Murine terminal deoxynucleotidyl transferase: cellular distribution and response to cortisone.

The mouse thymus contains two forms of terminal deoxynucleotidyl transferase (TdT) which are distinguishable by the salt concentration necessary to elute them from a phosphocellulose column, by their distrubtion among the thymocyte subpopulations, and by their sensitivity to cortisone treatment. In the whole thymus the later eluting peak (peak II) is the predominant one with about 3-10% of the total activity appearing in peak I. Both peak I and peak II activities are most sensitively assayed by the polymerization of dGMP onto an oligo(dA) primer. The minor population of thymocytes which is less dense and cortisone-resistant contains a higher specific activity of peak I TdT. The majority of TdT activity is, however, found in the major population of thymocytes which occurs in the center region of a bovine serum albumin gradient and is cortisone-sensitive. A very low level of an activity indistinguishable from peak II TdT activity is also detected in the mouse bone marrow. Other tissues, such as spleen, liver, heart, and brain lack detectable amounts of TdT activity.

New avenues of translational research in leukemia and lymphoma: outgrowth of a Leukemia Society of America-National Cancer Institute workshop.

The workshop was organized on the premise that truly innovative approaches are needed if we are to significantly change the clinical outcomes for leukemias and lymphomas. Several new concepts and pioneering approaches surfaced during the workshop discussions, as summarized above. The design and implementation of translational clinical trials that emphasize these innovative strategies were encouraged as a way to test the concepts put forward at the workshop. Representatives of the Leukemia Society of America and the National Cancer Institute plan to continue their dialogue to develop recommendations regarding the priorities for linked clinical-laboratory investigations on the hematopoietic malignancies and the optimal ways in which to foster such translational research. To this end, the explosion in basic science discoveries during the last two decades and especially during the last 5 years is producing a critical mass of knowledge. This knowledge, in turn, allows us to view leukemia from new perspectives that span molecular pathogenesis, epidemiology, early detection of minimal disease, and the selective targeting of critical leukemogenic mechanisms for therapeutic and ultimately preventive purposes. As in previous decades, during which leukemia has served as the testing ground for precedent-setting concepts of curative therapy (dose-intensity, non-cross-resistance-inducing combinations and aggressive therapy in the minimal residual disease state), leukemia should again serve as a clinical beacon for the identification and exploitation of new molecular targets for therapy. Any impact on curability and duration and quality of survival will be achieved only by building on the cumulative knowledge accrued at multiple levels--molecular and cellular levels as well as in the intact patient--and augmenting the momentum of the bidirectional exchange of information between the laboratory and the clinic that has characterized leukemia research from its incipience. The challenge is formidable and worthy of our most creative and concerted efforts.

The secondary leukemias: challenges and research directions.

Acute myelogenous leukemia (AML) arising following exposure to genotoxic agents has been recognized as a distinctive entity for more than 40 years. Secondary, or therapy-related, AML accounts for 10%-20% of all AML cases. This review addresses four overarching areas of investigation focused on secondary AMLs: 1) dissection of the molecular structure of the induced genetic lesions and identification of the functional consequences of these changes, thereby providing clues to the pathogenesis of secondary AML and potentially serving as a basis for innovative therapeutic interventions; 2) identification and characterization of mechanisms of DNA damage and the orderly repair of such damage; 3) identification and application of accurate biomarkers of leukemogenesis for the purpose of risk prediction and quantification, potentially allowing recognition of patients especially susceptible to the leukemogenic effects of chemotherapy (for genetic or acquired reasons) and allowing their treatment for cancer to be modified on the basis of this susceptibility; and 4) design and implementation of longitudinal clinical and genetic monitoring of high-risk populations (i.e., individuals under-going cytotoxic therapies for primary cancers). This review of the literature relating to these areas builds upon these themes and attempts to synthesize these seemingly disparate areas of research so that they can be more effectively utilized together to address the problem of secondary AML. Ultimately, the evaluation of these areas will improve our understanding of de novo leukemia and will serve as a springboard for the development of new concepts of therapy and prevention.

Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma.

Neoplastic cells from 253 patients with leukemia and 46 patients with malignant lymphoma were studied for the presence of terminal deoxynucleotidyl transferase (TdT) by biochemical and fluorescent antibody technics. TdT was detected in circulating blast cells from 73 of 77 patients with acute lymphoblastic leukemia, 24 of 72 patients with chronic myelogenous leukemia examined during the blastic phase of the disorder and in cell suspensions of lymph nodes from nine of nine patients with diffuse lymphoblastic lymphoma. Blast cells from six of 10 patients with acute undifferentiated leukemia were TdT positive, but the enzyme was found in only two of 55 patients with acute myeloblastic leukemia. TdT was not detected in other lymphocytic or granulocytic leukemias or in other types of malignant lymphomas. The fluorescent antibody assay for TdT permits rapid and specific identification of the enzyme in single cells. The TdT assay is clinically useful in confirming the diagnosis of acute lymphoblastic leukemia, evaluating patients with blastic chronic myelogenous leukemia, and distinguishing patients with lymphoblastic lymphoma, whose natural history includes rapid extranodal dissemination, from patients with other poorly differentiated malignant lymphomas.

