Synergistic Interaction Between L-8-D~Arabinofuranosylcytosine

Proc. NatL Acad. Sci. USA Vol. 78, No. 8, pp. 5132-5136, August 1981 Medical Sciences

Synergistic interaction between l-8-D~arabinofuranosylcytosine, thymidine, and hydroxyurea against human B cells and leukemic blasts in vitro (biochemical modulation/deoxycytidine triphosphate/isobologram analysis) JEROME A. STREIFEL AND STEPHEN B. HOWELL Department of Medicine and The Cancer Center, University of California at San Diego, La Jolla, California 92093 Communicated byJ. Edwin Seegmiller, March 9, 1981

ABSTRACT Isobologram analysis was used to examine the in- hance the phosphorylation ofAra-C in murine cells (12, 30-32) teraction between 1-fiD-arabinofuranosylcytosine (Ara-C), thy- and to markedly improve the therapeutic ratio ofAra-C against midine (dThd), and hydroxyurea. All three pairs ofdrugs, as well the murine L1210 leukemia in vivo (33). as the triple combination, were synergistic against a human B cell We used the technique of isobologram analysis (34) to in- line in vitro across a broad range of concentrations. Synergy was vestigate the interactions between Ara-C, dThd,. and HU associated with an increase in the Ara-C nucleotide pool and Ara- against a human B cell line in vitro and found that all three pairs C triphosphate concentration. dThd increased, and hydroxyurea and the triple combination of the drugs interacted synergisti- decreased, the incorporation ofAra-C into trichloroacetic acid-insoluble macromolecules per unit time. Hydroxyurea was more cally. On an equimolar basis, HU was more effective than dThd effective than dThd at equimolar concentrations in increasing the in increasing the Ara-C nucleotide pool in both the B cell line acid-soluble Ara-C pool. Maximal stimulation of Ara-C triphos- and in malignant blasts from six patients with acute leukemia. phate formation by dThd occurred at 1 mM and was associated with reduction of the deoxycytidine triphosphate pool to 31% of MATERIALS AND METHODS control. At the same concentration, hydroxyurea increased Ara- C triphosphate formation to a greater extent but increased de- Drugs. dThd was obtained from the Investigative Drug oxycytidine triphosphate to 116% of control. When tested at clin- Branch ofthe National Cancer Institute. Ara-C was purchased ically achievable concentrations on blasts from patients with acute from Upjohn, HU from Sigma, and [3H]Ara-C from New En- leukemia, hydroxyurea increased the Ara-C nucleotide pool in all gland Nuclear. six cases studied, whereas dThd decreased the Ara-C nucleotide Cells. The SB cell is a human B lymphocyte line transformed pool. These results indicate that in SB cells dThd and hydroxyurea by Epstein-Barr virus (35). SB cells in the logarithmic phase work by different mechanisms to augment the Ara-C nucleotide of growth were seeded at 2 x 105 cells per ml in RPMI-1640 pool and that hydroxyurea may be more effective than dThd as medium with 10% heat-inactivated fetal calf serum, 1% anti- a modulator of Ara-C activity in patients with acute leukemia. biotic solution (penicillin at 10,000 units/ml, Fungizone at 25 .ag/ml, and streptomycin at 10,000 /i/ml) (Flow Laboratories, 1-3-D-Arabinofuranosylcytosine (Ara-C) is one of the most im- McLean, VA), and 1% glutamine at 370C in 5% CO2. The final portant drugs in the treatment ofhuman acute leukemia (1-4). concentration of dThd in the medium contributed by the fetal A significant increase in the selectivity ofAra-C may have far- calf serum was 0.5 1LM. In growth assays the number of viable reaching consequences for the management ofthis disease. Ara- cells