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PubMed1991

VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

Blotting, Northern↗
PubMed2003

Analysis of the maturation process of dendritic cells deficient for TNF and lymphotoxin-alpha reveals an essential role for TNF.

Dendritic cells (DCs) generated from bone marrow (BM) precursor cells of C57BL/6 (B6.WT) mice and cultured in the presence of granulocyte macrophage-colony stimulating factor differentiate to mature BM-DCs spontaneously. These mature DCs are characterized by high levels of major histocompatibility complex (MHC) class II, CD40, and CD86 on their surface. To analyze the involvement of tumor necrosis factor (TNF) and the related cytokine lymphotoxin (LT)alpha in DC maturation, we studied the development of DCs from the BM of B6.TNF(-/-), B6.LTalpha(-/-), and B6.TNF/LTalpha(-/-) mice and compared it to B6.WT mice. Although the development of BM precursor cells to the level of immature DCs (CD11c(+), MHC class II(low), CD40(low), and CD86(low)) was equivalent in all genotypes, B6.TNF(-/-) and B6.TNF/LTalpha(-/-) cells showed an impaired capacity to differentiate to mature DCs. In contrast, mature BM-DCs generated from LTalpha-negative, immature DCs developed like B6.WT cells. Further studies revealed that once matured, the phenotype of all tested genotypes was comparable. They expressed high levels of CD40 and CD86, were exclusively positive for the chemokine receptor (CCR)7 but negative for CCR5 and CCR2, and were able to enter the paracortex of draining lymph nodes. The limited maturation of TNF-deficient BM-DCs could be restored by mixing TNF-negative with TNF-positive Ly5.1 BM cells, and maturation of B6.WT DCs could be blocked with an anti-TNF monoclonal antibody. The substitution of B6.TNF(-/-) BM cells with recombinant TNF revealed promotion or suppression of BM-DC maturation depending on the point of time of TNF addition.

Animals↗
PubMed2005

Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos.

The differentiation of hematopoietic progenitors into erythroid or myeloid cell lineages is thought to depend upon relative levels of the transcription factors gata1 and pu.1. While loss-of-function analysis shows that gata1 is necessary for terminal erythroid differentiation, no study has demonstrated that loss of gata1 alters myeloid differentiation during ontogeny. Here we provide in vivo evidence that loss of Gata1, but not Gata2, transforms primitive blood precursors into myeloid cells, resulting in a massive expansion of granulocytic neutrophils and macrophages at the expense of red blood cells. In addition to this fate change, expression of many erythroid genes was found to be differentially dependent on Gata1 alone, on both Gata1 and Gata2, or independent of both Gata factors, suggesting that multiple pathways regulate erythroid gene expression. Our studies establish a transcriptional hierarchy of Gata factor dependence during hematopoiesis and demonstrate that gata1 plays an integral role in directing myelo-erythroid lineage fate decisions during embryogenesis.

Animals↗
PubMed1985

Formation of methotrexate polyglutamates in purified myeloid precursor cells from normal human bone marrow.

Immature myeloid precursor cells were preferentially selected from normal human bone marrow by using immune rosette techniques that employed monoclonal antibodies against mature granulocytes, monocytes, T and B lymphocytes, and erythroid precursors (Mo5, M3, OKT3, B1, and EP1, respectively). We examined the formation, retention, and cytotoxic effects of methotrexate (MTX) polyglutamates (MTX-PGs) in these purified myeloid precursor cells. After 1- and 24-h exposures to MTX, with thymidine and deoxyinosine as rescue, the intracellular MTX-PG profile was examined by high-pressure liquid chromatography. Efflux patterns of MTX-PGs were also studied after additional 1- and 24-h incubations in drug-free media. Cytotoxic effects of retained MTX-PGs on bone marrow myeloid precursors were examined by colony formation in drug-free semisolid agar. Normal myeloid precursor cells converted MTX to MTX-PGs in a concentration- and time-dependent manner, preferentially retaining MTX-PGs with three to five glutamyl moieties. At low concentrations of MTX (1 microM), MTX-PG formation was insufficient to maintain saturation of the target enzyme dihydrofolate reductase after removal of drug from the incubation medium, and there was no decrease in myeloid colony formation. At higher concentrations of MTX (10 microM), formation of higher molecular weight polyglutamates was sufficient to allow for 24-h saturation of intracellular binding capacity after removal of extracellular drug and resulted in a 35% reduction in the formation of colony-forming units in culture. Comparison of MTX metabolism in normal bone marrow cells and the MTX-sensitive HL-60 human leukemia cell line showed twofold greater PG formation by these tumor cells after 24-h exposure to 1 or 10 microM MTX, and a marked (greater than 30-fold) increase in cytotoxicity for the HL-60 cells as compared with normal myeloid precursors, suggesting that the MTX polyglutamation may be important to its selective antitumor action.

