Biomedical subjects
Publications and source records attributed to R McCaffrey.
There is growing interest in the molecular events which govern response to therapy in the leukemias. Glucocorticoid therapy would appear to be a fertile area for study as much is known about mechanism of steroid hormone action. In this paper we review current knowledge of glucocorticoid receptor numbers and function in leukemic blast cells and their relationship to steroid treatment and outcome.
The clinical significance of initial DEAE chromatography of glucocorticoid binders in lymphoblastic disease was evaluated in an animal model. Domestic cats and dogs with lymphoblastic disease were treated with prednisone, 2 mg/kg/day, for 14 days, and the outcome of therapy was correlated with DEAE chromatograms of glucocorticoid binders, using 3H-triamcinolone as ligand. Six of 30 animals had a single-peak low-salt binder species, similar to that seen in a subset of human leukemia, and none of these responded. Of the 29 animals with chromatograms identical to normal tissues, 6 had a complete response and another 11 a partial response. This distribution of responders is statistically significant (p = 0.02). Thus, the leukemia-associated single-peak DEAE species appears to be associated with glucocorticoid resistance, as defined by clinical responsiveness. In contrast, the two-peak normal pattern is a necessary, but insufficient, criterion for defining responsive disease.
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Normal thymocyte and bone marrow terminal deoxynucleotidyl transferase (TdT) have distinguishing characteristics by phosphocellulose chromatography in Tris buffer: marrow TdT elutes as a single peak at 0.3 M salt, whereas thymocyte TdT separates into two forms, one at 0.3 M salt and one at 0.4 M salt. Since the majority of TdT-positive acute leukemias are anatomically bone marrow-derived, one would have predicted the presence of a bone marrow TdT-phosphocellulose chromatographic pattern in such patients. However, in 376 consecutive, untreated TdT-positive acute lymphoblastic leukemias (ALL) studied by us we have invariably encountered the two-peak thymocyte-type phosphocellulose pattern. The TdT patterns in the thymic-dependent, TdT-positive lymphoma of AKR mice, and the TdT-positive bone marrow-derived, thymic-independent Abelson virus leukemia of Balb/C mice duplicate the situation in human ALL: a thymocyte pattern is seen in both the marrow-derived and thymus-derived diseases. This chromatographic difference between leukemia-associated and normal marrow-associated TdT in both murine and human leukemia suggested that phosphocellulose-TdT patterns might be useful for monitoring residual marrow tumour cell burden in TdT-positive leukemia. This has not turned out to be the case: in eight patients studied in early relapse the blast cell TdT pattern was the single-peak 0.3 M species. Therefore, leukemic cell TdT cannot reliably be distinguished from normal marrow cell TdT. The chromatographic behaviour of TdT may be regulated by phosphorylation-dephosphorylation, the 0.3 M salt peak can be converted to the 0.4 M salt species by treatment with protein kinase and ATP, and the 0.4 M species can be converted to the 0.3 M form by exposure to alkaline phosphatase. Thus, apparently anatomic compartment-specific forms of TdT may only reflect differing cellular metabolic activity.
In normal tissues, 3H-triamcinolone acetonide (3H-TA) labeled glucocorticoid receptors can be resolved into 2 components by DEAE chromatography: peak I elutes at 0.04 M salt and peak II at 0.22 M salt. By glycerol gradient centrifugation, peak I is 3.5S and peak II is 8.5S. Peak I binds to DNA, while peak II does not. Blast cell 3H-TA-binding macromolecules in 27 of 62 cases of acute leukemia had DEAE binding characteristics identical to those of normal tissues; the remaining 35 cases were abnormal. In these cases there was either a single DEAE species eluting in the peak I area (30 cases) or multiple low-amplitude peaks eluting across the entire gradient (5 cases). The abnormal single peak material failed to bind to DNA in 5 cases (of 5 studied), whereas peak I material from 5 cases (of 5 studied), showing normal peak I-peak II ratios, bound normally to DNA. In 3 cases (of 3 studied), the abnormal single peak material had an S value of 2-2.5S, whereas in 5 cases with normal peak I-peak II ratios, the S values were 3.5S and 8.5S, respectively. We hypothesize that those leukemias with abnormal binder characteristics cannot respond to glucocorticoid therapy.
