Ronald McCaffrey

Antileukemic activity and mechanism of action of cordycepin against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells

The nucleoside analogue cordycepin (3-deoxyadenosine, 3-dA) is substantially more cytotoxic to terminal deoxynucleotidyl transferase positive (TdT+) leukemic cells than to TdT leukemic cells in vitro in the presence of an adenosine deaminase inhibitor, deoxycoformycin (dCF), and has been considered as a therapeutic agent for TdT+ leukemia. The intracellular metabolism of 3-dA was examined with HPLC, and the mechanism of its anti-TdT+ leukemic activity was analyzed. In the presence of dCF (2.5 microM), TdT+ leukemic cells (N = 5) were sensitive to the cytotoxic effect of 3-dA, whereas TdT (N = 6) cells were not. A high level of 3-dA-5-triphosphate (3-dATP) formation was detected in TdT+ NALM-6 cells (67 pmol/10(6) cells) and TdT- K562 cells (49 pmol/10(6) cells) when cultured with 1 microM [3-3H]-labeled 3-dA. A substantial level of 3-dATP was detected in TdT HUT-102 cells (27 pmol/10(6) cells), whereas the level of 3-dATP in TdT+ MOLT-4 cells was low (0.3 pmol/10(6) cells). The mean IC50 values of 3-dA against phytohemagglutinin (PHA)-activated and resting peripheral blood mononuclear cells (PBM) (N = 5) were 8 and 32 microM, respectively. There was a modest level of 3-dATP (7 pmol/10(6) cells) in PHA-PBM, whereas a lower level of 3-dATP was detected in resting PBM (2.5 pmol/10(6) cells). These data suggest that the presence of 3-dATP is not sufficient for the antileukemic effect of 3-dA, but that TdT positivity is essential, and that PBM are significantly less sensitive to the cytotoxicity of 3-dA in vitro. Further development of 3-dA as a potential antileukemic agent to treat patients with TdT+ leukemia is warranted.

Clinical activity of a cytotoxic fusion protein in the treatment of cutaneous T-cell lymphoma.

PURPOSEnA phase I trial in patients with refractory hematologic malignancies was performed at our institution to test the clinical relevance of the selective cytotoxic activity of the interleukin-2 (IL-2)-diphtheria toxin fusion protein, DAB486IL-2. A subset of five patients from this trial, all with cutaneous T-cell lymphomas (CTCL), forms the basis of this report.nnnPATIENTS AND METHODSnTwo treatment schedules were used. One patient received DAB486IL-2 at a dose of 0.075 mg/kg/d intravenous (i.v.) bolus over 15 minutes daily for 5 consecutive days. The other four patients received DAB486IL-2 at a dose of 0.1 mg/kg as an i.v. infusion over 180 minutes weekly for 5 consecutive weeks.nnnRESULTSnThree of the five CTCL patients achieved significant tumor responses. One patient attained a complete clinical and pathologic response (CR), which has been sustained without any interval treatment for 33+ months. Two other patients achieved partial responses (PRs) of 17+ and 4 months duration, respectively. Treatment was well tolerated. The most common adverse effect was a transient increase in hepatic transaminases experienced by all five patients.nnnCONCLUSIONnThe growth factor-cytotoxin fusion protein DAB486IL-2 demonstrated significant clinical activity with acceptable toxicity in a group of heavily pretreated patients with CTCL.

Treatment of malignant pleural mesothelioma with cisplatin, mitomycin C and alpha interferon.

From October 1990 to September 1991, 20 consecutive patients with histologically proven malignant pleural mesothelioma (MPM), secondary to environmental exposure to asbestos or erionite, were treated with cisplatin, mitomycin C and alpha interferon (cisplatin 50 mg/m2 i.v. on day 1 of a 21-day cycle; mitomycin C 10 mg/m2 i.v. day 1 of cycles 1,3 and 5; alpha-2b-interferon 10 x 10(6) units i.m., 4 h prior to cisplatin and 10 x 10(6) units i.v. immediately prior to cisplatin day 1 of each cycle). Eighty-two treatment cycles were administered to 19 evaluable patients. Two patients attained a partial response. Eleven patients had stable disease and 6 had disease progression. Toxicities included interferon-related fever and flu-like symptoms, and vomiting. Actuarial median survival was 15 months. Three patients are alive at 20+, 21+ and 27+ months. We conclude that while the addition of alpha interferon to cisplatin and mitomycin C did not result in an objective response higher than previously reported with the cytotoxic agents alone, the trend towards an improvement in median survival as compared to a well-matched historical group suggests some benefit from the inclusion of interferon.

