Fractions were collected, and the radioactivity was determined. The ability of cycloheximide to reverse the alterations in the polysome profile was determined by a modification of the method of Reichman and Penman (14). L1210 cells were divided into 4 cultures, of which 2 were control and 2 were exposed to 50 /¿M DG for 5 hr. One control and one DG culture were also exposed to cycloheximide (2 /ig/ ml) for the last 30 min of the incubation. The cells were chilled rapidly with HBSS, and the polysomes were displayed on sucrose gradients as described above. Initiation Complex Formation Assay. The formation of the 43S ribosomal preinitiation complex in drug-treated cells was determined the rate of 2 processes, i.e., the synthesis of mRNA and the addition of the poly(A)+ RNA tail, the RNA was labeled sepa rately with [14C]uridine to accurately measure only the synthesis of the body of the mRNA. Here again, the inhibition of protein synthesis by DG could not be explained by a depression in mRNA synthesis (Table 1). Polyribosome Sedimentation Profile Analysis. The polyribosome sedimentation profile from L1210 cells exposed to 50 120 by a modification of the method of Henshaw (5, 6). L1210 cells were labeled overnight with [3H]uridine (1 fiCi/ml) to uniformly label the RNA. The cells were resuspended in fresh medium and exposed to 50 ¡IM DG for 5 hr. The cells were centrifuged at 37°, and the pellet of 2 x 107 cells was resuspended in 0.5 ml of RPMI 1630 lacking methionine but containing DG. The cells were preincubated at 37°for 2 min and then labeled with [35S]methionine (200 ,uCi/ml) for 2 min. The reaction was stopped with 10 volumes of ice-cold HBSS, and an aliquot was removed to measure the incorporation of label into proteins by precip itation (see below). The rest of the sample of cells was lysed, and the postmitochondrial supernatant was isolated as described previously. The supernatant was layered over 12-ml linear 20 to 30% sucrose gradients made in 100 mM KCI, 2 mM magnesium acetate, 20 mM triethanolamine-HCI (pH 7.0), and 0.5 mM dithiothreitol. The gradients were centrifuged at 25,000 rpm for 16 hr at 4°in a SW 41 rotor. The 10~5 10'* 10'3 absorbance of the gradients was monitored, and the fractions contain ing the 40S to 45S ribosomal subunits were collected. Due to the poor resolving power of sucrose gradients, 40S subunits isolated from these gradients were contaminated by other ribonucleoprotein species and by [35S]methionine-labeled proteins. Chart 1. Effect of DG on macromolecular synthesis and viability. Cells were exposed to DG for 5 hr and then labeled for 1 hr with [3H]thymidine, [3H]uridine, or [3H]leucine. Incorporation of the isotopes into the acid-insoluble fraction was These species were separated further from each other with CsCI density gradients. To the fraction isolated from the sucrose gradients, 0.1 volume of 0.11 M morpholinopropane sulfonic acid-KOH (pH 7.0) determined, and the values presented are the mean of triplicate samples. The standard deviations were less than 2% of mean values. Top, effect of DG on cell viability, determined by the colony formation assay for the various drug concen trations. 4040 CONCENTRATION (M) CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. VOL. 42 Inhibition of Initiation by DG /IM DG for 5 hr showed increased numbers of monomers and small polysomes and a decrease in the number of large polysomes (Chart 2). The decrease in the polysome fraction, i.e., that fraction of total ribosomes engaged in translation, was dose-related (see Chart 2, inset), and the cytotoxic and polysomal effects of DG occurred at the same concentration range (10 to 50 fiM DG). One possible mechanism for this shift in the polysome profile caused by DG was fragmentation of active mRNA during ex traction. This possibility was examined by labeling the nascent polypeptides of cells (treated with 50 fiM DG) with [3H]leucine. The