Kenneth S. Zuckerman

A Pilot Study of Isotretinoin in the Treatment of Juvenile Chronic Myelogenous Leukemia

BACKGROUND Juvenile chronic myelogenous leukemia (CML) is a rare myeloproliferative disease of infants and young children for which there is no effective therapy other than allogeneic bone marrow transplantation. In vitro, isotretinoin (13-cis-retinoic acid) attenuates both the spontaneous proliferation of leukemic peripheral-blood progenitor cells (granulocyte-macrophage colony-forming units) and their selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). We conducted a pilot study to evaluate the clinical efficacy of isotretinoin in juvenile CML. METHODS To be eligible the patients had to have newly diagnosed untreated disease, leukocytosis with monocytosis, marrow with less than 25 percent blasts, hepatosplenomegaly, no chromosomal abnormalities, and negative viral cultures and antibody titers. Isotretinoin was administered orally in single daily doses of 100 mg per square meter of body-surface area. When possible, patients subsequently underwent bone marrow transplantation. RESULTS Ten children (median age, 10 months) were enrolled in the study. In all 10 there was spontaneous colony formation of leukemic progenitor cells in vitro. In the eight patients tested there was hypersensitivity to GM-CSF. The only toxic effect of isotretinoin therapy was cheilitis in two patients. Four children had disease progression. Two children had complete responses to isotretinoin (normalization of the white-cell count and disappearance of organomegaly), three had partial responses (more than a 50 percent reduction in the white-cell count and degree of organomegaly), and one had a minimal response (more than a 50 percent reduction in the white-cell count, but a 26 to 50 percent reduction in the degree of organomegaly). The median duration of response was 37 months (range, 6 to 83). Three of the four children who had a complete or partial response and who did not undergo bone marrow transplantation were alive 36 to 83 months after the diagnosis of juvenile CML. The spontaneous colony formation in vitro was reduced in samples from the five patients in whom this factor was reassessed during treatment. There was also a reduction in the hypersensitivity of leukemic progenitor cells to GM-CSF in the two patients retested. CONCLUSIONS Isotretinoin can induce durable clinical and laboratory responses in patients with juvenile CML.

Valproic acid-induced cytopenias: Evidence for a dose-related suppression of hematopoiesis

Valproic acid is an important agent in the management of complex seizure disorders. The drug is being used increasingly in higher doses as a single antieonvulsant agent that produces a good response and has an acceptable level of toxicity. 1 The hematologic side effect primarily reported has been thrombocytopenia, which is believed to be mainly of idiosyncratic or immunologic origin. 2 Neutropenia and erythrocyte aplasia have also been reported, but the cause is unknown. We report a patient with severe erythrocyte aplasia and mild neutropenia associated with valproate monotherapy. The cytopenias were temporally related to escalation of VPA dose. In vitro studies of normal adult bone marrow indicated that VPA levels in the therapeutic range for high-dose valproate monotherapy directly suppressed the growth of hematopoietic progenitors.

Ubiquitin/Proteasome-dependent Degradation of D-type Cyclins Is Linked to Tumor Necrosis Factor-induced Cell Cycle Arrest

Tumor necrosis factor-α (TNF) is well known for its cytotoxic effect on malignant cells. Its role in cell cycle control is relatively less known. In this study, we found that TNF induced G1 arrest of TF-1 and MV4–11 cells while simultaneously causing apoptosis. Treatment of the cells with TNF for 48 h caused cell cycle arrest, accompanied by dephosphorylation of pRb and reduction in D-type cyclin expression. The down-regulation of the D-type cyclins resulted in ∼50–80% decrease of the cyclin-dependent kinase activities. Cells treated with calpain-dependent inhibitor ALLN and apoptosis inhibitor zVAD-FMK suppressed degradation of IκBα and activation of caspase 3, respectively. However, treatment of cells with these two inhibitors was not able to prevent TNF-induced down-regulation of the D-type cyclins. In contrast, proteasome inhibitor MG-132 and lactacystin blocked both TNF-induced degradation of IκBα and down-regulation of D-type cyclins. These data suggest that down-regulation of D-type cyclins by TNF may be proteasome-proteolysis dependent. Additional support for this conclusion was obtained from experiments showing an increase of proteasome activity in TNF-treated cells and in vitro degradation of cyclin D3 by 26 S proteasome.

