Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2.

We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.

IL-1 receptor accessory protein is an essential component of the IL-1 receptor.

The recently described IL-1R accessory protein (IL-1R AcP) interacts with IL-1beta and the IL-1 type-IR (IL-1RI), but an essential requirement for IL-1R AcP in IL-1 signaling in vitro has not been established and its role in vivo has not been examined. In this study, IL-1R AcP-deficient mice and fibroblasts were produced and characterized. All IL-1 agonists bound to IL-1R AcP-deficient cells through the type I IL-1R, but failed to activate gene expression through either the nuclear factor-kappaB or AP-1-dependent signaling pathways. Absence of IL-1R AcP differentially affected the affinity for IL-1 ligands. IL-1R AcP-deficient fibroblasts bound murine IL-1alpha and human IL-1R antagonist protein (IL-1Ra) with only moderately reduced affinity when compared with wild-type cells, whereas murine IL-1beta affinity was reduced by 70-fold. IL-1 also failed to produce a biologic response in vivo in IL-1R AcP-deficient mice. These data demonstrate that a type I IL-1R/IL-1R AcP complex is required for signaling by all IL-1 agonists and for high affinity binding by IL-1beta. Finally, IL-1R AcP is an essential signal transducing component of the functional IL-1R and should represent a novel target for blocking IL-1 function in human disease.

Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.

IL-1alpha and IL-1beta are potent inflammatory cytokines that contribute to a number of normal physiologic processes and to the development of a number of inflammatory diseases. Two IL-1R, the type I and type II receptors, have been identified. This work describes the derivation and characterization of mice deficient in expression of the type I IL-1R (IL-1RI). IL-1RI-deficient mice were viable and fertile, but failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever. Similar to IL-1beta-deficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine. In contrast, IL-1RI-deficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes. These data demonstrate that the IL-1RI is essential for all IL-1-mediated signaling events examined, and that both IL-1alpha and IL-1beta are critical to the animals' response to injury and infection. These data also demonstrate that IL-1 function is not required for normal development or homeostasis.

Differential effect of E-selectin antibodies on neutrophil rolling and recruitment to inflammatory sites.

The selectins are inducible adhesion molecules critically important for the inflammatory response. We investigate here the functional effects of three monoclonal antibodies (MoAbs) raised against murine E-selectin (9A9, 10E6, and 10E9.6) on neutrophil recruitment in vivo, leukocyte rolling and circulating leukocyte concentrations in vivo, and adhesion of myeloid cells to E-selectin transfectants and recombinant E-selectin-IgG fusion protein in vitro. MoAbs 9A9 and 10E6 map to the lectin and epidermal growth factor (EGF)-like domains of murine E-selectin, whereas 10E9.6 binds to the consensus repeat region. 10E9.6 blocked neutrophil recruitment in a model of thioglycollate-induced peritonitis in Balb/c mice by more than 90% but had no effect in C57BL/6 mice. 9A9 and 10E6 blocked neutrophil recruitment in this assay only when combined with a P-selectin antibody, 5H1. Neither 9A9 nor 10E9.6 alone blocked leukocyte rolling in tumor necrosis factor-alpha-treated venules of Balb/c mice, but 9A9 almost completely inhibited leukocyte rolling when combined with the function-blocking murine P-selectin MoAb, RB40.34. In contrast, 10E9.6 had no effect on leukocyte rolling in RB40.34-treated Balb/c or C57BL/6 mice. 10E9.6 did not affect adhesion of myeloid cells to E-selectin transfectants or attachment, rolling, and detachment of myeloid cells to murine E-selectin-IgG fusion protein. However, adhesion was completely blocked in the same assays by 9A9. Taken together, these results indicate that E-selectin serves a function, other than rolling, that appears to be critically important for neutrophil recruitment to inflammatory sites in Balb/c mice.

Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice.

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)-12 in the pathogenesis of this disease by undertaking an analysis of IL-12-deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL-12. Concomitant decreases were observed in serum levels of pathogenic, collagen-specific IgG2a antibodies and collagen-induced secretion of interferon-gamma by immune splenocytes in vitro, consistent with an impaired T helper-1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL-12 in the pathogenesis of CIA, although it is not absolutely required for disease development.