Bone Marrow↗
PubMed2000

Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation.

Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.

Actins↗
PubMed1988

Purification of a factor inhibiting differentiation from conditioned medium of nondifferentiating mouse myeloid leukemia cells.

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-factor activity) was detected in conditioned medium of variant M1 cell clones that were resistant to differentiation inducers, and this I-factor activity was shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-factor was purified to apparent homogeneity from conditioned medium of resistant M1 cells. The purification procedure consisted of ammonium sulfate precipitation, CM-Sepharose CL-6B, Sephadex G-200, reverse-phase high performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The factor was analyzed by radioiodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The purified factor gave a single band of protein with a molecular weight of 68,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with its biological activity. The concentration of I-factor required for 50% inhibition of dexamethasone-induced differentiation of M1 cells was 24 pM. At its effective concentration it had no effect on cell proliferation, and even at 1.2 nM it did not inhibit colony formation of normal bone marrow cells, suggesting that it was distinct from the inhibitor of normal precursors of macrophages and/or granulocytes.

Animals↗
PubMed1984

GM 58/8: a monoclonal antibody that identifies a new lineage-specific determinant expressed by myeloid progenitors (CFU-GM) and their progeny.

A cytotoxic (IgM) monoclonal antibody (McAb) raised against human erythroleukaemia cell line HEL appears specific for the myelomonocytic lineage. Indirect immunofluorescence and fluorescence-activated cell sorter analyses of peripheral blood and bone marrow cells showed that this McAb (designated GM 58/8) reacts exclusively with more than 90% of the granulocytes, monocytes and myeloid precursors. The positive (fluorescent) fraction sorted from McAb-treated bone marrow buffy coat cells showed a marked enrichment of myeloid precursors, while nonmyeloid cells were recovered in the negative fraction. GM 58/8 specifically inhibited the growth of 90-100% of the committed myelomonocytic progenitors (CFU-GM) in five complement-mediated cytotoxicity experiments. GM 58/8 had virtually no effect on the erythroid progenitor (BFU-E and CFU-E) growth. Cell sorting experiments using this McAb resulted in recovery of 84% of CFU-GM among the fluorescent fractions. Comparisons with other anti-myelomonocytic McAbs reported so far suggest that GM 58/8 has more restricted reactivity and shows more efficient CFU-GM cytotoxicity. Patterns of reactivity with normal cells and established lines indicate that the antigenic determinant identified by GM 58/8 is different from that recognized by previously described anti-myelomonocytic McAbs. McAb GM 58/8 could be used to isolate or remove pure populations of progenitors and/or more differentiated cells of the myelomonocytic lineage for in vitro studies. This study also provides evidence that the HEL cell line, derived originally from an erythroleukaemia patient, expresses an antigenic determinant shared by the normal cells of myelomonocytic lineage.

Antibodies, Monoclonal↗
PubMed2004

C-terminal SRC kinase controls acute inflammation and granulocyte adhesion.

To establish whether the widely expressed regulator of Src family kinases Csk contributes to the control of acute inflammation in vivo, we inactivated csk in granulocytes by conditional mutagenesis (Cre/loxP). Mutant mice (Csk-GEcre) developed acute multifocal inflammation in skin and lung. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation with hyperinduction of integrins. This hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Hyperphosphorylation of key signaling proteins such as Syk and Paxillin in mutant granulocytes further supported breakdown of the activation threshold set by Csk. By enforcing the need for ligand engagement Csk thus prevents premature granulocyte recruitment while supporting the motility of stimulated cells through negative regulation of cell adhesion.