increased numbers of monosomes that resulted from DG treatment were not associated with a proportionate amount of [3H]leucine when compared to the amount of [3H]leucine as sociated with the polysomes (Chart 3). This indicated that the monosomes were nonfunctional and had not been derived directly from breakdown of active polysomes. This shift in the polysome profile caused by DG is similar to that resulting from impaired initiation of translation (14). Cycloheximide at low concentrations is known to inhibit the elonga tion step of translation without significantly affecting initiation, whereas at high doses both steps are inhibited (25). The slowing of elongation by low concentrations of cycloheximide would be expected to counteract the effect on the polysome Table 1 Effect of DG on polyW Mean ±S.D. 1Not significantly received 2 /ig cycloheximide per ml for the last 30 min of the incubation, and the polysome profile of each of these samples was determined. In control cells exposed to cycloheximide, the polysome sedimentation profile shows a reduction in the monosome peak and an increase in all sizes of the polysomes (Chart 4). Cyclo1.5 1.0 10 c 0.5 o to o l UJ 0.4 5 IT O î and poly(Ay RNA synthesis control) control) 84.5 ±7.3 101.0 ±5.7 79.8 ±5.7 91.5 ±7.16 10 Top 20 Bottom Chart 3. Effect of DG on labeling of nascent polypeptides. Cells were exposed to 50 /IM DG for 5 hr and during the last 3 min were pulse labeled with [3HJleucine. The polysomes were displayed on 15 to 40% sucrose gradients, fractions were collected, and radioactivities were determined. different from poly(A)* values. CONTROL CONTROL* VOLUME CYCLOHEXIMIDE (ml) Chart 2. Effect of DG on the polyribosome sedimentation profile. Cells were exposed to 50 JIM DG for 5 hr, and the polyribosomes were displayed on a 15 to 40% sucrose gradient. . drug-treated cells; , control cells. Inset, effect of DG on the polysome fraction. The polysome fraction was calculated by expressing the absorption of the polysomes (the area from dimers to bottom of gradient) as a percentage of the total ribosomal absorption (monomers to bottom of gradient). LD.»,,50% lethal dose. 1982 30 VOLUME (ML) — OCTOBER S 0.2 L1210 cells, exposed to 50 JIM DG for 5 hr. were labeled during the last hr of drug treatment with either [3H]adenosine or ("CJuridine. The RNA in the postmitochondrial supernatant was extracted and then separated into poly(A)* RNA and poly(Ar RNA fractions on oligodeoxythymidylic acid-cellulose columns. The incorporation of the isotopes into RNA was measured. poly(A)» RNA (% of poly(A) RNA (% of Label [3H]Adenosine ['"CJUridine profile of a drug which inhibits initiation, as was shown for 5azacytidine (14). The effects of a low concentration of cyclo heximide (2 /¿g/ml)on the polysome profile following DG treat ment were therefore examined. L1210 cells were divided into 4 cultures, 2 of which were untreated and 2 of which received 50 juM DG for hr. One control and one DG-treated culture VOLUME (ml) Chart 4. Effect of cycloheximide on the polyribosome sedimentation profile. Cells were exposed to 2 fig cycloheximide per ml for 30 min, and the polysomes were displayed on a 15 to 40% sucrose gradient. 4041 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. R. S. Rivest et al. heximide, which inhibited [3H]leucine incorporation into pro teins by 80% (16), prevented the analog-induced shift towards monosomes and returned the polysome fraction of DG-treated cells back toward that of control cells (Chart 5). In addition, although the relative size distribution of polysomes was not altered by cycloheximide in control cells, in DG-treated cells cycloheximide reversed the greater preponderance of smaller polysomes and increased the fraction of larger polysomes. The reversal by cycloheximide of the shift in the polysome sedi mentation profile from DG-treated cells further suggested that DG inhibited the initiation of