Augmentation of vincristine cytotoxicity by megestrol acetate

Abstractu2002Purpose:u2002To determine the effect of a semisynthetic progesterone, megestrol acetate (MA), on the cytotoxicity of various chemotherapeutic agents including vincristine, doxorubicin, actinomycin-D, taxol, vinblastine and colchicine in cell lines with or without P-gp expression. Methods:u2002Three cell lines with high P-gp expression (two colon cancer and one leukemia), and a control cell line with no P-gp expression were exposed to chemotherapeutic agents in the presence or absence of MA and drug sensitivity was determined using the MTT colorimetric assay. P-gp-170 expression was detected by flow cytometry using JSB-1 monoclonal antibody and the functionality of MDR expression was tested by rhodamine-123 uptake studies. In vitro drug accumulation studies were performed using [3H]-vincristine. The results were subjected to paired t-test analysis and 95% confidence intervals were determined in cytotoxicity tests. Results: MA augmented the cytotoxicity of vincristine, but not doxorubicin, actinomycin-D, taxol, vinblastine or colchicine in the three P-gp-expressing cell lines, whereas verapamil augmented the cytotoxicity of doxorubicin and vincristine. MA did not augment the cytotoxicity of vincristine in the P-gp-negative HUT-102 cell line. Conclusion: MA augmented vincristine cytotoxicity in P-gp-expressing cell lines. However, this phenomenon did not occur with the other classic MDR drugs. Therefore, the augmentation of vincristine cytotoxicity by MA can be explained either by involvement of a different mechanism that coexists with the mdr-1 phenotype or by the presence of a different affinity or binding site on the P-gp molecule for MA compared to that for the other classic MDR drugs and verapamil.

Peripheral blood hairy cell leukemia cells express only low affinity IL-2 receptors

Purified leukemia cells from three patients with hairy cell leukemia were studied for IL-2 receptor status. All samples expressed the p55IL-2 receptor subunit. However, none demonstrated sensitivity to the IL-2-diphtheria toxin conjugate, DAB486IL-2, which shows selective cytotoxicity for high affinity IL-2 receptor-bearing cells. In the one case studied with 125I-IL-2 binding, only low affinity IL-2 receptors were noted. These data suggest that hairy cell leukemia cells express only low affinity IL-2 receptors and are insensitive to DAB486IL-2.

Purine metabolism in leukemia.

The importance of purine metabolism in immune function was established by the discovery of associations of deficiencies of two purine salvage enzymes with hereditary immunodeficiency states: adenosine deaminase (ADA) deficiency with severe combined immunodeficiency, and purine nucleoside phosphorylase (PNP) deficiency with T cell dysfunctions. Patients studied had severe lymphopenia, decreased response to PHA, PWM, and ConA stimulation, and cellular depletion in lymphatic tissues. Accumulation of toxic nucleosides, deoxyadenosine and deoxyguanosine, which are normally broken down by ADA and PNP, was thought to be one of the mechanisms for lymphocytotoxicity.** Another important development during these years was the availability of enzyme inhibitors, especially the ADA inhibitors of varying potencies?.6 Use of these inhibitors in vitro prevented maturation of stem cells into mature T cells, inhibited tumor-directed cell-mediated cytotoxicity,8 and enhanced toxicity of adenosine, deoxyadenosine, and other adenosine analogues. Treatment of animals with these inhibitors produced syndromes similar to those of immunodeficient patients. The literature on ADA inhibitors has recently been reviewed: These observations indicated a regulatory role of these enzymes in lymphocytic growth, differentiation, and function, and provoked a number of important questions. Do lymphocytic populations differ in purine metabolism? If they do, is it then possible to characterize lymphoproliferative disorders based on these differences? Can this difference in purine metabolism be exploited as a therapeutic target site? In this paper we briefly review present knowledge of purine metabolism in lymphocytes, differences in metabolism in leukemic cells. and discuss how this information may be used to help answer the questions raised above.

PHA induces IL-2 receptors on B-CLL cells and is a potential biological response modifier for the LIL-2-diphtheria toxin, DAB486IL-2

DAB486IL-2 is an IL-2-diphtheria toxin conjugate which was developed to be specifically cytotoxic to cells bearing high affinity IL-2 receptors. The high affinity IL-2 receptor is a heterodimer comprising p55 and p75 subunits. While the p75 subunit appears to be ubiquitously expressed among the common North American leukemias and lymphomas, the p55 subunit is more restricted in its expression. To broaden the therapeutic relevance of the DAB486IL-2 we have sought physiologically feasible inducers of the p55 IL-2 receptor subunit. This report describes that PHA, in vitro, induces the p55 IL-2 receptor subunit on initially p55-negative B-CLL cells and converts toxin-insensitive leukemia cells to a toxin-sensitive state.

Inhibitors of Terminal Transferase: A New Strategy for the Treatment of Human Leukemia

Terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) is a unique DNA polymerase which catalyzes the polymerization of deoxyribonucleotides on the 3’-hydroxyl ends of preformed oligo- or polydeoxynucleotide initiators, in a template-independent manner (1). Its expression is restricted, in normal animals, to subsets of primitive lymphocytes, and, in disease states, to the blast cells of certain forms of acute leukemia and diffuse lymphoma (2). For immunobiologists TdT has emerged as a useful marker for characterizing subsets of pre-B and pre-T lymphocytes (3–7). For physicians caring for patients with leukemia and lymphoma, neoplastic cell TdT status has turned out to be a useful criterion for patient assignment to therapeutically meaningful categories (8).

Dysfunctional Glucocorticoid Receptors in Acute Leukemia

The obligatory role of specific cytoplasmic receptors as mediators of steroid hormone action is well established. In lymphoid cell culture systems the development or glucocorticoid resistance is almost always due to a mutation to a receptor negative state [1]. Therefore, if the assumption is that glucocorticoid-induced remission in leukemia is by a direct effect on leukemic blast cells, one would predict that the presence of absence of blast cell glucocorticoid receptors would be a major determinant or clinical steroid sensitivity. However, receptor quantization has not correlated with steroid responsiveness [2–5], and in particular, high receptor numbers are not uniformly associated with responsive disease [6].