Defective iron uptake and globin synthesis by erythroid cells in the anemia of the Belgrade laboratory rat

Erythroid cell iron uptake and globin synthesis were studied in the anemia of the Belgrade Laboratory rat (gene symbol, b), an autosomal recessive trait characterized by hypochromia and hyperferrinemia. Reticulocyte protein and globin synthesis, as measured in vitro by the incorporation of 3H-L-leucine, were significantly diminished in b/b animals, although no major imbalance between alpha-and beta-chain production was observed in b/b reticulocytes. The incorporation in vitro of 3H-L-methionine into marrow cell globin demonstrated no difference between b/b animals and +/? control animals in the proportion of alpha-to beta-chain production. The transfer of iron from plasma to reticulocytes, as measured in vitro by the uptake of 59Fe, was significantly decreased in b/b animals; Sephadex G 200 chromatography of b/b red cell lysates did not reveal the accumulation of 59Fe in nonhemoglobin fractions found when heme synthesis was inhibited with isoniazid. The magnitude of the reticulocyte iron-uptake defect was greater than the reticulocyte globin-synthesis defect, suggesting that the former is the primary lesion.     

Heterogeneity of T-cell lymphoblastic malignancies

To determine the T-cell lineage of the malignant lymphoblast in lymphoblastic lymphoma, tumor cells from nine patients were phenotyped employing a T-cell subset specific heteroantisera, TH2. The normal human peripheral blood T-cell compartment is composed of 80% TH2- negative and 20% TH2-positive T cells, as defined by reactivity with subset specific heteroantisera. Human suppressor cells are TH2 reactive, whereas helper cells are TH2 unreactive. Tumor cells from the majority of patients with lymphoblastic lymphoma were TH2 reactive in contrast to the lack of reactivity previously described in the majority of patients with T-cell acute lymphoblastic leukemia. Comparative clinical studies, including disease presentation and course, were correlated with the presence of the TH2 antigen on the tumor cell. These results provide evidence to support the notion of heterogeneity in the T-lymphoblastic malignancies and suggest that lymphoblastic lymphoma and T-cell acute lymphoblastic leukemia are probably not a single disease process.     

The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3- dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin- like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL- 13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.     

Circulating monoclonal B cells expressing P glycoprotein may be a reservoir of multidrug-resistant disease in multiple myeloma

Multiple myeloma is basically an incurable cancer. Most patients respond initially to chemotherapy with reduction in bone marrow (BM) plasma cells and monoclonal Ig levels, but the disease nearly always recurs and becomes refractory to therapy. The objective of this study was to characterize the expression of the multidrug transport pump, P- glycoprotein 170 (P-gp), in myeloma. The great majority of B cells from peripheral blood mononuclear cells (PBMCs) in myeloma express P-gp, detected by the monoclonal antibody MRK-16. P-gp+ blood B cells exhibit extensive DNA hyperdiploidy, suggesting replicative abnormality characteristic of malignant growth. We speculate these represent a stem cell population in myeloma. The proportion of B cells expressing P-gp was comparable among untreated myeloma patients and those treated with chemotherapy, biologic response modifiers, or off treatment. Among BM cells, P-gp was absent or low in untreated myeloma patients but was expressed at high levels on BM cells from patients previously treated with chemotherapy. For untreated patients the majority of B/plasma cells expressing P-gp are located in PBMCs, not the BM cells. Flow cytometric analysis of rhodamine 123 dye efflux indicated a functional P-gp that was efficiently blocked by verapamil or cyclosporin A (CsA). Both the CD11bhi CD19+ B cells and the T cells in myeloma PBMCs had active CsA-inhibited dye efflux, but monocytes lacked the ability to efflux dye. Nearly all CD38hi plasma cells from myeloma BM cells retained dye, indicating their lack of a functional transport pump. Thus, PBMC B cells may be the predominant set of drug-resistant tumor cells. Myeloma PBMC B cells were cultured with Adriamycin with or without CsA and drug toxicity evaluated by the induction of apoptosis, using flow cytometry to quantitate DNA disruption. No apoptosis was detectable at 0.01 microgram/mL adriamycin, the in vivo steady-state level, with or without CsA. With 0.1 microgram adriamycin, no apoptosis was detectable in the absence of CsA, but with CsA, 66% of B cells initiated DNA disruption, whereas most T cells were spared. This work suggests that currently used drug dosages are too low to effect P-gp+ B- cell death, even in the presence of CsA. We suggest that blood B cells comprise a highly drug-resistant subset of the myeloma B lineage that escapes conventional chemotherapy and may underlie the almost uniform fatal relapse in myeloma patients.     