translation. Initiation Complex Formation. The results obtained by the use of cycloheximide suggested but did not directly prove that DG inhibited the initiation of translation. In the subsequent experiments, formation of radiolabeled formylated methionine tRNA (the initiator aminoacyl tRNA species) and incorporation into the 43S and preinitiation complex served as the basis for the direct assay of initiating activity in DG-treated cells. L1210 cells therefore were grown overnight with [3H]uridine to uni The ratio of 35S to 3H counts associated 3000 - 1000 2000 o_ O - 500 — 1000 - formly label the RNA, were treated with 50 /XMDG for 5 hr, and then were exposed for 2 min to [35S]methionine. The 43S preinitiation complex thus contained the [35S]methionine, which had condensed with the initiator methionyl-tRNA. The 40 to 45S region isolated from sucrose gradients was then further displayed on CsCI gradients. The results of the effects of DG on the formation of the 43S preinitiation complex are shown in Chart 6. The various ribonucleoprotein species, shown in Chart 6, separated on the CsCI gradients are, from left to right, the 60S ribosomal subunit, the 80S ribosome and initiation complex, the 40S ribosomal subunit, and the 43S preinitiation complex. The 35S label was not associated with the first 3 species but was recovered around Fraction 30, which represents the 43S preinitiation complex, and around Fraction 40, which represents proteins. with the 43S peak decreased after DG treatment, indicating that DG inhibited the formation of the 43S preinitiation complex. These results, summarized in Table 2, show the agreement between the decrease in the incorporation of [35S]methionine 10 20 30 SAMPLE NUMBER 40 3000 - 2000 - O. O 1000 - 20 30 SAMPLE NUMBER DG Chart 6. Effect of DG on the formation of the 43S formylated methionine tRNA preinitiation complex. Cells labeled overnight with [3H]uridine were treated with 50 /IM DG for 5 hr and then were exposed for 2 min to [35S]methionine. The 40S DG+CYCLOHEXIMIDE subunits, isolated on sucrose gradients, were displayed on CsCI gradients. Fractions were treated as described in "Materials and Methods." The major tritium peaks represent, from left to right, the 60S, 80S, 40S (p = 1.52), and 43S (p = 1.44) ribonucleoprotein species, and the 35S activity was distributed mainly in 2 peaks, representing the 43S initiation complex (p = 1.44) and proteins (p = 1.30). Table 2 Inhibition by DG of formation of 43S preinitiation complex and of total protein synthesis LI 210 cells, labeled overnight with [3H]uridine, were treated with 50 ^M DG for 5 hr and then were exposed for 2 min to [35S]methionine. Aliquots were removed to measure [35S]methionine incorporation into proteins. The 40 to 45S region, isolated from sucrose gradients, was displayed on CsCI gradients. The ratios of 35S to 3H cpm in the 43S preinitiation complex fractions of the CsCI gradients were used as a measure of preinitiation complex formation. synthesis3 (% Experiment1 20 10 VOLUME (ml) of control)45 control)5133 30 Bottom Chart 5. Effect of cycloheximide on polyribosome sedimentation profile alter ations in DG-treated cells. Cells were exposed to 50 JIM DG for 5 hr and for the last 30 min were also exposed to 2 ng cycloheximide per ml. Polysomes were displayed on a 15 to 40% sucrose gradient. 4042 preinitiation com plex formation6 (% of 3749 2 3 4443S MeanProtein * [35S]Methionine incorporation into TCA-insoluble 0 35Scpm/3H cpm; TCA-precipitable material. CANCER 52 45 material. RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. VOL. 42 Inhibition of initiation by DG into total proteins and the diminished formation of the 43S preinitiation complex. Protein synthesis decreased to 44% of control, and the formation of the 43S preinitiation complex decreased to 45% of control after treatment with 50 /¿M DG. DISCUSSION DG inhibited protein synthesis and DMA synthesis in