Mechanism of Action of Inosine Dialdehyde (NSC 118994) in the Inhibition of Proliferation of Tumor Cells in Culture1

[CANCER RESEARCH 37, 2188-2195. July 1977]

Peter G. W. Plagemann, Jon C. Graff, and Marsha Behrens

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

SUMMARY entered phase I clinical trials (Ref 9; V. H. Bono and D. Abraham, personal communication). INOX inhibits the syn Inosine dialdehyde (INOX), the periodate oxidation prod thesis of DNA and RNA in cultured L12104 and Ehrlich uct of inosine, inhibited the proliferation of various tumor ascites cells (4), but its mode of action is still poorly under cell lines in suspension culture in a concentration-depend stood. Recently, it has been shown that INOX, like other ent manner. A concentration of about 1 mM was required to similar dialdehyde derivatives, inhibits the ribonucleotide completely inhibit the proliferation of Novikoff rat hepatoma reducÃaseactivity in cell-free extracts of Ehrlich ascites and mouse L-cells, whereas about 0.1 mM completely in cells, and it was suggested that the inhibition of the reduc- hibited the proliferation of L1210 and P388 mouse leukemia tase step is 1 of the major sites of inhibition by INOX (2, 4). and Chinese hamster ovary cells. INOX inhibited in a similar Our results with cultured Novikoff cells are not consistent time- and concentration-dependent manner the synthesis of with the view that an inhibition of the reductase plays any protein, RNA, and DNA, as measured by the incorporation specific role in the inhibition of tumor cell replication. On of labeled amino acid, uridine, and thymidine, into acid- the contrary, they indicate a general effect of INOX on insoluble material, without significantly affecting the incor macromolecular synthesis, since the syntheses of RNA, poration of these precursors into the acid-soluble pool. DNA, and protein are inhibited by INOX in a similar time- Flow microfluorometric analyses showed that many of the and concentration-dependent manner, and many of the INOX-treated cells became arrested in G-2+ M. The results cells become arrested in G-, + M. are consistent with the view that INOX affects multiple meta bolic steps. The effects of INOX were quite different from MATERIALS AND METHODS those caused by typical inhibitors of ribonucleotide reduc Ãase, hydroxyurea, and 2,3-dihydro-1H-pyrazolo(2,3- Cell Cultures. Wild-type and azaguanine-resistant (21) a)imidazole, which very rapidly inhibited DNA synthesis and Novikoff rat hepatoma cells (subline N1S1-67) and mouse L- caused arrest of the cells in G,, with minimal effects on RNA cells were propagated in suspension culture in Swim's Me and protein synthesis. dium 67 and enumerated and analyzed for cell volume by means of a Coulter counter as described previously (7, 20). Chinese hamster ovary cells and L1210 and P388 mouse INTRODUCTION leukemia cells were propagated in the same manner, except that the growth medium was Eagle's minimal essential me INOX2(NSC 118994) is the periodate oxidation product of dium for suspension culture (with spinner salts) supple inosine and is 1 of the more potent inhibitors of the prolifer mented with 10% (v/v) heat-inactivated (57Â°,30 min) fetal ation of various tumor cells in vivo and in vitro among a calf serum. The medium was prepared as follows. Pow number of periodate oxidation products of purine and pyrimidine ribonucleosides (1, 3, 4, 19).3~5It is therefore of dered Medium F-14 (52.9 g; Grand Island Biological Co., Grand Island, N. Y.) was dissolved in 4870 ml distilled water interest as a potential anticancer agent and has recently plus 125 ml of 8.14% (w/v) NaHCO:1and 5 ml of a solution containing 62.5 mg Penicillin G and 12.5 mg streptomycin sulfate per ml. The medium was sterilized by filtration 1This work was supported by USPHS Research Grant CA 16228 and by Contract NOI-CM-43788 from the Division of Cancer Treatment, National through a 0.2 Selas filter (bubble pressure, 25 Ibs/sq inch; Cancer Institute, NIH, Department of Health, Education, and Welfare. Selas Flotronics, Spring House, Pa.) and supplemented 2The abbreviations used are: INOX, inosine dialdehyde; imidazopyrazole, 2,3-dihydro-1H-pyrazolo(2,3-a)imidazole. aseptically with fetal calf serum. L1210 and P388 cells were 3Screening data, Division of Cancer Treatment, National Cancer Institute. generously supplied by Arthur Little and Co., Acorn Park, 4 Progress Reports 5 and 7, submitted by Dr. L. L. Bennet, Jr., and Dr. R. Cambridge, Mass., and Chinese hamster cells were sup W. Brockman of the Southern Research Institute, Birminghan, Ala., to the Division of Cancer Treatment under Contract NOI-CM-43784. plied by Dr. V. Ling, Ontario Cancer Institute, Toronto, * Personal communication from Dr. V. H. Bono and Dr. D. Abraham, Ontario, Canada. Molecular Biology and Methods Development Section, Laboratory of Precursor Incorporation. Cells were collected by centrif- Medicinal Chemistry and Biology, Division of Cancer Treatment, National Cancer Institute, NIH. ugation at about 200 x g for 1 to 2 min and suspended in a Received January 31, 1977; accepted April 15, 1977. basal medium, BM42B (16) or leucine-free BM42B, and

