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-in- soluble 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 col- umn, 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 pro- duced 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.