Childhood acute myeloid leukemia in mice with severe combined immunodeficiency

Primary bone marrow blasts from 4 children with t(8;21) acute myeloid leukemia (AML), 6 children with inv(16) AML, and 2 children with t(9;11) AML were injected intravenously or transplanted under the kidney capsule of sublethally irradiated mice with severe combined immunodeficiency (SCID). Leukemic cells from all AML patients infiltrated the SCID mouse thymus, suggesting that the thymic microenvironment supports the survival and growth of human AML blasts. Blasts from 1 of 4 t(8;21) AML patients and 4 of 6 inv(16) AML patients caused histopathologically detectable disseminated leukemia. Blasts from the remaining patients produced disseminated occult leukemia that was only detected by polymerase chain reaction. Occurrence of histopathologically detectable disseminated leukemia was dependent on intravenous injection of leukemic cells; none of the mice challenged with an inoculum transplanted under the kidney capsule developed overt leukemia. No obvious association was noted between occurrence of leukemia in SCID mice and clinical or laboratory features presented by patients, including age, sex, or leukocyte count at diagnosis. To our knowledge, this study is the first to show that leukemic blasts from children with newly diagnosed AML, especially inv(16) AML, can cause disseminated human leukemia in SCID mice without exogenous cytokine support. The SCID mouse model system may prove particularly useful for designing more effective treatment strategies against childhood AML.     

Cytogenetics of childhood T-cell leukemia

The karyotypes of 57 cases of childhood T-cell acute lymphoblastic leukemia (ALL) were analyzed to establish the cytogenetic profile in this disease. Three questions were of particular interest. Do the chromosomal changes in T-cell ALL preferentially affect bands where genes encoding the T-cell receptor for antigen (TCR) have been mapped? Do alterations involving the TCR gene regions appear with any notable frequency in B-progenitor ALL? Do chromosomal abnormalities in this disease relate to stage of T-cell ontogeny? A relatively high proportion of cases (65%) had a pseudodiploid karyotype at presentation, the majority (58%) characterized by a translocation. The overall frequency of translocations was 44%, comparable to that among all banded cases of ALL seen in our laboratory. Hypodiploidy and hyperdiploidy were exceedingly rare (only four of 57 cases); 16 cases (28%) had apparently normal karyotypes. In half the cases with a translocation (14 of 24), the breakpoints were in regions to which the alpha and beta chain TCR genes have been mapped. Chromosomal breakpoints that were consistently observed in the vicinity of TCR gene loci were 7q32-q36 (TCR beta chain; n = 8), 14q11-q13 (TCR alpha chain; n = 6); other frequent breakpoints were 9p13-pter (n = 8) and 6q15-qter (n = 9). Chromosomal alterations occurred near TCR gene loci significantly more often in T-cell cases than in a comparison group of 335 patients with B-cell precursor ALL (26% v 1.5%, P = .0001). Stage I thymocyte development (CD7+, CD2+, CD5+, CD1-, CD3-, CD4-, CD8-) was noted in 23 cases, stage II (CD7+, CD2+, CD5+, CD1+, CD3-, CD4 +/-, CD8 +/-) in 25 cases, and stage III (CD9+, CD2+, CD1-, CD5+, CD3+, and either CD4+ or CD8+) in nine cases. The only statistically significant associations between cytogenetic findings and T-cell ontogeny were a higher frequency of normal karyotypes in cases with stage I thymocytes, and of pseudodiploidy in stage II cases. There was no apparent relationship between particular translocations and level of thymocyte maturation. Our findings indicate that most children with T-cell ALL have pseudodiploid karyotypes, although a surprisingly high percentage lack demonstrable abnormal clones. Specific chromosomal changes do not appear to be related to discrete stages of T-cell ontogeny as defined in this study, but they occur preferentially in bands containing TCR genes.     

Reduced invasion and growth of Plasmodium falciparum into elliptocytic red blood cells with a combined deficiency of protein 4.1, glycophorin C, and p55

In this investigation, we have measured the invasion and growth of the malaria parasite Plasmodium falciparum into elliptocytic red blood cells (RBCs) obtained from subjects with homozygous hereditary elliptocytosis. These elliptocytic RBCs have been previously characterized to possess molecular defects in protein 4.1 and glycophorin C. Our results show that the invasion of Plasmodium falciparum into these protein 4.1 (-) RBCs is significantly reduced. Glycophorin C (-) Leach RBCs were similarly resistant to parasite invasion in vitro. The intracellular development of parasites that invaded protein 4.1 (-) RBCs was also dramatically reduced. In contrast, no such reduction of intracellular parasite growth was observed in the glycophorin C (-) Leach RBCs. In conjunction with our recent finding that a third protein termed p55 is also deficient in protein 4.1 (-) and glycophorin C (-) RBCs, the present data underscore the importance of the membrane-associated ternary complex between protein 4.1, glycophorin C, and p55 during the invasion and growth of malaria parasites into human RBCs.     

Prognostic importance of structural chromosomal abnormalities in children with hyperdiploid (greater than 50 chromosomes) acute lymphoblastic leukemia

Approximately one fourth of children with newly diagnosed acute lymphoblastic leukemia (ALL) have hyperdiploid (greater than 50 chromosomes) blasts and a relatively favorable prognosis. Nonetheless, a substantial proportion of these patients fail therapy. We studied 138 children (70 male, 68 female) with hyperdiploid greater than 50 ALL to assess initial clinical and cytogenetic features that might predict treatment failure. In 85 of these cases (62%), structural chromosomal abnormalities were also present; clinical and laboratory features in this group did not differ from those of the 53 cases with only numeric abnormalities. However, of the 28 failures seen at a median follow-up of 4 years, 22 occurred in cases with structural chromosomal abnormalities (P = .03 by Breslow test). In a multivariate analysis, only the presence of structural chromosomal abnormalities and male gender were independently associated with treatment failure. Structural chromosomal abnormalities in cases of ALL with greater than 50 chromosomes may define a biologically different form of leukemia characterized by increased likelihood of drug resistance.     

