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SUMMARY: BioJava is a mature open-source project that provides a framework for processing of biological data. BioJava contains powerful analysis and statistical routines, tools for parsing common file formats and packages for manipulating sequences and 3D structures. It enables rapid bioinformatics application development in the Java programming language. AVAILABILITY: BioJava is an open-source project distributed under the Lesser GPL (LGPL). BioJava can be downloaded from the BioJava website (http://www.biojava.org). BioJava requires Java 1.5 or higher. All queries should be directed to the BioJava mailing lists. Details are available at http://biojava.org/wiki/BioJava:MailingLists.
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Biología Computacional/métodos , Lenguajes de Programación , Conformación de Ácido Nucleico , Conformación Proteica , Análisis de SecuenciaRESUMEN
The recently defined ligand for the Mpl receptor, thrombopoietin (TPO), has been found to be the principal regulatory cytokine of megakaryocytopoiesis. Furthermore, it has been hypothesized that direct interaction between stroma or stroma-derived extracellular matrix (ECM) and human progenitor cells (HPC) may modulate megakaryocytopoiesis. For that purpose, we studied megakaryocytic development of HPC, obtained from patients with hematological malignancies in complete remission or solid tumors without bone marrow involvement, under the influence of megakaryocyte growth and development factor (MGDF, a pegylated, truncated molecule related to the endogenous Mpl ligand). The megakaryocytic development of HPC cultured in contact with a stromal layer (contact cultures) was compared with cultures in which HPC were grown separated from a stromal layer by a microporous membrane (non-contact cultures). A significantly lower number of megakaryocytes (CD41- and CD61-positive cells) was obtained from contact cultures compared to non-contact cultures. Furthermore, the expression of CD42b was higher in non-contact cultures, as compared to contact cultures, indicating an increase in megakaryocyte maturation in non-contact cultures. In contrast, no difference in clonogenic capacity was observed (CFU-GM, BFU-E, CFU-Mk). The possibility that direct contact between HPC and stroma induces the production of a soluble inhibitory cytokine as an explanation for the diminished megakaryocytic development in contact cultures, was excluded by performing transwell experiments in which HPC were cultured in direct contact and in a transwell simultaneously. The percentage of megakaryocytes raised from HPC present in the transwell did not decrease, irrespective of the presence of HPC simultaneous below the transwell in direct contact with stroma. It is concluded that both megakaryocytic development out of HPC and megakaryocytic differentiation is reduced in contact cultures, as compared to non-contact cultures. This is due to modulation by adhesive interactions with stroma, stroma-derived ECM or cytokines bound to the membrane of stromal cells, rather than resulting from the production of diffusible cytokines by stromal cells.
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Megacariocitos/citología , Células Madre/citología , Células del Estroma/fisiología , Trombopoyetina/fisiología , Animales , Comunicación Celular , Recuento de Células , Diferenciación Celular , División Celular/fisiología , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-kit/fisiología , Células del Estroma/citologíaRESUMEN
Bryostatin 5 is a macrocyclic lactone which activates protein kinase C (PKC). PKC activation has been implicated in leukemic cell differentiation. We have examined the effect of PKC activation by bryostatin 5 on human acute myeloid cell differentiation in the presence and absence of vitamin D3. In vitro treatment of 20 patient samples of acute myeloid leukemias in a 4 days culture system with 10 nM bryostatin 5 induced strongly adherent macrophage-like cells in all cases. Bryostatin 5 induced a significant (p = 0.00006) increment in esterase activity in a majority of the samples, which was further enhanced by vitamin D3. CD14 expression was significantly (p = 0.035) enhanced with the combination of bryostatin 5 and vitamin D3. Nitroblue tetrazolium (NBT) reducing ability was, however, nearly abolished (p = 0.0007). A loss of CD34 expression occurred during cell culture; this loss was enhanced by vitamin D3, but prevented partly by bryostatin 5. Together these findings indicate that exposure to bryostatin 5 leads to a strong macrophage-like cell differentiation in human myeloid leukemia and that VD3 has an additional effect. These findings strengthen the potential role of bryostatins as possible antileukemic agents.
