BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells.

Bruton's tyrosine kinase (BTK) is a member of the SRC-related TEC family of protein tyrosine kinases (PTKs). DT-40 lymphoma B cells, rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout, did not undergo radiation-induced apoptosis, but cells with disrupted lyn or syk genes did. Introduction of the wild-type, or a SRC homology 2 domain or a plecstrin homology domain mutant (but not a kinase domain mutant), human btk gene into BTK-deficient cells restored the apoptotic response to radiation. Thus, BTK is the PTK responsible for triggering radiation-induced apoptosis of lymphoma B cells, and its kinase domain is indispensable for the apoptotic response.

Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases.

B-cell precursor (BCP) leukemia is the most common form of childhood cancer and the second most common form of acute leukemia in adults. Human BCP leukemia was treated in a severe combined immunodeficient mouse model by targeting of the tyrosine kinase inhibitor Genistein (Gen) to the B cell-specific receptor CD19 with the monoclonal antibody B43. The B43-Gen immunoconjugate bound with high affinity to BCP leukemia cells, selectively inhibited CD19-associated tyrosine kinases, and triggered rapid apoptotic cell death. At less than one-tenth the maximum tolerated dose more than 99.999 percent of human BCP leukemia cells were killed, which led to 100 percent long-term event-free survival from an otherwise invariably fatal leukemia. The B43-Gen immuno-conjugate might be useful in eliminating leukemia cells in patients who have failed conventional therapy.

Intrinsic radiation resistance of primary clonogenic blasts from children with newly diagnosed B-cell precursor acute lymphoblastic leukemia.

The radiation sensitivity of primary clonogenic blasts from 44 children with newly diagnosed B-cell precursor acute lymphoblastic leukemia (ALL) was analyzed using leukemic progenitor cell (LPC) colony assays. The derived values for SF2 (surviving fraction at 200 cGy) and alpha (initial slope of radiation survival curves constructed according to the linear quadratic model) indicated a marked interpatient heterogeneity in intrinsic radiation sensitivity of LPC populations. The SF2 values ranged from 0.01 to 1.00 (median = 0.430; mean +/- SE = 0.47 +/- 0.04), and the alpha values ranged from 0.000 to 3.272 Gy-1 (median = 0.280 Gy-1; mean +/- SE = 0.430 +/- 0.093 Gy-1). When CD19+ CD34+ versus CD19+ CD34- immunophenotypes were compared, a trend toward higher SF2 and lower alpha values were observed in LPC from CD34+ patients, consistent with greater radiation resistance. When patients were divided into three approximately equal groups based on increasing levels of CD34 expression, a clear ordering effect was observed indicating that increased CD34 expression levels are associated with significantly higher radiation resistance at the level of B-lineage LPC. The highest CD34 expression group (> or = 75% positivity) had 1.4-fold higher SF2 (P = 0.05) and twofold lower alpha values (P = 0.06) than the lowest group (< 30% positivity). Furthermore, the CD34 positivity of radiation resistant (alpha < or = 0.2 and SF2 > or = 0.5) B-cell precursor ALL cases was greater than the CD34 positivity of radiation sensitive (alpha > 0.2 and/or SF2 < 0.5) cases (56 +/- 9% versus 34 +/- 9%, P = 0.09). Whereas only 6 of 16 (38%) of radiation sensitive cases were CD34+, 11 of 15 (73%) of radiation resistant cases expressed CD34 (P = 0.04). Our results offer new insights into the inherent and/or acquired radiation resistance of primary clonogenic blasts from B-cell precursor ALL patients.

Induction of apoptosis in multidrug-resistant and radiation-resistant acute myeloid leukemia cells by a recombinant fusion toxin directed against the human granulocyte macrophage colony-stimulating factor receptor.

