RESUMEN
Patients with acute myelogenous leukemia or myelodysplastic syndrome may respond to farnesyl transferase inhibitors (FTIs) with partial or complete response rates noted in about 30% of such patients. FTIs prevent the attachment of a lipid farnesyl moiety to dependent proteins prior to their insertion into the plasma membrane and thereby prevent activity of these prenylation-dependent proteins, but their mechanism of tumor suppression remains unknown. Many patients receiving FTIs do experience myelosuppression. In this work, the in vitro effects of the FTI, R115777 on normal and leukemic hematopoiesis have been examined as have its effects on apoptosis induction and cell cycle profile in both leukemic blasts and normal CD34+ cells. R115777 was inhibitory to normal CD34+ cell proliferation and to leukemic blast cells, but did not affect long-term culture initiating cell frequency nor NOD-SCID reconstituting capacity. No induction of apoptosis or cell cycle changes were noted in AML blasts. These data suggest that myelosuppression with R115777 occurs largely at the intermediate to late progenitor stage of hematopoiesis and that cyclic use might avoid long-term marrow suppression.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Hematopoyesis/efectos de los fármacos , Leucemia/tratamiento farmacológico , Quinolonas/farmacología , Animales , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Farnesiltransferasa , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/trasplanteRESUMEN
ABSTRACT Macrophage inflammatory protein-1alpha (MIP-1alpha) is a C-C chemokine which has antiproliferative effects on early hematopoietic progenitors and stimulatory effects on later progenitors. It also possesses chemotactic and activating properties for monocytes, macrophages, and T-cells. CD34+ progenitors isolated utilizing an avidin-biotin immunoadsorption column produced significant amounts of MIP-1alpha from 24 h onward when cultured in medium with 10% fetal calf serum (>200 pg/ml). Such production persisted through 96 h of culture and was greater when such progenitors were cocultured with a preformed marrow stromal layer (4000 pg/ml at 24 h). The production of MIP-1alpha declined over time of coculture with stromal layers, and stromal layers themselves produced minimal MIP-1alpha as detected by ELISA: <100 pg/ml. In contrast, CD34+ cells isolated by flow cytometry or by magnetic bead adsorption produced minimal MIP-1alpha (0-30 pg/ml). MIP-1alpha production also increased when cells isolated by these two methods were cocultured with stromal layers. The difference in MIP-1alpha production could not be accounted for by differences in purity of the CD34+ population between isolation methods nor on the basis of monocytic or lymphocytic contamination as assessed by the presence of CD14 or CD3 positive cells. CD34+ cells isolated by immune adsorption had increased expression of endothelial and mesenchymal associated antigens, however, suggesting that this subpopulation might account for the MIP-1alpha production observed. Freshly isolated CD34+ cells expressed MIP-1alpha message as assessed by RT-PCR and by in situ hybridization. Coculture of CD34+ cells isolated by any means with stromal cells increased transforming growth factor-beta (TGF-beta) production, in this case by the stromal layer itself. Both MIP-1alpha and TGF-beta have been found to influence cell cycle status and proliferation status of early hematopoietic progenitors, and both have potential effects on accessory cell function. These studies indicate that progenitor-stromal cell interactions may influence local cytokine output, thus potentially influencing progenitor cycling status and accessory cell activation. The method of isolation of CD34+ progenitors may influence secretion of certain cytokines and chemokines.
Asunto(s)
Antígenos CD34/metabolismo , Proteínas Inflamatorias de Macrófagos/biosíntesis , Células Madre/metabolismo , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Calcio/metabolismo , Comunicación Celular , Separación Celular/métodos , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Técnicas de Cocultivo , Regulación de la Expresión Génica , Humanos , Leucemia/patología , Proteínas Inflamatorias de Macrófagos/farmacología , ARN/genética , ARN/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células del Estroma/citología , Factor de Crecimiento Transformador beta/genética , Células Tumorales CultivadasRESUMEN
The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.
Asunto(s)
Antígenos de Neoplasias/genética , Quimiocina CCL5/inmunología , Quimiocinas/fisiología , Epítopos/genética , Inmunoglobulina G/genética , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Antígenos de Neoplasias/fisiología , Secuencia de Bases , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocinas/genética , Quimiotaxis , Endotelio Vascular/inmunología , Endotelio Vascular/fisiología , Epítopos/fisiología , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/metabolismo , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/inmunología , Ingeniería de Proteínas , Receptor ErbB-2/genética , Receptores CCR5/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/inmunología , Venas UmbilicalesRESUMEN
Stimulation of CD34(+)-enriched marrow or light density marrow with various growth factor combinations can generate granulocyte progenitors and mature neutrophils in vitro. In this work, we have examined the influence of irradiated marrow stromal layers on growth factor-induced myeloid and early multipotential progenitor expansion from enriched marrow CD34+ progenitors. We have also explored whether the addition of early-acting growth factors known to enhance myelopoiesis in long-term culture, such as fibroblast growth factor (b-FGF), insulin growth factor (IGF-1), c-kit ligand or stem cell factor (SCF), and flk-2flt-3 ligand (FL), can lengthen survival of CD34+ progenitors in these cultures. Stromal cell coculture resulted in greater numbers of total cells and CFU-GM at day 7 and day 14, but with the addition of multiple growth factors, these effects of stromal cell coculture were diminished. At day 14, generally < 1% of the expanded cells over stromal coculture conditions were CD34+, with up to 90% demonstrating CD15 positivity. Culture of CD34+ cells in the presence of early-acting growth factors did not cause significant expansion of CD34+ cells over a 14-day life span, even in the presence of marrow stromal cells. These data suggest that although stromal cell coculture for a period up to 14 days can enhance expansion of total cell numbers and CFU-GMs, stromal cell presence does not lead to expansion of CD34+ cells in these cultures and may diminish the number of clonogenic cells present when growth factors with differentiating capacity are present. Mature neutrophils harvested from such cultures are capable of chemotaxis, actin polymerization, and migration, suggesting a replete functional status.