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Antineoplásicos/farmacología , Colecalciferol/farmacología , Lactonas/farmacología , Leucemia Mieloide Aguda/patología , Adolescente , Adulto , Anciano , Antígenos CD/metabolismo , Antígenos CD34 , Antígenos de Diferenciación Mielomonocítica/metabolismo , Brioestatinas , Hidrolasas de Éster Carboxílico/metabolismo , Diferenciación Celular/efectos de los fármacos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/inmunología , Receptores de Lipopolisacáridos , Macrólidos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Persona de Mediana Edad , Proteína Quinasa C/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Células Tumorales Cultivadas/patologíaRESUMEN
In previous studies bryostatin has been shown to cause dose-dependent stimulatory or inhibitory effects on colony formation in acute myeloid leukemias. In this study we investigated the inhibitory effect of high dose bryostatin-1 (bryo-1) on normal human bone marrow mononuclear cells (BMNC) colony-forming capacity. Preculturing BMNC for 24 h with 250 nM bryo-1 reduced colony formation by 66 +/- 11% whereas this treatment did not reduce clonogenic capacity of highly purified CD34+ BMNC. When precultures with bryo-1 were performed in the presence of several cytokine neutralizing antibodies abrogation of the inhibitory effect could only be demonstrated by anti-TNF alpha. However, preculturing of BMNC or CD34+ cells with a wide range of TNF alpha concentrations as well as TNF alpha neutralization in supernatant of bryo-1-stimulated BMNC failed to affect the inhibitory effect on CD34+ cells. Both indicate that TNF alpha was not the only factor responsible for the inhibitory effect. Depletion of CD14+ cells from BMNC cultures showed that upon bryo-1 exposure the monocytes served as the main source of TNF alpha but not as a source of the inhibitory cytokine(s): in CD14+-depleted cultures the combination of exogenous added TNF alpha and bryo-1 resulted in an inhibition of colony formation comparable to that found in crude BMNC. In contrast, purified CD34+ cultures were not directly affected by bryo-1 and TNF alpha. However, clonogenic growth of purified CD34+ cells was inhibited if mononuclear cells were preincubated with TNF alpha alone for 24 h, and the supernatant of these cultures was used together with bryo-1. These results show that bryo-1-induced inhibition of clonogenic cell growth is not mediated by a direct effect of bryo-1 on CD34 cells but is a result of a process involving production of TNF alpha by CD14+ cells upon bryo-1 stimulation together with the induction of (a) secondary factor(s) by TNF alpha, which together with bryo-1 itself is inhibitory towards clonogenic cell growth.
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Antineoplásicos/farmacología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Lactonas/farmacología , Leucocitos Mononucleares/citología , Linfocitos T/citología , Factor de Necrosis Tumoral alfa/fisiología , Antígenos CD/análisis , Antígenos CD34/análisis , Brioestatinas , Antígenos CD2/análisis , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Receptores de Lipopolisacáridos/análisis , Macrólidos , Modelos Biológicos , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In view of cellular immunotherapy with cytotoxic monocytes in minimal residual leukemia we have studied the effects of monocytes on the growth and survival of leukemic cells from cell lines and from patients with acute myeloid leukemia (AML). Using highly purified and interferon-gamma (IFN gamma) activated human monocytes, monocyte-mediated cytotoxicity (MMC) was evaluated in an MTT-based colorimetric cytotoxicity assay against six human leukemic cell lines (U937, THP1, KG1, K562, HL60, and 1,25(OH)2D3 differentiated HL60 cells) and cells from AML patients. Leukemic cells from cell lines with an immature phenotype were found to be resistant to MMC, whereas leukemic cells with a more mature and monocytic phenotype were sensitive. This paralleled the sensitivity to tumor necrosis factor-alpha (TNF-alpha). AML cells from patients with an immature phenotype (FAB-M1/M5A) were significantly less sensitive to MMC as compared to more mature AML cells (FAB-M2/M4/M5B). The growth stimulatory effects of non-activated monocytes on immature AML cells could be abrogated in the presence of IFN gamma or IL-3 and GM-CSF. In addition, these cytokines further potentiated MMC, preferentially affecting cells with a more mature phenotype. AML cells with an immunologically immature phenotype (CD34(high), HLA-Dr(low), CD13(low), CD14(low)) were revealed as the least sensitive cells to MMC. The growth stimulatory effects of IL-3/GM-CSF with or without TNF-alpha on AML cells correlated with resistance to MMC. In addition, the cytolytic effects of TNF-alpha in the presence of IFN gamma correlated with an increased susceptibility of AML cells to MMC. In conclusion, our data strongly indicate that MMC is related to maturation in AML, which is correlated to the differential stimulatory and cytolytic effects of monocyte-derived cytokines such as IL-3, GM-CSF, and TNF-alpha.