2188 CANCER RESEARCH VOL. 37

supplemented with labeled precursors as indicated in the from Schwarz/Mann, Orangeburg, N. Y.; and 3H-labeled appropriate experiments. The suspensions were incubated reconstituted protein hydrolysate was from New England on a gyratory shaker at about 200 rpm, and duplicate 1-ml Nuclear, Boston, Mass. INOX (Wyeth, Lot 4467-63-1) and samples were analyzed for radioactivity in total cell material imidazopyrazole (NSC 51143) were supplied by the Division (acid-soluble plus acid-insoluble) or for radioactivity in acid- of Cancer Treatment, National Cancer Institute. [8-14C] insoluble material by washing the cells once in balanced INOX (4 mCi/mmole) was supplied by the Stanford Re salt solution or repeatedly in cold perchloric and trichloroa- search Institute, Menlo Park, Calif., through National Can cetic acid, respectively (14, 16). Cells labeled with amino cer Institute Contract NOI-CM-43788. The compositions of acids were heated in perchloric acid at 70Â°for30 min before Chromatographie Solvents 9, 18, 28, 30, and 34 have been further acid washes (hot acid-insoluble material; Ref. 20). described previously (14, 16). All values presented are averages of the duplicate samples. Samples of cells labeled with uridine or thymidine were also RESULTS extracted with perchloric acid, and the acid extracts were analyzed chromatographically with Solvent 28 as described Chart 1 illustrates the time- and concentration-dependent previously (14, 16). inhibition of the proliferation of Novikoff cells by INOX. Other Methods. Other methods used have been de Relatively high concentrations of INOX (>1 mM) were re scribed previously: fixation and RNase treatment of cells quired to inhibit cell proliferation rapidly and completely. and analysis in a flow microfluorometer (11), quantitation of The inhibition caused by higher concentrations of INOX was polyribosomes by centrifugation of cell extracts in sucrose- not reversed by removal of the drug from the medium. No density gradients (13), the extraction of cellular RNA, analy further cell replication was observed when, after 8 hr of sis of the RNA by zone sedimentation in sucrose-density incubation with 1.66 mM INOX, the cells were collected by gradients, alkali hydrolysis of the RNA, and electrophoretic centrifugation and resuspended in fresh medium. The cells separation of the nucleoside monophosphates (13, 14, 16). remained nonstainable by trypan blue for about 24 hr, but Materials. Unlabeled nucleosides, hydroxyurea, and cy- then the proportion of stainable cells increased progres tosine-/3-D-arabinoside were purchased from Sigma Chemi sively with further incubation (data not shown). The prolifer cal Co., St. Louis, Mo.; colcemid was obtained from Grand ation of an azaguanine-resistant [hypoxanthine-guanine Island Biological Co.; vinblastine sulfate (Velban) was from phosphoribosyltransferase deficient; (21)] subline of N1S1- Eli Lilly and Co., Indianapolis, Inc.; [5-3H]uridine and 67 cells was inhibited by INOX to about the same extent as [methyl-3H]thymidine were from Amersham/Searle Corp., that of wild-type cells (Chart 1). The average cell volume of Arlington Heights, III.; [8-3H]guanine and [3H]leucine were the cells increased markedly during incubation with INOX

Chart 1. Effect of INOX on the proliferation of wild- type (WT) and azaguanine-resistant (AG') N1S1-67 cells. Exponential-phase cultures of the appropriate cell line were supplemented with the indicated con centrations of INOX, incubated at 37Â°on a gyratory shaker at about 200 rpm, and monitored for cell density.

