Effect of a combined exposure to cytosine arabinoside, bryostatin 1, and recombinant granulocyte-macrophage colony-stimulating factor on the clonogenic growth in vitro of normal and leukemic human hematopoietic progenitor cells.

We have examined the effect of a combined 24 h exposure to cytosine arabinoside (ara-C) and the protein kinase C activator bryostatin 1, either alone or in conjunction with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), on the clonogenic growth of 14 primary samples from acute myelogenous leukemia (AML) patients, as well as normal human committed and early hematopoietic progenitors. Incubation of blasts with 1 microM ara-C and 12.5 nM bryostatin 1(+/- 1.25 ng/ml rGM-CSF) resulted in a heterogeneous pattern of inhibitory effects toward primary leukemic colonies, ranging from 32-98%, and subadditive to synergistic drug interactions. However, exposure of blasts to ara-C and bryostatin 1, either with or without rGM-CSF, eliminated leukemic cell self-renewal in 80-93% of samples, and very substantially reduced growth in the remainder. Exposure of normal human bone marrow mononuclear cells to identical concentrations of ara-C and byostatin 1 permitted the survival of 23% of committed myeloid progenitors (granulocyte-macrophage colony-forming units), and greater than 50% when rGM-CSF was included. Finally, exposure of bone marrow populations highly enriched for progenitor cells (CD34+, DR-, CD71-) to ara-C and bryostatin 1 +/- rGM-CSF for 24 h led to minimal reductions (e.g. 10-15%) in the survival of early hematopoietic progenitors (high proliferative potential colony-forming cells). Together, these findings indicate that combined exposure in vitro to ara-C and bryostatin 1, both with and without rGM-CSF, effectively inhibits the growth of leukemic cells with self-renewal capacity, while sparing a significant fraction of normal committed and primitive hematopoietic progenitors.     

Control of actin conformation in AML myeloblasts: the effects of bryostatin and TPA

Protein kinase C (PKC), an enzyme involved in signal transduction, is the receptor for both the tumor-promoting phorbol esters and the anti-neoplastic bryostatins. In many cells, phorbol esters and bryostatins cause similar effects; we have found that both agents increase actin polymerization in neutrophils. In some cells, however, the two agents result in different cell processes; we have found consistently different effects of these agents on actin conformation in myeloblasts obtained from leukemic patients. The patients tested all had increases in F-actin in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) and most had decreases in F-actin in response to bryostatin. The data suggests that leukemic myeloblasts have a different cytoskeletal response to a tumor promoter and an antineoplastic agent despite their common receptor.     

Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells

The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells. We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E. The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples. The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions. To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.     

Elimination of leukemic cells by the combined use of ether lipids in vitro

Two ether lipids, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)-n- propyl]-4-phenylpiperidine) and ET-18-OCH3 (racemic 1-O-octadecyl-2-O-methylglycero-3-phosphocholine) have been shown to possess antileukemic activity in vitro. To explore the possible use of these compounds for purging remission bone marrow cells of leukemic cells, we examined the cytotoxic effect of these compounds on normal hematopoietic progenitor cells and leukemic cell line cells (HL-60, K-562, KG-1a, KG-1, and Daudi) by using the clonogenic assay. When cells were treated with CP-46,665-1 or ET-18-OCH3 (50 micrograms/ml for 1 h), these compounds did not inhibit the growth of normal progenitors, whereas the growth of the clonogenic leukemic cells was inhibited with differences in their sensitivities to the cytotoxic effect of CP-46,665-1 and ET-18-OCH3. Incubation of leukemic cells (HL-60 and Daudi cells) with both CP-46,665-1 (50 micrograms/ml) and ET-18-OCH3 (50 micrograms/ml) for 1 h resulted in a greater reduction of clonogenic leukemic cells than treated with each compound alone. Approximately a 3 log killing of clonogenic HL-60 cells and a 5 log killing of Daudi cells was achieved; however, the combined treatment of normal bone marrow cells with CP-46,665-1 and ET-18-OCH3 did not alter the growth of normal progenitors. This combined treatment also selectively eliminated the leukemic cells (HL-60 and Daudi cells) from a mixture (1000:1) of normal bone marrow cells and leukemic cells. It is conceivable that the pronounced difference in sensitivity to this combined treatment can be exploited for the elimination of residual leukemic cells in autologous remission marrow grafts.     