Characterization of the inhibitory effects of the phytotoxic agent propachlor (alpha-chloro-N-isopropyl-acetanilide), on L1210 cell proliferation.

We recently reported that a variety of phytotoxic compounds are capable of inhibiting the proliferation of mammalian tumor cells. We now report that an additional herbicide, propachlor (alpha-chloro-N-isopropyl-acetanilide), has a strong inhibitory effect on the proliferation of L1210 mouse leukemia cells. When tested in vitro against L1210 cells, propachlor displayed an ID50 on cell proliferation of less than 3 x 10(-7) M. Propachlor also inhibited significantly the uptake of leucine, thymidine and uridine. Kinetic experiments indicate that the inhibitory effects on cell proliferation and precursor uptake are present after the first day of culture. Furthermore, the inhibitory effect of propachlor is largely reversible in that cells grown in propachlor and then washed free of the compound return to a nearly normal rate of proliferation. Finally, these effects of propachlor were dependent on cell density, with greater activity occurring at higher propachlor to cell ratios.

Chemotherapy screening assay using 3-dimensional cell culture.

A new alginate culture method (ACM) was used to test the effects of vincristine and 5-fluorouracil (5-FU) on the commonly used human colon carcinoma cell line HT-29. Colorimetric analysis of viability following treatment with physiologically tolerated levels of vincristine showed significant reduction of the surviving cell population. Hematoxylin and eosin staining of sections of treated organoid growths corroborated the colorimetric analyses. The ACM is inexpensive, rapid and extremely versatile. Many alginate beads containing "mini-tissue" growths of many cell lines can be used simultaneously to evaluate numerous chemotherapeutic agents in an "in vivo-like" environment.

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.

Use of 59Fe for sheep erythrocyte kinetic studies.

Erythrocyte (RBC) survival time as determined by in vivo 59Fe-labeled RBC in 6 adult sheep was 111.7 (SEM +/- 8.4) days. The plasma clearance (T/2) of 59Fe was 148 (+/- 17.3) minutes and the maximum RBC uptake of 59Fe was 52.4% (+/- 3.6%). Plasma iron turnover rate was 0.356 (+/- 0.016) mg/kg/24 hours, and RBC iron turnover rate was 0.186 (+/- 0.016) mg/kg/24 hours. Blood volume measurement was 53.5 (+/- 3.9) ml/kg of body weight.

Antifungal activity of 3'-deoxyadenosine (cordycepin).

The antifungal activity of the nucleoside analog 3'-deoxyadenosine (cordycepin) was studied in a murine model of invasive candidiasis. When protected from deamination by either deoxycoformycin or coformycin, both of which are adenosine deaminase inhibitors, cordycepin exhibited potent antifungal efficacy, as demonstrated by prolongation of survival and a decrease in CFU in the kidneys of mice treated with cordycepin plus an adenosine deaminase inhibitor. The antifungal effect was seen with three different Candida isolates: Candida albicans 64, a relatively fluconazole-resistant clinical isolate of C. albicans (MIC, 16 micrograms/ml), and the fluconazole-resistant Candida krusei. Cordycepin and related compounds may provide another avenue for the discovery of clinically useful antifungal drugs.

Eosinophilic cytoplasmic inclusions in fetal leukocytes: are Auer bodies a recapitulation of fetal morphology?

Among the most striking morphological features of acute nonlymphoblastic leukemias (ANLL) is the occurrence of eosinophilic cytoplasmic inclusions known as Auer rods on Auer bodies. We examined immature myeloid cells from the peripheral blood of 9 human fetuses of 16-19 wk gestation for the presence of such structures. Five of these 9 samples contained cytoplasmic inclusions, which were identical to the Auer rods typically seen in blast cells from patients with ANLL. The incidence of positive cells was low (1-5 cells/10,000 cells surveyed). The inclusions were azurophilic with Wright-Giemsa staining and were cytochemically positive with peroxidase, acid phosphatase, and Sudan black staining. We observed no inclusions in identically prepared control myeloid cells from the bone marrow of 5 patients with acute lymphoblastic leukemia in remission and 3 patients with chronic myelogenous leukemia in stable phase. Nor were they present in peripheral blood myeloid cells of 10 normal adults. Myeloid precursors in long-term bone marrow culture from 2 normal adult donors did not develop the inclusions during 24 hr of incubation with prostaglandin F2 (the abortifacient). These observations suggest that Auer rod formation is an occasional but normal phenomenon in fetal hematopoiesis.

