Gerald W. Crabtree

Site-Selective Cyclic AMP Analogs as New Biological Tools in Growth Control, Differentiation, and Proto-oncogene Regulation

The physiologic role of cyclic adenosine monophosphate (cAMP) in the growth control of a spectrum of human cancer lines, including leukemic lines, and v-rasH oncogene-transformed NIH/3T3 cells is demonstrated by the use of site-selective cAMP analogs. These cAMP analogs, which can select either of the two known cAMP binding sites of the cAMP receptor protein, induce potent growth inhibition, phenotypic change, and differentiation (leukemic cells) of cancer cells at micromolar concentrations with no sign of cytotoxicity. The growth inhibition parallels selective modulation of cAMP-dependent protein kinase isozymes, type I versus type II, and suppression of cellular proto-oncogene expression. Site-selective cAMP analogs thus provide new biological tools for investigating cell proliferation and differentiation and also for the improved management of human cancers.

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.

Pathways of nucleotide metabolism in Schistosoma mansoni—III: Identification of enzymes in cell-free extracts☆

Abstract A survey of purine anabolic and catabolic enzymes has resulted in identification of major pathways in schistosome nucleotide biosynthesis. It is shown that multiple pathways for the incorporation of purine bases and nucleosides exist. The evidence suggests that adenosine phosphoribosyltransferase (APRT) activity is about ten times greater than adenosine kinase activity. Furthermore adenosine is converted to AMP principally via the pathway of adenosine deaminase, followed by conversion of inosine to hypoxanthine. In this sequence hypoxanthine phosphoribosyltransferase (HPRT) activity is rate limiting. On the basis of enzyme activities determined, one can suggest candidates of nucleotide analogs which might be useful chemotherapeutic agents.

Pathways of nucleotide metabolism in Schistosoma mansoni—V Adenosine cleavage enzyme and effects of purine analogues on adenosine metabolism in vitro☆

Abstract Schistosoma mansoni extracts have been found to possess an active anabolic pathway for nucleotide biosynthesis in which adenosine is cleaved to adenine followed by conversion of adenine to AMP via adenine phosphoribosyltransferase. A significant fraction of labeled adenosine was found to enter the nucleotide pool by this pathway; howeverm, most of the nucleoside was converted to nucleotides by a pathway which employs adenosine deaminase, purine nucleoside phosphorylase and hypoxanthine phosphoribosyltransferase enzymes. Formycin A has been found to be a potent blocker of adenosine cleavage when tested in worm extracts. Arabinosyl-6-mercaptopurine and 6-thioguanosine are inhibitors of worm adenosine deaminase, and formycin B and 6-thioguanosine were found to inhibit the purine nucleoside phosphorylase of this parasite. Combinations of arabinosyl-6-mercaptopurine with either formycin A or formycin B result in substantial blockage of adenosine utilization for nucleotide synthesis. These studies thus suggest that adenosine analogues in combination might be useful in vivo for the chemotherapy of schistosomiasis.

Pathways of nucleotide metabolism in schistosoma mansoni—VII: Inhibition of adenine and guanine nucleotide synthesis by purine analogs in intact worms

Abstract Twelve analogs of adenosine or its related purines have been tested as single agents or in combinations as possible antischistosomal compounds. Measurements were made of the effect of these drugs on the anabolism and catabolism of [8- 14 C]adenosine by Schistosoma mansoni in vitro . Based on the degree of inhibition of biosynthesis of adenine and guanine nucleotides by intact worms. the best currently available purine analogs are 7-deaza-adenosine (tubercidin) and N 6 -phenyladenosine. These drugs reduced the total synthesis of nueleotides to 30 and 25 per cent, respectively, of controls. Blockade of the catabolic pathway (adenosine deaminase) by coformycin resulted in significantly increased synthesis of adenine nucleotides rather than the expected decrease. Thus, adenosine kinase must play a more prominent role in nucleotide synthesis than had been previously estimated. The implications of these findings in the development of new anti-schistosomal drugs are discussed.

Clinical pharmacokinetics of 3-day continuous infusion cisplatin and daily bolus 5-Fluorouracil

In-vitro and animal studies have indicated that the combination of 5-Fluorouracil (5-FU) and cisplatin may have a synergistic antitumor effect [1-3]. The synergism of combination regimens has been shown to occur clinically in the treatment of head and neck cancer [4] and colorectal carcinoma [5, 6]. In this report, we describe the pharmacokinetics of the combination regimen using a 3-day continuous infusion of cisplatin (25mg/m2/day) and bolus doses of 5-FU (400 mg/m 2) at 24, 48 and 72 h after the start of continuous infusion cisplatin. The clinical results of this trial have been reported [6]. A total of 7 patients with gastrointestinal malignancies (5 m, 2 f; ages 48-78 y) participated in the study. Cisplatin blood samples were drawn at times 0, 3, 24, 48, and 72 h during infusion and 10, 30, 60, 120 and 180 min post-infnsion. Four successive 24-h urine samples were collected for measurement of cisplatin excretion. 5-FU blood samples were drawn at times 5, 10, 20, 30, 45 and 60 min after the 24 and 72-h bolus 5-FU. Samples were assayed for total and filterable platinum by the method of Forastirere et al. [7] and for 5-FU by the method of Miller et al. [8]. Cisplatin measurements were made on samples from 6 patients (6 courses) and 5-FU measurements on samples from 5 patients (8 courses). Because of the unexpected result of prolonged elevated filterable platinum levels in plasma after drug administration, an animal study using rats was performed to confirm the clinical data. Sprague-Dawley rats (400600 g) had cisplatin (15 mg/m 2 in 0.5 ml saline) administered by tail vein injection i min before 5-FU (300 mg/m 2 in 0.5 ml saline) or saline (0.5 ml) injections. Rats were sacrified by exsanguination at various times after cisplatin administration (20, 60, 90 and 180 min). The blood was immediately collected for measurement of total and filterable plasma platinum levels [7]. Table 1 presents the results of 5-FU plasma measurements and the pharmacokinetics are in agreement with

Phase I trial of the combination of 6-methylmercaptopurine riboside and 5-fluorouracil.

Abstract6-Methylmercaptopurine riboside (MMPR) and 5-fluorouracil (5-FU) were administered sequentially to 12 patients in a phase I clinical trial. Toxicities included mild nausea and vomiting, as well as reversible leukopenia and thrombocytopenia. Maximal accumulation of 6-methylmercaptopurine ribonucleoside 5′-monophosphate (MMPR-P), the active metabolite of MMPR, in patients’ erythrocytes occurred between 2 and 6 h after the administration of MMPR and the degree of accumulation was dose-related. At 96 h after MMPR administration, MMPR-P was still detectable in patients’ erythrocytes. Although no clinical responses were documented, a modified dosage schedule of these drugs should be pursued based on the pharmacokinetic data obtained.