Multiagent chemotherapy regimens fail to cure more than one-half of the patients with acute myeloid leukemia (AML) because of the emergence of dominant multidrug-resistant subclones of leukemia cells. We have developed a recombinant diphtheria toxin-human granulocyte macrophage colony-stimulating factor chimeric fusion protein (DTctGMCSF) that specifically targets GMCSF receptor-positive AML cells. This novel biotherapeutic agent induced rapid apoptotic cell death of chemotherapy-resistant AML cell lines and primary leukemic cells from treatment-refractory AML patients. Our results suggest that DTctGMCSF may be useful in the treatment of AML patients whose leukemia has recurred and developed resistance to contemporary chemotherapy programs.

Cytotoxic activity of epidermal growth factor-genistein against breast cancer cells.

The receptor (R) for epidermal growth factor (EGF) is expressed at high levels on human breast cancer cells and associates with ErbB2, ErbB3, and Src proto-oncogene family protein tyrosine kinases (PTKs) to form membrane-associated PTK complexes with pivotal signaling functions. Recombinant human EGF was conjugated to the soybean-derived PTK inhibitor genistein (Gen) to construct an EGF-R-directed cytotoxic agent with PTK inhibitory activity. The EGF-Gen conjugate was capable of binding to and entering EGF-R-positive MDA-MB-231 and BT-20 breast cancer cells (but not EGF-R-negative NALM-6 or HL-60 leukemia cells) via its EGF moiety, and it effectively competed with unconjugated EGF for target EGF-R molecules in ligand binding assays. EGF-Gen inhibited the EGF-R tyrosine kinase in breast cancer cells at nanomolar concentrations, whereas the IC50 for unconjugated Gen was >10 microM. Notably, EGF-Gen triggered a rapid apoptotic cell death in MDA-MB-231 as well as BT-20 breast cancer cells at nanomolar concentrations. The EGF-Gen-induced apoptosis was EGF-R-specific because cells treated with the control granulocyte-colony stimulating factor-Gen conjugate did not become apoptotic. Apoptosis was dependent both on the PTK inhibitory function of Gen and the targeting function of EGF, because cells treated with unconjugated Gen plus unconjugated EGF did not undergo apoptosis. The IC50s of EGF-Gen versus unconjugated Gen against MDA-MB-231 and BT-20 cells in clonogenic assays were 30 +/- 3 nM versus 120 +/- 18 microM (P < 0.001) and 30 +/- 10 nM versus 112 +/- 17 microM (P < 0.001), respectively. Thus, the EGF-Gen conjugate is a >100-fold more potent inhibitor of EGF-R tyrosine kinase activity in intact breast cancer cells than unconjugated Gen and a >100-fold more potent cytotoxic agent against EGF-R+ human breast cancer cells than unconjugated Gen. Taken together, these results indicate that the EGF-R-associated PTK complexes have vital antiapoptotic functions in human breast cancer cells and may therefore be used as therapeutic targets.

CD5 antigen-positive B lymphocytes in human B cell ontogeny during fetal development and after autologous bone marrow transplantation.

We examined bone marrow and peripheral blood specimens from pediatric acute lymphoblastic leukemia (ALL) patients after autologous bone marrow transplantation (BMT) as well as fetal lymphohematopoietic tissues for the presence of CD5+ B lymphocytes. CD5+ B lymphocytes represented 23.6 +/- 0.7% of CD19+ fetal spleen B lineage lymphoid cells, 24.2 +/- 2.5% of CD19+ fetal bone marrow B lineage lymphoid cells and 18.1 +/- 1.7% of CD19+ fetal liver B lineage lymphoid cells. By comparison, in normal pediatric bone marrow samples, only 1.5 +/- 0.3% of lymphoid cells and 4.3 +/- 0.2% of CD19+ B lineage lymphoid cells expressed CD5 antigen. Similarly, very few CD5+CD19+ cells (< or = 2% of lymphoid cells) were found in day 30 and day 100 post-BMT bone marrow or peripheral blood specimens from B lineage ALL patients undergoing autologous BMT using autografts purged ex vivo with B43(anti-CD19)-PAP immunotoxin plus 4-hydroperoxycyclophosphamide (4-HC). Two bone marrow samples that were obtained and analyzed 1 year post-BMT contained only 2 to 4% CD5+CD19+ cells, accounting for 5 to 7% of the total CD19+ population. The fraction of CD5+ B lymphocytes in post-BMT bone marrow samples was not greater than the fraction of CD5+ B lymphocytes in normal healthy bone marrow.