Acute Disease↗
PubMed1995

Morphology, biology and biochemistry of cobalamin- and folate-deficient bone marrow cells.

B12- or folate-deficient haemopoietic cells display abnormalities in their morphology under both the light and electron microscope, their cell kinetics and their capacity to synthesize protein. These abnormalities are maximal in the last dividing cell class and in non-dividing cells, presumably because B12 and folate uptake is largely confined to the most immature erythroid and granulocyte precursors. In patients with moderate or severe anaemia due to B12 or folate deficiency, erythropoiesis is markedly ineffective; intramedullary cell death occurs mainly in the early and late polychromatic megaloblasts. The damaged erythroblasts appear to display neoantigens or normally-hidden antigens at their cell surface and these react with naturally occurring antibodies. The opsonised erythroblasts are then recognised by macrophages via their IgG-Fc receptors and phagocytosed. Marrow cells from B12- or folate-deficient patients show a subnormal suppression of 3H-thymidine incorporation after pre-incubation with nonradioactive deoxyuridine, suggesting that such cells suffer from an impairment of the 5,10-methylene-THF-dependent methylation of deoxyuridylate to thymidylate. However, the exact mechanism by which B12 deficiency causes a reduced supply of this folate coenzyme is uncertain. Methylcobalamin is required for the 5-methyl-THF-dependent methylation of homocysteine to methionine and an impairment of this reaction will result in both reduced conversion of 5-methyl-THF to THF and in reduced methionine synthesis. There is controversy as to whether the reduced supply of THF or methionine is responsible for the reduced availability of 5,10-methylene-THF. Currently, the balance of evidence favours the hypothesis that the reduced supply of methionine leads to reduced synthesis of formyl-THF and, eventually, of 5,10-methylene-THF. Despite the evidence for impaired thymidylate synthesis, the duration of the S phase of megaloblasts appears to be normal or only modestly increased. Data on rates of DNA strand elongation are inconsistent, with subnormal rates reported in PHA-stimulated B12- or folate-deficient lymphocytes and normal rates in B12- or folate-deficient bone marrow cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoptosis↗
PubMed1995

Evidence for a mechanism that can provide both short-term and long-term haemopoietic repopulation by a seemingly uniform population of primitive human haemopoietic precursor cells.

One of the controversies surrounding the repopulating capacities of haemopoietic stem cells is whether or not the same or different populations are responsible for short-term and long-term repopulation after transplantation. To address this question, we analysed results obtained from an in vitro model for the clonal production of granulocyte-macrophage colony-forming cells (CFU-GM) by individual primitive multilineage precursors in adult human bone marrow. The primitive precursors adhere to plastic and produce CFU-GM in a 1-week long 'delta' type culture. The clones that form are classified as having short maturation pathways (clones containing predominantly day 7 CFU-GM) or long maturation pathways (clones containing predominantly day 21 CFU-GM). The results indicate that individual primitive (P delta) cells produce clones that reach full maturity after different periods of time so that cells corresponding to a range of maturational stages can become available simultaneously. Consequently, transplanted stem cells may be able to provide both rapid and long-term mature cell recovery whilst at the same time reconstituting the stem cell pool. These results suggest that it might be possible to use highly purified stem cell populations, devoid of committed progenitors, for clinical transplantation.

Bone Marrow Cells↗
PubMed2000

Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells.

Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.

Animals↗
PubMed1983

Monoclonal antibody to human eosinophils recognizing 95 kD surface membrane antigen.

Mice were immunized with purified eosinophils obtained from patients with the idiopathic hypereosinophilic syndrome. A hybridoma initially producing an IgM antibody which switched to an IgG1 antibody was selected for cloning and further testing. This IgG1 antibody reacted with human eosinophils, granulocytes, monocytes and large granular lymphocytes, but did not react with T lymphocytes, B lymphocytes, platelets, erythrocytes, or a panel of human leukemia cells and cell lines. Bone marrow analysis revealed staining of myeloid precursor cells but not erythroid precursors or plasma cells. This IgG1 antibody had no effect on aggregation of granulocytes, lysozyme release, superoxide production, chemotaxis, or killing activity; however, there was some stimulation of beta-glucuronidase secretion. While the antibody did not augment the killing of Staphylococcus aureus by granulocytes, the antibody itself was bactericidal. By immunoprecipitation of granulocytes, eosinophils and monocytes, a molecule with a molecular weight of 95 kD was identified.

