Edward L. Schwartz

Effect of interferon on 5-fluorouracil-induced perturbations in pools of deoxynucleotide triphosphates and DNA strand breaks

Abstractu2002Interferon (IFN) augments the anabolism of 5-fluorouracil (5FU) to its active metabolite, fluoro-deoxyuridylate (FdUMP), which inhibits thymidylate synthase (TS). We sought to determine whether this resulted in greater perturbations of nucleotide pools and if so, whether this was associated with an increase in cell lethality, specifically focussing on the lethal cellular lesion, DNA double strand breaks (dsb). To determine whether combination therapy with 5FU+IFN resulted in greater depletion of thymidine nucleotide pools than 5FU alone, a highly sensitive DNA polymerase assay was used. In two human colon cancer cell lines, treatment with 5FU+IFN resulted in a rapid decrease in levels of dTTP by 95%. The addition of IFN to 5FU resulted in greater depletion of dTTP levels over treatment with 5FU alone by up to four fold, and markedly augmented the dATP/dTTP ratio. The addition of IFN to 5FU had no effect on 5FU-induced perturbations in dCTP, dGTP or dATP pools at 8 and 12u2005h. Measurement of DNA dsb demonstrated that treatment of HT-29 cells with 10u2005μM 5FU for 24u2005h did not increase DNA dsb versus control. The combination of 5FU+500u2005U/ml IFN, however, resulted in an increased number of dsb versus both 5FU and untreated control cells (P∼0.01), equivalent to 0.74±0.12 Gy. The addition of IFN to 5FU resulted in a selective further depletion of pools of dTTP and an increase in the number of DNA dsb versus 5FU treatment alone.

Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate synthase-inhibited cells using a modified DNA polymerase assay☆

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.

Development of two radioimmunoassays to detect paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids

Paclitaxel is an antimitotic agent isolated from the Pacific yew tree. It has demonstrated antitumor activity in several cancers and is the first of a new class of antineoplastic agents containing a taxane ring system. Its levels in serum and urine have been measured previously by high performance liquid chromatography (HPLC). In this study, the authors developed two competitive radioimmunoassay methods to determine whether they could reliably be used to measure levels of paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids.

Biologic agents as biochemical modulators: pharmacologic basis for the interaction of cytotoxic chemotherapeutic drugs and interferon

Biochemical modulation of cytotoxic cancer chemotherapeutic agents is one means of enhancing the activity and selectivity of antitumor drugs. Traditionally this approach has utilized detailed information regarding a particular enzymatic reaction or biochemical pathway to develop potential modulating agents. In contrast, the reported clinical therapeutic activity of IFN in combination with cytotoxic agents has prompted a reexamination of the biochemical actions of the cytokine. Interferon elicits a number of cellular actions that might contribute to its pharmacologic activity, including both direct antitumor effects and host-mediated actions. The best understood are those related to the cytotoxicity of the fluoropyrimidine antimetabolites and include enzymatic reactions involved in fluoropyrimidine metabolic activation, catabolism, and interaction with its target enzyme. However, even in this instance, a mechanistic association of a specific pharmacologic action with therapeutic activity remains to be determined. These studies demonstrate that cytokines and other biologic agents may exert specific biochemical modulations that augment (or potentially attenuate) the activity of the cytotoxic chemotherapeutic agents.

An ECOG phase II study of amonafide in unresectable or recurrent carcinoma of the head and neck (PB390)

The purpose of this study was to determine the efficacy and toxicity of amonafide in unresectable or recurrent head and neck cancer and to determine if the degree of toxicity with amonafide correlated with the acetylator phenotype of the patient. Thirty patients were registered on the study and received amonafide, 300 mg/m2, over two hours each day for five consecutive days every 21 days. There was one partial response [39] which lasted four months. The dose limiting toxicity was myelosuppression. Acetylator phenotype was determined prior to treatment using HPLC to quantitate caffeine metabolites in urine samples after administration of caffeine. This pharmacokinetic evaluation was performed in 21 patients and revealed that (17/21) 81% of the patients were slow acetylators and 19% of the patients were rapid acetylators. No association was found between acetylator phenotype and toxicity in our patient population. Based on this study, it appears that amonafide given at 300 mg/m2 for 5 consecutive days every 21 days is not active in squamous cell carcinoma of the head and neck, and that acetylator status does not correlate with toxicity.

Phase I clinical and pharmacokinetic study of menogaril (7-con-O-methylnogarol) in previously treated patients with acute leukemia

SummaryFifteen patients with relapsed or refractory acute leukemia were treated in this phase I study of menogaril (7-con-O-methylnogarol), a nogalamycin anthracycline derivative. Doses ranged from 50 mg/m2/day to 130 mg/m2/day, administered daily for 5 days. Pharmacokinetic studies were performed at each dose level and confirmed the findings of pharmacokinetic data derived from previous studies in patients with solid tumors. All patients experienced grade 4 hematologic toxicity and the dose limiting toxicity was mucositis. Two patients, one with acute myeloid leukemia and one with acute lymphoid leukemia, achieved complete responses. The AML complete response lasted 10 months and the ALL patient died in CR at 2 + months. Both patients were treated at a dose of 100 mg/m2/day for five days. At this dose, a second induction or consolidation course could be given without severe mucositis, and this is the dose recommended for further phase II studies in leukemia using this schedule.

Inhibition of c-myc expression in human promyelocytic leukemia and colon adenocarcinoma cells by 6-thioguanine

A rapid decrease in expression of the oncogene c-myc has been associated with the induction of differentiation of HL-60 human leukemia cells. In this manner, the treatment of a hypoxanthine phosphoribosyltransferase (HPRT)-deficient HL-60 variant (HL-60/var) with 6-thioguanine (TG) was accompanied by lower c-myc mRNA levels. This occurred in the absence of 6-thioguanosine 5-monophosphate (TGMP) synthesis and without alterations in cellular nucleotide pool sizes. Paradoxically, inhibition of c-myc expression in the wild type HL-60 (HL-60/wt) cell, which is only weakly induced to differentiate by TG, was 5-fold more sensitive to the thiopurine (IC50 = 35 microM). Furthermore, inosine, which blocks the formation of TGMP and enhances the extent of differentiation of HL-60/wt cells, decreased the sensitivity of c-myc expression in the HL-60/wt to TG. These actions of TG and inosine on c-myc were also observed in the human colon carcinoma cell line COLO 320, further dissociating some of the effects of TG on c-myc expression from granylocytic differentiation. The hematopoietic granulocyte-macrophage colony stimulating factor (GM-CSF) elevated c-myc expression and antagonized the actions of TG on c-myc in the HL-60 cells. GM-CSF more readily antagonized the inhibitory action of TG in the HL-60/var cell line when compared to the HL-60/wt cells, restoring c-myc levels to that of the untreated controls. Hence, TG inhibited c-myc expression by two distinct mechanisms in cells which express high levels of the oncogene: a TGMP-dependent, differentiation-independent process with an IC50 of 35 microM, and a TGMP-independent action with an IC50 of 175 microM that was associated with induction of differentiation and was reversed more readily by GM-CSF.