present after 72 hr was determined by trypan blue dye C behaves as an analog of deoxycytidine (dCyd) that, when exclusion. Leukemic human blasts were recovered from the phosphorylated to 1-,3-D-arabinofuranosylcytosine triphos- marrow or peripheral blood ofpatients with acute leukemia by phate (Ara-CTP), is a potent inhibitor of DNA polymerase Ficoll/Hypaque separation (36) and washed twice before in- (5-7) and is also incorporated into DNA (7-9). The activity of cubation with drugs. Ara-C is determined by the amount ofAra-CTP formed within [3H]Ara-C and [3H]Uridine Incorporation. SB cells (0.2 x the cell, and the concentration of the natural substrate deoxy- 106 cells per ml) and leukemic blasts (0.5-2.0 x 10r cells per cytidine triphosphate (dCTP), with which Ara-CTP must com- ml) were exposed-simultaneously to [3H]Ara-C or [3H]uridine pete for binding to DNA polymerase (5, 6, 10, 11). with either HU or dThd for 4 hr. The acid-soluble fraction of Phosphorylation of Ara-C is normally constrained by feed- [3H]Ara-C and its metabolites was calculated as the difference back inhibition ofdCTP on dCyd kinase, the enzyme catalyzing between the radioactivity in whole cells after washing and the the rate-limiting step in the dCyd salvage pathway (12, 13). radioactivity precipitable with trichloroacetic acid. The fraction Thymidine (dThd) has been shown to increase the toxicity of of [3H]uridine incorporated into RNA was determined by al- Ara-C in vitro and in vivo against several murine (14-17) and kaline hydrolysis of the RNA. human tumors (16). This is in part due to the ability ofthymidine Measurement of [3H]Ara-CTP and dCTP. After a 4-hr ex- triphosphate (dTTP) to inhibit ribonucleotide reductase (18), posure to [3H]Ara-C, cells were extracted with 0.4 M perchloric which catalyzes an obligatory step in the die novo synthesis of acid, clarified by centrifugation, and neutralized with Alamine/ dCTP (19, 20) and depletes the cellular dCTP pool (15, 18, 19, Freon, as described by Khym (37). [3H]Ara-CTP was separated 21-26). Hydroxyurea (HU) is also an inhibitor of mammalian from other Ara-C nucleotides by high-pressure liquid chro- ribonucleotide reductase (27), but one that binds at a different matography usinga Waters instrument (Waters Associates) with site than dTTP (28, 29). HU has also been demonstrated to en- a Partisil-10 SAX solumn (Whatman). Extract from 8 X 105 cells

The publication costs ofthis article were defrayed in part by page charge Abbreviations: Ara-C, 1-f3-D-arabinofuranosylcytosine; Ara-CTP, 1-f3- payment. This article must therefore be hereby marked "advertise- D-arabinofuranosylcytosine triphosphate; Ara-U, I-P-D-arabinofura- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. nosyluracil; HU, hydroxyurea. 5132 Downloaded by guest on September 27, 2021 Medical Sciences: Streifel and Howell Proc. Natl. Acad. Sci. USA 78 (1981) 5133

was injected onto the column and eluted isocratically at 30'C 3, with 0.4 M ammonium phosphate/acetonitrile (10:1, vol/vol) at a flow rate of 2 ml/min (38). Fractions were collected and 2 A B radioactivity was measured by liquid scintillation counting. dCTP concentration was determined in cell extracts by the Ei method of Garrett and Santi (38). Extract from 50 X 106 cells E- 1 was injected on the high-pressure liquid chromatography column, using the same conditions as for the measurement ofAra- CTP. Column output was monitored by absorbance at 280 nm 0 50 100 150 200 0 50 100 150 200 and the peak areas were electronically integrated. External Ara-C, nM Ara-C, nM standards of dCTP were used for calibration. FIG. 2. Isobolograms depicting the interaction between Ara-C and dThd (A), and Ara-C and HU (B) at concentrations that kill 90% of the RESULTS SB cells (cytotoxic). See Fig. 1 for explanation. Isobologram Analysis. SB cells were grown in the presence ofiztrnl nonrtu rif annh arrantn nlannn r%.r Asrithrlrtc ;1 UV VdiIUUa . 