L1210 cells in culture, whereas RNA synthesis was not decreased. The effect of DG on DMA synthesis and the lack of effect of DG on RNA synthesis have been reported in L1210 and Chinese hamster ovary cells (20, 23), and it has been concluded that the effects of the drug on DNA synthesis were most closely associated with cytotoxicity (8). Although DG did not selectively decrease protein synthesis in Chinese hamster ovary cells (20), the inhibition of protein synthesis that we observed in L1210 cells correlated both with the dose and length of drug exposure (15). In addition, the inhibition of protein synthesis after treatment of the cells with 50 UM DG, as measured by the incorporation of [3H]leucine, [3H]tyrosine, and [3H]lysine into the acid-insoluble fraction, was 60, 59, and 55% of control, respectively (16). In later experi ments with [35S]methionine (see Table 2), a comparable inhi bition of protein synthesis was seen. Since no selective inhibi tion of any of the major cytoplasmic proteins from DG-treated cells could be observed by polyacrylamide gel electrophoresis (15) and since poly(A)+ RNA synhesis was not decreased by DG treatment, this suggested that the inhibition of protein synthesis was a function of an alteration in the process of translation. The shift in the polysome profile caused by DG was similar to that resulting from impaired initiation of translation. The possibility that the shift in the polysome profile was a result of fragmentation of active mRNA during extraction was elimi nated. The nascent polypeptides of cells treated with DG were labeled with [3H]leucine, and the increased numbers of monosomes that resulted from DG treatment were not associated with a proportionate increase in the amount of [3H]leucine when compared to the polysemes. This indicated that the monomers had not been derived directly from the breakdown of active polysomes. The slowing of elongation by low concentrations of cycloheximide would be expected to counteract the effect on the polysome profile of a drug which inhibits initiation, as was shown for 5-azacytidine (14). Treatment of control cells with cycloheximide indicated an increase in the loading of all mRNAs with ribosomes due to the decreased rate of elongation. Once on the mRNA, each ribosome took a longer time to travel along the message before it dissociated, leading to a uniform increase in the amount of ribosomal RNA associated with each size of mRNA. For DG-treated cells later exposed to cyclohex imide, the shift from lighter to heavier polysomes suggested that mRNAs had not been fully loaded with ribosomes in the presence of DG. When ribosomes attached at substantially lower rates than in controls because of a block in initiation, the slowing of elongation by cycloheximide then allowed more time for messages to become fully loaded with ribosomes. These results, which suggested that DG inhibited the initia tion of translation, were confirmed by the demonstration of the inhibition of the formation of the 43S preinitiation complex. The agreement between the decrease in the formation of the initi OCTOBER 1982 ation complex and the decrease in protein synthesis suggests that DG inhibits protein synthesis by inhibition of the initiation step of translation. In addition, the inhibition of the 80S initiation complex has also been demonstrated by us (17). However, the precise mechanism for the inhibition of the formation of the 43S preinitiation complex has not yet been ascertained. A block in translational initiation could result from interfer ence in the role of GTP which is an essential requirement for initiation. This is possible since DG needs to be metabolized to its nucleotide form for activity (9, 21) and since DG interferes with de novo purine biosynthesis (26). Therefore, the formation of deaza-GTP or a reduction in GTP concentrations could lead to an inhibition of initiation. The presence of deaza-GTP