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Leucemia Mieloide/inmunología , Linfocitos/inmunología , Monocitos/inmunología , Enfermedad Aguda , Adolescente , Adulto , Anciano , Antígenos CD/análisis , Comunicación Celular , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocinas/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia Mieloide/patología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
Recognition events between hematopoietic progenitor cells (HPC) and bone marrow endothelial cells (BMEC) initiate homing of HPC to the bone marrow. The chemokine SDF-1 is present on BMEC and plays a crucial role in bone marrow engraftment. We studied the role of proteoglycans (PGs) on BMEC in binding and presentation of SDF-1. SDF-1 mRNA was present in three human BMEC cell lines. Competition experiments showed that 125I-SDF-1 alpha binding to the BMEC cell line 4LHBMEC was inhibited by heparins, heparan sulfate (HS) intestinal mucosa, chondroitin and dermatan sulfate (CS/DS), but not by HS bovine kidney. Pretreatment of 4LHBMEC with glycosaminoglycan (GAG)-degrading enzymes or sodium chlorate demonstrated that SDF-1 bound to both HSPGs and CS/DSPGs in a sulfation-dependent manner, as determined with an SDF-1 antibody recognizing the CXCR4-binding site. 4LHBMEC bound four-fold more SDF-1 than HUVEC. Isolated endothelial PGs did not bind SDF-1 in a filter or microplate-binding assay, suggesting the necessity of membrane association. In flow adhesion experiments, endothelial arrest of CXCR4+ KG-1 and not of CXCR4- KG-1a cells increased significantly when SDF-1 was presented on 4LHBMEC. In conclusion, SDF-1 is produced by BMEC and binds to the BMEC cell surface via HS and CS/DS-GAGs, thereby presenting its CXCR4 binding site to HPC contributing to their arrest.
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Células de la Médula Ósea/metabolismo , Quimiocinas CXC/metabolismo , Endotelio Vascular/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteoglicanos de Heparán Sulfato/fisiología , Animales , Bovinos , Quimiocina CXCL12 , Quimiocinas CXC/genética , Cloratos/farmacología , Sulfatos de Condroitina/farmacología , Cartilla de ADN/química , Dermatán Sulfato/farmacología , Citometría de Flujo , Proteoglicanos de Heparán Sulfato/farmacología , Heparitina Sulfato/farmacología , Humanos , Reacción en Cadena de la Polimerasa , Unión Proteica , Células del Estroma/metabolismoRESUMEN
OBJECTIVE: Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. MATERIALS AND METHODS: Human CD34(+) cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. RESULTS: After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the beta(1) integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. CONCLUSION: In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.
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Adhesión Celular/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Megacariocitos/citología , Células del Estroma/citología , Animales , Anticuerpos Bloqueadores/farmacología , Antígenos CD/biosíntesis , Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Fibroblastos/citología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Inmunofenotipificación , Integrina alfa4beta1 , Integrinas/antagonistas & inhibidores , Integrinas/metabolismo , Ratones , Modelos Biológicos , Receptores de Fibronectina/antagonistas & inhibidores , Receptores de Fibronectina/metabolismo , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Receptores Mensajeros de Linfocitos/metabolismo , Factor de Células Madre/farmacología , Trombopoyetina/farmacologíaRESUMEN
Little is known about the precise ways in which monocytes and macrophages recognize tumor cells and how they exert their cytolytic and/or cytostatic effects. By a functional, morphologic, and flow-cytometric approach, we have studied monocyte/macrophage- and cytokine-mediated cytotoxicity against U937 cells, a human histiocytic lymphoma cell line. A rapid decrease in cell viability of U937 cells (MTT assay) could be observed at an effector-to-target cell (E:T) ratio of 10 in the presence of interferon (IFN)-gamma-activated monocytes. Light and electron microscopic examination showed the characteristic features of apoptosis of U937 cells after incubation with either monocytes or tumor necrosis factor (TNF)-alpha. TNF-alpha-induced apoptosis (10(4) U/mL) as measured by multiparameter flow cytometry (propidium iodide [PI]) paralleled the functional decrease in cell viability (MTT assay) of 20 +/- 3% after 24 hours up to a maximum of 50 +/- 4% after 48 hours. Apoptosis could be confirmed by the detection of DNA degradation into multiples of 200-bp subunits by agarose gel electrophoresis. After prolonged incubation times, monocyte-mediated leukemic cell death could be quantified as apoptosis by flow cytometry, whereas no decrease in net cell viability of tumor cells relative to the initial cell number could be observed by MTT spectrophotometry. In conclusion, our data provide evidence that apoptosis is the major mode of TNF-alpha-dependent monocyte-mediated cytotoxicity against U937 cells. Furthermore, multiparameter flow-cytometric analysis offers a sensitive method to quantify cytokine- and cell-induced apoptosis in leukemia.