4ia agentL or i combination to.nenraiosdetermine the concentrations.i .U to examined across a range of concentrations from those that re- required produced the net production of SB cells to zero (cytostatic) to those duce the saine degree of effect. Because for cancer chemo- that killed 90% of the cells after 72 hr (cytotoxic). Fig. 1 shows therapeutic agents the drug concentrations that are of most in- the isobolograms for the interaction ofeach pair ofdrugs at con- terest are those that produce cell killing, drug interactions were centrations producing cytostasis. The intercepts on the ordinate and abscissa indicate the cytostatic concentration of each drug 1.2- used alone. Ifthere were no interaction between the drugs and LA the agents were purely additive, then the data points for the 1.0- A combinations of two drugs causing cytostasis would fall on a 0.8- straight line connecting the intercepts. If the interaction were antagonistic, the line joining the intercepts would be convex. 0.6. In this case, the data points were found to describe a concave curve, which indicates synergy. For Ara-C in combination with 0.4- either dThd or HU, synergy was also observed at drug concen- 0.2- trations reducing the number of viable cells to as little as one 1/10th the number at the start of the culture (cytotoxic). The degree ofsynergy, as reflected by the degree ofconcavity ofthe 0.1 0.2 0.3 0.4 0.5 isobologram, was greater at cytotoxic drug concentrations than Ara-C ,uM at cytostatic concentrations (Fig. 2). 60 The isobologram for the cytostatic triple combination ofAra- 50 B C, dThd, and HU is shown in Fig. 3. On this plot, complete absence of any interaction would be reflected by a plane inter- 40 secting each of the three axes at the intercepts. Antagonism 30

20 0 0 10 dThd

0.1 0.2 0.3 0.4 0.5 Ara-C, AM 1.2 4 1.0 C 0.8 - I -0"0 0.6- "0 0.4- 0.2 -

20 40 60 80 HU, AuM Ara-C FIG. 1. Isobolograms depicting the interaction between Ara-C and dThd (A), Ara-C and HU (B), and dThd and HU (C) at concentrations FIG. 3. Isobologram depicting the interactions among Ara-C, that reduce the growth rate of SB cells to zero (cytostatic). The inter- dThd, and HU in triple combination. The intercepts represent the con- cepts represent the concentration of each agent required to produce centration of each agent required to produce cytostasis when used cytostasis when used alone, and the data points represent those com- alone, and the curved surface represents those combinations of the binations of two agents that when present together produced the same three agents used together that produced the same effect. The data effect. points have been omitted for clarity. Downloaded by guest on September 27, 2021 5134 Medical Sciences: Streifel and Howell Proc..NatL- Acad. Sci. USA 78 (1981)