Effect of deoxycytidine on the metabolism and cytotoxicity of 5-aza-2'-deoxycytidine and arabinosyl 5-azacytosine in normal and leukemic human myeloid progenitor cells

The effect of deoxycytidine (dCyd) on the inhibitory effects of two antileukemic nucleoside analogs, 5-aza-2'-deoxycytidine and ara-5-aza-Cyd, toward the clonogenic growth of normal human bone marrow progenitors (CFU-GM) and leukemic blast progenitors (L-CFU) was examined. Continuous exposure of cells to 10(-6)-10(-5) M 5-aza-deoxycytidine or 10(-5)-5 x 10(-5) M ara-5-aza-Cyd in conjunction with a 10- 100-fold excess of dCyd resulted in significantly greater restoration of CFU-GM growth than L-CFU colony formation at each dose relationship. Normal bone marrow mononuclear cells exposed to 10(-3) M dCyd for 4 hr (along with 5-aza-dCyd or ara-5-aza-Cyd) exhibited intracellular deoxycytidine triphosphate (dCTP) pools 20-fold higher than their leukemic counterparts. However, this finding was not associated with enhanced analog incorporation into leukemic cell DNA. These results suggest that high concentrations of dCyd preferentially protect normal versus leukemic progenitor cells from the inhibitory actions of 5-aza-dCyd and ara-5-aza-Cyd. They also raise the possibility that this in vitro selectivity may be related to enhanced expansion of dCTP pools in normal bone marrow elements and involves factors other than differential short-term analog incorporation into DNA.     

Prognostic implications of blast self-renewal capacity in acute myelocytic-leukemia

The in vitro culture studies have revealed that the infinite proliferation of leukemic cells in acute myelocytic leukemia (AML) is supported by leukemic blast progenitors with self-renewal capacity. The self-renewal of blast progenitors is assessed by either secondary blast colony-formation in methylcellulose culture or clonogenic cell recovery in suspension culture. The patients whose leukemic blast progenitors had high self-renewal ability were more difficult in achieving complete remission and survived for shorter term than those with leukemic blast progenitors of low self-renewal capacity. Namely, the self-renewal capacity of leukemic blast progenitors was highly predictive of not only remission induction outcome but also survival duration of AML patients. Among several antileukemic agents tested, only a few drugs such as cytosine arabinoside were effective against blast self-renewal. To cure AML, a therapy that aims to eradicate self-renewing leukemic blast progenitors should be developed.     

Influence of humoral regulators on proliferation and maturation of normal and leukemic cells.

Factors that influence the proliferation of marrow elements can be detected in sera. To determine the function and to compare the effect of these factors, cells were obtained from patients with normal and leukemic bone marrows. The effects of drug-induced stimulatory and inhibitory sera and leukemic pretreatment sera over time (0 to 6 days) on proliferation and granulocytic morphology of normal and leukemic bone marrow cells in culture were evaluated. Increased proliferation was associated with stimulatory sera, while inhibitory and leukemic pretreatment sera retarded proliferation of both normal and malignant cells. Exposure of normal proliferative cells to inhibitory or leukemic pretreatment sera yielded the greatest increase in mature granulocyte forms. In contrast, although the proliferative response of leukemic cells to these sera was similar to normal, maturation was minimal. These data suggest that leukemic pretreatment sera are similar to inhibitory sera and are not leukemogenic. Both retard proliferation of normal and leukemic bone marrow cells while enhancing maturation of normal cells. Leukemic myeloblasts, however, cannot be made to mature by these humoral regulators.     

Cytogenetic study of maturing granulocytes in bone marrow of patients with acute myelogenous leukemia.

To determine the cytogenetic origin of maturing granulocytes in the bone marrow of patients with acute myelogenous leukemia, bone marrow cells were studied using a modified cytogenetic technique, which does not disrupt the cell membrane, in conjunction with periodic acid-Schiff (PAS) staining. In four cases successfully studied, myeloblasts were PAS-negative and granulocytes were PAS-positive. In three cases successfully studied following 0-2 days of culture, metaphase spreads with abnormal karyotypes characteristic of the patients' leukemic clones were seen in five of five, six of nine, and four of four PAS-positive cells successfully studied. These patients' bone marrows were AN, AA, and AA, respectively, by standard cytogenetic study. Therefore, the cytogenetic status of PAS-positive cells did not necessarily correlate with presence or absence of normal metaphases determined by standard cytogenetic study. Bone marrow cells which underwent full and partial granulocytic maturation in suspension culture were studied following 2 weeks of culture. Abnormal karyotypes were seen in five of five and two of two metaphases in PAS-positive cells successfully studied in two patients. Therefore, we have demonstrated that when acute myelogenous leukemia cells undergo myeloid maturation in culture, the mature cells may be definitely proven to derive from leukemic progenitors rather than from normal stem cells.     