Insertion of a structural domain of interleukin (IL)-1 beta confers agonist activity to the IL-1 receptor antagonist. Implications for IL-1 bioactivity.

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the beta-bulge (QGEESN) of IL-1 beta at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the beta-bulge was co-expressed with a second substitution (His-54 --> Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

Enhanced antitumor efficacy in mice by combination treatment with interleukin-1 alpha and interferon-alpha.

The antitumor efficacy of recombinant murine interleukin-1 alpha (rMuIL-1 alpha) was evaluated either alone or in combination with recombinant human hybrid interferon alpha A/D (IFN-alpha A/D) against the murine B16 F10 malignant melanoma. Treatment of subcutaneous tumor-bearing mice intraperitoneally with rMuIL-1 alpha resulted in a dose-dependent inhibition of tumor growth with the greatest activity obtained with the maximum tolerated dose of rMuIL-1 alpha (10 micrograms per treatment). Augmented tumor inhibition comparable to that seen in mice treated with a high dose of rMuIL-1 alpha was observed in subcutaneous tumor-bearing mice injected with the combination of IFN-alpha A/D and a low dose of rMuIL-1 alpha. Similar inhibition of subcutaneous tumor growth was obtained in T-cell-deficient nude or natural killer cell-deficient beige mice. In contrast, treatment of mice bearing B16F10 experimental pulmonary metastases with rMuIL-1 alpha resulted in no decrease in the number of metastases, and rMuIL-1 alpha did not potentiate the antimetastatic activity of IFN-alpha A/D. A synergistic induction of IL-6 was induced in mice treated with the combination of rMuIL-1 alpha plus IFN-alpha A/D but the level of IL-6 induced was not correlated with inhibition of tumor growth because this elevation of IL-6 was not observed in tumor-bearing nude mice. No direct antiproliferative activity was demonstrable in vitro against B16 F10 cells with rMuIL-1 alpha, IL-6, or rMuIL-1 alpha plus IL-6, and addition of these cytokines did not enhance the antiproliferative activity of IFN-alpha A/D.(ABSTRACT TRUNCATED AT 250 WORDS)

A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-kappa B p65 causes sequence-specific immune stimulation.

Antisense oligonucleotides have proved effective in achieving targeted inhibition of gene expression. In such experiments, sense oligonucleotides have frequently been used as a control for nonspecific effects, but the results have been variable, raising questions about the reliability of sense oligomers as a control. It is possible that some of the effects of sense oligonucleotides may be specific. We have shown that phosphorothioate antisense oligonucleotides to the p65 subunit of NF-kappa B, a transcription factor, cause a block in cell adhesion. In our efforts to test the efficacy of NF-kappa B p65 oligonucleotides in vivo, we unexpectedly observed that the control p65-sense, but not the p65-antisense, oligonucleotides caused massive splenomegaly in mice. In the current study we demonstrate a sequence-specific stimulation of splenic cell proliferation, both in vivo and in vitro, by treatment with p65-sense oligonucleotides. Cells expanded by this treatment are primarily B-220+, sIg+ B cells. The secretion of immunoglobulins by the p65-sense oligonucleotide-treated splenocytes is also enhanced. In addition, the p65-sense-treated splenocytes, but not several other cell lines, showed an upregulation of NF-kappa B-like activity in the nuclear extracts, an effect not dependent on new protein or RNA synthesis. These results demonstrate that phosphorothioate oligonucleotides can exert sequence-specific effects in vivo, irrespective of sense or antisense orientation.

Interleukin 1: an important mediator of host resistance against Pneumocystis carinii.