Innovative treatment programs against cancer. I. Ras oncoprotein as a molecular target.

Modulation of Ras function may provide a novel means by which cancer cells with oncogenic mutations can be sensitized to chemotherapeutic or radiotherapeutic regimens. Moreover, cancer cells without ras oncogene mutations can also be eliminated by compounds that interfere with the mevalonate pathway, which is more fundamental to mitogenesis because it allows the synthesis of sterol and nonsterol lipids and without which many Ras-related proteins and nuclear lamins would not be prenylated and functional.

Innovative treatment programs against cancer: II. Nuclear factor-kappaB (NF-kappaB) as a molecular target.

Nuclear factor-kappaB (NF-kappaB) activity affects cell survival and determines the sensitivity of cancer cells to cytotoxic agents as well as to ionizing radiation. Preventing the protective function of NF-kappaB may result in chemo- and radio-sensitization of cancer cells. Therefore, NF-kappaB has emerged as one of the most promising molecular targets in rational drug design efforts of translational cancer research programs.

Engagement of the CD19 receptor on human B-lineage leukemia cells activates LCK tyrosine kinase and facilitates radiation-induced apoptosis.

As presently reported, both ionizing radiation and engagement of the CD19 receptor are capable of inducing apoptosis in B-lineage acute lymphoblastic leukemia (ALL) cells. In both instances, activation of tyrosine kinases appears to be a proximal and mandatory step, since it can be prevented by the tyrosine kinase inhibitor genistein. This common biochemical signaling pathway involves the rapid activation of the Src family tyrosine kinase LCK (p56lck), which is physically associated with the CD19 receptor, and enhanced tyrosine phosphorylation of multiple substrates leading to stimulation of phosphoinositide turnover, and activation of protein kinase C. Importantly, engagement of the CD19 receptor promoted radiation-induced apoptosis in radiation-resistant B-lineage ALL cells in a cell type-specific fashion. Our results prompt the hypothesis that clonogenic B-lineage ALL blasts with an inherent or acquired resistance to radiation could be radiosensitized in clinical settings using anti-CD19 MoAb B43 or its homoconjugate as adjuncts.

Differential effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the radiation sensitivity of normal versus leukemic bone marrow progenitor cell populations.

We examined the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), interleukin 3 (rhIL-3) and interleukin 6 (rhIL-6) on the radiation sensitivity of normal and leukemic bone marrow progenitor cell populations. Conditioning of leukemic progenitor cells (LPC) from acute lymphoblastic leukemia (ALL) patients with rhG-CSF enhanced their radiation sensitivity, whereas conditioning with rhIL-3 or rhIL-6 had the opposite effect. In contrast to its effects on LPC derived from ALL patients, rhG-CSF reduced the radiation sensitivity of normal myeloid progenitor cells as well as LPC from acute myeloblastic leukemia (AML) patients. Differential modulation of the radiation sensitivity of LPC by rhG-CSF may provide the basis for better total body irradiation (TBI) regimens for ALL patients undergoing autologous bone marrow transplantation (BMT).

In vivo radiosensitizing effects of recombinant interleukin 6 on radiation resistant BCL-1 B-lineage leukemia cells in a murine syngeneic bone marrow transplant model system.

The ability of total body irradiation (TBI) to eradicate clonogenic leukemia cells from B-lineage acute lymphoblastic leukemia patients prior to bone marrow transplantation (BMT) is greatly hampered by their inherent or acquired radiation resistance. The radiorefractory nature of these cells is believed to contribute to the high relapse rate subsequent to TBI and BMT in patients with B-lineage acute lymphoblastic leukemia (ALL). A method by which clonogenic leukemia cells could be radiosensitized in vivo could be clinically beneficial. In the present study, we used a highly radiation resistant subclone of the murine B-lineage leukemia cell line BCL-1 in a syngeneic BMT model to investigate if any of the B-cell stimulatory cytokines interleukin 2, interleukin 4, interleukin 5, or interleukin 6 could have radiosensitizing effects. All untreated BALB/c mice (N = 33) inoculated with 1 x 10(6) BCL-1 cells died of disseminated leukemia within 24 days with a median survival of 13.3 days. TBI (700 cGy = LD100/30 for BALB/c mice) followed by syngeneic BMT (N = 70) extended the median survival to 23.6 days (P < 0.001 by log-rank test). A single intraperitoneal bolus injection of 100 ng, 500 ng, or 2500 ng recombinant murine interleukin 6(rmIL-6) 2-4 hours before TBI extended the median survival to 32.5 days, 31.0 days, and 30.5 days, respectively (P < 0.01 by log-rank test for all dose groups). The improved survival was not due to any direct anti-leukemic activity of rmIL-6 and all control BALB/c mice (N = 15) that received the same doses of rmIL-6 but did not undergo TBI and BMT died of BCL-1 leukemia within 28 days with a median survival of 13.6 days. In contrast to rmIL-6, recombinant murine interleukin 5 (rmIL-5) had minimal radiosensitizing effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of tyrosine phosphorylation in radiation-induced cell cycle-arrest of leukemic B-cell precursors at the G2-M transition checkpoint.

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34cdc2. We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34cdc2 amino-terminal region or (c) recombinant human p34cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49 WEE1 HU and enzymes which augment the tyrosine kinase activity of p49 WEE 1HU.

Granulocyte-macrophage colony-stimulating factor receptor-targeted therapy of chemotherapy- and radiation-resistant human myeloid leukemias.

Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1-10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed > 99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.

A recombinant fusion toxin targeted to the granulocyte-macrophage colony-stimulating factor receptor.

Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and differentiation of myeloid lineage hematopoietic cells, and may participate in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of human granulocyte-macrophage colony-stimulating factor and diphtheria toxin (DT) to produce a recombinant fusion toxin, DTctGMCSF, that targets diphtheria toxin to high affinity GMCSF receptors expressed on the surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DTctGMCSF was specifically immunoreactive with antidiphtheria toxin and anti-GMCSF antiseras, and exhibited the characteristic catalytic activity of diphtheria toxin, catalyzing the in vitro ADP-ribosylation of purified elongation factor 2. The cytotoxic effects of DTctGMCSF were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium (MTT) bromide assay of cell viability and in vivo assays of protein synthesis inhibition. DTctGMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptors for human GMCSF with IC50 of 10(-9) to 10(-11) M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) receptors. In receptor positive cells, cytotoxicity can be specifically blocked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. THough DTctGMCSF inhibited granulocyte-macrophage colony formation by committed myeloid progenitor cells (CFU-GM), it did not significantly affect erythroid burst formation by committed erythroid progenitor cells (BFU-E), or mixed granulocyte-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DTctGMCSF holds promise for the treatment of myeloid lineage malignancies, and is a useful reagent to study hematopoiesis.

Comparative analysis of the in vivo radioprotective effects of recombinant granulocyte colony-stimulating factor (G-CSF), recombinant granulocyte-macrophage CSF, and their combination.

The purpose of the present study was to evaluate and compare the in vivo radioprotective effects of pre-total body irradiation (TBI) conditioning with recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF) in a large series of lethally and supralethally irradiated mice. Also analyzed were the radioprotective effects of simultaneous as well as sequential combinations of rG-CSF and rGM-CSF. Our findings in 1,180 mice provide direct evidence that in vivo administration of rG-CSF or rGM-CSF before TBI protects a significant fraction of mice from the lethal effects of LD100/30 TBI. At equivalent doses, rG-CSF displayed a more potent radioprotective activity than rGM-CSF. Not only was rG-CSF radioprotective at much smaller doses than rGM-CSF, the survival rate after lethal TBI was also significantly higher in mice receiving optimally radioprotective doses of rG-CSF as compared with mice receiving optimally radioprotective doses of rGM-CSF. Pretreatment of mice with rGM-CSF markedly attenuated the radioprotective affects of rG-CSF in lethally as well as supralethally irradiated mice. Pretreatment with rG-CSF followed by rGM-CSF was slightly more effective than rG-CSF alone in supralethally irradiated mice but not in lethally irradiated mice. Notably, marked differences among different strains of mice were noted regarding the optimally radioprotective doses of rG-CSF or rGM-CSF as well as probability of survival and median survival time after lethal or supralethal TBI. This report confirms and extends previous studies concerning the potential of cytokines in prevention or therapy of lethal radiation injury.

In vivo radioprotective effects of recombinant human granulocyte colony-stimulating factor in lethally irradiated mice.

The purpose of this study was to investigate the in vivo radioprotective effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) in lethally irradiated BALB/c mice. We initially analyzed the effects of increasing doses of rhG-CSF on survival of mice receiving 700 cGy (LD100/30) single dose total body irradiation (TBI). While 1 microgram/kg to 100 micrograms/kg doses of rhG-CSF were not radioprotective, a dose-dependent radioprotection was observed at 200 micrograms/kg to 4,000 micrograms/kg rhG-CSF. We next compared four different rhG-CSF treatment regimens side by side for their radioprotective effects in LD100/30 irradiated mice. One hundred percent of control mice receiving phosphate buffered saline died within 21 days after TBI with a median survival of 14 days. The median survival was prolonged to 20 days and the actuarial 60-day survival rate was increased to 27% when mice received 2,000 micrograms/kg rhG-CSF 24 hours before TBI (P = .0002; Mantel-Peto-Cox). Similarly, the median survival time was prolonged to 24 days and the actuarial 60-day survival rate was increased to 33%, when mice were given 2,000 micrograms/kg rhG-CSF 30 minutes before TBI. Optimal radioprotection was achieved when 2,000 micrograms/kg rhG-CSF was administered in two divided doses of 1,000 micrograms/kg given 24 hours before and 1,000 micrograms/kg given 30 minutes before TBI. This regimen prolonged the median survival time of LD100/30 irradiated mice to more than 60 days and increased the actuarial 60-day survival rate to 62% (P = .0001; Mantel-Peto-Cox). By comparison, no survival advantage was observed when mice received rhG-CSF 24 hours post-TBI. Similar radioprotective effects were observed when mice were irradiated with 650 cGy (LD80/30). The presented findings provide conclusive evidence that rhG-CSF has significant in vivo radioprotective effects for mice receiving LD100/30 or LD80/30 TBI.

Physical and functional interactions between Lyn and p34cdc2 kinases in irradiated human B-cell precursors.

Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

In vitro and in vivo activity of topotecan against human B-lineage acute lymphoblastic leukemia cells.

Topotecan [(S)-9-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride; SK&F 104864-A, NSC 609699], a water soluble semisynthetic analogue of the alkaloid camptothecin, is a potent topoisomerase I inhibitor. Here we show that topotecan stabilizes topoisomerase I/DNA cleavable complexes in radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, causes rapid apoptotic cell death despite high-level expression of bcl-2 protein, and inhibits ALL cell in vitro clonogenic growth in a dose-dependent fashion. Furthermore, topotecan elicited potent antileukemic activity in three different severe combined immunodeficiency (SCID) mouse models of human poor prognosis ALL and markedly improved event-free survival of SCID mice challenged with otherwise fatal doses of human leukemia cells at systemic drug exposure levels that can be easily achieved in children with leukemia.