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Apoptosis , Citotoxicidad Inmunológica , Linfoma de Células B Grandes Difuso/patología , Macrófagos/inmunología , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/farmacología , ADN de Neoplasias/metabolismo , Electroforesis en Gel de Agar , Citometría de Flujo , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Macrófagos/ultraestructura , Microscopía Electrónica , Monocitos/ultraestructura , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.
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Médula Ósea/metabolismo , Endotelio Vascular/metabolismo , Heparitina Sulfato/análisis , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Especificidad de Órganos , Venas/metabolismoRESUMEN
Bone marrow endothelial cells are likely to play an important role in the homing of hematopoietic progenitor cells. In view of analyzing the interactions between endothelial cells and hematopoietic progenitor cells, we studied several methods of isolating endothelial cells from human bone marrow, including fluorescence activated cell sorting (FACS) and separation by immunomagnetic beads. FACS sorting gave the best results as contamination with other cells did not occur. After density-gradient centrifugation of bone marrow aspirates, the mononuclear cell (MNC) fraction was depleted for T cells, B cells, and myeloid cells by immunomagnetic separation. Further enrichment of endothelial cells was achieved by FACS sorting using BNH9 or S-Endo1 monoclonal antibodies (MAbs). These MAbs, in contrast to several other endothelial-cell reactive MAbs, were found to react highly specifically with sinus endothelial cells as tested by immunohistochemistry on bone marrow tissue sections and cell culture preparations and by double-colored FACS analysis on bone marrow MNCs (BMMNC). Sorted cells, which formed 0.05% of the MNC fraction, showed strong intracytoplasmic von Willebrand factor positivity. Ultrastructural analysis revealed cells with endothelial characteristics. Cells were cultured in fibronectin-coated, 24-well culture plates in endothelial-cell culture medium or long-term bone marrow culture medium. After 1 to 3 weeks of culture, a monolayer of spindle-shaped cells developed expressing endothelial cell antigens. Cells could be kept in culture for 4 to 6 weeks. In conclusion, the method described provides highly purified preparations of human bone marrow endothelium that may permit in vitro adhesion experiments with normal and leukemic hematopoietic progenitor cells.
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Células de la Médula Ósea , Separación Celular/métodos , Anticuerpos Monoclonales , Células Cultivadas , Centrifugación por Gradiente de Densidad , Medios de Cultivo , Endotelio/citología , Citometría de Flujo , Humanos , Inmunohistoquímica , Separación Inmunomagnética , Microscopía ElectrónicaRESUMEN
In this study we describe a semiautomated clonogenic assay in which human leukemic cell lines (U937 and HL60) are cultured in a semisolid 96-well microtiter culture system and clonogenicity is measured spectrophotometrically in a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT)-assay and sulforhodamine (SRB)-assay. Culture medium with 0.6% (wt/vol) methylcellulose and 10% (vol/vol) fetal calf serum (FCS) appeared to provide optimal culture conditions with a low background absorbance in both colorimetric assays and an optimal linearity between cell number and optical density (OD). An excellent correlation between clonogenic growth of U937 and HL60 cells in the conventional colony-forming unit assay (CFU-assay) and the microtiter CFU-assay was observed. The value of this microtiter CFU-assay was assessed by modulating U937 and HL60 cells with either 1,25(OH)2D3 or cytosine arabinoside (Ara-C). Additionally, the number of living cells was quantitated spectrophotometrically in both MTT- and SRB-assays. Results obtained by either method did not differ significantly (p < 0.001). In liquid culture, however, significantly (p < 0.001) less reduction of cellular growth was observed with 1,25(OH)2D3-modified cells. No significant differences between the liquid and semisolid assay systems could be observed in the presence of Ara-C. In conclusion, the microtiter clonogenic assay provides a rapid and objective way of measuring clonogenic capacity of (leukemic) cell lines. The semiautomated clonogenic assay will be useful to assess large series of immune modulations and/or cytostatic drug screening.