in tumor cells following treatment with DG has been reported (23), and direct measurements of DG-induced alterations of the pools of purine nucleotides are pending. However, another guanine analog, 8-azaguanine, has been shown by us to inhibit initiation in a manner similar to that of DG (17, 18), and its actions do not appear to be related to the levels of nucleotide triphosphates (10, 13, 19, 27). Thus, it is doubtful that the effect of DG in interfering in the initiation process is due to an effect involving deaza-GTP or diminished levels of GTP. DG could also block initiation by incorporation into any of the RNA species so as to alter their structure and function. DG was reported to be incorporated into nucleic acids (23), but this incorporation did not inhibit the synthesis of RNA in our system. There is no direct evidence to suggest that DG affects the function of mRNA either through incorporation into the body of the message or through interference with the 7-methylguanosinyl-5' cap modification of mRNA. However, 8-aza guanine, which also inhibits the initiation of translation in a manner indistinguishable from that of DG (17), probably acts by its incorporation into mRNA. The antitumor activity of 8azaguanine is most likely related to its ability to inhibit protein synthesis. However, DG was different from 8-azaguanine in that DG had a specific effect on DNA synthesis as well as protein synthesis. Classical inhibitors of protein synthesis in eukaryotes also decrease the rate of DNA synthesis with less of an immediate effect on RNA synthesis. In mammalian cells, cycloheximide does not inhibit total RNA synthesis at concentrations many times higher than those eliciting maximal depression of protein synthesis, and the RNA effect is delayed. The pattern of inhi bition of DNA synthesis resembles that of protein synthesis more closely (24). Low concentrations of emetine produce no appreciable depression of RNA synthesis, whereas protein and DNA formation are reduced to 10 and 20% of control values, respectively (4). Puromycin inhibits protein synthesis more than that of nucleic acids, and DNA synthesis appears to be more depressed than RNA synthesis (12). Of the 3 drugs, the pattern of inhibition of macromolecular biosynthesis produced by DG qualitatively resembles most closely that of emetine. The exact role of the inhibition of initiation of protein synthesis by DG in its antitumor action or even in the concurrent inhibition of DNA synthesis and subsequent cell death is still unresolved. REFERENCES 1. Allen, L. B.. Huffman. J. H.. Cook, P. D., Meyer. R. B., Robins, R. K., and Sldwell, R. W. Antiviral activity of 3-deazaguanine, 3-deazaguanosine, and 3-deazaguanylic acid. Antimicrob. Agents Chemothers., 12: 114-119. 1977. 2. Chu, M. Y., and Fischer, G. A. The incorporation of [3H]cytosine arabinoside 4043 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. R. S. Rivest et al. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. and its effect on murine leukemia cells (L5178Y). Biochem. Pharmacol., Õ7. 753-767, 1968. Cook, P. D., Allen, L. B.. Streeter, D. G., Huffman, J. H.. Sidwell. R. W., and Robins, R. K. Synthesis and antiviral and enzymatic studies of certain 3deazaguanines and their imidazolecarboxamide precursors. J. Med. Chem., 21: 1212-1218, 1978. Grollman, A. P. Inhibitors of protein biosynthesis. V. Effect of emetine on protein and nucleic acid biosynthesis in HeLa cells. J. Biol. Chem., 243: 4089-4094, 1968. Henshaw, E. C. CsCI equilibrium density gradient analysis of native ribosomal subunits(and ribosomes). Methods Enzymol., 59. 410-421, 1979. Henshaw. E. C. Assay of the binding of the ternary complex met-tRNAf. elF-2-GTP to the 40S ribosomal subunit by sucrose gradient and CsCI gradient analysis. Methods Enzymol., 60. 