Expression of the human monocyte membrane antigen gp55 by murine fibroblasts after DNA-mediated gene transfer

Human DNA sequences that contain the gene encoding gp55, a cell surface glycoprotein expressed exclusively on mature human monocytes and monocytic leukemia cells, were isolated in a mouse genetic background. DNA from mature human monocytes was cotransfected with DNA from a molecularly cloned feline sarcoma virus containing the v-fms oncogene into NIH-3T3 cells. Transformed mouse fibroblasts that expressed gp55, based on their reactivity with the MY4, B44.1, or LeuM3 monoclonal antibodies, were selected by fluorescence-activated cell sorting. Regardless of which antibody was used for selection, equivalent binding of all three antibodies was observed for positive transformants. Secondary and tertiary mouse cell transformants were obtained after additional rounds of transfection and cell sorting with the use of DNA from primary and then secondary transformants. Southern blot analysis of the cellular DNA from two independently derived tertiary subclones revealed a limited complement of human sequences, thus indicating that the gene encoding gp55 is included in fewer than 50 kilobases of human DNA. Independently derived tertiary subclones displayed concordant patterns of reactivity with 13 monocyte-specific monoclonal antibodies, thus indicating that each recognized an epitope on the product (gp55) of a single human gene. The 55-kilodalton cell surface polypeptide was specifically immunoprecipitated with a representative monoclonal antibody, 26if, from lysates of enzymatically radioiodinated peripheral blood monocytes and tertiary transformants. We conclude that gp55 is highly immunogenic and that a large number of independently derived monoclonal antibodies specific for human monocytes react with epitopes on this one molecule.     

Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.     

Metabolic dependence of protein arrangement in human erythrocyte membranes. I. Analysis of spectrin-rich complexes in ATP-depleted red cells

The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS- polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase- deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.     

Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures

We studied the erythroid burst-promoting activity (BPA) of recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) and Interleukin-3 (IL-3) with two experimental approaches. First we studied the effects of polyclonal antisera prepared against human GM-CSF and gibbon IL-3 on colony formation from 1,000 bone marrow null cells/dish in serum-containing culture. Both GM-CSF and IL-3 independently enhanced erythroid burst formation; however, IL-3 showed more BPA activity than GM-CSF. These data are in agreement with an emerging view that the primary targets of IL-3 are primitive progenitors and that the targets of GM-CSF are intermediate progenitors, including erythroid burst-forming units (BFU-E). The proliferation of one population of BFU- E was independent of GM-CSF or IL-3. To characterize this population of BFU-E further, we developed a serum-free culture assay for the purified progenitors by incorporating insulin-like growth factor-1 (IGF-1) to the serum-free medium. The development of erythroid bursts was supported by IL-3, IGF-1, and erythropoietin (Ep) in a serum-free culture system and to a lesser extent by the combination of GM-CSF, IGF- 1, and Ep. Although the burst-promoting ability of GM-CSF and IL-3 was again demonstrated in this system, unlike serum-containing culture Ep alone did not support burst formation. These results indicate that when fetal calf serum (FCS) is present, the culture system contains BPA that is not GM-CSF or IL-3.     

Synergistic factors for stem cell proliferation: further studies of the target stem cells and the mechanism of stimulation by interleukin-1, interleukin-6, and granulocyte colony-stimulating factor

Serial observations of blast cell colony development from spleen cells of mice treated with 5-fluorouracil (5-FU) four days earlier revealed that either form of human interleukin-1 (IL-1 alpha or IL-1 beta) hastens the emergence of interleukin-3 (IL-3)-dependent blast cell colonies. This activity was essentially indistinguishable from the effect of interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G-CSF) in the same system, an effect that we have ascribed previously to a shortening of the G0 period of the dormant stem cells. We also analyzed the time courses of colony formation from cultures of day-2 post-5-FU marrow cells supported by IL-1 alpha, IL-6, or G-CSF alone or in combination with IL-3. In the presence of IL-3, G-CSF and IL-6 but not IL-1 alpha hastened the development of colonies and increased the numbers of multilineage colonies relative to cultures of IL-3 alone. This observation, together with our previous data from the human system, suggests that the synergistic effect of IL-1 is likely due to induction of secondary growth factors, including IL-6 and G-CSF, by accessory cells in culture. The effect of IL-6 on G0 was confirmed by analysis of the cycling status of progenitor cells in short-term culture. While neither IL-3 nor IL-6 alone had any effect on the cycling status, the combination of factors resulted in a rapid recruitment of quiescent cells into cell cycle (within 48 hours) as represented by a twofold increase in the numbers of multipotential progenitors and a significant increase in the sensitivity of these cells to 3H-thymidine with high specific activity. Combinational testing of all of these synergistic factors revealed that the target cell populations for the IL-1, IL-6, and G-CSF overlap considerably, suggesting that they all may act through a common mechanism. This is further supported by our finding that cells from blast cell colonies grown in the presence of a combination of any one of the synergistic factors with IL-3 replate with higher efficiency and yield more multilineage secondary colonies than those from colonies grown in IL-3 alone. These findings provide further evidence that IL-1, IL-6, and G- CSF serve to integrate the immediate host responses to infection through augmentation of effector cells and antibody production as well as the longer term host responses by recruitment of dormant hemopoietic stem cells into active cell cycling.