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Células Tumorales Cultivadas/patología , Calcitriol/farmacología , Células Clonales , Medios de Cultivo , Citarabina/farmacología , Humanos , Técnicas In Vitro , EspectrofotometríaRESUMEN
In order to shorten the pancytopenic period following high-dose melphalan 140 mg/m2 (HDM) treatment of multiple myeloma patients, we studied the effects of re-infusing granulocyte colony stimulating factor (G-CSF) [Filgrastim, Neupogen]-primed unprocessed whole blood. 30 patients with multiple myeloma were treated with HDM. One litre of blood after 5 or 6 days stimulation with G-CSF (10 micrograms/kg) was drawn, kept unprocessed for 1 day and re-infused 24 h after chemotherapy. Time to granulocyte recovery (> 0.5 x 10(9)/1) and platelet recovery (> 20 x 10(9)/1) were assessed as well as length of hospital stay, number of transfusions and antibiotic use. These 30 patients were compared with 20 historical control patients who were similarly treated but without stem cell support. The response rate was 75% (21/28) including a complete remission (CR) rate of 29% (8/28). Two early deaths due to Aspergillus pneumonia were observed. The median overall survival after HDM has not been reached after a median follow-up of 14 months. 10 patients showed progression at a median of 7 months. Currently, 23 patients are alive with a median follow-up time of 14 months. Haematological recovery was significantly faster in the study group as compared to the historical control group. The neutrophil count reached 0.5 x 10(9)/1 at a median of 14 days after infusion of 1 litre of unprocessed whole blood compared with 38 days in the historical control group. A platelet count of 20 x 10(9)/1 was reached at a median of 26 days compared with 36 days in the historical control group. Length of hospital stay decreased from a median of 43 to 18.5 days. The number of days with antibiotics was reduced from a median of 21 to 6 days. HDM is effective therapy for multiple myeloma. Toxicity of the regimen is considerably reduced by the use of G-CSF-stimulated unprocessed whole blood, an easy to perform and cheap technique to mobilise and collect stem cells.
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Antineoplásicos Alquilantes/uso terapéutico , Transfusión de Sangre Autóloga , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Melfalán/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Adulto , Antineoplásicos Alquilantes/efectos adversos , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Melfalán/efectos adversos , Persona de Mediana Edad , Pancitopenia/inducido químicamente , Pancitopenia/prevención & control , Proteínas Recombinantes , Tasa de SupervivenciaRESUMEN
PURPOSE: Estimation of the relative biological effectiveness of electron emissions of 67Gallium for cell growth delay and inactivation. METHODS AND MATERIALS: Human myeloid HL60 cells were incubated in vitro with 0.74 MBq/mL, 1.48 MBq/mL, or 2.96 MBq/mL of 67Gallium for 4 days. Proliferation (vital cell counts and colorimetric tetrazolium assay), clonogenic survival, and cell-cycle effects were compared with responses of HL60 cells externally irradiated with 0.78, 10.37, and 13.22 Gy by an external 67Gallium source. Dosimetric calculations were performed by Monte Carlo simulations (10,000 events). RESULTS: Proliferation of cells was equally inhibited after 67Gallium incubation with 1.48 and 2.96 MBq/mL compared with 10.37 and 13.22 Gy external irradiation. Irradiation with 10.37 and 13.22 Gy caused a 81% and 89% reduction of Colony Forming Units, compared with 34%, 66%, and 80% reduction after 67Gallium incubation with 0.74, 1.48, and 2.96 MBq/mL, respectively. Peak values for G2/M accumulation were reached on day 4 for the cells externally irradiated with 10.37 and 13.22 Gy (47.5% and 56.7%) and on day 5 after 67Gallium incubation with 0.74 MBq/mL, 1.48 MBq/mL, and 2.96 MBq/mL (26.7%, 43.4%, and 58.2%). CONCLUSIONS: 67Gallium incubation exerts a significant cytotoxic effect on human HL60 cells, which, on the basis of dosimetric studies, may be mainly ascribed to conversion electrons (80 KeV) and 8 KeV Auger electrons. Low energy (< 1 keV) Auger electrons do not contribute significantly. The relative biological effectiveness of 67Gallium compared with external low dose rate gamma irradiation is about 1.0 for clonogenic survival and approximately 1.8 and 1.5 for proliferation inhibition and G2 arrest, respectively. For in vivo therapy, this might implicate that higher doses of 67Gallium than 131Iodine or 90Yttrium are necessary for the same biological effect.