275-280, 1979. Khwaja, T. A., Kigwana, L., Meyer. R. B., Jr., and Robins, R. K. 3-Deazaguanine, a new purine analog exhibiting antitumor activity. Proc. Am. Assoc. Cancer Res.. »6:162, 1975. Khwaja, T. A., Momparler, L., Varven, J. C., and Mian, A. M. Studies on mechanism of antitumor activity of 3-deazaguanine (3DG). Proc. Am. Assoc. Cancer Res., 20. 152, 1979. Khwaja, T. A., and Varven. J. C. 3-Deazaguanine (3-DG), a potent inhibitor of mammary adenocarcinoma R3230AC and mammary adenocarcinoma 13762. Proc. Am. Assoc. Cancer Res., 17: 200, 1976. Klubes, P., and Mandel. H. G. Effects of purine nucleoside triphosphates on amino acid incorporation in a cell-free system from 8-azaguanine treated Bacillus cereus. Biochim. Biophys. Acta. Õ29. 594-600, 1966. Mandel, H. G., Connors, T. A., Melzack, D. H., and Merai, K. Studies on the mechanism of action of 5-aziridinyl-2,4-dinitrobenzamide in tumor cells. Cancer Res., 34: 275-280, 1974. Nathan, 0. Puromycin. In: D. Gottlieb and P. D. Shaw (eds.). Antibiotics I. Mechanism of Action, pp. 259-277. Berlin: Springer-Verlag, 1967. Nelson. J. A., Carpenter, J. W., Rose, L. M., and Adamson, D. J. Mechanisms of action of 6-thioguanine, 6-mercaptopurine, and 8-azaguanine. Cancer Res.. 35. 2872-2878. 1975. Reichman, M., and Penman. S. The mechanism of inhibition of protein synthesis by 5-azacytidine in HeLa cells. Biochim. Biophys. Acta, 324: 282- 4044 289. 1973. 15. Rivest. R. S.. Irwin, D., and Mandel, H. G. Inhibition of macromolecular synthesis by 3-deazaguanine (3DG) in mammalian tumor cells in vitro. Proc. Am. Assoc. Cancer Res., 21: 279. 1980. 16. Rivest, R. S., Irwin, D., and Mandel, H. G. Mechanism of protein synthesis inhibition by 3-deazaguanine (3DG) and related purine analogs in L1210 cells m vitro. Proc. Am. Assoc. Cancer Res., 22. 214, 1981. 17. Rivest, R. S., Irwin, D., and Mandel, H. G. Purine analogs revisited: interfer ence in protein formation. Adv. Enzyme. Regul., 20: 351-373, 1982 18. Rivest, R. S., Irwin, D., and Mandel, H. G. Inhibition of initiation of translation in L1210 cells of 8-azaguanine. Biochem. Pharmacol.. in press, 1982. 19. Roy. J. K.. Kvam, D. C., Dahl. J. L., and Parks, Jr., R. E. Effect of triphosphate nucleosides of 8-azaguanine, 6-thioguanine, and 6-mercaptopurine on amino acid incorporation in vitro into microsomal protein. J. Biol. Chem., 236. 1158-1162, 1961. 20. Saunders, P. P.. and Chao, L. Y. Mechanisms of action of 3-deazaguanine and 3-deazaguanosine in mammalian cells in vitro. Proc. Am. Assoc. Cancer Res., 20. 222, 1979. 21. Saunders, P. P., Chao, L. Y., Loo. T. L.. and Robins, R. K. Actions of 3deazaguanine and 3-deazaguanosine on variant lines of Chinese hamster ovary cells. Biochem. Pharmacol., 30. 2374-2376, 1981. 22. Saunders. P. P., Chao, L. Y., and Robins. R. K. Action of 3-deazaguanine in Escherichia coli. Mol. Pharmacol., 15: 691-697, 1979. 23. Schwartz, P.. Hammond, D.. and Khwaja, T. A. Biochemical pharmacology of 3-deazaguanine (3-DG). Proc. Am. Assoc. Cancer Res., 18: 153, 1977. 24. Sisler, H. D., and Siegel, M. R. Cycloheximide and other glutarimide anti biotics. In: D. Gottlieb and P. D. Shaw (eds.), Antibiotics I, Mechanism of Action, pp. 283-307. Berlin: Springer-Verlag, 1967. 25. Stanners, C. P. The effect of cycloheximide on polyribosomes from hamster cells. Biochem. Biophys. Res. Commun., 24: 758-764. 1966. 26. Streeter, D. G., and Koyama, H. H. D. Inhibition of purine nucleotide biosynthesis by 3-deazaguanine. its nucleoside and 5'-nucleotide. Biochem. Pharmacol., 25. 2413-2415, 1976. 27. Zimmerman, E. F., and Greenberg, S. A. Inhibition of protein synthesis by 8azaguanine. I. Effects on polyribosomes in HeLa cells. Mol. Pharmacol.. ÃŽ.-113-125, 1965. CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research. 42 3-Deazaguanine: Inhibition of Initiation of Translation in L1210 Cells Rachel S. Rivest, David Irwin and H. George Mandel Cancer Res 1982;42:4039-4044. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/42/10/4039 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.