60 0 TIME (HR)

JULY 1977 2189

(Chart 2). For instance, the intact cells remaining after 23 hr strate, therefore, that INOX caused a similar time- and con incubation with 1.66 rriM INOX were on the average about centration-dependent inhibition of protein, RNA, and DNA twice as large as untreated cells. Some INOX-treated cells, synthesis. In several additional experiments, however, the however, had lysed at this time as indicated by the presence incorporation of thymidine and uridine into acid-insoluble of small fragments in the suspension (50 to 500 cu Â¿im)and material was slightly more inhibited by INOX than was leu- a decrease in cell density (see Chart 1A). cine incorporation (see, for instance, Chart 6, presented Chart 3, A to C, illustrates the time- and concentration- later). It should also be noted that precursor incorporation dependent inhibition by INOX of the incorporation of amino was measured in medium not containing INOX, but the acids, uridine, and thymidine into macromolecules (acid- extent of the inhibition was about the same when INOX was insoluble material). The data in Chart 3D, on the other hand, present during the labeling period (data not shown). Thus show that the incorporation of amino acids and uridine into the inhibition of macromolecular synthesis was not readily the acid-soluble pool was not significantly affected by INOX, reversed by removal of the INOX from the medium. Further even after 7 hr incubation. The incorporation of thymidine more, after 6 hr incubation with 1.66 mw INOX, over 90% of into the acid-soluble pool was reduced by the INOX the polyribosomes in the cell had been dissociated, and few treatment (Chart 3D), but this effect is typical of all inhibi reformed after removal of INOX from the medium (data not tors of DNA replication (see Chart 4 and Refs. 15 and 16) shown). and reflects the limited size and expandability of the dTTP The effects of INOX on macromolecular synthesis were pool as well as the rapid turnover of dTTP. Most of the quite different from those observed with typical inhibitors of radioactivity in the acid-soluble pool of uridine- and thymi- ribonucleotide reducÃase, such as hydroxyurea (12) and dine-labeled cells was associated with UTP plus UDP-hex- imidazopyrazole." These inhibitors had little effect on leu- oses and dTTP, respectively, (data not shown), just as in cine and uridine incorporation, even after 6.5 hr incubation untreated cells (14-16). Thus the inhibitions of amino acids, with the inhibitors (the data for hydroxyurea are shown in uridine, and thymidine incorporation into acid-insoluble Chart 4, A and B), whereas the incorporation of thymidine material by INOX reflected inhibitions of protein, RNA, and into DNA was inhibited by these inhibitors within a few min DNA synthesis, respectively. The results in Chart 3 demon of treatment (Chart 4C). The selective inhibition of DNA

,/ : \ 0.33 088 \ .... 083 050 \:\- 1.66 020 'Â«'â€¢ \ â€¢. \ \\\

IOOO 2000 3000 4000 CELL VOLUME (cuum)

Chart 2. Effect of INOX on the volume of wild-type N1S1-67 cells. The details of the experiment are described in the legend to Chart 1. After 23 hr propagation in the presence of the indicated concentrations of INOX (see growth curves, Chart ^A), samples of cells were incubated with trypsin and then analyzed for cell volume distribution by means of a Coulter counter attached to a channelizer (7).