100 B 10-5M soluble pools with HU was independent of the Ara-C concen- a) tration, reaching a mean (± SD) percent increase of 440% ± - , .10-6 f M 72% at 10 mM HU, whereas the percent increase in the Ara- 10 - , 1- CTP pool was greater at 0.01 ,M Ara-C (750%) than at 10 AM , M Ara-C (339%). Neither dThd nor HU altered the percent ofAra- v C nucleotides present as Ara-CTP, which averaged (± SD) 85% ¢ 1.0 F- 1,+~~~~~10-7 lo-,,m ± 5% at all concentrations. Likewise, neither dThd nor HU cd enhanced the cellular retention ofAra-C nucleotides measured 0.1; for up to 4 hr after cells were transferred to Ara-C-free medium. While dThd and HU had similar effects on incorporation into 10-6 10-5 1O-4 10-3 10-2 0 10 1o-4 10-3 10-2 the acid-soluble pools, they had opposite effects on the rate of incorporation of[3H]Ara-C into acid-insoluble macromolecules, dThd, M HU, M almost all of which represent incorporation into DNA (9, 15). per unit FIG. 4. Effect of various concentrations of dThd (A) and HU (B) As shown in Fig. 6, dThd enhanced the incorporation on the [3H]Ara-C acid-soluble pool in SB cells in the presence of Ara- time, and the degree of enhancement closely paralleled the Cat the.concentration indicatedbeside each curve for 4 hr at 3700. The changes in the Ara-CTP concentration, being most marked at measurements of [3H]Ara-C include all its acid-soluble metabolites. 10 nM Ara-C and less pronounced at 10 AM Ara-C. In contrast, Bars represent SD. HU reduced the rate of incorporation into acid-insoluble material, the effect being greater in the presence of 10 nM than in the presence of 10 ,uM Ara-C. For both dThd and HU, these would produce a convex surface, and synergism a concave sur- changes in the, rate of incorporation do not necessarily reflect face. The isobologram for the interaction of Ara-C, dThd, and changes in the amount of [ H]Ara-C incorporated per unit of Hu clearly describes a concave surface, indicating that all three newly synthesized DNA, because both agents and Ara-CTP it- agents were synergistic when present together and that the selfinfluence the rate of DNA synthesis. HU did not inhibit the combination of both ribonucleotide reductase inhibitors was incorporation of [3H]uridine into RNA (data not shown). more effective in augmenting the activity of Ara-C than either dCTP Content. The effect of 1 mM dThd and HU on the one alone. dCTP content of SB cells after 4 hr of drug exposure was de- Incorporation of [3H]Ara-C in SB Cells. Increasing concen- termined three times on separate cultures ofcells. Control cells trations of dThd in the range 0.5-1000 ,M in the presence of contained (mean ± SD) 23.3 ± 4.4 pmol per 106 cells. dThd Ara-C concentrations ranging between 0.01 and 10 AM caused reduced dCTP content to 7.2 ± 0.2 pmol per 106 cells (31%), a progressive expansion ofboth the total acid-soluble Ara-C pool whereas HU increased dCTP to 27.0 ± 2.1 pmol per 106 cells (Fig. 4A) and the Ara-CTP pool (Fig. 5A). However, when the (116%). dThd concentration was raised to 10 mM both the total acid- [3H]Ara-C Incorporation in Leukemic Blasts. Table 1 com- soluble pool and the Ara-CTP were reduced. The maximum pares the effects ofdThd and HU on the incorporation of [3H]- percent expansion of the total acid-soluble Ara-C pool induced Ara-C into the acid-soluble pool and acid-insoluble material of by dThd was independent of the Ara-C concentration, and at SB cells and leukemic blasts freshly harvested from one patient 1 mM dThd the total acid-soluble Ara-C pool was increased to with acute myelomonocytic leukemia and five patients with a mean (±SD) of 205% ± 51%. The maximum percent increase acute lymphoblastic leukemia. Cells were exposed to the. clin- in the Ara-CTP pool was similar in the presence of0.01 AM Ara- ically achievable concentrations of 1 mM dThd (39-41) and HU C (269%) and in the presence of 10 ,M Ara-C (200%). The per- (42), and 0.1 ,uM Ara-C (43). The effects of dThd were quali- cent increase in the pools per 10-fold increase in drug concen- tatively and quantitatively different in SB cells than in the leu- trations was greater for HU than for dThd (Figs. 4B and SB), kemic