Reversal of cytosine arabinoside (ara-C) resistance by the synergistic combination of 6-thioguanine plus ara-C plus PEG-asparaginase (TGAP) in human leukemia lines lacking or expressing p53 protein

Background: Sequence-specific combinations of purine analogs, such as fluda rabine or 6-mercaptopurine (6-MP), administered prior to cytosine arabinosi de (ara-C) have been shown to abrogate ara-C resistance in human leukemia c ells in vitro and in patients with relapsed acute myeloid or lymphoblastic leukemias. The two-drug combination of 6-MP plus ara-C results in greater c ytotoxicity than that achieved with either ara-C or 6-MP alone. Further pre clinical investigations have shown that the addition of PEG-asparaginase (P EG-ASNase) to the combination of 6-MP plus ara-C (6-MP + ara-C + PEG-ASNase ) results in 15.6-fold synergism over that achieved with the two-drug regim en. This is due to increased DNA damage leading to apoptotic cell death. Pu rpose: Since the intravenous preparation of 6-MP is no longer available and since oral 6-thioguanine (6-TG) provides higher levels of intracellular th ioguanine nucleotides than an isotoxic dose of oral 6-MP, we investigated t he potential drug synergism of 6-TG plus ara-C plus PEG-ASNase (TGAP) in my eloid (HL60/S, HL60/SN3, U937) and lymphoblastic (CEM/0, CEM/ara-C/B, CEM/a ra-C/I, MOLT-4) leukemia cell lines. The CEM clones, MOLT-4 and HL60/SN3 ce ll lines expressed functional or measurable p53 protein, while the other ce ll lines did not. Methods: The MTT and trypan blue dye exclusion assays wer e used to determine drug cytotoxicity. In addition, cellular apoptosis and cellular p53, p21/waf-1 and bcl-2 protein concentrations were determined by FACS analysis and ELISA assays. Results: Sequential exposure to 6-TG (24 h ) plus ara-C (24 h) plus PEG-ASNase (24 h) produced 1.3- to 18.3-fold drug synergism over the two-drug combination of 6-TG plus ara-C. The molecular m echanism of synergism was due to the fact that the three-drug combination w as capable of downregulating bcl-2 oncoprotein levels in these cell lines e ven when p53 was absent. Conclusion: These studies strongly demonstrate tha t the TGAP regimen is highly synergistic in p53-null and p53-expressing leu kemia cell lines. We conclude that this combination regimen is collaterally sensitive with ara-C and further evaluation in an investigational phase I trial in relapsed leukemia patients is warranted.