Effect of pharmacologically relevant concentrations of mitoxantrone on the in vitro growth of leukemic blast progenitors

The inhibitory effects of a 1 h exposure to pharmacologically relevant concentrations of the anthracenedione mitoxantrone were assessed with respect to the in vitro clonogenic growth of leukemic myeloblasts obtained from patients with acute myelogenous leukemia (AML). A steep mitoxantrone dose-response curve was observed, and increases in drug concentrations at both low and high dose ranges were associated with a significant potentiation of anti-leukemic effects. In particular, mitoxantrone concentrations of 1 microM or greater were highly inhibitory to leukemic blast progenitor cell growth, and were also effective in reducing or eliminating leukemic cell self-renewal capacity in those samples assayed. These findings suggest that mitoxantrone mediated lethal effects towards leukemic cells are dose-related within a pharmacologically achievable range, and raise the possibility that an increase in plasma levels through dose escalation might lead to enhanced leukemic cell killing in vivo.     

In vitro growth response to G-CSF and GM-CSF by bone marrow cells of patients with acute myeloid leukemia.

The in vitro growth response of bone marrow and blood cells to granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) was studied in 18 acute myeloid leukemia (AML) patients using semisolid and suspension cultures. In 80% of the cases growth of leukemic progenitor cells was stimulated by GM-CSF and/or G-CSF, as judged by colony or cluster formation. In acute promyelocytic leukemia [t(15;17)], G-CSF stimulated and maintained the leukemic progenitors only transiently but fully stimulated the residual normal granulocyte/macrophage colony-forming units (CFU-GM). In some cases of M2 and M4 leukemia, G-CSF enhanced markedly the production of mature but cytochemically abnormal neutrophils. In some cases of M1 leukemia, neither CSF stimulated leukemic progenitors but instead stimulated only residual normal granulopoiesis. Spontaneous colony formation was observed in 20% of cases and was correlated with high-grade leukemic growth in vivo and a poor response to chemotherapy. The differing effects of the CSFs upon leukemic cells and residual normal granulopoiesis may have some implications for the clinical use of GM-CSF and G-CSF to overcome infectious complications.     

Induction of apoptosis and differentiation by fludarabine in human leukemia cells (U937): interactions with the macrocyclic lactone bryostatin 1.

We have examined interactions between the purine nucleoside analog fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine) and the macrocyclic lactone bryostatin 1 in the human monocytic leukemic cell line U937. Fludarabine exerted dose-dependent effects on U937 cell viability and growth which were associated with both induction of apoptosis, as well as cellular maturation. Incubation of cells with bryostatin 1 (10 nM; 24 h) after, but not before a 6-h exposure to 10 microM fludarabine resulted in a modest but significant increase in apoptosis, and was associated with greater than a 1 log reduction in clonogenicity. Subsequent exposure to bryostatin 1 also increased the percentage of fludarabine-treated cells displaying differentiation-related features (eg plastic adherence, CD11b positivity) compared to cells exposed to fludarabine alone. Bryostatin 1 did not increase the retention of the active fludarabine metabolite, F-ara-ATP, nor did it increase 3H-F-ara-A incorporation into DNA. Despite its capacity to trigger cellular maturation, fludarabine exposure (either with or without bryostatin 1) failed to induce the cyclin-dependent kinase inhibitors (CDKls) p21WAF1/CIP1 and p27KIP1. Nevertheless, dysregulation of p21 (resulting from stable transfection of cells with a p2lWAF1/CIP1 antisense construct) reduced fludarabine-mediated differentiation, while inducing a corresponding increase in apoptosis. Enforced expression of Bcl-2 partially protected cells from fludarabine-related apoptosis, an effect that was overcome, in part, by subsequent exposure of cells to bryostatin 1. Interestingly, Bcl-2-overexpressing cells were as or in some cases, more susceptible to differentiation induction by fludarabine (+/- bryostatin 1) than their empty vector-containing counterparts. Collectively, these results indicate that the antiproliferative effects of fludarabine toward U937 leukemic cells involve both induction of apoptosis and cellular maturation, and that each of these processes may be enhanced by bryostatin 1.     

Potentiation of the activity of 1-beta-D-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: association with enhanced fragmentation of mature DNA.