blasts. Whereas dThd increased the acid-soluble pool of and rather than peaking at 1 mM HU, the total acid-soluble pool Ara-C in SB cells, it uniformly decreased these pools in leu- and the Ara-CTP pool continued to increase as HU was raised kemic cells. Whereas dThd increased the rate of [ H]Ara-C in- to 10 mM. As with dThd, the percent increase in the total acid- corporation into acid-insoluble macromolecules in both types ofcells, the effect was quite marked in the SB cells and marginal blasts. In the effect of HU was 1000 r A 1000 B in the leukemic contrast, qual- 10-5 M itatively similar in both types of cells in that it increased the CA 1o-5 M soluble pool and decreased incorporation into insoluble mate- -4 _- 1-a2) 100; 100 Q To0 10 r- 0 10 F 10 F E 0. 10-8M toc) 1.0 Q L.( 1.0 h 0 cd'a E 0.1 ¢- 10-8M C. 0.2 0.1 a..qi, 10-2 0 10-5 10-4 10-3 10-2 10-7 10-6 10-5 10-4 10-3 0.01 dThd, M HU, M 10-6 10-5 10-4 10-3 1o-2 0 10-5 1o-4 10-3 1o-2 dThd, M HU, M FIG. 5. Effect of various concentrations of dThd (A) and HU (B) on the Ara-CTP pool in SB cells at the two, indicated Ara-C concen- FIG. 6. Effect of dThd (A) and HU (B) on the incorporation of [3H1 trations. Cells were simultaneously incubated with [3HIAra-C and Ara-C into the acid-insoluble pool of SB cells as a function of Ara-C dThd or HU for 4 hr at 370C, and Ara-CTP was measured by high-pres- concentration. Cells were simultaneously incubated with [3H]Ara-C sure liquid chromatography of acid extracts of the cells. and dThd or HU for 4 hr at 370C. Downloaded by guest on September 27, 2021 Medical Sciences: Streifel and Howell Proc. Natl. Acad. Sci. USA 78 (1981) 5135

Table 1. Effect of dThd and HU on incorporation of [3H]Ara-C pool. The uniform decrease in the acid-soluble Ara-C and Ara- into SB cells and leukemic blasts CTP pool at dThd concentrations above 1 mM can readily be [3H]Ara-C incorporation, % of control explained by the ability ofdThd at high concentrations to com- petitively inhibit the transport system shared by dCyd and Ara- dThd HU C (12) and by the ability ofhigh levels ofdTTP to inhibit dCyd Cell type* Soluble Insoluble Soluble Insoluble kinase (55). SB 190 206 450 26 The mechanism by which HU causes an expansion ofthe Ara- AMML 11 92 304 45 CTP pool and the reason why its effect is more pronounced than ALL 1 38 101 156 78 that ofdThd are uncertain (12, 30-32). Although HU is a potent 2 82 133 178 82 inhibitor of ribonucleotide reductase (27), the major effect of 3 37 116 199 50 this inhibition is a decrease in the dATP and dGTP pools (21, 4 25 127 127 69 57, 58). Studies of the effect of HU on dCTP concentration in 5 60 110 173 39 various nonhuman mammalian cells have yielded conflicting Mean ALL results (21, 57-59). Our results indicate that, under conditions ± SD 48 ± 23 115 ± 12 167 ± 27 64 ± 18 in which Ara-CTP was increased to more than 500% of control Freshly harvested blasts were incubated with 1 mM dThd or HU for (10 nM Ara-C, 1 mM HU), HU did not decrease dCTP, and thus 4 hr in the presence of [3H]Ara-C at a final concentration of 0.1 gM. enhanced phosphorylation must be due to some mechanism Controls were not exposed to dThd or HU. other than reduced feedback inhibition ofdCyd kinase by dCTP * AMML, acute myelomonocytic leukemia; ALL, acute lymphoblastic (54, 55). No significant conversion of [3H]Ara-C to [3H]Ara-U leukemia. was detected in SB cells in the presence or absence of HU, so that inhibition ofdCyd deaminase cannot account for increased Ara-CTP. Agarwal et al. (30, 31) have also reported that HU does rial; quantitatively the HU effect was more pronounced in SB not block deamination