Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in haematopoietic cells.

Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

A model of human acute lymphoblastic leukemia in immune-deficient SCID mice.

A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies.

Erythroid colony growth in congenital hypoplastic anemia.

Four children with congenital hypoplastic anemia (Diamond-Blackfan syndrome) and 30 control children with normal erythropoiesis were studied by a cell culture method in which human marrow, grown in a plasma clot, responds to added erythropoietin (EPO) with the appearance of discrete colonies of nucleated erythroid cells. The colonies arise from EPO-responsive stem cells and are not related to the number of morphologically identifiable marrow erythroids plated. Results of studies on control marrow indicated that without EPO there was little or no colony formation. Increasing EPO doses or nucleated marrow cells per culture resulted in a linear increase in colony numbers. The optimal EPO concentration of 2.5 U/ml yielded a mean of 158 +/- 79 colonies/1 x 10(5) nucleated cells on day 7 of incubation. Even in the absence of recognizable erythroids, marrows of all four patients with anemia grew erythroid colonies. Two patients on no therapy had decreased colony numbers. The other two, on prednisone, had normal numbers. Sera from patients did not inhibit colony formation from either autologous or control marrow. In contrast, serum from an adult with acquired pure red cell aplasia produced striking inhibition of colony growth. It appears that the red cell failure in this disorder is not due to an absence of erythroid stem cells, and a serum inhibitor to erythropoiesis as seen in the acquired disease is unlikely.

Regulation of interferon receptor expression in human blood lymphocytes in vitro and during interferon therapy.

Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. The binding characteristics of IFN to human lymphocytes were studied using IFN alpha 2 labeled with 125I to high specific activity. The specific binding curves generated were analyzed by the LIGAND program of Munson and Rodbard to determine receptor numbers. The number of receptors in peripheral blood lymphocytes (PBL) and tonsillar B-lymphocytes (TBL) from normal individuals were 505 +/- 293 (n = 10) and 393 +/- 147 (n = 3) respectively. When these cells were preincubated in vitro with unlabeled IFN alpha 2, the receptor number decreased to 82 +/- 45 and 61 +/- 16 respectively. Receptor binding activities recovered gradually over a period of 72 h when the cells were incubated in IFN-free medium. This recovery of receptors could be blocked by the addition of actinomycin D to the incubation medium. A similar decrease in receptor expression was observed in vivo in PBL from patients being treated daily with 5 X 10(6) units/m2 per d of IFN alpha 2 by subcutaneous injection, for acute lymphoblastic leukemia or papilloma virus infections. Receptor numbers in PBL in vivo were further reduced concurrent with the progression of IFN therapy. Thus the reduction in IFN receptor expression observed in vitro can be demonstrated in vivo. These studies indicate that monitoring IFN receptor expression in vivo can provide information regarding the availability of IFN receptors at the cell surface for the mediation of IFN actions during the course of IFN therapy.

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin.

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.

Growth requirements for human acute lymphoblastic leukemia cells: refinement of a clonogenic assay.

Since freshly obtained acute lymphoblastic leukemia (ALL) cells rarely replicate spontaneously in vitro in a sustained way, development of a useful clonogenic assay for ALL blast progenitors is dependent on identifying the cellular growth requirements. Thus, marrows from 25 ALL cases were cultured in methylcellulose to determine the optimal conditions for cell growth. Blast colonies were confirmed as leukemic by morphology, cytochemistry, surface markers, and cytogenetics. Irradiated (7000 rads) normal peripheral blood feeder cells were an absolute requirement and produced number-dependent increases in ALL colonies; added growth factors enhanced the feeder cell effect. ALL cell-feeder cell contact was essential since their physical separation in a two-layer culture system drastically interfered with colony growth. Feeder cells from various donors, including new and relapsed cases of ALL, yielded colony numbers that differed widely when tested on the same marrow with and without added growth factor; thus, identification of a "good" feeder cell donor was key to an optimal assay. Neither recombinant interleukin-2 nor recombinant GM-CSF had ALL growth-promoting properties when tested alone or in combination but in the presence of feeder cells they moderately enhanced the feeder cell effect. The most effective growth factors were derived from cells exposed to phytohemagglutinin (PHA) for 72 h. In order of magnitude for colony growth-promoting activity, PHA-T cell conditioned medium (CM) was more stimulatory than PHA-blast cell CM followed by PHA-leukocyte CM; removal of PHA from CM by affinity chromotography did not alter the results. The most potent PHA-TCM was prepared from T-cells from a phlebotomized hemochromatosis patient; PHA-TCM from transfused thalassemia patients and normal donors were less active. Concanavalin-A blast cell CM had modest colony promoting properties whereas CM prepared with other B-cell mitogens and supernatants from ALL blasts in liquid culture had none. Our studies illustrate the complex and fastidious growth needs of ALL cells. The data have allowed us to refine a clonogenic blast progenitor assay that should facilitate study of proliferative properties of B and T lineage leukemias. The assay could be adapted further for detection of residual leukemia cells in marrow samples used for autologous transplantation, and in patients during complete hematological "remission."

