Todd M. Savarese

5′-methylthioadenosine phosphorylase—I: Substrate activity of 5′-deoxyadenosine with the enzyme from Sarcoma 180 cells

Abstract 5′-Deoxy-5′-methylthioadenosine phosphorylase (MTA phosphorylase), an enzyme involved in the salvage of adenine moieties from 5′-deoxy-5′-methylthioadenosine (MTA) produced primarily during polyamine biosynthesis, is present in Sarcoma 180 cells (0.0026 ± 0.0002 μ M units/mg cytosol protein). 5′-Deoxyadenosine (5′-dAdo), an adenosine analog previously thought not to be metabolizable, has been shown [D. Hunting and J.F. Henderson, Biochem . Pharmac . 27 , 2163 (1978)] to have a number of biochemical effects on Ehrlich ascites cells. We have now found that 5′-dAdo is a substrate for the MTA phosphorylase from Sarcoma 180 cells, yielding free adenine and 5-deoxyribose-1-phosphate. The reaction was reversible and totally dependent upon phosphate. Evidence that MTA phosphorylase is responsible for 5′-dAdo phosphorylase activity includes the following: (1) Sarcoma 180 MTA phosphorylase preparations did not show additive rates of adenine production in the presence of saturating concentrations of both 5′-dAdo and MTA; (2) double-reciprocal plots of the rates of adenine formation from 5′-dAdo by Sarcoma 180 enzyme preparations in the presence of MTA displayed a pattern characteristic of alternative, competing substrates; (3) the rate of depletion of 5′-dAdo by Sarcoma 180 preparations was inhibited by the presence of MTA; (4) the K i value of a competitive inhibitor of Sarcoma 180 MTA phosphorylase, 5′-deoxy-5′-chloroformycin, was the same when either MTA or 5′-dAdo was employed as substrate; and (5) the apparent K m values of phosphate for both MTA and 5′-dAdo phosphorylase activities were identical (3.5mM). The K m of Sarcoma 180 MTA phosphorylase for MTA is 4 μM; the K m for 5′-dAdo is 23 μM ( V max relative to MTA = 180 per cent). Incubation of Sarcoma 180 cells with either 5′-dAdo or MTA caused profound elevations of adenine nucleotides, as well as an inhibition of 5-phosphoribosyl-l-pyrophosphate (PRPP) accumulation. The reaction of 5′-dAdo with MTA phosphorylase to yield free adenine, which is then salvaged to adenine nucleotides, can account for many of the previously reported biochemical effects of 5′-dAdo, such as inhibitions of PRPP accumulation, purine de novo synthesis, and glycolysis that have previously been attributed to the unmetabolized nucleoside. The other product of this reaction, 5-deoxyribose-l-phosphate, may also contribute to these effects.

Reversal by L-cysteine of the growth inhibitory and glutathione-depleting effects of N-methylformamide and N,N-dimethylformamide

N-Methylformamide and N,N-dimethylformamide, which can induce differentiation in selected malignant cell lines, are known to increase doubling times, inhibit clonigenicity in agar, and to effect responses against particular human colon carcinomas in vivo. At concentrations which inhibit growth and clonigenicity, N-methylformamide (170 mM) and N,N-dimethylformamide (103 mM) deplete total intracellular glutathione levels of DLD-1 Clone A human colon carcinoma cells in a dose and time dependent manner. In the presence of 0.5 mM 1-cysteine, both the growth and glutathione levels of polar-solvent treated DLD-1 Clone A cells are restored. 1-Cysteine also reverses the inhibition of clonigenicity mediated by NMF. The mechanism of action of N-methylformamide and N,N-dimethylformamide against this cell line, at least in vitro, is therefore related to its effects on cysteine/glutathione metabolism. Furthermore, this evidence suggests that glutathione plays a key role in regulating the growth of these cells.

Posttranscriptional regulation of c-myc proto-oncogene expression and growth inhibition by recombinant human interferon-β ser17 in a human colon carcinoma cell line

SummaryRecombinant human interferon-β ser17 (IFN-β ser17), a cytokine that exhibits both antiviral and antiproliferative activity against a wide variety of cell types, causes a time- and dose-dependent inhibition of monolayer growth and of the expression of the c-myc proto-oncogene in DLD-l Clone A human colon-carcinoma cells. The suppression of c-myc expression mediated by IFN-β ser17 is due to a posttranscriptional destabilization of c-myc mRNA rather than to an inhibition of c-myc mRNA transcription. There is evidence suggesting that the selective reduction in the half-life of c-myc mRNA in IFN-β ser17-treated cells occurs through an increase in the activity of the 2′,5′-oligoadenylate synthetase/RNase L [2′,5′-oligo (A) synthetase] pathway in DLD-1 Clone A cells. Cotreatment of these cells with IFN-β ser17 and the anticancer agentN-methylformamide leads to the partial abrogation of 2′,5′-oligo (A) synthetase activity and the stabilization of c-myc mRNA. These findings suggest that there is a correlation between the IFN-β ser17-mediated suppression of c-myc expression and the induction of 2′,5′-oligo (A) synthetase activity in DLD-1 clone A cells.

5′-Halogenated Formycins as Inhibitors of 5′-Deoxy-5′-methylthioadenosine Phosphorylase: Protection of Cells Against the Growth-Inhibitory Activity of 5′-Halogenated Adenosines

Abstract A series of 5′-halogenated formycins, including the chloro-, bromo- and iodo- derivatives, were synthesized. These compounds are competitive inhibitors of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAPase) with Ki values in the range of 10−7 M, making them the most potent inhibitors of MTAPase reported to date. These compounds protect cells from the growth-inhibitory action of 5′-halogenated adenosines, which must be activated by MTAPase. The syntheses of 5′-halogenated formycin B derivatives, which inhibit purine nucleoside phosphorylase, are also described.

Acycloadenosine Derivatives as Inhibitors of 5′-Deoxy-5′-Methylthioadenosine Phosphorylase (MesADo PASE)

Abstract Four classes of acyclo amlogs of 5′-methylthioadenosine were synthesized and tested as inhibitors of mammalian methylthicadenosine phosphorylase. Halogenated dihydroxypropyl acycloadenosires were most potent, i.e. Ki = 0.2 - 0.7 uM.