2190 CANCER RESEARCH VOL. 37

synthesis by cytosine arabinoside, by a mechanism other activity from [8-14C]INOX would be via its degradation by than an inhibition of the reducÃase(8), had equally little nucleoside phosphorylase to hypoxanthine and the dial- effect on protein and RNA synthesis (Chart 4, A to C). The dehyde derivative of ribose. Such degradation occurs very average volume of hydroxyurea-treated cells also became rapidly in the case of inosine and deoxyinosine in Novikoff greater than that of untreated cells, probably due to the cells (16) but seems to be minimal for INOX. The results in synthesis of RNA and protein in the absence of DNA synthe Chart 7 are additional evidence that any degradation of sis, and Colcemid, which arrests cells in metaphase, had a INOX, if it occurs, must be very slow when compared to that similar effect (data not shown). The results raised the possi of inosine or deoxyinosine. Suspensions of cells were in bility that INOX might cause an arrest of the cells shortly cubated with or without 250 /AM cordycepin or 1.66 mM before cell division. This conclusion was confirmed by di INOX, and, at 0 time and after 1 hr of incubation at 37Â°, rect analysis by flow microfluorometry of the DNA content samples of the suspensions were monitored for the incorpo of individual cells (Chart 5). After 6 hr incubation with 0.83 ration of [3H]guanine (at 0.5 /AM) into total cell material. rriM INOX, a large proportion of cells had become arrested Cordycepin, like adenosine and deoxyadenosine (16), is in G2 + M (Chart 50). The proportion of S-phase cells, rapidly deaminated by Novikoff cells and further degraded however, was also slightly increased when compared to the to hypoxanthine and 3-deoxyribose-1-phosphate (P. G. W. control cells (Chart 5A). After a similar time of exposure to Plagemann, unpublished data). This formation of hypo Colcemid, 80 to 90% of the cells possessed a 2 S DNA xanthine resulted in a rapid inhibition of the incorporation content, and the remainder of the cells were in S phase of labeled guanine (Chart 7), mainly due to the competition (Chart 5C). Incubation with 1 /J.Mvinblastine sulfate had of hypoxanthine with guanine in the phosphoribosyltrans- about the same effect (data not shown). In contrast, most of ferase reaction. INOX, on the other hand, had little effect the cells possessed a 1 S DNA content after treatment with on the incorporation of [3H]guanine (Chart 7). hydroxyurea and thus were arrested in G, (Chart 5D). We also investigated the effect of INOX on the prolifera The inhibition of macromolecular synthesis by INOX, tion of a number of other cell lines. Mouse L-cells exhibited however, was not only a consequence of an arrest of the about the same sensitivity to INOX as Novikoff cells, cells in M phase as caused by Colcemid or other spindle whereas Chinese hamster ovary cells and L1210 and P388 inhibitors. First, the effect of spindle inhibitors is readily mouse leukemia cells were about 10 times more sensitive. reversible, whereas the inhibition of INOX was not. Second, Replication of the latter 3 cell lines was inhibited 80 to 90% the incorporation of leucine into protein was little affected by 0.083 mM and completely by 0.33 mM INOX. Neverthe by a 6-hr incubation with 1 /UM Colcemid or vinblastine less, limited experiments with P388 and L1210 cells demon sulfate, and the incorporation of uridine was inhibited far strated that the pattern of INOX inhibition of precursor less than by a 6-hr incubation with INOX (Chart 6, A and B). incorporation in these cells was similar to that observed Even thymidine incorporation was reduced less by Col with Novikoff cells, and a similar proportion of the cells cemid and vinblastine sulfate than by INOX treatment (Chart became arrested in G2 + M, except that the effects were 6C), even though most of the cells were arrested in M phase observed with lower concentrations of INOX (data not (see Chart 5C). Thymidine incorporation by these cells must shown). have been due to the proportion of cells still progressing through S (see Chart 5C) and to repair synthesis. The time DISCUSSION courses of incorporation of leucine, uridine, and thymidine into the acid-soluble pool by Colcemid- or vinblastine sul- Our finding that INOX inhibits in a similar time- and con fate-treated cells were about the same as those of untreated centration-dependent manner the synthesis of protein, cells (data not shown; see Chart 30). RNA, and DNA by a number of tumor cell lines in culture and Attempts to determine the fate of the INOX added to cell causes the arrest of a large proportion of the cells in G2+ M suspensions by the use of [8-14C]INOX (25 /J.M, 8 cpm/ is inconsistent with the view that the inhibition of prolifera pmole) were somewhat equivocal. About 4% of the total tion is mainly due to an inhibition of ribonucleotide reduc radioactivity was rapidly incorporated into acid-soluble plus Ãase.An about equal inhibilion of RNA and DNA synlhesis acid-insoluble components by the cells within the 1st hr of by INOX has also been observed in L1210" and Ehrlich incubation, and the labeling patterns of nucleotides in the asciles cells (4), and prolein synthesis was also inhibited in acid-soluble pool and of GMP and AMP in cellular RNA's L1210 cells.4 The periodale oxidalion produci of /3-D-ribo- (data not shown) were similar to those obtained when label syl-6-methyllhiopurine also causes an Â¡nhibilion of protein, ing with hypoxanthine or inosine (16). However, we attri RNA, and DNA synlhesis in L1210 cells when propagaled in bute this initial incorporation to the incorporation of small mice (1). A G2+ M block caused by INOX has been observed amounts of labeled inosine and/or hypoxanthine present as with additional cell lines (V. H. Bono and D. Abraham, contaminants rather than of [14C]INOX, because very little personal communicalion). Our evidence indÃcales, how subsequent further incorporation of radioactivity occurred, ever, lhal Ihe inhibilion of macromolecular synlhesis is not and no significant alterations in the Chromatographie prop only a consequence of an arresi of the cells in G2 + M. Cell erties of labeled INOX in 5 solvent systems (Solvents 9, 18, death seems lo be a consequence of these effects on mac 28, 30, and 34) were observed. Since Novikoff cells lack romolecular synthesis and is accompanied by cell lysis. inosine kinase (Ref. 16; P. G. W. Plagemann, unpublished The mechanism by which INOX causes these multiple data), the only pathway for the incorporation of radio inhibilions, however, has noi been elucidaled. Because of