Characterization of malignant peripheral blood cells of juvenile chronic myelogenous leukemia.

Characterization studies were performed on the malignant peripheral blood cells from three patients with juvenile chronic myelogenous leukemia (JCML) by allowing the cells to increase numerically in liquid cultures. JCML cells proliferated rapidly and excessively in the absence of an added humoral growth factor, whereas control peripheral blood cells declined in number when cultured under identical conditions. Clonality of JCML cells was proven by cytogenetic analysis of the proliferating population. JCML cells were exclusively of monocytic lineage as determined by morphology, staining characteristics, and monoclonal antibody identification of cell-specific surface antigens, but cytochemical and functional studies identified aberrant properties indicating defective differentiation. Striking differences from control cells in ultrastructure and topography were also observed by transmission and scanning electron microscopy. These data provide new information on the cellular origin of JCML and form the basis for further study of leukemic cell biology in this disease.

Relationship of DNA topoisomerase II alpha and beta expression to cytotoxicity of antineoplastic agents in human acute lymphoblastic leukemia cell lines.

The levels of expression of topoisomerase II alpha and topoisomerase II beta were investigated in six established cell lines of human childhood acute lymphoblastic leukemia (ALL) as a function of doubling time, cell cycle distribution, and of sensitivity to the antineoplastic agents Adriamycin and etoposide. The slowest growing cell line, ALL-G, was most sensitive to both drugs, whereas the fastest growing cell line, ALL-C, was 15.3- and 6.4-fold more resistant than ALL-G to Adriamycin and etoposide, respectively. Furthermore, ALL-W, the second most rapidly dividing cell line, was most resistant to both Adriamycin (22.8-fold) and etoposide (14.1-fold). Expression of topoisomerase II alpha varied inversely with doubling time, whereas no correlation was found between topoisomerase II beta levels and doubling time. Expression of topoisomerase II beta varied inversely with that of topoisomerase II alpha. The level of topoisomerase II alpha correlated directly with the percentage of cells in S and G2-M phases, whereas topoisomerase II beta expression varied directly with the number of cells in G1. An inverse correlation was found between the level of expression of topoisomerase II beta and resistance to Adriamycin, whereas a direct correlation was observed between the level of expression of topoisomerase II alpha and resistance to Adriamycin. Studies with etoposide, although not statistically significant, were consistent with the pattern observed with Adriamycin. These findings suggest that in ALL cells, cytocidal activity of Adriamycin and etoposide may be mediated, at least in part, by topoisomerase II beta.

Autocrine interleukin-1beta production in leukemia: evidence for the involvement of mutated RAS.

Interleukin (IL)-1beta is constitutively expressed in many leukemias and operates as an autocrine growth factor. To study the cellular basis for this aberrant production, we analyzed two cell lines, B1 (acute lymphoblastic leukemia) and W1 (juvenile chronic myelogenous leukemia), which express high levels of IL-1beta and have mutations in the K-RAS and N-RAS genes, respectively. Electromobility shift assays demonstrated transcription factor binding at multiple IL-1beta promoter elements [nuclear factor (NF)-IL6/CREB, NFB1, NFkappaB, and NF-IL6], consistent with the activation of an upstream signaling pathway. To determine whether activated Ras was involved, two structurally distinct classes of farnesyltransferase (FTase) inhibitors (the monoterpenes and a peptidomimetic) and an adenoviral vector expressing antisense targeted to K-RAS were used to specifically interfere with Ras function and/or expression. Treatment with the FTase inhibitors resulted in a concentration-dependent decrease in both NF-IL6/CREB binding to the IL-1beta promoter and IL-1beta protein levels, without a significant change in total cellular protein levels. Furthermore, exposure of the B1 cells to antisense against K-RAS resulted in an approximately 50% reduction in both p21Ras and IL-1beta protein levels. Growth suppression was observed after FTase inhibitor or antisense exposure, an effect that was partially reversible by the addition of recombinant IL-1beta to the cultures. Our observations suggest that mutated RAS genes may mediate autocrine IL-1beta production in some leukemias by stimulating signal transduction pathways that activate the IL-1beta promoter.