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Fase G2/efectos de la radiación , Radioisótopos de Galio/uso terapéutico , Leucemia Promielocítica Aguda/patología , Leucemia Promielocítica Aguda/radioterapia , Recuento de Células , Ciclo Celular/efectos de la radiación , División Celular/efectos de la radiación , Células Clonales/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta en la Radiación , Galio/farmacocinética , Radioisótopos de Galio/química , Humanos , Leucemia Promielocítica Aguda/metabolismo , Dosis de Radiación , Planificación de la Radioterapia Asistida por Computador , Sales de Tetrazolio , Tiazoles , Células Tumorales CultivadasRESUMEN
PURPOSE: The purpose of the present study was to investigate if there were differences between U715 spheroids and single cells in the radiotoxic effect of 67Ga on cell growth and clonogenic capacity in vitro and to generate dosimetric approaches for the multicellular tumor model. METHODS AND MATERIALS: Human lymphoma U715 cells were cultured in vitro as single cells and multicellular spheroids, grown with the use of a combination of fibrin clot technique, spinner flasks, and liquid-overlay culture. Cells were incubated with 2.96-8.88 MBq/ml 67Gallium for 4 days. Spheroids were dispersed to single cells by treatment with plasmin. Residual proliferative and clonogenic capacity after 67Ga incubation were assayed using the MTT-test and clonogenic test, respectively. Autoradiography was performed with 1 microm sections and Ilford L4 liquid photographic emulsion. Dosimetric approaches were made, based on the MIRD-approach. RESULTS: During 67Ga incubation proliferation was inhibited. The residual proliferative or clonogenic capacity was inhibited by 8.88 MBq/ml for 39 and 88%, respectively. For single cells with 6.66 MBq/ml these inhibitions were 64 and 96%, respectively. Autoradiography showed an homogeneous distribution of 67Ga in spheroids and single cells. In single cells a 2.1-3.5 times higher 67Ga uptake/cell than in spheroids produced an equitoxic effect. The uptake parameters were implemented in new dosimetric approaches and showed that the efficacy of intracellular 67Ga was two times higher in spheroid clusters than in single cells due to energy deposition of internal conversion electrons within the cell clusters with a mean diameter size of nine cells. Both for proliferative and clonogenic capacity the exponential survival curves were superimposed. CONCLUSIONS: With the new approaches made in our dosimetric model the discrepancy found between 67Ga accumulation and radiotoxic effect in spheroids as compared to single cells can be explained by additional effects of the crossfire of internal conversion electrons within clusters of about nine cells in diameter in spheroids. Only twice as much 67Ga was needed to reach equitoxic absorbed doses in spheroids than was needed in single cells. Such might be important for the use of 67Ga treatment of small metastasis of malignant lymphoma.
Asunto(s)
Radioisótopos de Galio/farmacología , Radioisótopos de Galio/farmacocinética , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de la radiación , Autorradiografía , Recuento de Células/efectos de la radiación , División Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Humanos , Linfoma/metabolismo , Linfoma/patología , Dosis de Radiación , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
We examined the effect of bryostatin-5 (bryo-5) with and without a combination of myeloid growth promoting factors on human acute myeloid leukemia (AML) cell growth, maturation, and primary plating efficiency. In vitro treatment of AML samples with bryo-5 induced a macrophage-like cell differentiation as evidenced by morphological changes, esterase staining, and cell surface expression of CD11a and CD18. AML cells exposed to growth factors doubled their cell numbers following culture, this increase being abrogated by co-exposure to bryo-5. An antiproliferative effect, as well as the antagonistic interaction of bryo-5 with growth factors, was confirmed in methylcellulose clonogenic assays. Together, these findings indicate that the compound bryo-5 exerts an anti-proliferative effect on AML cells and counteracts growth factor induced leukemic proliferation.