JULY 1977 2191

B [3HJ URIDINE

l HR 4HR

60 0 30 30 60 TIME(MIN)

0 30 TIME (MINI Chart 3. Effect of INOX on the incorporation of labeled amino acids (A), undme (6), and thymidine (C) into acid-insoluble material and into the acid-soluble pool (D). Samples of an exponential-phase culture of N1S1-67 cells (at 1.5 * 10scells/ml) were supplemented with the indicated concentrations of INOX and further incubated at 37Â°.After1, 4, and 7 hr incubation, samples of cells were collected by centrifugation from 20 ml of each suspension and suspended in 20 ml of BM42B (without INOX) containing 0.5 fid (350.000 cpm) of a mixture of 3H-labeled amino acids (reconstituted protein hydrolysate) per ml (A), 5 fiM [3H]uridine (70 cpm/pmole) (B), or 0.5 Â¿IM[3H]thymidine(740 cpm/pmole) (C). The suspensions were incubated at 37Â°,andsamples of each suspension were analyzed for radioactivity in hot (A) or cold (8 and C) acid-insoluble material, or for radioactivity in total cell material. The amounts of radioactivity in the acid- soluble pool (D) were calculated by subtracting the amounts of radioactivity in acid-insoluble material from the total radioactivity associated with the cells. After 7 hr incubation with 0, 0.33, 0.83, and 1.66 mM INOX, the densities of the suspensions were 2.1, 1.85, 1.65, and 1.3 x 10' cells/ml, respectively.

2192 CANCER RESEARCH VOL. 37

C.THYMIDINE O HR 6.5 HR

ACID- INSOLUBLE

0 30 60 0 30 60 TIME (MIN)