Inhibition of Normal Murine Hematopoiesis by Leukemic Cells

Inhibition of normal mouse hemopoietic stem cells by leukemic cells (C1498) was observed with use of in vitro agar and in vivo diffusion-chamber cultures. The C1498 cells were unresponsive to colony-stimulating activity, and, above a critical threshold, they inhibited normal granulocyte progenitors in agar culture. C1498 cells added to normal marrow in diffusion chambers progressively reduced granulocyte progenitors. The larger, more rapidly growing, C1498 cells showed the most inhibitory effect. Transmembrane culture of C1498 cells adjacent to normal marrow in double diffusion chambers for five to 14 days led to reduction of recovery of granulocyte progenitors (72 +/- 7 per cent of control) and pluripotent stem cells (45 +/- 7 per cent of control) from the normal marrow chambers. These results indicate that leukemic mouse cells inhibit normal mouse-marrow stem cells by releasing a diffusible substance, and this inhibition occurs primarily at the level of the pluripotent stem cell.

Tumor Necrosis Factor-α-induced Proliferation of Human Mo7e Leukemic Cells Occurs via Activation of Nuclear Factor κB Transcription Factor

Tumor necrosis factor-α (TNF-α) stimulates proliferation of Mo7e, CMK, HU-3, and M-MOK human leukemic cell lines. We report here the signal transduction pathway involved in TNF-α-induced Mo7e cell proliferation. Mo7e cells spontaneously die in the absence of growth factors, but treating the cells with interleukin (IL)-3, IL-6, thrombopoietin, granulocyte/macrophage colony-stimulating factor, or TNF-α promotes their survival and proliferation. Although most of these factors activate MAP kinase and Jun NH2-terminal kinase/signal transducer and activators of transcription signaling pathways, TNF-α fails to activate either pathway. When Mo7e cells were treated with TNF-α, nuclear factor κB (NF-κB) was activated transiently. The activated NF-κB consisted of heterodimers of p65 and p50 subunits. The degradation of IκBα coincided with activation of NF-κB in TNF-α-treated cells. To investigate the role of activated NF-κB in TNF-α-induced Mo7e proliferation, a cell-permeable peptide (SN50) carrying the nuclear localization sequence of p50 NF-κB was used to block nuclear translocation of activated NF-κB. Pretreating Mo7e cells with SN50 blocked TNF-α-induced nuclear translocation of NF-κB and inhibited TNF-α-induced Mo7e cell survival and proliferation. A mutant SN50 peptide did not affect TNF-α-induced Mo7e cell growth. SN50 had no effects on IL-3- or granulocyte/macrophage colony-stimulating factor-induced Mo7e cell proliferation. The results indicate that activation of NF-κB is involved in TNF-α-induced Mo7e cell survival and proliferation.

Characterization of a unique factor-independent variant derived from human factor-dependent TF-1 cells: a transformed event

A factor-independent variant (TF-1a) has been isolated from the factor-dependent TF-1 cell line. The subline has been grown continuously in culture for > 1.5 years without added cytokines. The cells retain the ability to respond to multicytokines, with a different response pattern from its parental cell line. The TF-1 cells appeared singly in liquid culture. In contrast. TF-1a cells formed aggregates which increased markedly in size and in number upon TGFbeta1 treatment and showed a diminished TGFbeta-mediated growth inhibition. TF-1a, but not TF-1 cells, formed colonies in soft agar culture in the absence of any added growth factors, and developed the capacity to generate an invasive tumor(s) in nude mice. There was a constitutive activation of MAPK and MEK in TF-1a but not in TF-1 cells, which may be one of the mechanisms leading to factor-independent growth of TF-1a cells. Phenotypically, TF-1 cells were CD34+ /CD38+, whereas TF-1a cells were CD34+ /CD38-. This suggests that TF-1a may represent a less mature hematopoietic cell than TF-1. In conclusion, TF-1a is different from TF-1 in many important aspects which are associated with neoplastic transformation. The variant appears to be an excellent model for studying the process of progressive malignant transformation of myeloid cells and for studying signal pathways involved in the spontaneous and factor-induced growth of the cells.

Adult Chediak-Higashi parkinsonian syndrome with dystonia.