Animals↗
PubMed1986

Experimental chemotherapy and concepts related to the cell cycle.

Scheduling of chemotherapy is limited by damage to normal tissues, and tolerated schedules are dependent on normal tissue recovery. Most anticancer drugs are more toxic to proliferating cells and the fall and recovery of granulocyte counts after chemotherapy may be explained by the effect of drugs on rapidly proliferating precursor cells in the bone marrow. It is argued that serious toxicity due to myelosuppression most often occurs because of damage to proliferating precursors that may be recognized in bone marrow rather than to stem cells. In contrast, therapy that is aimed at producing cure or long-term remission of tumours must be directed at killing tumour stem cells. The evidence that tumours contain a limited population of cells which can repopulate the tumour after treatment (and are therefore tumour stem cells) is reviewed critically. While there is quite strong evidence for a limited population of target cells, evidence from studies on metastases suggests that the tumour cells which may express this stem cell property may change with time. The stem cell concept has major implications for predictive assays. Although colony-forming assays appear to have a sound biological background for predicting tumour response, technical problems prevent them from being used routinely in patient management. Cells in tumours are known to be heterogeneous and at least three types of heterogeneity may influence tumour response to drug treatment: the development of subclones with differing properties including drug resistance; variation in cellular properties due to differentiation during clonal expansion; and variation in properties due to nutritional status and micro-anatomy. Heterogeneity in drug distribution within solid tumours may occur because of limited drug penetration from blood vessels, and nutrient-deprived cells in solid tumours may be expected to escape the toxicity of some anticancer drugs as well as being resistant to radiation because of hypoxia. This may occur both because nutrient-deprived cells have a low rate of cell proliferation, and also because of poor drug penetration to them. There is a need for improved understanding of the mechanisms that lead to cell death in tumours. If these mechanisms were understood, it might be possible to simulate them by therapeutic manoeuvres. Recent research from our laboratory suggests that the combination of low extracellular pH and hypoxia may be very toxic to cells in nutrient-deprived regions. Drugs which limit the cell's ability to survive in regions of acid pH may provide strategy for therapy of nutrient-deprived cells.

Animals↗
PubMed1991

Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

Animals↗
PubMed2004

Antineutrophil autoantibodies and their target antigens in systemic lupus erythematosus.

Various autoantibodies have been identified in sera from patients with systemic lupus erythematosus (SLE) and autoantibodies against neutrophil have been reported. It was suggested that antineutrophil autoantibodies might be involved in the pathogenesis of neutrocytopenia; however, the role of autoantibodies against neutrophil precursors and their specific target autoantigen(s) remained further characterized. The objective was to investigate the target antigens and clinical associations of autoantibodies against neutrophils and neutrophil precursors in patients with SLE. Sera were collected from 92 patients with SLE and renal biopsy proven lupus nephritis. Cell lysates of peripheral neutrophils (as mature neutrophils) from a normal blood donor and white blood cells from a patient with blast crisis of chronic granulocytic leukemia (CGL) (as neutrophil precursors) were used as antigens in Western blot analysis to detect autoantibodies in sera from patients with SLE. The clinical significance of antineutrophil autoantibodies that recognized different antigens were further analysed. Using normal peripheral neutrophils as antigens, two bands could be blotted: 64 kD (33/92, 35.9%) and 50 kD (13/92, 14.1%). The prevalence of anti-64 kD autoantibody in patients with positive rheumatic factor was significantly higher than that in patients without (54.5 versus 18.8%, P < 0.05). Using CGL white cells as antigen, five bands could be blotted: 60 kD (34/92, 37.0%), 50 kD (32/92, 34.8%), 29 kD (27/92, 29.3%), 42 kD (19/92, 20.7%) and 18 kD (16/92, 17.4%). The prevalence of anti-60 kD autoantibody was significantly higher in patients with neutrocytopenia than that in patients without neutrocytopenia (100 versus 48.3%, P < 0.01). The prevalence of anti-29 kD autoantibody was significantly higher in patients with alopecia than that in patients without alopecia (45.8 versus 20.8%, P < 0.05). Furthermore, the prevalences of anti-60 kD, anti-50 kD and anti-42 kD autoantibodies were significantly higher in patients with anti-Ro autoantibody than those in patients without; the prevalences of anti-29 kD and anti-18 kD autoantibodies were significantly higher in patients with anti-Sm autoantibody than those in patients without. We conclude that there are heterogeneous autoantibodies against both neutrophils and their precursors in sera from patients with SLE. Different autoantibodies may have different clinical significance.