We have examined the interaction between 1-beta-D-arabinofuranosylcytosine (ara-C) and the macrocyclic lactone protein kinase C activator bryostatin 1 in the human promyelocytic leukemia cell line HL-60. Preexposure of cells to 10 nM bryostatin 1 for 24 h, followed by an additional 24-h incubation with 10 microM ara-C, resulted in greater than additive inhibitory effects toward clonogenic HL-60 cells. In a series of alkaline elution assays, cells preincubated with bryostatin 1 and prelabeled with [3H]thymidine exhibited a significant increase in DNA fragmentation following exposure to ara-C in comparison to cells exposed to ara-C alone. This increase in DNA damage was apparent at both neutral and alkaline pH and was not protein associated. In contrast, studies using cells pulse-labeled with [3H]thymidine immediately before analysis suggested that bryostatin 1 pretreatment did not increase the ability of ara-C to interfere with DNA replicative intermediates. Additional studies demonstrated that the increase in DNA fragmentation induced by bryostatin 1 and ara-C preceded both loss of cell membrane integrity (as determined by trypan blue exclusion) as well as depletion of intracellular ATP and NAD pools. Furthermore, the enhanced inhibitory effects of bryostatin 1 and ara-C toward clonogenic HL-60 cells did not appear to result from the induction of cellular differentiation. Finally, agarose gel electrophoresis of DNA obtained from cells exposed to both bryostatin 1 and ara-C revealed a pattern of integer multiples of 180- to 200-base pair fragments commonly associated with endonucleolytic cleavage; the extent of this fragmentation was considerably greater than that observed in cells exposed to ara-C alone. Taken together, these findings suggest that exposure of HL-60 cells to bryostatin 1 renders them more susceptible to ara-C-related DNA damage and that this phenomenon contributes to the cytotoxic effects of this drug combination. They also raise the possibility that bryostatin 1, perhaps through modulation of intracellular signaling events in leukemic cells, has the capacity to potentiate ara-C-related apoptosis or programmed cell death.     

Selective killing of malignant cells from leukemic patients by alkyl-lysophospholipid

We studied the effects of the alkyl-lysophospholipid, 1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine (ALP), on human leukemia cells from 56 patients with various leukemias and on normal bone marrow progenitors in order to assess the application of ALP as an in vitro marrow-purging agent. The tumoricidal activity was analyzed by the elimination of clonogenic leukemia cells (leukemic colony-forming cells), by the inhibition of the proliferative capacity [( 3H]thymidine incorporation) of leukemia cells, and by the elimination of viable leukemia cells measured with flow cytometry. The tumoricidal activity of ALP was dose and incubation time related, as, although to a lesser extent, held true for normal marrow progenitors. For some leukemias the ALP dose necessary for the elimination of 100% of the leukemic colony-forming cells is probably too toxic for normal marrow cells. The results of this study strongly support the possibility that ALP is a promising purging agent in the majority of patients with leukemias.     

Interactions between retinoic acid and colony-stimulating factors affecting the blast cells of acute myeloblastic leukemia

The responses to retinoic acid (RA) of acute myeloblastic leukemia (AML) blasts and normal hemopoietic progenitors was examined under defined growth factor conditions. For the leukemic cells marked patient to patient variation was seen; blast colony formation by cells from some patients was stimulated by RA without growth factors or in the presence of recombinant granulocyte colony-simulating factor (rG-CSF), recombinant granulocyte-macrophage-CSF rGM-CSF and recombinant interleukin-3 (rIL-3); for other populations inhibition was observed under the same conditions. Some blast cells were stimulated by RA in the presence of rGM-CSF and rIL-3 and inhibited when cultured with RA and rG-CSF. Supernatants prepared from blasts cultured with RA and growth factors did not show activities that were not readily explained by the carry-over of growth factors; this result did not provide evidence that RA and growth factors interact to produce factors. Titrations of RA showed that activity was first observed at concentrations of 10(-9) M and was maximum at concentrations of 10(-7) M. Different effects of RA in combination with rG-CSF compared with rGM-CSF or IL-3 were not seen when the cells were tested in suspension culture rather than in methylcellulose, a finding that may be interpreted to mean that the interaction between RA and factors affects terminally-dividing blast cells. Three normal bone marrow samples were cultured with RA and growth factors. Colony formation was stimulated by RA in the presence of rGM-CSF or rIL-3 but inhibited by RA with rG-CSF. Thus a differential effect of RA in combination with growth factors occurs in normal hemopoietic cells and persists in some AML populations.     