Cimetidine significantly slowed metastatic development and prolonged survival in tumour-bearing mice, in association with inactivation of suppressor cells. Pharmacological blockade of the suppressor cell system may represent a new strategy for successful immunotherapy of human neoplasia.
Interest in the DNA-synthetic enzyme terminal deoxynucleotidyl transferase (TdT) has developed from two sets of observations: first, in normal animals, it occurs only in immature thymic lymphocytes and in a subpopulation of bone marrow lymphocytes; second, it is present in the blast cells of almost all patients with acute lymphoblastic leukemia. A prospective trial to evaluate blast cell TdT as a predictor of responsiveness to vincristine and prednisone in 30 Philadelphia chromosome-positive patients with blastic chronic myelogenous leukemia was undertaken. Eleven of 16 TdT-positive patients responded, whereas only one of 14 TdT-negative patients showed improvement. Among TdT-positive patients under the age of 50 years, the response rate was 78%. Enzyme-negative patients under the age of 50 had an 11% response rate. Blast cell morphology (i.e., lymphoblastic versus myeloblastic) had no significant correlation with either responsiveness or TdT activity. These results suggest that blast cell TdT activity may identify leukemic patients who are likely to respond to vincristine and prednisone irrespective of their conventional classification.
We studied the activity of 14 compounds, all of which have been shown to interfere in plant cell division, in two animal tumor cell cultures, EL-4 and L1210. Four compounds [propham, chlorpropham, bensulide S-(O,O-diisopropylphosphorodithioate) ester of N-(2-mercaptoethyl)benzenesulfonamide), and siduron] had a 50% inhibitory dose less than 10(-4) M; six [2,3,5-triiodobenzoic acid, (2,4-dichlorophenoxy)acetic acid, bromacil, (2,4,5-trichlorophenoxy)acetic acid, naptalam, and (4-chloro-2-methylphenoxy)acetic acid] had a 50% inhibitory dose between 10(-4) and 10(-3) M, and the remaining four 2,3:4,6-di-O-isopropylidene-2-keto-L-gulonate, eptam, maleic hydrazide, and 4-(methylsulfonyl)-2,6-dinitro-N,N,-dipropylaniline] had a 50% inhibitory dose at higher than 10(-3) M. There was a significant correlation between the effect on the two cell lines as well as between the inhibition of cell proliferation and that of thymidine and leucine uptake. More detailed study of cell proliferation and leucine and thymidine uptake for bensulide and 2,3,5-triiodobenzoic acid revealed a dose-response pattern of inhibition starting shortly after exposure of the cells to the compounds. These results indicate that some inhibitors of plant cell division are capable of inhibiting the proliferation of animal tumor cells.
3H-triamcinolone acetonide labeled glucocorticoid receptors in normal lymphoid tissues can be resolved into two component by DEAE chromatography: peak I elutes at 0.04 M salt and peak II is 0.22 M salt. By glycerol gradient centrifugation, peak I is 3.5S and peak II 8.5S. Peak I binds to DNA and chromatin, while peak II binds to neither. After heat activation, peak II alters its coefficient of sedimentation to 3.5S and on DEAE rechromatography changes its elution position to 0.04 M salt (peak I area) and acquires affinity for DNA. Glucocorticoid receptors in lymphoblastic leukemia cells can now be characterized using these techniques and compared to receptors in normal lymphoid cells.
