Robert Silber

Heterogeneity of 5′-Nucleotidase Activity in Lymphocytes in Chronic Lymphocytic Leukemia

The specific activity of 5-nucleotidase was determined in lymphocyte plasma membranes from 14 normal subjects and 10 patients with chronic lymphocytic leukemia (CLL). Whereas the enzyme was present in the preparation from normal lymphocytes, in 7 out of 10 CLL patients the membranes had markedly decreased or no detectable 5-nucleotidase activity. The lack of this activity from the lymphocytes of most patients with CLL constitutes an alteration in a plasma membrane enzyme from the normal cell. The presence of the enzyme in the lymphocytes of some patients with CLL and its decrease in others provide further evidence for biochemical heterogeneity among patients with this disorder.

Human lymphocytes: 5'-nucleotidase-positive and -negative subpopulations.

The enzyme, 5-nucleotidase (5N) (E.C.-3.1.3.5) is present in lymphocytes isolated from the blood of normal subjects. This activity is markedly decreased or not detectable in the cells from three-quarters of patients with chronic lymphocytic leukemia (CLL), while supranormal levels are found in less than 10% of the cases. To determine whether the decreased 5N value in CLL represents a lower activity per cell or fewer enzyme-containing cells than in the normal, conditions were established for the histochemical measurement of 5N in human lymphocytes. It was found that the cells isolated from the blood of normal subjects or patients with CLL consist of 5N-positive and 5N-negative subpopulations. Normal subjects who had high 5N specific activity were shown to have a greater percentage of 5N-positive cells than individuals with low 5N activity. Patients with CLL who had no activity by standard chemical assay had no 5N-positive cells, while the exceptional patient with CLL with a higher than normal specific activity showed an percentage of 5N-positive cells. It is suggested that the selective proliferation of 5N-positive and 5N-negative populations may account for the heterogeneity of 5N in CLL.

[2] Glutathione dehydrogenase (ascorbate)

Publisher Summary This chapter describes assay, purification, and properties of glutathione dehydrogenase—that is, dehydroascorbate reductase. The enzyme catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbic acid to ascorbic acid. Dehydroascorbate reductase has been detected in various animal cells using a coupled assay which monitors the oxidation of nicotinamide adenine dineucleotide phosphate (NADPH) by glutathione reductase. The spectrum of ascorbic acid has a peak at 265 nm, while dehydroascorbic acid has no absorbance at that wavelength. The activity of dehydroascorbate reductase is assayed spectrophotometrically by measuring the change in absorbance at 265 nm associated with the generation of ascorbic acid. A unit of enzyme activity is the amount of enzyme which reduces 1 μmol of L-dehydroascorbic acid in 1 min at 37°. Specific activity is expressed as units per milligram of protein. The nonenzymatic reduction of dehydroascorbic acid by GSH occurs rapidly at pH 7.5–8.0; therefore despite the pH optimum of the enzymatic reduction of 7.5, the assays should be performed between pH 6.3 and 6.8.

[53] Cytidine deaminase from leukemic mouse spleen

Publisher Summary This chapter describes the purification procedure of cytidine deaminase enzyme from leukemic mouse spleen. Cytidine deaminase is widely distributed among mammalian tissues. This enzyme is responsible for the inactivation of cytosine arabinoside––one of the most useful chemotherapeutic agents available for the treatment of acute myeloblastic leukemia. In the mouse spleen, a marked increase in the specific activity of cytidine deaminase occurs following infection with Friend leukemia virus. Elevated levels of cytidine deaminase are also found in regenerating mouse liver, in human leukemic cells, following the treatment of the patient with cytosine arabinoside, and in HeLa cells cultured in the presence of cytosine arabinoside. The activity of cytidine deaminase is assayed by measuring the amount of labeled uridine formed by the deamination of 2-14C-labeled cytidine. High levels of cytidine deaminase are found in human polymorphonuclear leukocytes.

15 DNA Topoisomerases in Clinical Oncology

I. INTRODUCTION There is a compelling reason for studying human disease as the ultimate experimental system. This approach, highly practical and direct, can use already available information correlating clinical status, pathology, and pharmacology. This data base may help investigators to reevaluate current trends and to develop new directions in therapy. In this chapter, DNA topoisomerase research is discussed in the relation to biology and chemotherapy of selected malignancies. It was recently recognized that some of the most valuable anticancer drugs interact with topoisomerase II in cell-free systems, as well as in intact cells (for review, see Chen and Liu 1986; Potmesil 1988). In addition, topoisomerase I was identified as a target for the plant alkaloid camptothecin (Hsiang et al. 1985; Hsiang and Liu 1988). The interactions between the drug and topoisomerase involve mostly natural products of microbial or plant origin and their analogs. This results in a complex of a topoisomerase molecule bound covalently to cleaved DNA (Chen and Liu 1986), which is, most likely, the initial event leading to cell death. Drug interaction with either topoisomerase I or II plays a crucial role in drug-exerted cytotoxicity. II. ANTICANCER DRUGS INHIBITING TOPOISOMERASE I In the early 1970s, camptothecin, a plant alkaloid isolated from Camptotheca acuminata of the Nyssaceae plant family (Wall et al. 1966), was briefly tested in phase-I and -II clinical trials (Gottlieb et al. 1970; Moertel et al. 1971; Muggia et al. 1972). Myelosuppression was the dose-limiting toxicity in cancer patients treated with camptothecin sodium salt, and cystitis...