Asunto(s)
Antineoplásicos/farmacología , Sustancias de Crecimiento/farmacología , Lactonas/farmacología , Leucemia Mieloide Aguda/patología , Adulto , Anciano , Antígenos CD/metabolismo , Brioestatinas , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Clonales/citología , Activación Enzimática , Femenino , Humanos , Integrinas/metabolismo , Macrólidos , Macrófagos/citología , Masculino , Persona de Mediana Edad , Proteína Quinasa C/metabolismo , Células Tumorales CultivadasRESUMEN
The potent anti-neoplastic actions displayed in vitro by bryostatins have led to the introduction of short-term bryostatin-1 infusions in phase I clinical trials. Because bryostatin (bryo) undergoes a rapid clearance in vivo, we were interested in its scheduling effects on acute myeloid leukemia (AML) clonogenicity. Therefore, we assessed the primary plating efficiency (PE1) of AML samples in response to several bryo concentrations after various preincubation periods in a semi-solid colony forming assay. Whereas continuous exposure to 10 nM bryo during the assay period reduced the PE1 in nearly all samples tested, preincubation of eight AML patients' specimens for 1, 2, 3 or 4 days with bryo in a dose range of 0.1-10 nM showed a heterogenous PE1 response. Stimulatory as well as inhibitory effects on leukemic clonogenic growth were seen within individual specimens depending on dose and preincubation time with no single incubation time or concentration that caused unequivocal common overall inhibition of clonogenic growth in most or all of the samples. Higher doses of bryo also failed to result in specific inhibition of leukemic cells: 4/8 samples showed an increased clonogenic response to 250 nM bryo whereas normal bone marrow progenitor cells were consistently inhibited in their clonogenicity at this dose. A marked similarity, i.e. undulatory effects with increasing bryo concentrations, was found for HL60 leukemic cells. In conclusion, the effects of bryo on clonogenic leukemia cell growth are strongly dependent on scheduling and dose varying between and within individual AML samples. These results caution against in vivo bryo pulse therapy, as currently applied, for treatment of AML.
Asunto(s)
Antineoplásicos/farmacología , Lactonas/farmacología , Leucemia Mieloide/patología , Enfermedad Aguda , Adulto , Anciano , Antineoplásicos/administración & dosificación , Brioestatinas , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Lactonas/administración & dosificación , Leucemia Mieloide/sangre , Macrólidos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Ensayo de Tumor de Célula MadreRESUMEN
In this study we explored the effects of bryostatin-5 on the clonogenic response of normal bone marrow mononuclear (BM) cells and HL60 myeloid leukemia cells. Leukemic HL60 colony formation was strongly inhibited by bryostatin-5 depending on dose and schedule. An inhibitory effect on HL60 colony formation was readily demonstrated after 1 h of exposure, reaching a maximal inhibitory effect at 96 h. Normal BM cells differed in their clonogenic response: short-term exposure to bryostatin-5 resulted in increased clonogenicity while longstanding exposure to bryostatin-5 permitted the survival of a substantial fraction of committed progenitors. This differential modulation of normal and leukemic myeloid clonogenicity by bryostatin-5 suggests a possible role for bryostatin-5 in the treatment of acute myeloid leukemia.
Asunto(s)
Antineoplásicos/farmacología , Hematopoyesis/efectos de los fármacos , Lactonas/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Brioestatinas , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/patología , Macrólidos , Estimulación Química , Células Tumorales CultivadasRESUMEN
Promising clinical results are obtained with radiolabeled antibodies in leukemia patients. 67Gallium (67Ga) is a radionuclide that accumulates in many malignant tissues without need for a monoclonal antibody. For this reason, the use of 67Ga as a therapeutic agent is appealing. In the present we study, we report data about the radiotoxicity of 67Ga on peripheral blast cells of 23 patients with acute myelogenous leukemia (AML) in vitro. Isolated blast cells were incubated for 4 days with 0.74 MBq/ml (20 microCi/ml), 1.48 MBq/ml (40 microCi/ml) or 2.96 MBq/ml (80 microCi/ml) 67Ga. Compared with non-irradiated control cells proliferation during incubation was almost abolished. Clonogenic survival was measured by a colony forming unit assay (CFU-assay). In 13 of the 23 patients (56%) sufficient colony growth was observed for evaluation. The mean clonogenic survival of blasts after incubation with 0.74 MBq/ml and 2.96 MBq/ml 67Ga was 22.5, 11.3 and 3.5%, respectively. In some cases colony growth was completely abolished after incubation with only 0.74 MBq/ml 67Ga. No correlation was found between cellular 67Ga-uptake, (micro)dosimetry and transferrin receptor density (CD-71) via which 67Ga enters the cell. In vitro the blasts received a dose of > 10 Gy in 9 of the 2.96 MBq/ml, in 3 of the 1.48 MBq/ml and in 2 of the 0.74 MBq/ml incubations. In one patient, even a radiation dose > 40 Gy was reached. Low dose rate irradiation is known to arrest cells in G2/M-phase of the cell cycle, but no such arrest was observed during incubation with 67Ga. Thus, 67Ga induces clonogenic cell death in leukemic blasts. Cellular uptake of 67Ga in vitro varies between patients and can be very high in some patients. The easy availability, low costs and absence of immunological problems warrant further investigation of the therapeutic potential of 67Ga in refractory or relapsed AML patients.