Chart 4. Effect of hydroxyurea, cytosine arabinoside, and Â¡midazopyrazoleon the incorporation of leucine (A), uridine (B), and thymidine (C) by N1S1-67 cells. Samples of an exponential-phase culture at 1.4 x 10" cells/ml were supplemented where indicated with 0.5 mw hydroxyurea (HU), 50 Â¿Â¿Mcytosine arabinoside (ara-C) or 0.4 mM imidazopyrazole (IPZ) and further incubated at 37Â°.At0 time and after 6.5 hr further incubation at 37Â°,cellswere collected from samples of 20 ml of each suspension and suspended in 20 ml of leucine-free BM42B (A) containing 0.5 /id [3H]leucine per ml or of BM42B (fl and C) containing 5 IÂ¿M[3H]uridine(60 cpm/pmole) or 0.5 IÂ¿M[3H]thymidine(680 cpm/pmole) plus the same concentration of drug as present in the preincubation medium. The suspensions were incubated at 37Â°and monitored for radioactivity in total cell material (open symbols) and acid-insoluble material (closed symbols). the lack of Â¡nosinekinase in Novikoff cells, INOX probably and ribonucleotide reducÃase(2, 4) by various dialdehyde does not become phosphorylated, and since it is not metab derivatives of purine nucleosides might have a similar olized in any other manner, or at least only very slowly, the cause, and other enzymes might be subject to the same observed effects are probably due to INOXper se and not to kind of covalent interaction. The time dependence of the a metabolic product of INOX. As we found with [14C]INOX, inhibition of macromolecular synthesis and of cell prolifera the periodate oxidation product of ['"evÃ¡denosme has been tion as well as the irreversibility of this inhibition and of the reported to be rapidly incorporated into nucleic acids, and inhibition of enzymes (4) are consistent with this view. The an incorporation into RNA was considered to cause an cytotoxicity of other dialdehyde compounds (5, 6) may have inhibition of ribosomal RNA processing (3), but the possibil a similar basis, but the purine moieties of the dialdehyde ity that the incorporation of radioactivity was due to residual nucleosides and nucleotides probably play some role in the labeled adenosine was not ruled out. If a compound under overall inhibitory effects, since they influence the relative investigation is not metabolized, the presence of small effect of these substances on various enyzmes (2, 4, 10,18, amounts of a metabolizable contaminant can readily lead to 19)." Also enzymes found to be inhibited by these com misleading results. pounds are involved in the synthesis or metabolism of nu We consider it more likely that the mode of action of INOX cleic acids or precursors thereof. Not all enzymes with and other aldehyde derivatives of nucleosides and nucleo- nucleotides or nucleosides as substrates or cofactors, how tides is a cross-linking of various proteins by the formation ever, are inhibited by INOX or similar nucleoside deriva of Schiffs bases with functionally critical amino groups of tives. For instance, the finding that the incorporation of these proteins. For instance, the inhibition of RNase A by uridine, thymidine, and guanine into the nucleotide pool by the dialdehyde derivative of 6-methylmercaptopurine ribo- Novikoff cells is not significantly inhibited by INOX suggests nucleoside seems to be due to the formation of Schiffs that uridine, thymidine, UMP, dTMP, and dTDP kinase, and base of this compound with the e-amino group of lysine hypoxanthine-guanine phosphoribosyltransferase are rela residues of the enzyme (17). The inhitition of thymidylate tively resistant to INOX. Similarly, enzymes involved in thy kinase (10), various RNA and DNA polymerases (10, 18,19), midine and guanine incorporation as well as deoxycytidine

JULY 1977 2193

A. C.+COLCEMID CONTROL ( IpM)

or UJ m Chart 5. DNA content of individual cells in N1S1-67 populations incubated with INOX, Colcemid, or hydroxyurea. Samples of an exponential-phase culture at 1.4 x 10' cells/ml were supplemented as indicated with 0.83 rriM INOX. 1 /Â¿MColcemid. 1 mM hydroxyurea (HU), or 1 JIM vinblastine u sulfate (data for latter not shown). After 6 B + INOX D.+HU hr further incubation the cells were enum (083mM) ImM) erated in each suspension and analyzed for DNA content in a flow microfluorometer. The average DNA contents of G, and G2 + M cells are indicated on the abscissa by 1 and 2, respectively. The cell densities (per ml) at 6 hr were (A) 1.9 x 106, (B) 1.3 x 10Â».(C)1.4 x 108, and (0) 1.4 x 10Â« for the vinblastine sulfate-treated culture.