Chediak‐Higashi syndrome (CHS) is a rare autosomal‐recessive disorder characterized by immune deficiency, partial oculocutaneous albinism, and large eosinophilic, peroxidase‐positive inclusion bodies in granule‐containing cells. The adult form of CHS manifests during late childhood to early adulthood and is marked by various neurologic sequelae, including parkinsonism, dementia, spinocerebellar degeneration, and peripheral neuropathy. We report the case of a 29‐year‐old man with adult CHS who exhibited a progressive asymmetric parkinsonism, including rest tremor, and axial, cervical, and appendicular dystonia. The diagnosis was confirmed by the presence of characteristic large peroxidase‐positive granules within leukocytes and markedly decreased natural killer cell function. Levodopa/carbidopa and amantadine provided benefit for tremor. CHS, although rare, should be considered in the differential diagnosis of young adult parkinsonism.

Transforming growth factor β inhibits growth of more differentiated myeloid leukemia cells and retinoblastoma protein phosphorylation at serine 795

Transforming growth factor beta (TGF-beta) has been shown to be a specific inhibitor of early human myeloid progenitors. We show here that TGF-beta1 potentially inhibited not only the growth of primitive but also more mature myeloid leukemic cells. Surprisingly, those apparently more mature progenitor cells, such as MV4-11 and Mo7e cells, are very sensitive to the action of TGF-beta. The addition of TGF-beta1 to liquid cultures of these cells significantly inhibited their proliferation, with as much as 72% inhibition of growth of MV4-11 cells. The suppressive effect by TGF-beta1 was not reversed or prevented by granulocyte-macrophage colony-stimulating factor or interleukin 3 used to promote cell growth in TF-1a and MV4-11 cells. TGF-beta1 completely abolished the clonal growth of MV4-11 cells in soft agar and inhibited Mo7e, KG-1, K562, TF-1, and TF-1a colony growth by 99%, 90%, 63%, 53%, and 43%, respectively. The cells treated with TGF-beta1 showed progressive accumulation in the G1 phase of cell cycle. Maximal G1 arrest (93%) was observed in MV4-11 cells. Using anti-retinoblastoma protein (pRb) and anti-specific phosphorylated-pRb antibodies, we demonstrated that TGF-beta1 greatly inhibited pRb phosphorylation at serine 795 in MV4-11 and Mo7e cells. Taken together, our data suggest that the sensitivity of myeloid leukemic progenitor cells to growth inhibition by TGF-beta may not be inversely correlated with their maturation stage, and the inhibition of the cells appeared to be linked to the suppression of pRb phosphorylation at serine 795.

Cloning and sequencing of the cDNAs encoding two alternative splicing-derived variants of the alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor

Two distinct cDNA clones, corresponding to alternative mRNA splicing variants of the alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R alpha) were isolated from human blood mononuclear cells by RT-PCR. The first one lacks 179 bp, and the second one lacks 136 bp of the major variant, GM-CSF-R alpha 1. Both of variants result in shifts in the reading frame and are expected to encode 377- and 285-amino-acid membrane anchoring and soluble receptor isoforms, respectively.

Differential effects of transforming growth factor on cell cycle regulatory molecules in human myeloid leukemia cells.

In this report we have studied the mechanism by which Transforming Growth Factor beta (TGFβ) inhibits growth of human myeloid leukemia cell lines. TGFβ1 arrested cells in G1 phase and significantly downregulated the expression of cyclin D2, cyclin D3, cdk4, cyclin A, and cdk2. The downregulation of the molecules resulted in approximately 50–90% decrease of the molecule-dependent kinase activity, varying with each molecule. Although treatment of cells with TGFβ1 up-regulated accumulation of p27kip1 in both nucleus and cytoplasm, the association of the p27kip1 with cdk2, cyclin A, cyclin D2, cyclin D3, and cdk4 was markedly down-regulated, suggesting that p27kip1 is not responsible for the downregulation of the kinase activity. In contrast, TGFβ1 upregulated cyclin E-associated p27kip1 with no effect on the expression of cyclin E. p27kip1-immunodepletion upregulated cyclin E-dependent kinase activity by more than 10-fold in TGFβ1-treated cells but not in proliferating cells; whereas immunodepletion of p27kip1 from cdk2-immunoprecipitates markedly downregulated cdk2 kinase activity in the lysates extracted from both proliferating and TGFβ-treated cells. Consistent with this observation, TGFβ1 and p27kip1 antisense cDNA had a synergistic or additive inhibitory effect on cdk2 but not cyclin E-dependent kinase activity. Our data suggest that (1) TGFβ1-mediated growth inhibition is accomplished through multiple pathways and (2) p27kip1 has opposing effects on cdk2 and cyclin E activity in response to TGFβ1.