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Human globulin messenger RNA, purified by oligo(dT)-cellulose column chromatography, is reproducibly separated into two bands by polyacrylamide gel electrophoresis in the presence of 99% formamide. The more rapidly migrating (fast) band is somewhat more abundant than the slow band in normal (nonthalassemic) total reticulocyte globin messenger RNA. In alpha-thalassemic (Hb H disease) messenger RNA, the slow band is 6.5 times more abundant than the fast band, whereas in beta-thalassemic messenger RNA the fast band is three times more abundant than a second band, which has a slightly greater mobility than the slow band of normal and alpha-thalassemic RNA. The RNA bands of nonthalassemic globin messenger RNA were eluted from the gel and efficiently transcribed into DNA copies by use of the RNA-dependent DAN polymerase of avian myeloblastosis virus. Hybridization of these copy DNAs to fast and slow band RANs and to nonfractionated normal, alpha-thalassemic, and geta-thalassemic messenger RNAs revealed that the eluted fast band RNA contains predominantly alpha-chain specific sequences, whereas the eluted slow band RNA contains predominantly beta-chain specific sequences. Nucleotide sequence analysis of 32-P-labeled RNA transcribed from the slow band copy DNA also indicated that the slow band RNA is beta messenger RNA.

The use of 51Cr for sheep red blood cell survival studies.

The red blood cell half-life as determined by 51Cr-labelled autologous cells in five adult sheep was 13.7 days. In contrast, the red blood cell life span measured by cohort 59Fe-labelling in six adult sheep was 111.7 days. The rapid loss of 51Cr-activity from the circulation appears to result from rapid elution of the label from the circulating red cells. 51Cr does not appear to be a suitable isotope for erythrokinetic studies in sheep.

A technique for the flow cytometric analysis of lymphocytes bearing histamine receptors.

Histamine receptors have been demonstrated on lymphocyte membranes by a variety of techniques. We now report a method that allows for the flow cytometric analysis of histamine receptors on human peripheral T cells. Histamine is conjugated to fluoresceinated human albumin by the coupling agent ECDI. This conjugated histamine compound (FHA-his) binds to approximately 45% of T cells. Fluoresceinated human albumin alone (FHA), not conjugated to histamine, does not bind to T cells. In addition, unconjugated histamine can inhibit completely the binding seen with FHA-his. We conclude that this technique demonstrates specific FHA-his binding to histamine receptors on T cells and can be used to determine the number of cells bearing such receptors. In addition, the reagent could be used with a cell sorter to isolate distinct histamine-receptor-bearing (HR+) cells for further immunologic study.

Terminal deoxynucleotidyl transferase positive lymphoblastic lymphoma: a study of 15 cases.

The investigation was undertaken to define the features of lymphoblastic lymphoma. Fifteen lymph node biopsies from a group of 82 specimens studied for the enzyme terminal deoxynucleotidyl transferase (TdT) fulfilled morphological criteria for this diagnosis. These criteria required a diffuse infiltrate of relatively uniform, immature lymphoid cells with basophilic cytoplasm; round, oval or lobulated nuclei with evenly dispersed chromatin; rare or inconspicuous nucleoli; and numerous mitotic figures. Examination of 1-micron thick, plastic-embedded, Giemsa-stained tissue sections revealed convoluted nuclei in more than 50% of neoplastic cells in four cases: in six specimens there was an admixture of cells with grooved, hyperlobulated, and round nuclei, and in five the round or oval nuclei were non-convoluted. Specimens from all 15 patients were positive for TdT by fluorescent antibody and biochemical assays. The percentage of cells from involved nodes reacting by indirect immunofluorescence with an antiserum against bovine TdT ranged from 4 to 90% (mean of 52%), and the mean level of biochemically measured enzyme activity was 8.7 units/g of tissue (range of 1.9 to 27.5). Cytochemical stains for acid phosphatase were positive in 13 of the 15 cases. In eight samples more than 50% of cells formed rosettes with sheep erythrocytes, while the E rosettes varied from 14 to 38% in the other seven. The percentage of cells with complement receptors varied widely (range of 6 to 80), but cells bearing surface immunoglobulin or IgGfc receptors were not increased. All patients presented with supradiaphragmatic lymphaedenopathy, eight with an anterior mediastinal mass. Two-thirds of the patients were male, and the mean age was 20 years (range 4 to 46 years). None were leukemic at the time of diagnosis, but eight patients subsequently developed acute lymphoblastic leukemia. Involvement of the central nervous system was observed in four of the 15, and of the testes in two. Ten patients have died of their disease with a median survival of 8 months (range 4 to 20), and five are alive 3--8 months after diagnosis. We observed no differences in clinical findings at presentation, incidence of mediastinal involvement or leukemic dissemination, content of TdT, acid phosphatase staining, or immunologic cell surface characteristics between the convoluted and non-convoluted types of lymphoblastic lymphoma. Distinctive morphologic, cell surface, biochemical, and clinical features of lymphoblastic lymphoma can be identified irrespective of the presence or absence of convoluted nuclei.

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.