The importance of endogenous interleukin 1 (IL-1) in resistance to Pneumocystis carinii infection was examined in a SCID mouse model. Naturally acquired pulmonary infection of P. carinii in SCID mice was completely cleared by reconstitution of the infected mice with immunocompetent spleen cells. IL-1 activity in the lung homogenate supernatant of these mice increased significantly after reconstitution and returned to baseline level after the clearance of P. carinii. Treatment of reconstituted SCID mice with 35F5, a monoclonal antibody against murine type I IL-1R almost completely inhibited the clearance of P. carinii. In contrast, treatment with control rat immunoglobulin G had no detectable effect. Further study revealed that for the complete clearance of P. carinii, IL-1 must be present at the early stage of immune responses induced by reconstitution, since clearance could be blocked by a single injection of 35F5 into SCID mice at 2 d, but not at either 8 or 13 d postreconstitution. Furthermore, pulmonary recruitment of neutrophils, macrophages, and lymphocytes was significantly inhibited in mice that received 35F5 treatment. These findings strongly suggest that, in reconstituted SCID mice, endogenous IL-1 is important in host resistance to P. carinii infection and that IL-1 may function early in the host response possibly by the recruitment of inflammatory cells into the lungs.

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue. Neither addition of the C-terminal spacer arm nor biotinylation affected affinity of these analogs for GRF antibody. Relative to hGRF(1-44)-NH2 (hGRF44: potency = 1.0), the biotinylated analogs were equipotent in vitro to their nonbiotinylated, parent compounds: [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2 (4.7) = [Ala15]hGRF29-GGC-(tpBiocytin)-NH2 (3.9) greater than hGRF29-GGC-(tpBiocytin)-NH2 (0.8). Based upon cumulative GH release data in vivo (0-60 min postinjection), [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2, [Ala15]hGRF29-GGC-(tpBiocytin)-NH2, and hGRF29-GGC-(tpBiocytin)-NH2 displayed 8.6, 5.5, and 0.8 times, respectively, the potency of hGRF44. These in vivo potency values were not significantly different from the corresponding parent compounds (i.e., with or without the C-terminal spacer arm). In summary, biotinylated hGRF analogs have been developed that retain full immunoreactivity and potent bioactivity (in vitro and in vivo), thus permitting their use in GRF receptor isolation, ELISA, and histochemical procedures.

Inhibition of interleukin 1 (IL-1) binding and bioactivity in vitro and modulation of acute inflammation in vivo by IL-1 receptor antagonist and anti-IL-1 receptor monoclonal antibody.

Recombinant human interleukin 1 receptor antagonist (IL-1ra) and 35F5, a neutralizing monoclonal antibody (mAb) to the type I mouse IL-1 receptor, were examined for their ability to bind to IL-1 receptors (IL-1Rs) on various types of mouse cells and to block immune and inflammatory responses to IL-1 in vitro and in mice. IL-1ra competed for binding of 125I-IL-1 alpha to type I IL-1R present on EL-4 thymoma cells, 3T3 fibroblasts, hepatocytes, and Chinese hamster ovary cells expressing recombinant mouse type I IL-1R. The IC50 values for IL-1ra binding (ranging from 2 to 4 ng/ml) were similar to those of IL-1 alpha. In contrast, IL-1ra bound with very low affinity (IC50 values ranging from 10 to 200 micrograms/ml) to cells expressing type II IL-1R, i.e., 70Z/3 pre-B cell line and polymorphonuclear leukocytes (PMN) derived from bone marrow and acute inflammatory exudates. The mAb 35F5 bound specifically to type I IL-1R; no inhibition of 125I-IL-1 alpha binding to cells having type II IL-1R was observed with very high concentrations of antibody. While neither IL-1ra nor 35F5 had intrinsic activity in bioassays using T helper D10.G4.1 cells and mouse thymocytes, both agents blocked the ability of IL-1 to stimulate proliferation of these cells. The effects of IL-1ra and 35F5 on acute inflammatory responses in mice were also evaluated. IL-1ra and 35F5 blocked the local accumulation of PMN after intraperitoneal injection of rIL-1 alpha. The response to IL-1 was inhibited when IL-1ra or 35F5 was administered simultaneously with or before administration of IL-1. IL-1ra and 35F5 also blocked PMN accumulation after intraperitoneal injection of lipopolysaccharide or proteose peptone, suggesting IL-1 is important in mediating responses to these agents. In addition, IL-1ra and 35F5 significantly blocked the ability of IL-1 to stimulate egress of PMN from bone marrow, to induce a transient neutrophilia, and to elevate serum levels of hepatic acute phase proteins, IL-6, and corticosterone. Thus, IL-1ra and 35F5 competitively inhibit the binding of IL-1 to the IL-1R on certain cell types. These two IL-1 receptor antagonists act to inhibit biological responses induced by IL-1 and other inflammatory agents.