Asunto(s)
Radioisótopos de Galio/toxicidad , Leucemia Mieloide/patología , Células Tumorales Cultivadas/efectos de la radiación , Enfermedad Aguda , Transporte Biológico , Ciclo Celular/efectos de los fármacos , División Celular/efectos de la radiación , Radioisótopos de Galio/metabolismo , Radioisótopos de Galio/uso terapéutico , Humanos , Técnicas In Vitro , Leucemia Mieloide/radioterapia , Receptores de Transferrina/metabolismoRESUMEN
Several studies have pointed out that L-selectin on CD34-positive cells plays a role in haematopoietic reconstitution after peripheral blood stem cell (PBSC) transplantation. Since it is known that a decrease in L-selectin expression in lymphocytes and granulocytes can be induced by a variety of stress situations, we have investigated in this study whether the freeze-thawing procedure, used in PBSC transplantation, would affect L-selectin expression on CD34+ stem cells. Flow cytometry was performed by labelling the cells with anti-CD34 (HPCA2 PE) and anti-CD62L (FMC46 FITC). The leucapheresis procedure itself caused a slight decrease of L-selectin expression on CD34 cells in 11 out of 12 cases (mean decrease of the percentage of positive cells 11.9; range 6-23). A much larger decrease was found upon freeze-thawing: a mean of 39% (range 4-78% in 27 cases) compared to fresh material. To determine if L-selectin expression might be up-regulated after cryopreservation, thawed transplant samples (n = 11) were incubated at 37 degrees C in RPMI with 10% FCS at 5% CO2. Already early in the course of incubation two CD34-positive populations appeared in the blast region, characterized by either a low or high forward scatter. Simultaneous viability staining with the DNA dye 7-Amino Actinomycin D and the DNA/RNA dye Syto16 revealed that the population with low forward scatter was apoptotic while the population with the high forward scatter was non-apoptotic. The latter population is considered to be most relevant for transplantation. In this population an increase of L-selectin expression after overnight incubation was observed in 8/11 samples up to values of 46-120% of the values of the fresh nonfrozen samples. In addition, the mean fluorescence intensity was significantly increased in 10/11 cases. Kinetic experiments with shorter incubation times revealed that only part of the leucapheresis samples (two out of 8) showed an increase of L-selectin expression within 4 h. In addition, a decrease of L-selectin expression was found upon CD34 purification from fresh leucapheresis material by magnetic isolation (decrease ranging from 59 to 92%, n = 5). In contrast to frozen samples, L-selectin reappearance was seen already within 4 h of incubation in all samples. Both the loss of L-selectin expression on CD34 cells occurring upon freeze-thawing, the emergence of apoptosis, as well as the recovery of L-selectin expression on non-apoptotic cells varies largely between individual leucapheresis samples, and therefore it is concluded that such processes should be considered when correlations with clinical outcome after transplantation are made.
Asunto(s)
Antígenos CD34/metabolismo , Criopreservación , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Selectina L/metabolismo , Movimiento Celular , Supervivencia Celular , Citometría de Flujo , Congelación , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , LeucaféresisRESUMEN
Growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) is effective at progenitor release into the peripheral blood. After high-dose chemotherapy haematopoietic reconstitution occurs after reinfusion of these peripheral blood progenitor cells (PBPC). However, the collection by leukapheresis and further processing of PBPC are very time consuming and expensive. We have studied the transplantation potential of a small volume of unprocessed autologous whole blood after G-CSF mobilisation. Six patients with plasma cell disorders received G-CSF 10 micrograms/kg sc during 6 days. Subsequently 11 of whole blood was collected by phlebotomy, kept unprocessed at room temperature and reinfused 24 h after high-dose melphalan 140 mg/m2. CFU-GM content was 845 per ml blood (median, range 320-3472) and CD34+ cells rose to a median percentage of 0.9 (range 0.4-2.0). Haematological recovery was significantly faster in the study group compared with the control group of 20 patients who received the same dose of melphalan without reinfusion of PBPC. The neutrophil count reached 0.5 x 10(9)/l at a median of 12.5 days after infusion of PBPC vs 38 days in the control group (p = 0.0003). The platelet count reached 20 x 10(9)/l after a median of 23.5 days vs 38 days (p = 0.0218). The shortened recovery was reflected by less transfusions, less antibiotic use and shortening of hospital stay (19 days vs 43 days, p = 0.0003). We conclude that this easy technique of mobilisation and collection of PBPC is very effective for hastening haematologic recovery after high-dose chemotherapy.