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In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.

The cell cycle perturbation effect on leukemic cells of the Brown Norway myelocytic leukemia (BNML) after high dose Ara-C injection was used as the rationale for chemotherapy studies. A one log leukemic cell load reduction as determined by means of the leukemic colony forming units-spleen (LCFU-S) assay was observed when the second Ara-C injection was administered during a period of induced accumulation of cells in S phase. evidence was obtained that Ara-C was also cytotoxic for G1 phase cells. By comparing a continuous Ara-C infusion during 24 h with two Ara-C injections at the same total dosage given 12 h apart, it was found that the tumor load was significantly more reduced in the latter group. For the drug combination Ara-C/Adriamycin, maximal LCFU-S reduction of one log was observed when Adriamycin (7.7 mg/kg) was given 12 h after Ara-C. From the increase in survival time after the schedule of 6 X Ara-C plus 1 X Adriamycin (all injections given at 12 h intervals), it could be extrapolated that the tumor load was reduced by 6.7 logs; this was in agreement with the theoretically expected reduction of 6.2 logs based on the LCFU-S experiments. The schedule of 6 X Ara-C plus 1 X Adriamycin reduced the normal haemopoietic stem cell compartment by 2.5 logs. This therapeutic gain is attributed to the specific recruitment-synchronization inducing effect of Ara-C on leukemic cells in the G0 phase.

For 14 patients with acute leukemia flow cytometry was used to determine the percentage of cells in S-phase flushed out of a bone marrow biopsy compared with the percentage in a bone marrow aspirate; there was a statistically significant difference (p less than 0.01) between the two. The percentage dilution of the bone marrow aspirate by peripheral blood was then calculated, according to Holdrinet et al. [1], in order to correct the percentage S-phase cells in the aspirate. The data presented show that when the percentage S-phase cells in the aspirate is corrected for blood dilution, it closely approaches the percentage S-phase cells in the biopsy (p greater than 0.10).

The effect of high-dose cytosine arabinoside (Ara-C; 1-beta-D-arabinofuranosyl cytosine) injections (200 mg/kg i.v.) on cell cycle perturbation was investigated in a slowly growing rat leukemia (BNML) which is a realistic model for human acute myelocytic leukemia. Flow cytometric analysis showed an initial decrease of cells in S phase from 26 to 13% and a subsequent accumulation of up to 50% at 10-14 h after injection. The low number of S phase cells during the first 8 h might be due to a combination of cell kill in S phase and a block at the G1/S boundary. The results make it very likely that the origin of the accumulated S phase cells is the resting compartment and that these recruited cells enter the proliferation phase as a synchronized cell population. By repeating the Ara-C injection at the time of accumulation of cells in S phase, a similar synchronized wave of recruited cells to that after the first Ara-C injection was observed. Flow cytometric analysis after Adriamycin (7.7 mg/kg i.v.) treatment, which has been shown to be cytotoxic for BNML cells, showed no changes in cell cycle distribution. It was concluded that Adriamycin might have the same toxicity for cells in all of the different cell cycle phases. The application of these data with respect to effective tumor load reduction is discussed in a second report.