Fanconi anemia genes act to suppress a cross-linker-inducible p53- independent apoptosis pathway in lymphoblastoid cell lines

Hypersensitivity to cross-linking agents such as mitomycin C (MMC) is characteristic of cells from patients suffering from the inherited bone marrow failure syndrome. Fanconi anemia (FA). Here, we link MMC hypersensitivity of Epstein-Barr virus (EBV)-immortalized FA lymphoblasts to a high susceptibility for apoptosis and p53 activation. In MMC-treated FA cells belonging to complementation group C (FA-C), apoptosis followed cell cycle arrest in the G2 phase. In stably transfected FA-C cells, plasmid-driven expression of the wild-type cytoplasmic FAC protein relieved MMC-dependent G2 arrest and suppressed p53 activation. However, in both FA and non-FA lymphoblasts, p53 seemed not to be instrumental in the induction of MMC-dependent apoptosis, since overexpression of a dominant-negative p53 mutant failed to affect cell survival. In addition, no differences in the level of Bcl-2 expression, an inhibitor of apoptosis, were detected between FA and non- FA cells either in the absence or presence of MMC. Our findings suggest that FAC and the other putative FA gene products may function in a yet to be identified p53-independent apoptosis pathway.     

Localization of the G-CSF gene on chromosome 17 proximal to the breakpoint in the t(15;17) in acute promyelocytic leukemia

The human granulocyte-colony stimulating factor gene (G-CSF) is localized at 17q11.2-q21, the region of one of the breakpoints in the 15;17 chromosome translocation specific for acute promyelocytic leukemia (APL). As G-CSF induces differentiation and loss of tumorigenicity in myeloid leukemic cells or cell lines, it was possible that the translocation in APL involved the DNA of the G-CSF coding region or its regulatory region. In situ hybridization to chromosomes with the t(15;17) from patients with the APL translocation using a G- CSF cDNA clone revealed that the coding region of this gene is proximal to the t(15;17) breakpoint on chromosome 17. Southern analysis of DNA from patients with the APL translocation showed no differences in hybridization between normal and leukemic cells. These results indicate that the G-CSF coding sequence is not disrupted by the chromosomal rearrangement characteristic of APL.     

A collaborative, double-blind randomized study of cetiedil citrate in sickle cell crisis

We have recently completed a double-blind, placebo-controlled, noncrossover study, the goal of which was to determine whether cetiedil citrate (cetiedil) could affect the course of vaso-occlusive crises in sickle cell disease. Patients, who presented to the emergency room at least 4 but no more than 24 hours after the onset of a painful vasoocclusive crisis severe enough to require hospitalization, were considered candidates for the study. Each patient received either placebo or cetiedil at one of the following three dosages: 0.2, 0.3, or 0.4 mg/kg body weight. The assigned drug dosage was given as a 30 minute intravenous infusion every 8 hours for 4 consecutive days. A total of 67 patients was enrolled in the study. Cetiedil, at its highest dosage (0.4 mg/kg body weight), was found to be significantly superior to placebo both in reducing the number of painful sites present on all 4 treatment days and in shortening the total time in crisis. No serious adverse reactions were observed during the course of the study. We conclude that cetiedil, given at a dosage of 0.4 mg/kg body weight, is therapeutically advantageous for sickle cell crisis.