RELATIVE DNA CONTENT/CELL

"A. PH] LEUCINE B PH] URIDINE THYMIDINE -OCONTROL â€¢-+INOX X Ã›-+COLCEMI Z CL A-+VIN- Chart 6. Comparison of the effects of INOX, O Colcemid, and vinblastine sulfate on macromo- BLASTIN lecular synthesis. The details of the experiment are described in the legend to Chart 5. After 6 hr in cubation with and without the inhibitors, samples of cells were collected by centrifugation from 30 ml of each suspension and suspended in 20 ml leucine-free BM42B (A) containing 0.5 /iCi [3HJ- o leucine per ml or of BM42B (B and C) contain z ing 5 Â¡IM[3H]uridine (60 cpm/pmole) or 0.5 /Â¿M [3H]thymidine (600 cpm/pmole) plus the same ÃœJ concentration of drug as present in the preincubation medium. The suspensions were incubated < at 37Â°and monitored for radioactivity in total cell ce material (data not shown) and for radioactivity in fe acid-insoluble material. CD

i IO

30 60 0 30 60 0 30 60

TIME (MIN)

2194 CANCER RESEARCH VOL. 37

pressive and Antitumor Activity of the Periodate Oxidation Product of fi- D-Ribosyl-6-methylthiopurine. Cancer Res., 28: 782-787, 1968. 2. Cory, J. G., and Marseli, M. M. Studies on Mammalian Ribonucleotide ReducÃaseInhibition by Pyridoxal Phosphate and the Dialdehyde Deriva tives of Adenosine, Adenosine S'-monophosphate, and Adenosine 5'- triphosphate. Cancer Res., 35: 390-396, 1975. 3. Cory, J. G., Mansell, M. M., George, C. B., and Wilkinson, D. S. Inhibi tion of Nucleic Acid Synthesis in Ehrlich Tumor Cells by Periodate- Oxidized Adenosine and Adenylic Acid. Arch. Biochem. Biophys., 160: 495-503, 1974. 4. Cory, J. G., Mansell, M. M., and Whitford, T. W., Jr. Inhibition of Ribonu cleotide Reductase Activity and Nucleic Acid Synthesis in Tumor Cells by the Dialdehyde Derivatives of Inosine (NSC 118994) and Inosinic Acid. Cancer Res., 36: 3166-3170, 1976. 5. Dvonch, W., Fletcher, H., Ill, Gregory, f. J., Healy, E. M. H., Warren, G. H., and Alburn, H. E. Antitumor Activity of Periodate-Oxidation Products O-CONTROL, of Carbohydrates and Their Derivatives. Cancer Res., 26: 2386-2389, 1966. â€¢-+CORDY-, 6. French, F. A., and Freedlander, B. L. Carcinostatic Action of Polycar- bonyl Compounds and Their Derivatives. I. 3-Ethoxy-2-ketobutyralde- CEPIN hyde and Related Compounds. Cancer Res., 18: 172-175, 1958. 7. Graff, J. C., and Plagemann, P. G. W. Alanosine Toxicity in Novikoff Rat r-I-INOX Heptatoma Cells due to Inhibition of the Conversion of Inosine Mono- phosphate to Adenosine Monophosphate. Cancer Res., 36: 1428-1440, 1976. 8. Graham, F. L., and Whitmore, G. F. Studies in Mouse L-Cells on the Incorporation of 1-/3-D-Arabinofuranosylcytosine into DNA and on the Inhibition of DNA Polymerase by 1-/3-D-Arabinofuranosylcytosine 5'-triphosphate. Cancer Res., 30: 2636-2640, 1970. 9. Kaufman, J. H., and Mittelman, A. Phase I Study of Inosine Dialdehyde (Diglycoaldehyde, NSC 118994). Proc. Am. Assoc. Cancer Res., 76: 202, 1975. 10. Kimball, A. P., Wilson, M. J., Bell, J. P., and LePage, G. A. Inhibition of Thymidylate Kinase and DNA Polymerase by Periodate Oxidation Prod uct of /3-D-Ribosyl-6-methylthiopurine. Cancer Res., 28: 661-665, 1968. 11. Marz, R. O., Zylka, J. M., Plagemann, P. G. W., Erbe, J., Howard, R., and Sheppard, J. R. G2+ M Arrest of Cultured Mammalian Cells after Incor poration of Tritium-Labeled Nucleosides. J. Cellular Physiol., 90: 1-8, 1977. 