Lipid fluidity and membrane protein monitoring using 1,6-diphenyl-1,3,5-hexatriene.

Experiments have been designed to challenge the use of stedy-state fluorescence polarizaiton with 1,6-diphenyl-1,3,5-hexatriene as an evaluator of the fluidity of cell plasma membranes. We used paraffinic systems, of defined structure and composition (liquid paraffin, soap bilayers and phospholipid liposomes--with and without incorporated proteins), to demonstrate that corresponding polarizaiton values cannot be interpreted in terms of the overall fluidity of the labeled medium. In homogeneous structured paraffinic media (lipid bilayers), knowledge of the location of the probe is essential for a consistent interpretation of the observed fluorescence polarization. Due to the highly polarizable electronic structure of the diphenylhexatriene molecule, the presence of heterogeneities with potential sites for interaction (e.g., C18-coated Si particles, albumin molecules, etc.) can lead to relatively high polarization values, even in isotropic media. In cellular systems, translocation experiments from labeled cells to added proteins show a rather localized peripheral distribution of the probe as well as its high affinity for hydrophobic sites of proteins. This and other arguments presented here suggest that although cellular polarization values represent an intricate average over all labeled hydrophobic regions of the cell (phospholipid bilayers, membrane proteins, etc.), these values might reflect, to a large extent, interactions of the probe with proteins from the inner periphery of the cell.

The value of intensive combination chemotherapy for juvenile chronic myelogenous leukemia.

Nine children with juvenile chronic myelogenous leukemia (JCML) were diagnosed in an 8-year period from 1977 to 1984. The clinical courses and outcomes of five patients who received minimal or no chemotherapy were compared with that of four patients who were treated with intensive acute nonlymphoblastic leukemia (ANLL) combination chemotherapy. None of the five patients in the former group achieved clinical remission and their survivals were 1, 4, 4, 7, and 29 months, respectively. All four patients in the latter group achieved clinical remissions that lasted 11, 21, 21, and 27 + months, respectively. The durations of their survival (21, 26, 30, and 32 + months) were significantly better than the five patients who received minimal or no chemotherapy (P less than .05). Despite hospitalizations for chemotherapy and for treatment of chemotherapy-associated complications, the clinical status and quality of life of the children who achieved clinical remission were superior to those who remained in relapse. Although intensive chemotherapy induced lengthy remissions, three of the four patients have relapsed. Cytogenetic and cell culture data indicated that the monocytic-macrophage cells characteristic of JCML appeared to be suppressed during remission rather than totally eliminated. We recommend that ANLL-type combination chemotherapy be used as the initial treatment of JCML because of its promptness in effecting clinical remissions. Improved maintenance and consolidation protocols have to be developed to produce durable remissions and cures. Alternatively, bone marrow transplantation may be a useful option soon after remission is achieved with chemotherapy.

The induction of CD10 on a pre-B leukemia cell line occurs with progression of the disease in scid mice.

We have previously demonstrated the engraftment and dissemination of human pre-B acute lymphoblastic leukemia (ALL) cells into scid mice. In the current study, the temporal pattern of infiltration of a CD10- pre-B leukemia line (G2) in various murine tissues and the progression of the disease in the whole animal were monitored by quantifying human CD44 mRNA expression by the polymerase chain reaction (PCR). Irradiated scid mice were injected intravenously with 10(6) G2 cells and killed 3 days to 10 weeks later. After 2 weeks, leukemic cells were found mostly in bone marrow, but also in lung. At 6 to 7 weeks, spleen and lung contained 30% human RNA, while peripheral blood, liver, and kidney contained 2-3%. Infiltration to brain and thymus was observed at 8-9 weeks. In terms of the whole animal, spleen and liver were the major sites of tumor burden. The induction of CD10 expression was previously observed in transplanted CD10- G2 leukemic cells recovered from scid thymus at 10-12 weeks, which corresponds to the terminal stage of disease. In this study, the CD10 expression on the leukemic cells was monitored at earlier time points by flow cytometry and quantitative PCR. Induction of CD10 was first observed in bone marrow, spleen, peripheral blood, and liver at 6-7 weeks (10-fold), at the time of the onset of dissemination of the leukemia. Despite the presence of 30% human RNA in lung at 6-7 weeks, CD10 induction was not significant in that site before 10 weeks. Increased levels of CD10 were seen in all tissues between 8 and 10 weeks; the highest levels were observed in leukemic cells proliferating in thymus (113-fold) and in those found in circulation. These findings suggest that initial induction of CD10 occurs in hematopoietic tissues at the time of rapid proliferation of the leukemic cells and their infiltration of several tissues. At later time points, the increase in CD10 expression is seen on the leukemic cells found in all peripheral organs suggesting an association with disease progression.

