John S. Holcenberg

THE ROLE OF SEROTONIN (5-HYDROXYTRYPTAMINE) IN MAST CELLS*

In the text following, we shall consider the question of the role of serotonin in mast cells. In order to do this, we shall proceed as follows: First, we shall indicate the reasons why our laboratory has been interested in mast cells; second, we shall review briefly the evidence for the presence of 5-hydroxytryptamine in mast cells of mammals; third, we shall consider, on the basis of evidence at hand, what role serotonin may play in the biological economy of those species in which it is present in the mast cells.

Glutaminase induced prolonged regression of established Ehrlich carcinoma

Abstract Glutaminase treatment of mice bearing an asparaginase-resistant Ehrlich carcinoma resulted in complete regression of one week established tumors in 67 to 80 percent of animals and more than 200 percent prolongation of survival time to date. Combination of glutaminase with asparaginase did not produce a better therapeutic effect than glutaminase alone.

Optimal Asparaginase Therapy

Editor’s Note: In the April issue of the journal, Volume 26, Number 4, Avramis and colleagues presented data to show that in a selected group of patients with Acute Lymphoblastic Leukemia being treated on a CCG trial that the presence of silent antibodies (that is clinically undetectable) potentially rendering the therapy ineffective is present in a significant number of patients (1). Given the importance of the enzyme in treatment of patients with ALL, as evidenced by finding that a lack of enzyme activity may correlate with outcome, questions such as “how best give the enzyme?” “how to monitor its effect?” and “how to prevent the development of antibodies?” seem crucial both to the patient as well as the physician-scientist evaluating the efficacy of the treatment. That is it would seem important to know why therapy failed or why was it toxic as we move towards maximizing/individualizing therapy. John Holcenberg is an acknowledged expert both in the use of enzymes as drugs and in the pharmacology of anti-cancer agents. Rather than having me pose questions and raise issues about how to incorporate Asparaginase in treatment plans, I simply asked Dr. Holcenberg to prepare a commentary for this issue. The results are more informative and the opinion more authoritative than mine.

New insights on asparaginase

I t has been almost 40 years since J. D. Broome identified asparaginase as the antileukemic factor in guinea serum and showed that sensitivity to this enzyme was due to insufficient asparagine synthetase (ASY) activity. ASYactivity was found to be low in most normal cells but capable of induction under conditions of asparagine deprivation. Mouse lymphoma and lymphocytic leukemic cells killed by asparaginase treatment had a defective ASY enzyme that could not be induced to adequate levels. Recent observations have questioned this simple explanation for asparaginase sensitivity. Stams et al showed that leukemic cells from children with TEL/AML1+ lymphocytic leukemia have higher baseline mRNA expression of ASY than other ALL cells or normal lymphoid cells. This is despite the clinical observation that this subtype of leukemia is more sensitive to asparaginase in vitro and has a better cure rate with asparaginase-containing regimens than other ALL subtypes. Krejci et al confirmed this observation and showed that expression of ASY mRNA was increased by asparaginase treatment in vitro in both TEL/AML1-positive and -negative ALL cells. They hypothesized that the increased ASY expression is due to an accumulation of TEL/AML1 cells in the G1/G0 phase of the cell cycle. Fine et al used cDNA microarrays to quantify multiple genes in ALL cell lines and in cells from untreated children with ALL. As expected, the cell lines showed a correlation between in vitro sensitivity to asparaginase and baseline expression of ASY. Furthermore, the asparaginase-resistant cell lines increased expression of ASYafter in vitro exposure to asparaginase, while the asparagine-sensitive cell lines did not increase this expression. In contrast, the levels of ASY did not correlate with asparaginase sensitivity in the clinical samples, and there was no clear relationship with levels of ASYexpression after exposure to asparaginase in vitro and sensitivity to asparaginase. In the March issue of Leukemia, Broome et al presented surprising evidence that expression of ASY mRNA in two asparaginase-sensitive ALL cell lines was increased by coculture of these cells with macrophages or supporting cells. The increased expression was associated with changes in acetylation of histone H3 and methylation of the ASY promoter. This increased ASY expression allowed the cocultured cells to grow in asparagine-free medium. This change persisted as long as the cells remained in contact with the supporting cells. Constant cell contact was required since removal of the leukemic cells to fresh medium caused ASY expression to fall to baseline levels. If induction of ASY expression in lymphoblasts by surrounding cells is a general phenomenon, it might explain the disparate results between ASY expression in clinical samples and in vitro asparaginase sensitivity. Different compartments like the bone marrow, testes, or meninges might provide various cell contacts with leukemic cells that could alter their local sensitivity to asparaginase and lead to sanctuaries of asparaginase-resistant populations. The increased in vivo sensitivity of TEL-AML1 cells might be due to their inability to induce ASYexpression by this type of cell contact. What might this observation mean to asparaginase treatment? Current regimens combine asparaginase with vincristine, a glucocorticoid, and an anthracycline. None of these agents primarily affects gene expression. Combination with dactinomycin or mithramycin, inhibitors of DNA-directed RNA synthesis, might prevent induction of ASYexpression by supporting cells. Discovery of the mechanism by which cell contact leads to ASY induction may lead to more specific therapy to block this potential cause of resistance to asparaginase. It might also be interesting to test the effects of agents that affect global methylation or acetylation patterns on this induction of ASY expression. I look forward to further experiments to address these potential new therapeutic approaches. In this regard, Broome et al also speculate that this expression and demethylation of ASY gene may be a characteristic of lymphoid cells, since similar patterns are seen with interferon gamma and IL-4. Perhaps the expression of other genes is affected by contact of lymphoblasts with supporting cells. It would be interesting to analyze these lymphoblast cell lines before and after co-culture for expression of proteins needed for activation of mercaptopurine, methotrexate, and cytosine arabinoside. Received for publication April 5, 2005; accepted April 6, 2005. From the Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington. Reprints: John Holcenberg, MD, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98105 (e-mail: jholce@ chmc.org). Copyright 2005 by Lippincott Williams & Wilkins

Active enzyme sedimentation of antitumor asparaginase and glutaminase enzymes

Abstract Active enzyme sedimentation of five asparaginase and glutaminase-asparaginase enzymes with antitumor activity was studied. The catalytically active species of each enzyme appeared to have a molecular weight greater than 100,000 g/mole. Gel filtration and disc gel electrophoresis confirmed the absence of catalytically active smaller species.