Stimulation of hematopoiesis and antibacterial resistance by interleukin-1.

The beneficial effects of IL-1 and other cytokines on hematopoiesis and on resistance to infection are profound. IL-1 stimulates proliferation of bone marrow cells in normal mice and potentiates the recovery of peripheral blood neutrophils in mice with drug-induced neutropenia. Prophylactic cytokine administration provides an elevated level of natural resistance to infections which is correlated with increased numbers of phagocytic leukocytes. These studies suggest that IL-1 has potential clinical application as a therapy to limit bone marrow dysfunction and immunosuppression and to augment hematopoiesis and natural immunity. Further research will continue to elucidate the mechanisms whereby interleukins and colony-stimulating factors act, and interact, to promote restoration of leukocyte production and to enhance host resistance.

Enhancement of antibacterial resistance of neutropenic, bone marrow-suppressed mice by interleukin-1 alpha.

The effect of recombinant human interleukin-1 alpha (IL-1) on the resistance of normal and bone marrow-suppressed mice against bacterial infection was evaluated. IL-1 induced neutrophilia and enhanced the resistance of normal mice against acute, systemic intraperitoneal infection with Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus. Mice with cyclophosphamide-induced bone marrow suppression were neutropenic and exhibited increased susceptibility to infection. Treatment of neutropenic C57BL/6 and C3H/HeJ mice with IL-1 before infection accelerated recovery of peripheral neutrophil counts and stimulated resistance against infection. Increases in neutrophils and enhancement of resistance induced by IL-1 were both dose and time dependent. Both neutrophilia and augmented resistance to infection were eliminated by a second dose of cyclophosphamide administered during the IL-1 treatments. Bone marrow-suppressed mice treated with IL-1 showed, at 4 h postinfection, greater increases in peripheral blood neutrophils and in numbers of peritoneal exudate neutrophils than suppressed mice treated with vehicle. The data suggest that the IL-1-stimulated recovery of myelopoiesis is an important factor in the enhancement of antibacterial resistance in bone marrow-suppressed, neutropenic mice. These findings indicate that IL-1 may be efficacious in limiting the duration of the neutropenia and of the increased risk for the development of bacterial infection associated with bone marrow suppression.

Blastogenesis of large granular lymphocytes in nonlymphoid organs.

High numbers of large granular lymphocytes (LGL) accumulate in the livers and peritoneal cavities of mice during the course of viral infection. Accumulation of natural killer (NK) cells at day 3 postinfection (p.i.) was shown to be radiation-sensitive, implying that proliferation was required for this response. Accumulation occurred in splenectomized mice, indicating that the spleen, known to be an organ for mature NK cell proliferation, was not the major source for liver and peritoneal NK/LGL. Significant percentages (greater than 25%) of the LGL found in the liver and peritoneal cavity following viral infection or interferon induction with poly-inosinic:poly-cytidylic acid were defined morphologically as blasts (large cells with prominent nucleoli and intensely basophilic cytoplasms containing azurophilic granules). Most blast LGL at day 3 p.i. were sensitive to administration of anti-asialo GM1 serum in vivo, were Lyt-2-, and were enriched in populations that lysed NK cell-sensitive targets in vitro, indicating that these were NK/LGL. At day 3 p.i., leukocytes from the liver and peritoneal cavity incorporated 3H-thymidine and bound to and killed NK cell-sensitive targets in single-cell cytotoxicity assays. These data suggest that NK/LGL undergo at least one round of division in the liver and peritoneal cavity during viral infection. In contrast, blast LGL at day 7 p.i. were resistant to in vivo treatments with anti-asialo GM1 serum, were Lyt-2+, and were enriched in populations of cells that killed virus-infected histocompatible targets, indicating that they were cytotoxic T lymphocytes (CTL). These results suggest that both NK/LGL and CTL/LGL are capable of blastogenesis and presumed proliferation at sites of virus infection, providing a means for the in situ augmentation of a host's cell-mediated antiviral defenses.