of Ara-C. cells. The finding of a synergistic interaction between dThd and HU was of interest because it suggests the possibility of an al- DISCUSSION losteric interaction between the subunits ofmammalian ribonu- These results demonstrate a synergistic interaction between cleotide reductase. Mammalian ribonucleotide reductase ap- HU and Ara-C against human B cells in vitro and confirm the pears to be very similar to the Escherichia coli enzyme, which synergistic interaction between dThd and Ara-C reported by has two subunits (60), one of which binds dTIP (29) and the others (12, 14-17, 44, 45). Synergy was demonstrable across a other of which interacts with HU (28). The action of one of the broad spectrum ofdrug effect, ranging from concentrations that drugs may influence the binding or effect of the other. Alter- reduced the net growth rate to zero to concentrations that killed natively, dThd and HU may interact synergistically by altering 90% ofthe cells in the culture. These concentrations encompass the concentration of one of the other nucleotides that regulate a range that is readily achieved in patients (39-43). In addition, the activity of ribonucleotide reductase (61). the results indicate that dThd and HU interact synergistically, The formation ofadequate Ara-CTP is a necessary, although so that the triple combination of Ara-C, dThd, and HU is not always sufficient requirement for the sensitivity ofleukemic synergistic. blasts (5, 16, 46, 62-68). Although dThd was effective in en- The biochemical basis for synergistic interaction of Ara-C hancing the phosphorylation ofAra-C in the human B cell line with both dThd and HU probably resides in the ability of both SB, at 1 mM it significantly reduced phosphorylation in all six these drugs to increase the intracellular concentration of Ara- samples ofleukemic blasts. Thus, these results raise the concern CTP relative to dCTP (12, 14, 15, 17, 45). At the end of a 4-hr that dThd may impair the activity ofAra-C in some patients with exposure to 10 nM Ara-C, the Ara-CTP-to-dCTP ratio in control acute leukemia. HU, on the other hand, enhanced the phos- cells was 0.015; addition of 1 mM HU increased the ratio to phorylation of Ara-C both in the SB cells and in the leukemic 0.068, and addition of 1 mM dThd raised it to 0.123. A large blasts of all six patients. Because recent clinical trials indicate number of other drugs that enhance the activity of Ara-C in that 1 mM dThd may be no less toxic than 1 mM HU when experimental systems also have the effect of augmenting the maintained for equivalent periods of time (69-71), HU may be formation of Ara-CTP (12, 32, 46-53). The fact that dThd and the preferred agent with which to modulate the activity ofAra- HU had similar effects on the acid-soluble pool of[3H]Ara-C but C. These results also indicate that that opposite effects on the rate ofincorporation into acid-insoluble combinations of agents material also suggests that modulation ofAra-CTP content was enhance the phosphorylation of Ara-C by different means may the basis for synergy. However, because dThd and HU may be more effective than single agents. On the basis ofreports that change the rate of DNA synthesis both directly and through dThd enhanced the phosphorylation ofAra-C in several murine enhanced formation of Ara-CTP, these experiments do not ex- B cell tumors and the Novikoffhepatoma (12, 14, 15), the com- clude the possibility that, per unit ofDNA synthesized, HU may bination of dThd and Ara-C has been introduced into clinical have increased [3H]Ara-C incorporation into DNA as effectively trial for the treatment