Histamine membrane receptors are defined as either H1 (blocked by diphenhydramine-like antagonists) or H2 (blocked by cimetidine-like agents). We now report the solubilization, separation, and partial characterization of specific H1 and H2 membrane receptors from calf thymocytes. Membrane fragments were incubated with [3H]histamine either alone or with unlabeled histamine, diphenhydramine, or cimetidine. Maximal specific binding occurred with incubation at 37 degrees C for 2 h at a concentration of 5 x 10(-6) M [3H]histamine. Labeled receptors were solubilized from membranes with 0.3 M KCl and 1% Nonidet 40. Chromatography of the solubilized labeled receptors on ion exchange columns revealed two classes of receptor. One class bound to DEAE-cellulose and eluted as a sharp peak at 0.15 M NaCl/Pi. The other bound to phosphocellulose and eluted as a sharp peak at 0.55 M NaCl/Pi. Initial incubation of the membranes in the presence of the H1 receptor antagonist diphenhydramine virtually abolished the DEAE-cellulose peak, while incubation with cimetidine, the H2 receptor antagonist, blocked the phosphocellulose peak. We conclude that H1 and H2 histamine receptors are physically separable and can be defined by their ability to bind to either DEAE-cellulose or phosphocellulose.
We undertook a prospective trial to evaluate terminal deoxynucleotidyl transferase activity as a predictor of responsiveness to vincristine and prednisone in 22 Philadelphia-chromosome-positive patients with blastic chronic myelogenous leukemia. Thirteen patients were transferase positive, and nine negative. None of the nine negative patients responded, whereas eight of the 13 positive (P = 0.004) responded with complete clearing of peripheral blood blast cells and a return of normal marrow cellularity with less than 5 per cent blast cells. Among transferase-positive patients under 50 years of age the response rate was 78 per cent. Blast-cell morphology (i.e., lymphoblastic versus myeloblastic) had no significant correlation with either responsiveness or terminal transferase activity. The results of this study suggest that responsiveness to vincristine and prednisone in blastic chronic myelogenous leukemia is confined to patients whose leukemic cells are transferase positive.
Fifty consecutive patients with blastic chronic myelogenous leukemia were evaluated clinically, morphologically, biochemically, and therapeutically. Forty-five patients had a preceding stable phase (38 Ph'+, 7 Ph'-); five patients presented with de novo Ph+ blast crisis. The most frequent clinical signs of impending blast crisis were weakness, fatigue, increasing splenomegaly, anemia, thrombocytopenia, marrow fibrosis, and a rising neutrophil alkaline phosphatase. Fever (unrelated to infection), skin infiltration, lymphadenopathy, hepatomegaly, thrombocytosis, and basophilia were much less common. The development of aneuploidy occurred in less than one-half of the total group. Myeloblastic morphology at blastic transformation was most frequent with occasional lymphoblastic, promyelocytic, and undifferentiated cases seen. Terminal deoxynucleotidyl transferase was present in one-third of the patients, but had no clear-cut relationship to the morphology. Response to treatment was generally disappointing (two complete and 15 partial remissions in 45 treated patients).
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Peripheral leukocytes from patients with and without leukemia were assayed for presence of terminal deoxynucleotidyl transferase. Activity of this enzyme was detected in circulating leukemic cells from 11 to 13 patients with acute lymphoblastic leukemia, and in one of four with chronic myelogenous leukemia in blast crisis, but not in leukocytes from patients with other kinds of leukemia or in normal leukocytes. Its presence in a patient with chronic myelogenous leukemia in blast crisis lends biochemical support to the suggestion that some patients with chronic myelogenous leukemia undergo a lymphoblastic rather than a myeloblastic crisis. The thymocyte and leukemic-cell enzyme have the same substrate and primer preference. Normal thymocytes and leukemic cells contain two forms of terminal deoxynucleotidyl transferase that can be separated by phosphocellulose chromatography. The enzyme may provide a means for classifying leukemic cells on a biochemical basis independently of classic morphologic and clinical criteria.
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Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells.