Transgenic mice expressing Tel-FLT3, a constitutively activated form of FLT3, develop myeloproliferative disease.

Evidence is continuing to accumulate that the FMS-like tyrosine kinase 3 (FLT3) receptor plays an important role in acute leukemias. Acute myeloid leukemia patients often express constitutive active mutant forms of the receptor in their leukemic cells. A t(12;13)(p13;q12) translocation between Tel and the FLT3 receptor was recently described in a patient with myeloproliferative disease (MPD). Here a Tel-FLT3 construct mimicking this fusion protein was used to generate transgenic mice. The fusion protein was previously found to constitutively activate FLT3 signaling and transform Ba/F3 cells. Expression of the fusion protein in the transgenic mice was found in all tissues assayed including spleen, bone marrow (BM), thymus and liver. These mice developed splenomegaly and had a high incidence of MPD with extramedullary hematopoiesis in the liver and lymph nodes. Spleens also had increased dendritic and natural killer cell populations. In vitro analysis of the hematopoietic progenitor cells derived from Tel-FLT3 transgenic mice showed a significant increase in the number of CFU-GM in the BM, and CFU-GM, BFU-E and CFU-GEMM in the spleen. BM also showed significant increases of in vivo CFU-S colonies. Thus, transgenic mice expressing constitutively activated Tel-FLT3 develop MPD with a long latency and also result in the expansion of the hematopoietic stem/progenitor cells.

Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor.

FLT3 is a receptor tyrosine kinase that may play a role in a significant proportion of leukemias. In addition to being aberrantly expressed in acute leukemias, activating mutations of the FLT3 gene have been found in patients with AML, myelodysplastic syndrome (MDS) and more rarely, ALL. Internal tandem duplications (ITDs) of the FLT3 gene have been detected in 17-34% of patients with AML and portend a poor prognosis for these patients. FLT3 receptors containing ITD mutations (FLT3/ITDs) are constitutively activated in the absence of FLT3 ligand (FL) stimulation leading to the activation of downstream signaling proteins, including ERK and STAT 5. FLT3 activity, therefore, is a logical target for therapeutic intervention. AG1296 is a tyrosine kinase inhibitor of the tyrphostin class that shows inhibitory activity for wild-type FLT3, in addition to the PDGF and c-KIT receptors. We examined the inhibitory effects of AG1296 on FLT3/ITDs isolated from AML patients in the IL-3-dependent cell line, Ba/F3, as well as in primary leukemia samples from AML patients. Immunoprecipitation and immunoblotting analyses demonstrated that FLT3/ITDs were constitutively phosphorylated in the absence of FL. The auto-phosphorylation of FLT3/ITDs was inhibited by AG1296 with an IC(50) of approximately 1 microM. FLT3/ITDs were associated with constitutive phosphorylation of ERK, STAT 5A, STAT 5B, CBL, VAV and SHP2 in Ba/F3 cells. The phosphorylation of these downstream signaling molecules was suppressed in a dose-responsive fashion by AG1296. AG1296 inhibited IL-3 independent growth and induced apoptosis in Ba/F3 cells transformed by FLT3/ITDs. AG1296 also inhibited FLT3 auto-phosphorylation, and induced a cytotoxic effect, in primary AML cells. These findings suggest that inhibiting the activity of FLT3 may have a therapeutic value in some leukemias expressing FLT3/ITDs.

Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3.

Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias.

Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor.

FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17-27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.

A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations.

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 have been found in 20% to 30% of patients with acute myeloid leukemia (AML). These mutations constitutively activate the receptor and appear to be associated with a poor prognosis. Recent evidence that this constitutive activation is leukemogenic renders this receptor a potential target for specific therapy. In this study, dose-response cytotoxic assays were performed with AG1295, a tyrosine kinase inhibitor active against FLT3, on primary blasts from patients with AML. For each patient sample, the degree of cytotoxicity induced by AG1295 was compared to the response to cytosine arabinoside (Ara C) and correlated with the presence or absence of a FLT3/ITD mutation. AG1295 was specifically cytotoxic to AML blasts harboring FLT3/ITD mutations. The results suggest that these mutations contribute to the leukemic process and that the FLT3 receptor represents a therapeutic target in AML. (Blood. 2001;98:885-887)

Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation.

Aberrant expression of FLT3 has been found in most cases of B-lineage ALL and AML, and subsets of T cell ALL, CML in blast crisis and CLL. In 20% of patients with AML the receptor has small internal tandem duplications of the juxtamembrane region which appear to contitutively activate the receptor. To investigate whether FLT3 activation could play a role in leukemia, we generated a constitutively activated FLT3 by fusing its cytoplasmic domain to the helix-loop-helix domain of TEL in analogy to the fusion that occurs with TEL-PDGFR in CMML. In vitro translation assays demonstrated oligomerization and intrinsic tyrosine kinase activity of the TEL-FLT3 chimeric receptor. Constitutively activated TEL-FLT3 conferred IL-3 independence and long-term proliferation to transfected Ba/F3 cells. Immunoblot analyses showed that JAK 2, STAT 3, STAT 5a, STAT 5b and CBL were tyrosine-phosphorylated in TEL-FLT3 expressing Ba/F3 cells in the absence of IL-3. These data suggest a possible role for the JAK/STAT pathway in FLT3 signaling. Transplantation of TEL-FLT3 expressing Ba/F3 cells into syngeneic mice caused mortality in all mice by 3 weeks after injection. Histopathologic analysis demonstrated a massive infiltration of mononuclear cells in the liver, spleen and bone marrow. The mimicking of naturally occurring TEL fusions provides an approach to assess aspects of the biology of activated FLT3, or other receptor-type tyrosine kinases (RTKs) in leukemic transformation.

Suppression of hematopoietic support function is associated with overexpression of interleukin-4 and transforming growth factor-beta 1 in LP-BM5 murine-leukemia-virus-infected stromal cell lines.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus (MuLV) is a disease with many similarities to human AIDS. Our previous studies demonstrated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures could be attributed to a defective hematopoietic stroma. We report now the generation of permanent stroma cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by the polymerase chain reaction for detecting viral genome expression of the p12 envelope glycoprotein. The ability of these cell lines to support in vitro hematopoiesis was evaluated. The results demonstrated that when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic progenitors, whereas in virus-infected progenitors was suppressed. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for interleukin (IL)-1 beta, IL-2, IL-3, IL-6, IL-7, IL-10, interferon, tumor necrosis factor-alpha and stem cell factor. However, infection was associated with higher expression of IL-4 and transforming growth factor-beta 1. These findings demonstrate that infected stomal cell lines generate a defective hematopoietic microenvironment to produce altered cytokine expression and faulty hematopoiesis. Further characterization of these defective cell lines should assist elucidation of the mechanism(s) whereby retroviruses alter hematopoiesis ultimately leading to the generation of immunodeficiency.

Effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) on hematopoiesis administered to retrovirus-infected immunodeficient mice receiving dose-escalation zidovudine (AZT).

We have previously demonstrated that continuous administration of dose-escalation zidovudine (AZT) in either normal or LP-BM5 MuLV immunodeficient virus-infected mice (MAIDS) was associated with the development of anemia, neutropenia, and thrombocytopenia. Hematopoietic growth factors/cytokines are being evaluated to determine their efficacy in ameliorating the hematopoietic toxicity associated with AZT. In normal mice receiving AZT, an increase in only plasma erythropoietin and not GM-CSF, Meg-CSF or TNF-alpha has been reported. This article describes studies that investigated the effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) administered in normal non-viral, viral-infected, and viral-infected C57BL6 mice receiving dose-escalation AZT, i.e. 0.1 mg/ml, 1.0 mg/ml, and 2.5 mg/ml placed in the drinking water. Non-viral control mice responded to IL-3/GM-CSF by increasing erythropoiesis, myelopoiesis and platelet production measured by increased bone marrow and spleen derived erythroid, myeloid and platelet precursor stem cells cultured in semi-solid media. Virus-infected control mice not receiving IL-3/GM-CSF developed pancytopenia. Administration of IL-3/GM-CSF to virus-infected mice receiving dose-escalation AZT did not ameliorate the peripheral pancytopenia associated with immunodeficiency disease and AZT treatment, even though erythroid, myeloid and platelet precursor progenitor cells were increased at certain times when compared to either normal or viral-infected mice receiving IL-3/GM-CSF. These results indicate that the combination use of IL-3 and GM-CSF in vivo is only a partially effective growth factor/cytokine treatment to ameliorate the hematopoietic toxicity associated with the use of the anti-viral drug zidovudine.

Effect of lithium in immunodeficiency: improved blood cell formation in mice with decreased hematopoiesis as the result of LP-BM5 MuLV infection.

Lithium salts have been demonstrated to induce the production of hematopoietic cells following administration in vivo and to minimize the reduction of these cells following treatment with either radiation, chemotherapeutic or antiviral drugs. We have previously demonstrated that lithium, when administered in vivo to immunodeficient mice infected with LP-BM5 MuLV (MAIDS) significantly reduced the development of lymphadenopathy, splenomegaly, and the lymphoma associated with late-stage immunodeficiency disease in this model, and increased the survival of these animals compared to virus-infected controls not receiving lithium. We report here the results of in vivo studies in the MAIDS model that determined the effect of lithium on peripheral blood indices and the number of myeloid (CFU-GM), erythroid (BFU-E) and megakaryocyte (CFU-Meg) hematopoietic progenitors from bone marrow and spleen harvested from immunodeficient mice receiving lithium carbonate (1 mM) placed in their drinking water compared to virus-infected controls not receiving lithium. Time-points evaluated were at weeks 1, 5, 9, 13, 17, and 21 postviral infection. Virus-control mice not receiving lithium demonstrated all the signs that are characteristic of MAIDS, i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, reduced hematopoiesis, and death. Infected mice receiving lithium demonstrated diminished presence of splenomegaly, lymphadenopathy, hypergammaglobulinemia, no suppression of hematopoiesis nor mortality. Enhanced hematopoiesis was demonstrated by neutrophilia, lymphocytosis, thrombocytosis, and erythrocytosis that was evident by increased myeloid, erythroid, and megakaryocyte progenitor cells cultured from bone marrow and spleen. These studies further demonstrate that lithium influences the disease process in the MAIDS model and restricts the development of hematopoietic suppression that develops in this retroviral animal model of immunodeficiency.

Increased hematopoietic toxicity following administration of interferon-a with combination dideoxynucleoside therapy (zidovudine plus ddI) administered in normal mice.

Because of the urgency to develop drugs which will effectively combat HIV infection, many combination therapies which have proved effective against HIV in vitro have undergone, or are undergoing clinical trial. Unfortunately many of drugs are being used without rigorous and exhaustive preclinical evaluation to assess their potential to develop hematopoietic toxicity. We report here the results of two in vivo studies performed to analyze the effect of combined zidovudine (AZT) plus didanosine (ddI) therapy, either with or without interferon-a (IFN-a), on murine hematopoiesis. Normal C57BL/6 female mice were administered AZT (1.0 mg/ml) plus dose-escalation ddI (0.1, 1.0 and 2.5 mg/ml) placed in their drinking water. Control mice received IFN-å (100 units/ml) alone. Mice were serially bled and sacrificed over a six-week period for assessment of hematopoietic toxicity measured by peripheral blood indices and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. AZT plus dose-escalation ddI decreased the hematocrit and white blood cell count when administered to normal mice compared to untreated controls during the six-week examination period. Marrow derived BFU-E, CFU-GM, and CFU-Meg were all reduced, however an increase was observed from the spleen for all three progenitor cell types. Use of IFN-a, in addition to combination AZT plus ddI further decreased the hematocrit, white blood cells and platelets. Marrow derived CFU-GM and CFU-Meg were increased slightly and only marginally for BFU-E with a similar response observed from the spleen. These results demonstrate that combination AZT plus ddI when used in vivo may produce synergistic hematopoietic toxicity, and that the addition of IFN-a to this treatment regimen increases this toxicity. These data indicate caution when this therapeutic approach is suggested for patients infected with HIV. If used, these patients will require careful monitoring for blood cell toxicity.

Stromal cell lines derived from LP-BM5 murine leukemia virus-infected long-term bone marrow cultures impair hematopoiesis in vitro.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus is a disease with many similarities to human AIDS. Previous studies indicated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures may be attributable to a defect of hematopoietic stroma. We report here the generation of permanent stromal cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by polymerase chain reaction for viral genome. The ability of these cell lines to support in vitro hematopoiesis was studied. Results indicated that, when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic precursors, whereas viral-infected cell lines induced suppression of both normal and viral-infected progenitors. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for stem cell factor and tumor necrosis factor alpha. However, infection was associated with higher levels of interleukin-4 and transforming growth factor beta 1 transcript expression. These findings suggest that infected stromal cell lines exhibit a defective hematopoietic microenvironment that produced altered cytokine expression resulting in faulty hematopoiesis. Further characterization of the defective cell lines should prove valuable for studies of the pathogenesis of murine AIDS.

Prevention of hematopoietic myeloid and megakaryocyte toxicity associated with zidovudine in vivo in mice with recombinant GM-CSF.

We studied the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the suppression of hematopoiesis associated with the use of the antiviral drug zidovudine (AZT) administered in vivo to normal mice, as determined by measuring peripheral blood indices, and assays of hematopoietic progenitors, i.e. erythroid (CFU-E/BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) from bone marrow and spleen. Previous studies from this laboratory have established that dose-escalation zidovudine induced a dose-dependent decrease in hematocrit, WBC, and platelets with altered populations of bone marrow and splenic erythroid, myeloid and megakaryocyte progenitors when administered to normal mice. Daily administration of GM-CSF (10 micrograms/kg/bw) was associated with altered peripheral blood indices and progenitor cells. Dose-escalation AZT, i.e. 0.1, 1.0 and 2.5 mg/ml, was associated with a comparable reduction in all indices, i.e. hematocrit, WBC, and platelets during the 6-week examination period. GM-CSF reduced zidovudine-induced myeloid toxicity (concentration < 2.5 mg/ml) which was associated with an increase in bone marrow and splenic CFU-GM. High concentration, i.e. 2.5 mg/ml still produced myelosuppression irreversible with GM-CSF. GM-CSF induced a reduction in circulating platelets following zidovudine treatment at weeks 2 and 4 with the 1.0 mg/ml and 2.5 mg/ml treatment groups respectively, compared to a persistent decrease in platelets in the presence of zidovudine alone. GM-CSF BFU-E were elevated indicating the restriction in erythoid differentiation was still present. These studies demonstrate GM-CSF influences myeloid and megakaryocyte recovery, but not the erythoid suppression associated with the antiviral drug zidovudine.

Effect of combination interleukin-1 and erythropoietin in ameliorating the hematopoietic toxicity associated with the use of zidovudine administered to normal mice.

Use of the anti-viral drug zidovudine in the treatment of acquired immunodeficiency syndrome (AIDS) has been associated with the development of hematopoietic toxicity. Several hematopoietic growth factors have been investigated in their ability to modulate such toxicity; however, no single factor has been demonstrated to produce restoration of hematopoiesis following use with zidovudine. We report results describing the effect of combination interleukin-1 (IL-1) and erythropoietin (Epo) in their ability to modulate the hematopoietic toxicity associated with dose-escalation zidovudine administered in normal mice. When administered over a six-week period, IL-1 and Epo raised the packed red cell volume, white blood cell and platelet counts in control mice and mice receiving dose-escalation zidovudine. These effects were attributed in part to the ability of combination IL-1 and Epo to increase erythroid, myeloid and megakaryocyte progenitor stem cells from bone marrow and spleen. These results indicate that use of combined IL-1 and Epo may be efficacious in ameliorating the hematopoietic toxicity associated with the use of zidovudine.

Sustained zidovudine treatment on hematopoiesis in immunodeficient mice.

Zidovudine (AZT) has been the drug of choice in the treatment of human AIDS; however, associated with the use of zidovudine has been the development of hematopoietic toxicity, the mechanism of which is not clearly defined. We report here studies designed to evaluate dose-escalation of zidovudine, i.e. 0.1 and 1.0 mg/ml placed in the drinking water on hematopoiesis in C57BL/6 normal and LP-BM5 immunodeficiency virus-infected mice. Over a 6-week evaluation period, compared to normal, non-virus-infected controls, murine immunodeficiency (MAIDS) infection was associated with reduced hematopoietic progenitors, i.e. CFU-E, BFU-E, CFU-GM, and CFU-Meg from bone marrow and spleen. Following zidovudine treatment, further suppression of marrow-derived progenitors was observed, while increased numbers of progenitors were obtained from the spleen. Spleen-derived erythroid progenitors, i.e. CFU-E, were increased by 950% (P < 0.001) from MAIDS-infected animals receiving 1.0 mg/ml of drug following 4-weeks exposure compared to non-drug-treated MAIDS control animals. Splenic BFU-E were increased 654% following 6-weeks exposure compared to non-drug-treated MAIDS-infected mice. This study suggests that the bone marrow is particularly sensitive to zidovudine toxicity which, at least early in exposure, appears to be compensated by splenic-derived hematopoiesis, in particular, erythropoiesis. Overt toxicity develops when, at least in this immunodeficiency model, the spleen is unable to provide progenitors in response to continued zidovudine exposure in vivo.

Lithium and anti-viral drug toxicity: II. Further studies on the ability of lithium to modulate the hematopoietic toxicity associated with the anti-viral drug zidovudine (AZT).

Lithium is an agent capable of influencing many aspects of blood cell production, in particular, the formation of granulocytes. Because of this property, lithium has been demonstrated to be an effective agent whenever granulocyte production is either faulty or inadequate. The anti-viral drug zidovudine (AZT) has used been extensively in the treatment of acquired immune deficiency syndrome (AIDS). However, its effectiveness is limited because of the myelosuppression and bone marrow toxicity associated with its use. We have previously demonstrated that lithium, when combined with AZT in vitro with normal bone marrow cells or when administered in vivo to mice receiving dose-escalation AZT, reduced the myelosuppression and marrow toxicity of AZT significantly. We report here further studies designed to evaluate the extent of lithium's capacity to modulate AZT toxicity by investigating the ability of lithium to influence blood cell production when administered to normal mice during an initial exposure to AZT. C57BL6 were administered dose-escalation AZT (1.0 mg/ml and 2.5 mg/ml) for a period of 4-weeks in the presence or absence of lithium carbonate (1 mM). This was followed by an additional 4-week period during which mice received only AZT. Animals were analyzed on a weekly basis for their peripheral blood indices. Animals receiving dose-escalation AZT demonstrated anemia, thrombocytopenia, and neutropenia which was dose-related. During the period when animals received combination lithium/AZT, there was significantly less anemia, thrombocytopenia, and neutropenia as compared to the AZT controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to establish long-term marrow cultures from immunodeficient mice (MAIDS): effect of zidovudine in vitro.

We report here the results of studies examining the ability of zidovudine (AZT) to influence the establishment and maintenance of long-term marrow cultures (LTMC) using marrow from murine immunodeficient mice (MAIDS). Normal C57BL6 mice were infected with LP-BM5 (MuLV) immunodeficiency virus (10 micrograms total protein) intraperitoneally. Five weeks after viral infection, mice were sacrificed and marrow was harvested from normal non-virus-infected and virus-infected animals. LTMC were established in the presence or absence of dose escalation of AZT, that is, 10(-6), 5 x 10(-7), and 10(-7) M in vitro. Compared with controls prepared from normal bone marrow, LTMC using MAIDS-infected marrow failed to establish and subsequently release supernatant-derived mononuclear cells. The addition of AZT was ineffective in either establishing LTMC or consistently producing mononuclear cells. Measurements of erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) precursors were all depressed and none were observed after 5 weeks of culture. Treatment with AZT failed to reverse this depression of stem cell progenitors. Microscopic examination of cultures at 10 weeks demonstrated a failure of MAIDS-LTMC to establish an adequate stromal layer compared to LTMC prepared form non-virus-infected controls. This data indicate that LP-BM5 MuLV infection alters the establishment of a normal functioning hematopoietic microenvironment or stroma. Acknowledging that important differences between MAIDS and human AIDS exist, the implications of these findings concerning the establishment of the immunodeficiency disease state in human immunodeficiency virus infection is discussed.

Modulation of the haematopoietic toxicity associated with zidovudine in vivo with lithium carbonate.

The drug zidovudine (AZT), a synthetic thymidine analogue, has been used in the treatment of acquired immunodeficiency syndrome (AIDS). Clinical use of zidovudine has induced haematopoietic toxicity manifested by anaemia, neutropenia, and overall bone marrow suppression. The monovalent cation lithium has been shown to be an effective agent capable of modulating several aspects of haematopoiesis such as the induction of neutrophilia, thrombopoiesis, and protection against suppression of hematopoietic progenitor stem cells following exposure to anti-cancer drugs and/or radiation at doses commonly used in the treatment of malignant disease. We report here the result of studies designed to evaluate the effectiveness of lithium in reversing zidovudine-induced haematopoietic suppression when administered to normal mice in vivo in the presence of dose-escalation zidovudine. Lithium carbonate (Li2CO3) reversed zidovudine toxicity as measured by increases in peripheral WBC, platelets, and CFU-GM and CFU-Meg haematopoietic progenitors; however lithium was insufficient in reversing the reduction of erythropoiesis associated with zidovudine use in vivo. These results further confirm the effective use of lithium to reverse the development of myelosuppression and thrombocytopenia associated with the anti-viral drug zidovudine, but is less effective in ameliorating the induction of anaemia.

Comparison of dideoxynucleoside drugs (DDI and zidovudine) and induction of hematopoietic toxicity using normal human bone marrow cells in vitro.

The drug zidovudine (AZT), a synthetic thymidine analog, has been used in the treatment of acquired immunodeficiency syndrome (AIDS). Clinical use of zidovudine has induced hematopoietic toxicity manifested by anemia, neutropenia and on occasion thrombocytopenia. Such toxicity has stimulated the development of alternative dideoxynucleoside drugs capable of exerting anti-viral potency while minimizing the risk for inducing organ toxicities. One such alternative dideoxynucleoside drug is 2',3'-dideoxyinosine (ddI). Recent therapeutic anti-viral strategy, now undergoing clinical trial, is the evaluation of combined zidovudine ddI treatment. Unfortunately a complete assessment of their potential toxicity using this drug regimen has not been thoroughly examined. We report here the results of studies comparing the toxicity profile of zidovudine versus ddI on their ability to influence several classes of hematopoietic progenitor stem cells, e.g. granulocyte--macrophage (CFU-GM), megakaryocyte (CFU-Meg) and erythroid (CFU-E/BFU-E) following in vitro co-culture with normal human bone marrow. Since the main clinical toxicity associated with zidovudine in vivo is the development of anemia, additional in vitro studies compared the dose-escalation effect of erythropoietin in the presence of combined zidovudine and ddI. CFU-GM, CFU-Meg, CFU-E and BFU-E were all reduced (P < 0.05) following incubation with either zidovudine or ddI thus determining their ID50 concentrations for these classes of hematopoietic progenitors; however, the extent of toxicity associated with ddI was lower than what was observed with zidovudine. More importantly, dose-escalation of erythropoietin was effective in reversing the inhibition observed for ddI on erythroid progenitors CFU-E and BFU-E (P < 0.05), an effect not reported with zidovudine in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

In-vivo effect of interleukin 3 and erythropoietin, either alone or in combination, on the hematopoietic toxicity associated with zidovudine.

We studied the effect of erythropoietin (EPO) and interleukin 3 (IL-3), either alone or in combination, on the hematopoietic toxicity associated with zidovudine in vivo, as determined by peripheral blood indices, and assay of hematopoietic progenitors, i.e. erythroid (CFU-E/BFU-E), myeloid (CFU-GM) and megakaryocyte (CFU-Meg) from bone marrow and spleen. Previous studies from this laboratory have established that dose escalation of zidovudine to normal mice induced a dose-dependent decrease in hematocrit, white blood cells and platelets with altered populations of marrow and splenic erythroid, myeloid and megakaryocyte progenitors. Daily administration of EPO (50 U/animal, i.p.) and/or IL-3 (5 U/animal, i.p.) was associated with altered peripheral blood indices and progenitor cells. In general, use of EPO and IL-3 alone reduced zidovudine-induced toxicity, notably in erythropoiesis; however, combination EPO/IL-3 was associated with enhanced toxicity with an observed rebound only with the use of < 2.5 mg/ml drug; 2.5 mg/ml drug in the presence of combination EPO/IL-3 accelerated zidovudine-erythroid toxicity. A similar response was noted with circulating platelets and megakaryocyte progenitors. Use of EPO or IL-3, either alone or in combination, failed to reverse zidovudine-induced neutropenia. These studies demonstrate that use of EPO or IL-3, either alone or in combination may serve as an effective adjuvant therapy to modulate the erythroid toxicity associated with lower doses of zidovudine; however, this cytokine therapy was ineffective modulating zidovudine-induced myelosuppression when used in vivo. A reversal in zidovudine-induced myeloid toxicity, therefore may require the use of a myelopoiesis inducing cytokine.

Prevention of the hematopoietic toxicity associated with zidovudine in vivo with IL-1 alone or in combination with GM-CSF administered to normal mice.

We studied the effect of interleukin-1 (IL-1 alpha) either alone or administered with GM-CSF on the induction of hematopoietic toxicity associated with zidovudine (AZT) in vivo, as determined by peripheral blood indices, and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. Previous results reported from this laboratory have established dose-escalation of zidovudine to normal mice induced a dose-dependent decrease in hematocrit, WBC, and platelets with altered populations of marrow and splenic erythroid, myeloid and megakaryocyte progenitors. Daily administration of IL-1 alpha (recombinant murine, 5 u/animal) with or without GM-CSF (recombinant murine (10 micrograms/kg/bw) was associated with reduced AZT-toxicity as measured by increases in peripheral blood indices and progenitor stem cells, i.e., CFU-GM, CFU-Meg and BFU-E cultured from either bone marrow and spleen. The presence of GM-CSF amplified the effect observed with IL-1 especially with respect to myelopoiesis. These results demonstrate IL-1 with or without GM-CSF reverses AZT-hematopoietic toxicity when used in vivo.