RESUMEN
APRIL, a proliferation-inducing ligand, is a member of the tumor necrosis factor (TNF) family that is expressed by various types of tumors and influences their growth in vitro and in vivo. Two receptors, transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), bind APRIL, but neither is essential for the tumor-promoting effects, suggesting that a third receptor exists. Here, we report that APRIL specifically binds to heparan sulfate proteoglycans (HSPG) on the surface of tumor cells. This binding is mediated by the heparin sulfate side chains and can be inhibited by heparin. Importantly, BCMA and HSPG do not compete, but can bind APRIL simultaneously, suggesting that different regions in APRIL are critical for either interaction. In agreement, mutation of three lysines in a putative heparin sulfate-binding motif, which is not part of the TNF fold, destroys interaction with HSPG, while binding to BCMA is unaffected. Finally, whereas interaction of APRIL with HSPG does not influence APRIL-induced proliferation of T cells, it is crucial for its tumor growth-promoting activities. We therefore conclude that either HSPG serve as a receptor for APRIL or that HSPG binding allows APRIL to interact with a receptor that promotes tumor growth.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Mutación/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Bryostatin-1 belongs to the family of macrocyclic lactones isolated from the marine bryozoan Bugula neritina and is a potent activator of protein kinase C (PKC). Bryostatin has been demonstrated to possess both in vivo and in vitro anti-leukaemic potential. In samples derived from chronic myeloid leukaemia (CML) patients, it has been demonstrated that bryostatin-1 induces a macrophage differentiation, suppresses colony growth in vitro and promotes cytokine secretion from accessory cells. We investigated the effect of bryostatin-1 treatment on colony-forming unit-granulocyte macrophage (CFU-GM) capacity in the presence of accessory cells, using mononuclear cells, as well as in the absence of accessory cells using purified CD34-positive cells. Cells were obtained from 14 CML patients as well as from nine controls. Moreover, CD34-positive cells derived from CML samples and controls were analysed for stem cell frequency and ability using the long-term culture initiating cell (LTCIC) assay at limiting dilution. Individual colonies derived from both the CFU-GM and LTCIC assays were analysed for the presence of the bcr-abl gene with fluorescence in situ hybridization (FISH) to evaluate inhibition of malignant colony growth. The results show that at the CFU-GM level bryostatin-1 treatment resulted in only a 1.4-fold higher reduction of CML colony growth as compared to the control samples, both in the presence and in the absence of accessory cells. However, at the LTCIC level a sixfold higher reduction of CML growth was observed as compared to the control samples. Analysis of the LTCICs at limiting dilution indicates that this purging effect is caused by a decrease in output per malignant LTCIC combined with an increase in the normal stem cell frequency. It is concluded that bryostatin-1 selectively inhibits CML growth at the LTCIC level and should be explored as a purging modality in CML.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Lactonas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Macrófagos/efectos de los fármacos , Brioestatinas , Activación Enzimática/efectos de los fármacos , Proteínas de Fusión bcr-abl/genética , Granulocitos/citología , Humanos , Hibridación Fluorescente in Situ , Macrólidos , Macrófagos/citología , Ensayo de Tumor de Célula MadreRESUMEN
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.
Asunto(s)
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
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
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
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
The suppressive effect of the glucocorticoid dexamethasone (DEX) on purified CD4+ T cells was found to depend on the activation pathway. In contrast to anti-CD3- or PHA-induced T cell proliferation, the alternative pathway of T cell activation, i.e., through anti-CD2 and anti-CD28, appeared largely resistant to DEX. By titrating anti-CD28 or the protein kinase C (PKC) activator PMA in the DEX-sensitive systems, it was demonstrated that inhibition by DEX could be abrogated by enhancing the CD28 signal or by stimulation of the PKC-dependent pathway. Supraoptimal concentrations of PMA were inhibitory for proliferation and this effect was partly prevented by DEX. These data suggest that the outcome of the effect of DEX on CD4+ T cells is dependent on the activation pathway, in particular the role and composition of the transcription factor AP-1.