In vitro purging of clonogenic leukemic cells from human bone marrow by heat: simulation experiments for autologous bone marrow transplantation.

In order to apply a simple purging method by heat to autologous bone marrow transplantation (ABMT), we have revaluated the ability to purge clonogenic leukemic cells from the simulated marrow mixture of normal marrow cells and leukemic cell lines (HL-60, Molt-3 and HEL) in vitro by heat, using two different clonogenic assays for normal granulocyte-macrophage progenitors (CFU-GM) and leukemic cell lines. It appeared that in vitro hyperthermia (42 degrees C for 120 min) is able to selectively remove clonogenic leukemic cells from simulated tumor cell-normal marrow mixtures even when leukemic cell concentrations are increased up to 3 x 10(6) cells/ml in vitro, and results in a 4-6 log destruction of clonogenic leukemic cells/ml according to a limiting dilution assay, while leaving half of normal CFU-GM surviving. The hyperthermic purging of clonogenic leukemic cells was not affected in the presence of normal marrow cells in vitro. This high level of clonogenic leukemic cell depletion by heat correlated with that of immunologic and pharmacologic studies. These results suggest that in vitro hyperthermia could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous marrow grafts before ABMT.     

Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells.

Overexpression of the detoxifying enzyme cytidine deaminase (CDD) renders normal and leukemic hematopoietic cells resistant to cytarabine (1-beta-D-arabinofuranosylcytosine), and studies on murine cells have suggested transgenic CDD overexpression as a way to reduce the substantial myelotoxicity induced by the deoxycytidine analogs cytarabine and gemcitabine (2',2'-difluorodeoxycytidine). We now have investigated CDD (over-)expression in the human hematopoietic system. Retroviral gene transfer significantly increased the resistance of CDD-transduced cord blood and peripheral blood-derived progenitor cells for doses ranging from 20-100 nM cytarabine and 8-10 nM gemcitabine. Protection was observed for progenitors of erythroid as well as myeloid differentiation, though the degree of protection varied for individual drugs. In addition, significant selection of CDD-transduced cells was obtained after a 4-day culture in 30-100 nM cytarabine. Thus, our data demonstrate that overexpression of CDD cDNA results in significant protection of human progenitors from cytarabine- as well as gemcitabine-induced toxicity, and allows in vitro selection of transduced cells. This strongly argues for a potential therapeutic role of CDD gene transfer in conjunction with dose-intensive cytarabine- or gemcitabine-containing chemotherapy regimen.     

A comparison of the abilities of nitrobenzylthioinosine, dilazep, and dipyridamole to protect human hematopoietic cells from 7-deazaadenosine (tubercidin).

Nitrobenzylthioinosine, dilazep, and dipyridamole are potent inhibitors of equilibrative transport of nucleosides that may have pharmacological applications in modulating the therapeutic index of nucleoside antimetabolites used in cancer chemotherapy. We have compared the relative abilities of these inhibitors to reduce the toxicity of in vitro exposures to tubercidin against clonogenic progenitor cells of normal human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two leukemic human cell lines (HL-60/C1, CCRF-CEM) that differ in their expression of transporter subtypes. Short (1-h) exposures to 1 microM tubercidin alone inhibited colony formation (a) of normal human hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and (b) of HL-60/C1 and CCRF-CEM cells by > 90%. Pretreatment (30 min) with nitrobenzylthioinosine, dilazep, or dipyridamole followed by simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoietic progenitors and cell lines. Greater reductions of toxicity were consistently seen with bone marrow progenitors and CCRF-CEM cells than with HL-60/C1 cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in tubercidin toxicity of 50-100% were achieved at these concentrations: > or = 0.1 microM (nitrobenzylthioinosine); > or = 0.1 microM (dilazep); and > or = 3.0 microM (dipyridamole). Pretreatment (30 min) followed by simultaneous treatment (1 h) with any of the transport inhibitors (> or = 0.1 microM) and 0.1 microM [3H]-tubercidin blocked the uptake of radioactivity completely in CCRF-CEM cells and only partially in HL-60/C1 cells. These effects, which were consistent with the nucleoside transport phenotypes of CCRF-CEM cells (inhibitor-sensitive) and HL-60/C1 cells (inhibitor-sensitive and inhibitor-resistant), suggested that protection was due to the inhibition of tubercidin uptake via equilibrative nucleoside transport system(s). Light-density mononuclear cells from human bone marrow, of which the clonogenic progenitors represented only a minor (< 0.01%) subpopulation, possessed far fewer nitrobenzylthioinosine-binding sites (2 x 10(4) sites/cell, Kd = 0.7 nM) than either HL-60/C1 cells (1.7 x 10(5) sites/cell, Kd = 0.9 nM) or CCRF-CEM cells (3.3 x 10(5) sites/cell, Kd = 0.5 nM). Initial rates of uptake of 1 microM [3H]adenosine (0-6 s, 20 degrees C) by human bone marrow mononuclear cells were reduced partially by 0.1 microM inhibitor (nitrobenzylthioinosine > dipyridamole > dilazep) and completely by 10 microM inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of recombinant human tumor necrosis factor on the self-renewal capacity of leukemia blast progenitors in acute myeloblastic leukemia