Tumor formation by a human pre-B leukemia cell line in scid mice is enhanced by matrigel and is associated with induction of CD10 expression.

We have previously demonstrated the engraftment of human pre-B acute lymphoblastic leukemia (ALL) cells injected intravenously into irradiated scid mice. We now report on the ability of the reconstituted extracellular matrix, Matrigel, to promote the formation of subcutaneous tumors in non-irradiated scid mice by a CD10- pre-B ALL cell line termed G2. Lymphatic tumors infiltrating the dermis were seen in all eight mice sacrificed 10-13 weeks after the co-injection of G2 cells and Matrigel but in only 2/8 mice injected with leukemic cells alone. Infiltration of bone marrow, spleen, thymus, lung and liver was observed earlier and was more extensive in the Matrigel-treated group. The tumor cells derived from Matrigel-treated mice could be passaged in vitro and their colony-forming ability was higher than that of the original G2 line. When re-injected intravenously into non-irradiated scid mice, the tumor cells invaded the thymus earlier than did the G2 cells. The expression of CD10/neutral endopeptidase was induced at high levels in all tumors, in Matrigel or non Matrigel-treated animals. This up-regulation was transient as the tumor variants grown in vitro or in vivo lost expression of CD10. However, 6-8 weeks later, induction of CD10 was observed on both tumor variants and parental G2 cells growing in the thymus and at a lower level on cells in bone marrow and spleen. Culturing G2 cells in vitro at high density or in the presence of documented growth-promoting cytokines such as IL-3, IL-6, IL-7, and GM-CSF did not stimulate the expression of CD10 mRNA. The induction of this surface endopeptidase was thus associated with growth of leukemic cells in the specific microenvironments provided by the lymphoid tumors and the thymus in scid mice. The function of CD10 might be related to the hydrolysis of peptides which are critical in regulating interactions between adjacent pre-B cells, the stromal microenvironment and the transduction of growth and/or differentiation signals.

Malignant myeloid transformation with isochromosome 7q in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome is an autosomal recessive disorder characterized by exocrine pancreatic dysfunction, bony metaphyseal dysostosis, various degrees of cytopenia, and a striking tendency to develop myelodysplastic syndrome and acute myeloblastic leukemia. Isochromosome 7 [i(7q)] is a rare non-random cytogenetic abnormality of myeloid cells in hematological malignancy. We report two cases of Shwachman-Diamond syndrome in which patients developed myelodysplastic syndrome and i(7q), detected by G-banding karyotype analysis and fluorescence in situ hybridization. Three other children have been previously reported to have myelodysplastic syndrome in association with i(7q); two of them had Shwachman-Diamond syndrome. Isochromosome 7q may be a fairly specific marker of myeloid malignant transformation in this syndrome and play a role in its pathogenesis.

B-lineage lymphoid blast crisis in juvenile chronic myelogenous leukemia: II. Interleukin-1-mediated autocrine growth regulation of the lymphoblasts.