of patients with acute leukemia. as did dThd. The putative mechanism of the dThd effect is that expansion We are grateful to Ivor Royston, John Mendelsohn, Susan Mansfield, of the intracellular dTTP pool by high extracellular concentra- and Michele Uke for their assistance with this work and to Judy Fitzner tions of dThd simultaneously reduces the dCTP concentration and Judith Glick for preparation of the manuscript. This investigation (12, 14, 26) by inhibiting de novo formation via ribonucleotide was supported by Grants CA 23334 and CA 09290 from the National reductase (18), and interferes with feedback inhibition of dCyd Cancer Institute and a grant from the Clayton Foundation, California kinase by dCTP, thus enhancing the phosphorylation of Ara-C Division. S. B. H. is a Clayton Foundation Investigator. dTTP can also inhibit dCMP deaminase How- (54-56). (12, 24). 1. Bodey, G. P., Freireich, E. J., Monto, R. W. & Hewlett, J. W. ever, the role ofsuch inhibition is less certain, because although (1969) Cancer Chemother. Rep. 53, 59-66. decreased deamination of Ara-CMP may increase Ara-CTP, 2. Clarkson, B., Dowling, M. D., Gee, T. S., Cunningham, I. B. decreased deamination of dCMP may also expand the dCTP & Burchenal, J. H. (1975) Cancer 36, 775-795. Downloaded by guest on September 27, 2021 5136 Medical Sciences: Streifel and Howell Proc. Natl. Acad - Sci - USA 78 (1981)

3. Frei, E., III, Bickers, J. N., Hewlett, J. S., Lane, M., Leary, W. 39. Ensminger, W. D. & Frei, E., III (1978) Clin. Pharmacot Ther. V. & Talley, R. W. (1969) Cancer Res. 29, 1325-1332. 24, 610-615. 4. Preisler, H. D., Rustum, Y., Henderson, E. S., Bjornsson, S., 40. Howell, S. B., Chu, B. C. F., Mendelsohn, J., Carson, D. A., Creaven, P. J., Higby, D. J., Freeman, A., Gailani, S. & Nalher, Kung, F. H. & Seegmiller, J. E. (1980) J. Nati. Cancer Inst. 65, C. (1979) Blood 53, 455-464. 277-284. 5. Furth, J. J. & Cohen, S. S. (1968) Cancer Res. 28, 2061-2067. 41. Zaharko, D. S., Bolten, B. J., Chuiten, D. & Wiernik, P. H. 6. Graham, F. L. & Whitmore, G. F. (1970) Cancer Res. 30, 2636- (1979) Cancer Res. 39, 47774781. 2644. 42. Haas, C. D., Belt, R. J., Kennedy, J. & Taylor, S. (1979) Proc. 7. Momparler, R. L. (1969) Biochem. Biophys. Res. Commun. 34, Am. Assoc. Cancer Res. 20, 143 (abstr.). 465-471. 43. Ho, D. H. W. & Frei, E., III (1971) Clin. Pharmacol Ther. 12, 8. Momparler, R. L. (1972) Mol. Pharmacol. 8, 362-370. 944-954. 9. Zahn, R. K., Muller, W. E. G., Forster, W., Maidhof, A. & Be- 44. Martin, D. S., Stolfi, R. L., Sawyer, R. C. & Spiegelman, S. yer, R. (1972) Eur. J. Cancer 8, 391-396. (1980) Proc. Am. Assoc. Cancer Res. 21, 303 (abstr.). 10. Kim, J. H. & Eidinoff, M. L. (1965) Cancer Res. 25, 698-702. 45. Reiter, H. (1980) Proc. Am. Assoc. Cancer Res. 21, 150 (abstr.). 11. Muller, W., Yamzaki, Z., Sogtrop, H. & Zahn, R. (1972) Eur.J. 46. Chou, T. C., Arlin, Z., Clarkson, B. D. & Philips, F. S. (1977) Cancer 8, 421428. Cancer Res. 37, 3561-3570. 12. Plagemann, P. G. W., Marz, R. & Wohlhueter, R. M. (1978) 47. Mills-Yamato, C., Lauzon, G. J. & Paterson, A. R. P. (1978) Cancer Res. 38, 978-989. Biochem. Pharmacol. 27, 181-186. 13. Momparler, R. L. & Fisher, G. A. (1968) J. Biol. Chem. 243, 48. Cadman, E. C., Dix, D. E. & Handschumacher, R. E. (1978) 4298-4304. Cancer Res. 38, 682-688. 14. Harris, A. W., Reynolds, E. C. & Finch, L. R. (1979) Cancer 49. Cadman, E. & Eiferman, F. (1979)J. Clin. Invest. 64, 788-797. Res. 39, 538-541. 50. Lauzon, G. J., Paran, J. H. & Paterson, A. R. P. (1979) Cancer 15. Grant, S., Lehman, C. & Cadman, E. (1980) Cancer Res. 40, Res. 38, 1723-1729. 1525-1531. 