Antibodies, Heterophile↗
PubMed2003

iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors: a mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B.

Our previous data indicated that C3, its bioactive product iC3b, and the iC3b ligand CD11b are critical for ultraviolet-induced immunosuppression. We thus hypothesized that iC3b is an important skin-based factor regulating CD11b+ monocytic cell function in the acute post-ultraviolet period. Although monocytic cell migration peaked at 1-3 d after ultraviolet exposure of skin, dermal CD1c dendritic cells underwent a rapid and prolonged depletion that did not recover until day 7. Because ultraviolet-induced iC3b deposits are reciprocally maximal on day 3, but fade by day 7, we next hypothesized that iC3b can be responsible for the delay in differentiation into dendritic cells of monocytic cells migrating into ultraviolet-exposed skin. Analysis of dermal cells derived from keratome biopsies suggested that iC3b exposure could inhibit the development of CD1c+ dermal cells. To model newly immigrating blood monocytes entering ultraviolet-exposed, iC3b-containing dermis, purified monocytes from human blood were induced with granulocyte-macrophage colony stimulating factor to generate a population of dendritic cell precursors expressing CD1c. Incubation with iC3b markedly inhibited the appearance of CD1c+ cells (p<0.05) and induced CD1c-CD14+ cells. This inhibition was reversed by coincubation with an anti-CD11b antibody that blocks the iC3b binding site. Other functions associated with dendritic cell maturation were also inhibited by iC3b, such as interleukin-12p70 production as well as CD80 and CD40 expression. Restimulation of monocytes for DC maturation revealed that iC3b induced a temporary inhibition of DC differentiation. Thus, a human skin response in which iC3b is transiently (3-7 d) generated in dermis, such as ultraviolet, can arrest monocytic skin-infiltrating cells from undergoing dendritic cell precursor differentiation.

Animals↗
PubMed1984

Direct relationship of marrow cell growth and 1-beta-D-arabinofuranosylcytosine metabolism.

To define the relationship between perturbed cell growth and intracellular metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) in sensitive human cells, growth kinetic, and biochemical pharmacological determinants were examined in normal human bone marrow populations in vitro in normal serum and in the presence of drug-induced humoral stimulatory activity (HSA). Cells cultured in HSA demonstrated both increased proliferation and greater ara-C-related inhibition of DNA synthesis than did cells maintained in normal serum, as measured by [3H]thymidine incorporation into DNA and [3H]thymidine granulocyte precursor labeling index. Parallel measurements of [3H]ara-C incorporation into DNA demonstrated similar behavior in HSA-perturbed cells. When these cultured cells were exposed to 1 and 10 microM ara-C, intracellular formation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate over 3 hr and retention of this active form during 1 subsequent hr in drug-free medium were both increased in HSA-stimulated cells relative to cells cultured in normal serum. These studies demonstrate coupling of induced cell growth kinetics with enhanced intracellular metabolism of the S-phase-specific antimetabolite ara-C in normal human marrow cells. The close direct relationship between growth kinetic perturbation and augmentation of intracellular ara-C activation in this normal hematopoietic model provides a basis for comparison with leukemic cell populations, in which uncoupling of growth kinetics and pharmacokinetics may signify divergence from normal drug-sensitive cell behavior and, thus, resistance to ara-C cytotoxicity.

Bone Marrow↗