A pre-B acute lymphoblastic leukemia (ALL) cell line with monosomy 7 was established from a child with juvenile chronic myelogenous leukemia (JCML) in lymphoid blast crisis. Analysis of the growth properties of the cell line, termed 'W1' showed an interleukin-1 (IL-1) mediated autocrine pattern of cell proliferation with the following features: W1 colony growth without added growth factor was density-dependent and colony growth was augmented with serum-free autologous cell culture supernatant; exogenous IL-1 beta had a growth-promoting effect on W1 colony numbers when cells were seeded at low density; W1 cells constitutively expressed mRNA for IL-1 beta, and high levels of IL-1 beta were measured in W1 cell lysates; anti-IL-1 beta antibodies as well as IL-1 receptor antagonist markedly suppressed W1 colony growth when either was added to cultures of cells seeded without growth factors at low density; anti-GM-CSF antibodies and anti-IL-3 antibodies had no inhibitory effect on W1 colony growth. Whereas W1 colony growth was also augmented by adding IL-3, IL-4, IL-6, IL-7, GM-CSF, Steel factor and erythropoietin individually to the cultures, W1 cells did not constitutively express mRNA for any of these cytokines. W1 colony growth was markedly suppressed by exogenous TNF-alpha which contrasts sharply with the autocrine growth promoting effect of TNF-alpha on myelomonocytic elements of JCML in 'chronic' phase. The inhibitory effect of TNF-alpha on W1 cells was not due to downregulation of IL-1 production. The IL-1-dependent growth of W1 cells appeared to be unique because none of five other pre-B lineage ALL cell lines established as controls showed an autocrine growth loop via IL-1. W1 cells provide a valuable opportunity to examine the relationship of monosomy 7, B-lineage acute lymphoblastic leukemia, aberrant genetic expression of cytokines and their receptors, and IL-1 mediated autocrine cell growth in cancer.

Effect of deferoxamine on DNA synthesis, DNA repair, cell proliferation, and differentiation of HL-60 cells.

The ribonucleotide reductase inhibitors deferoxamine and hydroxyurea induce monocyte-macrophage cell differentiation in the leukemic cell line HL-60 as judged by the expression of cell surface antigens, nonspecific esterase activity, and morphological changes. Treatment of HL-60 cells with deferoxamine results in inhibition of DNA synthesis and irreversible loss of colony-forming ability. In addition, both deferoxamine and hydroxyurea caused an increase in the number of DNA strand breaks in HL-60 cells. A DNA methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, also caused cellular differentiation in HL-60 cells associated with DNA strand breaks. These observations are consistent with a role for DNA damage or for inhibition of DNA synthesis and repair in the differentiation process of HL-60 cells.

Lymphoid blast crisis of B-lineage phenotype with monosomy 7 in a patient with juvenile chronic myelogenous leukemia (JCML).

We studied a patient with juvenile chronic myelogenous leukemia (JCML) whose terminal course was characterized by transformation to acute lymphoblastic leukemia. Karyotypic studies identified monosomy 7 in leukemic myelomonocytic marrow cells during the chronic phase and in the lymphoblasts during the transformation phase. Our ability to sustain the transformed lymphoblasts in culture allowed us to characterize them further. CD19, HLA-DR, and CD10 were present, consistent with a pre-B acute lymphoblastic leukemia phenotype. CD14 (My-4) and CD13 (My-7) were negative. Rearrangement of immunoglobulin heavy- and light-chain genes identified monoclonal populations of cells of the B lineage. This case provides further evidence that JCML is a clonal disease of pluripotent stem-cell origin.

Differential kinetics of engraftment and induction of CD10 on human pre-B leukemia cell lines in immune deficient scid mice.

The sensitivity of the scid mouse model was assessed by comparing the growth of two pre-B acute lymphoblastic leukemia (ALL) cell lines, A1 and G2, established from patients at relapse. When cell numbers varying from 10(4) to 10(7) were injected intravenously into scid mice, advanced growth and dissemination of leukemia was observed at 10-12 weeks with the G2 cells. Bone marrow, spleen and thymus contained high levels of human leukemic cells and infiltration into lung, kidney, liver, and brain was observed. Two of three mice grafted with only 100 cells showed high levels of infiltration at 15 weeks, suggesting that 100 G2 cells was near the limiting cell number that could produce disseminated leukemia. With the A1 line, a minimum of 10(5) cells was needed to obtain dissemination to liver, lung, brain, and kidney; a low level of spleen infiltration occurred and thymus invasion was not observed. In vitro, both lines showed a density dependent growth in clonogenic assays but the cloning efficiency of the A1 line was 10-fold higher than for G2 cells. These results indicate that G2 and A1 lines have a dissimilar aggressiveness in vivo which does not correlate with clonogenic assay in vitro. Neither G2 nor A1 lines, growing in vitro, expressed CD10/CALLA on their surface, despite low levels of antigen on the freshly obtained relapse samples. Although A1 cells remained CD10-negative in the scid mice, G2 cells showed detectable levels of CD10, particularly on those cells found in the thymus. Several subclones of the G2 line were derived from isolated colonies in vitro; they were found to be CD10- in vitro, but to become CD10+ when proliferating into scid mouse thymus, suggesting the induction of CD10 by the murine microenvironment.