Natural killer cells are not required for interferon-mediated prophylaxis against vaccinia or murine cytomegalovirus infections.

Natural killer (NK) cell-depleted or control mice were treated prophylactically with polyinosinic: polycytidylic acid (polyI: polyC) or purified beta interferon (IFN) and then infected with either vaccinia virus or murine cytomegalovirus. NK cell depletion alone enhanced virus titres in the spleen and peritoneal cavity. However, poly I: polyC and IFN inhibited virus replication equally well in control and NK cell-depleted mice. This suggests that prophylactic IFN treatment mediates antiviral effects independently of NK cells.

Accumulation of natural killer and cytotoxic T large granular lymphocytes in the liver during virus infection.

The immunologic mechanisms involved in virus-induced hepatitis were examined by measuring the cytotoxic capabilities and the morphologic and antigenic phenotypes of leukocytes isolated from livers of virus-infected mice. Large granular lymphocytes (LGL) of both natural killer (NK) cell and cytotoxic T lymphocyte (CTL) phenotypes were found to accumulate in livers of mice infected with either the nonhepatotropic Armstrong strain of lymphocytic choriomeningitis virus (LCMV-ARM) or the hepatotropic WE strain (LCMV-WE). Between days 1 and 5 postinfection (p.i.), both viruses induced a three- to fourfold increase in NK cell lytic activity in the livers of C3H/St mice and a three- to fourfold increase in the number of LGL in the organ. These LGL were characterized as NK cells on the basis of cell surface antigens, kinetics of appearance, target cell range, and morphology. By day 7 p.i., virus-specific, H-2-restricted, Thy-1+, Lyt-2+, CTL activity was present in the liver, and its appearance correlated with a second wave of LGL accumulation. CTL activity, total leukocyte number, and CTL/LGL number were at least fivefold higher in the livers of mice infected with LCMV-WE than with LCMV-ARM. The dramatic LCMV-WE-induced day 7 increases in total leukocytes and LGL were absent in athymic nude (nu/nu) mice, suggesting that the increases were T cell-dependent. LCMV-ARM infection of C57BL/6 mice induced significant spleen CTL activity but little liver CTL activity, whereas LCMV-WE infection resulted in significant liver CTL activity but minimal spleen CTL activity. Mice infected with the cytopathic hepatotropic viruses, mouse hepatitis virus (MHV) and murine cytomegalovirus (MCMV), experienced much greater increases in liver NK/LGL by day 3 p.i. than did mice infected with LCMV or injected with the interferon-inducer poly(I-C). MHV-infected mice homozygous for the beige (bg/bg) mutation also exhibited significant increases in liver NK/LGL cell number and activity, although the activity was less than heterozygote controls, and the morphology of the LGL granules was aberrant. These data show that the LGL accumulate in virus-infected organs, in this case, the liver. An early NK/LGL influx is most pronounced during infection with cytopathic hepatotropic viruses. This initial influx of NK/LGL is followed later by an influx of CTL also possessing LGL morphology. The CTL/LGL response in the liver is significantly greater during hepatotropic virus infections, even when a strong CTL response in the spleen is lacking.

Exquisite specificity of adoptive immunization in arenavirus-infected mice.

Lymphocytic choriomeningitis virus (LCMV)-infected mice can be adoptively immunized with T cells from immune mice sharing MHC compatibility in H-2K or D, suggesting direct cytotoxic effects of T cells in vivo. However, T cells, upon recognition of an appropriate target, secrete lymphokines which may be capable of mediating antiviral effects nonspecifically. In this report we show that LCMV-immune cells reduced LCMV spleen titers in mice infected with LCMV alone or with LCMV and Pichinde virus (PV), but had no effect on PV titers in these mice or in mice infected with PV alone. Titers of PV were reduced by PV-immune cells transferred into mice infected with PV alone or with PV and LCMV, while LCMV titers were not altered. PV and LCMV antigens were shown by fluorescence microscopy to be in proximity in the spleen prior to cell transfer. These data suggest that adoptive immunization against these arenaviruses involves direct cytotoxicity or an extremely localized effect of nonspecific soluble factors elaborated following antigen recognition, rather than generalized nonspecific antiviral effects of a more systemic nature.