51. Grant, S. & Cadman, E. (1980) Clin. Res. 28, 451A (abstr.). 16. Breitman, T. R. & Keene, B. R. (1979) Proc. Am. Assoc. Cancer 52. Ho, D. H. W., Carter, C. J., Brown, N. S., Hester, J., Mc- Res. 20, 89 (abstr.). Credie, H., Benjamin, R. S., Freireich, E. J. & Bodey, G. P. 17. Danhauser, L. L. & Rustum, Y. M. (1980) Cancer Res. 40, 1274- (1980) Cancer Res. 40, 2441-2446. 1280. 53. Roberts, D., Peck, C. & Hilliard, S. (1980) Proc. Am. Assoc. 18. Bjursell, G. & Reichard, P. (1973)J. Biol. Chem. 248, 3904-3909. Cancer Res. 20, 179 (abstr.). 19. Reichard, P., Cannellakis, Z. N. & Cannellakis, E. S. (1961) J. 54. Durham, J. P. & Ives, D. H. (1970) J. Biol. Chem. 245, 2776- Biol Chem. 236, 2514-2519. 2284. 20. Reichard, P. (1972) Adv. Enzyme Regul 10, 3-16. 55. Ives, D. H. & Durham, J. P. (1970) J. Biol. Chem. 245, 2285- 21. Adams, R. L. P., Beryman, S. & Thamson, A. (1971) Biochim. 2294. Biophys. Acta 240, 455-462. 56. Chabner, B. A., Johns, D. G., Coleman, C. N., Drake, J. C. & 22. Lowe, J. K. & Grindey, G. B. (1976) Mol. Pharmacol. 12, 177- Evans, W. H. (1974)J. Clin. Invest. 53, 922-931. 184. 57. Skoog, L. & Nordenskjold, B. (1971) Eur. J. Biochem. 19, 81-89. 23. Roller, B. & Cohen, G. H. (1976)J. Virol. 18, 58-64. 58. Walters, R. A., Tobey, R. A. & Ratcliff, R. L. (1973) Biochim. 24. Jackson, R. C. (1978) J. Biol. Chem. 253, 7440-7446. Biophys. Acta 319, 336-347. 25. Grindey, G. B., Wang, M. C. & Kinahan, J. J. (1979) Mol. Phar- 59. Clarkson, J. M. (1978) Mutat. Res. 53, 273-284. macol. 16, 601-606. 60. Eriksson, S. L., Thelander, L. & Akerman, M. (1979) Biochem- 26. Reynolds, E. C., Harris, A. M. & Finch, L. R. (1979) Biochim. istry 18, 2948-2952. Biophys. Acta 561, 110-123. 61. Thelander, L. & Reichard, P. (1979) Annu. Rev. Biochem. 48, 27. Krakoff, I. H., Brown, N. C. & Reichard, P. (1968) Cancer Res. 133-158. 28, 1559-1565. 62. Cohen, S. S. (1976) Med. Biol. 54, 299-326. 28. Atkin, C. L., Thelander, L., Reichard, P. & Lang, G. (1973)J. 63. Rustum, Y. M. (1978) Cancer Res. 38, 543-549. Biol. Chem. 248, 7464-7472. 64. Drahovsky, D. & Kreis, W. (1970) Biochem. Pharmacol. 19, 940- 29. Engstrom, Y., Eriksson, S., Thelander, L. & Akerman, M. (1979) 944. Biochemistry 18, 2941-2948. 65. Durham, J. P. & Ives, D. H. (1969) Mol Pharmacol. 5, 358-375. 30. Walsh, C. T., Craig, R. W. & Agarwal, R. P. (1979) Fed. Proc. 66. Kessel, D., Hall, T. C. & Wodinsky, I. (1967) Science 156, 1240- Fed. Am. Soc. Exp. Biol. 38, 370 (abstr.). 1241. 31. Agarwal, R. P., Walsh, C. T. & Craig, R. W. (1980) Proc. Am. 67. Momparler, R. L., Brent, T. P., Labitan, A. & Krygier, V. (1971) Assoc. Cancer Res. 21, 13 (abstr.). Mol. Pharmacol. 7, 413-419. 32. Plagemann, P. G. W. & Erbe, J. (1974)J. Cell. Physiol. 83, 337- 68. Smyth, J. R., Robins, A. B. & Leese, C. L. (1976) Eur.J. Cancer 344. 12, 567-573. 33. Schabel, F. M., Jr., Laster, W. R., Jr. & Trader, M. W. (1971) 69. Woodcock, T., Damin, L., O'Henhir, M., Hansen, H., Andreeff, Proc. Am. Assoc. Cancer Res. 12, 67 (abstr.). M. & Young, C. (1979) Proc. Am. Assoc. Cancer Res. 20, 114 34. Berenbaum, M. C. (1977) Clin. Exp. Immunol. 28, 1-18. (abstr.). 35. Royston, I., Smith, R. W., Buell, D. N., Huang, E.-S. & Pagano, 70. Chuiten, D. F., Wiernik, P. H., Zaharko, D. S. & Edwards, L. J. S. (1974) Nature (London) 251, 745-746. (1979) Proc. Am. Assoc. Cancer Res. 20, 76 (abstr.). 36. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21, 77-89. 71. Howell, S. B., Taetle, R. & Mendelsohn, J. (1980) Blood 55, 505- 37. Khym, J. X. (1975) Clin. Chem. 21, 1245-1252. 510. 38. Garrett, C. & Santi, D. V. (1979) Anal. Biochem. 99, 268-273. Downloaded by guest on September 27, 2021