12. Moore, E. C. The Effects of Ferrous Ion and Dithioerythritol on Inhibition by Hydroxyurea of Ribonucleotide Reductase. Cancer Res., 29: 291-295, 1969. 13. Plagemann, P. G. W. On the Mechanism of Phenethyl Alcohol-Induced Loss of Polyribosomes and Their Reformation after Reversal in Rat Hepatoma Cells. J. Biol. Chem., 243: 3029-3037, 1968. i 2 14. Plagemann, P. G. W. Nucleotide Pools of Novikoff Rat Hepatoma Cells Growing in Suspension Culture. I. Kinetics of Incorporation of Nucleo sides into Nucleotide Pools and Pool Sizes during Growth Cycle. J. TIME (HR) Cellular Physiol., 77: 213-240, 1971. Chart 7. Effect of INOX and cordycepin on the incorporation of 15. Plagemann, P. G. W., and Erbe, J. Thymidine Transport by Cultured [3H]guanine. Samples of a suspension of 2 x 10s N1S1-67 cells per ml of Novikoff Hepatoma Cells and Uptake by Simple Diffusion and Relation BM42B were supplemented where indicated with 250 /IM cordycepin or 1.66 ship to Incorporation into Deoxyribonucleic Acid. J. Cell Biol., 55: 161- HIM INOX. At 0 time and after 1 hr incubation at 37Â°,samples of each 178, 1972. suspension were supplemented with 0.5 /Â¿M[3H]guanine(400 cpm/pmole) 16. Plagemann, P. G. W., and Erbe, J. Intracellular Conversions of Deoxyri- and monitored for the incorporation of 3H into total cell material. bonucleosides by Novikoff Rat Hepatoma Cells and Effects of Hydroxyu rea. J. Cellular Physiol., 83: 321-336, 1974. 17. Spoor, T. C., Hodnett, J. L., and Kimball, A. P. The Molecular Mecha deaminase of Ehrlich tumor cells were not found to be nism of Action of Methylthioinosinedicarboxaldehyde on Ribonuclease A inhibited by INOX or the dialdehyde derivative of IMP (4). as a Model System. Cancer Res., 33: 856-858, 1973. Thus further work is required to elucidate the possible site- 18. Spoor, T., and Kimball, A. P. Mammalian DNA-dependent RNA Polymer ase: Inhibition of a Mammalian Enzyme by Methylinosine Dialdehyde. directed inactivation of certain enzymes by these dialde Proc. Soc. Exptl. Biol. Med., Ã•36:605-607, 1971. 19. Van Der Velde, G., Loy, P. R., and Kimball, A. P. Dimethyladenosine hyde derivatives of nucleosides and nucleotides. Dialdehyde: Inhibition of Leukemia 1210 DNA Polymerase. Proc. Soc. Exptl. Biol. Med., 134: 831-834, 1970. ACKNOWLEDGMENTS 20. Ward, G. A., and Plagemann, P. G. W. Fluctuations of DNA-dependent RNA Polymerase and Synthesis of Macromolecules during the Growth We thank John Erbe for the microfluorometric analyses of samples of cells Cycle of Novikoff Rat Hepatoma Cells in Suspension Culture. J. Cellular and Cheryl Thull for competent secretarial help. Physiol., 73: 213-231, 1969. 21. Zylka, J. M., and Plagemann, P. G. W. Purine and Pyrimidine Transport by Cultured Novikoff Cells. Specificities and Mechanism of Transport REFERENCES and Relationship to Phosphoribosylation. J. Biol. Chem., 250: 5756- 1. Bell, J. P., Faures, M. L., LePage, G. A., and Kimball, A. P. Immunosu- 5767, 1975.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. Mechanism of Action of Inosine Dialdehyde (NSC 118994) in the Inhibition of Proliferation of Tumor Cells in Culture

Peter G. W. Plagemann, Jon C. Graff and Marsha Behrens

Cancer Res 1977;37:2188-2195.

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