Influence of cell cycle phase-specific agents on simian fetal hemoglobin synthesis.

To determine the influence of cell cycle-specific agents on primate hematopoiesis and fetal hemoglobin production, two juvenile cynomolgus monkeys (Macaca fascicularis) were repeatedly bled to maintain their hemoglobins at approximately 6.5 g/dl and fetal hemoglobin levels at 3-5%. Six separate 5-d courses of hydroxyurea at 100 mg/kg per d were then administered over the next 200 d while phlebotomy was continued. These courses of hydroxyurea progressively raised the fetal hemoglobin levels to 17 and 18%, respectively. The drug had very little effect on the frequency of immature erythroid progenitors (BFU-E) in the bone marrow, but caused a marked reduction in the frequency of later progenitors (CFU-E) and a transient fall in the reticulocyte count. Following the courses of hydroxyurea, the number of F cells and the fetal hemoglobin level fell to base line over a period of 4 wk. Two control animals which were not phlebotomized showed no detectable increase in F cells or fetal hemoglobin when treated with the same regimen of hydroxyurea. A 5-d course of 5-azacytidine at 8 mg/kg per d was then given to each of the phlebotomized animals. This produced a more profound, albeit transient, reticulocytopenia, a fall in the CFU-E/BFU-E ratio, and a prompt increase in the fetal hemoglobin to levels even higher than were seen following a single 5-d course of hydroxyurea at 100 mg/kg/d. Subsequently, the animals were given a single dose of vinblastine at 0.4 mg/kg which reduced reticulocytes and CFU-E to the same extent as hydroxyurea; however, vinblastine at this dose had no effect on hemoglobin F (HbF) production. In contrast, when vinblastine was administered to the phlebotomized monkeys as a 5-d course at 0.2 mg/kg/d, prolonged reticulocytopenia followed by dramatic F cell and HbF responses were seen. Combinations of single dose vinblastine and a 5-d course of hydroxyurea were subsequently administered using two different schedules. When the animals received vinblastine on the first day of a 5-d course of hydroxyurea, the F cell response was double that seen following hydroxyurea treatment alone. In contrast, when vinblastine was administered on the final day of hydroxyurea treatment, the magnitude of the F cell response was the same as that which occurred following hydroxyurea treatment alone, but the onset of the rise was delayed for 4 d and HbF/F cell response was much higher. These results establish several important features of the fetal hemoglobin response to cytotoxic agents in the primate model. The response requires accelerated erythropoiesis and is preceded by transient reticulocytopenia. The response is produced by S phase- and M phase-specific agents when given in sufficient doses and at appropriate schedules. Passage of erythrocyte progenitors through M phase appears to be necessary for expression of the effect produced by S phase agents. The fetal hemoglobin response induced by cytotoxic drug administration occurs during the recovery of erythropoiesis following marrow suppression.

Augmentation of fetal-hemoglobin production in anemic monkeys by hydroxyurea.

The increase in fetal-hemoglobin synthesis in patients with beta-thalassemia or sickle-cell anemia induced by 5-azacytidine has been attributed to hypomethylation of DNA in the region of the gamma-globin genes. To determine whether hydroxyurea, a cytotoxic/cytostatic drug that does not influence DNA methylation, might stimulate fetal-hemoglobin synthesis, we phlebotomized two juvenile cynomolgus monkeys to induce anemia and reticulocytosis and then treated them with hydroxyurea. Immediately after phlebotomy was initiated, there was a rise in the level of F cells, which stabilized at an average value of 13 per cent in one animal and 20 per cent in the other during a two-month control period. Fetal hemoglobin gradually rose from undetectable values before bleeding to 3 per cent in one animal and 5 per cent in the other. Sixty-two days after initiation of phlebotomy, hydroxyurea (50 mg per kilogram of body weight per day for five days) induced only a small and transient increase in F cells and fetal hemoglobin. Two weeks later, however, a similar course (100 mg per kilogram per day) resulted in a prompt and dramatic increase in both indexes. These results strongly suggest that S-phase-specific cytotoxic/cytostatic drugs increase fetal hemoglobin by a mechanism that does not involve inhibition of DNA methylation.