The effects of human recombinant tumor necrosis factor type alpha (rTNF alpha) on the blast progenitors from 14 acute myeloblastic leukemia (AML) patients and 1 chronic myelogenous leukemia patient in blastic crisis were studied in methylcellulose and suspension cultures. Blast progenitors renew themselves and/or undergo terminal divisions. Plating efficiency of primary colony formation (PE1) in methylcellulose, which is considered to reflect the terminal divisions of blast progenitors, was suppressed by rTFN alpha in a dose-dependent manner in all cases. Plating efficiency of secondary colony formation (PE2) and the recovery of clonogenic cells in suspension culture, which are considered to reflect the self-renewal capacity of blast progenitors, were also suppressed by rTNF alpha in a dose-dependent manner in almost all cases. rTNF alpha also inhibited both PE2 and clonogenic cells in suspension culture, even in relapsed AML patients who were very refractory to intensive chemotherapies. The results demonstrate that rTNF alpha inhibits not only terminal divisions but also the self-renewal capacity of leukemic blast progenitors. The finding that rTNF alpha suppressed the self-renewal capacity of leukemic blast progenitors proposes the utility of rTNF alpha to AML therapy.     

Transplantable cell lines generated with NUP98-Hox fusion genes undergo leukemic progression by Meis1 independent of its binding to DNA.

Hox genes have been identified in chromosomal translocations involving the nucleoporin gene NUP98. Though the resulting chimeric proteins directly participate in the development of leukemia, the long latency and monoclonal nature of the disease support the requirement for secondary mutation(s), such as those leading to overexpression of Meis1. Models to identify such events and to study leukemic progression are rare and labor intensive. Herein, we took advantage of the strong transforming potential of NUP98-HOXD13 or NUP98-HOXA10 to establish preleukemic myeloid lines from bone marrow cells that faithfully replicate the first step of Hox-induced leukemogenesis. These lines contain early granulomonocytic progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity and low propensity for spontaneous leukemic conversion. We exploit such lines to show that Meis1 efficiently induces their leukemic progression and demonstrate a high frequency of preleukemic cells in the cultures. Furthermore, we document that the leukemogenic potential of Meis1 is independent of its direct binding to DNA and likely reflects its ability to increase the repopulating capacity of the preleukemic cells by increasing their self-renewal/proliferative capacity. The availability of lines with repopulating potential and capacity for leukemic conversion should open new avenues for understanding progression of Hox-mediated acute myeloid leukemia.     

No preferential sensitivity of clonogenic AML cells to ASTA-Z-7557

ASTA-Z-7557, an in vitro active metabolite of cyclophosphamide, has recently been introduced to purge autologous bone marrow grafts of patients with AML. The rationale of this approach assumes a relatively higher sensitivity of leukemic cells to the drug as compared to that of normal marrow precursors. We have investigated in direct comparison the sensitivity to ASTA-Z-7557 of normal bone marrow progenitors (GM-CFC and BFU-E) and clonogenic leukemic cells (L-CFC). Normal bone marrow cells and purified leukemic blast cells were exposed to varying concentrations of the drug. Dose-response relationships did not indicate a selective cytotoxic susceptibility of L-CFC to ASTA-Z-7557. The recovery of bone marrow precursors following exposure to ASTA-Z-7557 depended on the cell concentration during exposure and was higher for 2 X 10(7) cells/ml than for 1 X 10(6)/ml. To mimic minimal residual leukemia cell mixtures of 95% irradiated normal bone marrow cells with 5% leukemic blast cells were exposed to ASTA-Z-7557. In this mixture killing of L-CFC was largely decreased. These data suggest that in vitro incubation of autologous bone marrow grafts of patients with minimal residual leukemia with ASTA-Z-7557 might not offer a therapeutic advantage.