Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

BACKGROUND: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. RESULTS: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. CONCLUSIONS: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF

Activités biologiques de l'interleukine-3 et de l'interleukine-6 sur l'hématopoïèse humaine et sur la thrombopénie secondaire à l'administration de chimiothérapie anticancéreuse

Les facteurs de croissance hématopoïétiques (FCH) sont les régulateurs physiologiques de la prolifération et de la différenciation des cellules hématopoïétiques. Ils sont administrés, à dose pharmacologique, pour protéger de la toxicité médullaire de la chimiothérapie et de la radiothérapie anticancéreuses de deux façons : - dans le décours de la chimiothérapie, ils stimulent la prolifération et la différenciation des cellules non détruites et raccourcissent ainsi la période dangereuse de myélotoxicité. - comme mobilisateurs des cellules souches hématopoïétiques périphériques (CSHP), ils permettent la récolte d’un greffon composé de cellules qui assurent une reprise hématopoïétique plus rapide qu’après greffe de moelle. En 1991, les FCH actifs sur la myélopoïèse réduisaient déjà la neutropénie mais étaient inefficaces pour diminuer la thrombopénie et c’est à ce problème que nous nous attaquions alors. Plusieurs FCH actifs sur al lignée mégacaryocytaire in vitro et chez l’animal s’avéraient promotteurs. A cette époque, nous avons dès lors choisi l’interleukine (IL)-3 et l’IL-6 afin d’en étudier la toxicité et l’effet protecteur chez des patients recevant de la chimiothérapie. Ensuite, nous nous sommes attachés à mesurer l’effet sur la reprise hématopoïétique après greffe des combinaisons de FCH mobilisant les CSHP. avant d’entre dans le vif su sujet, je rappellerai succinctement l’intérêt de la chimiothérapie) haute dose ainsi que la biologie du système hématopoïétique et de la mégacaryocyopoïèse, puis je situerai ma contribution personnelle par rapport aux études cliniques similairesThèse de doctorat en sciences biomédicales (oncologie) -- UCL, 199

Transfert de gène de chimiorésistance dans les cellules souches hématopoïétiques : promesses et controverses

La principale limitation à l'administration de chimiothérapie à haute dose est sa toxicité hématologique. Récemment, l'étude des mécanismes de résistance à la chimiothérapie des cellules tumorales a permis d'identifier les gènes codant pour les protéines impliquées dans ces phénomènes. Ces gènes de résistance peuvent être transférés dans les cellules souches hématopoïétiques, afin de limiter l'hémato-toxicité des cytostatiques, grâce aux techniques de thérapie génique. Cela vise à permettre d'administrer de hautes doses de chimiothérapie pour un meilleur effet antitumoral. En outre, la sélection in vivo des cellules génétiquement modifiées par l'administration de chimiothérapie devrait augmenter l'efficacité du transfert de gène. Les modèles murins ont montré la faisabilité de cette approche. L'application clinique de cette option thérapeutique requiert encore des progrès tant dans le domaine de la construction des vecteurs que des conditions nécessaires au transfert de gène

IL-3 and peripheral blood stem cell harvesting.

Transplantation of blood-derived stem cells is increasingly performed because when used alone or when combined with autologous bone marrow grafting, it can demonstrably shorten myelosuppression following multi-agent chemotherapy. Hematopoietic growth factors can mobilize peripheral blood stem cells from the bone marrow to therapeutically intervene in accelerating hematologic recovery. Interleukin 3 (IL-3), whose hematopoietic activities were first described some ten years ago, is one of several candidate growth factors that may prove useful in enhancing this mobilization in order to obtain adequate yields of circulating stem cells for transplantation. IL-3 used in conjunction with synergistically acting cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) have already yielded interesting results, while new factors like stem cell factor are in the process of clinical evaluations and appear promising. However, further issues remain to be clarified to confirm the general applicability of cytokine-augmented peripheral blood stem cells in improving on-schedule delivery of high-dose myelosuppressive chemotherapy in patients with malignancies

Chemoprotection and selection by chemotherapy of multidrug resistance-associated protein-1 (MRP1) transduced cells

Utilization of chemotherapy for treatment of tumors is mainly limited by its hematological toxicity. Because of the low level expression of drug resistance genes, transduction of hematopoietic progenitors with multidrug resistance 1 (MDR1) or multidrug resistance-associated protein 1 (MRP1) genes should provide protection from chemotherapy toxicity. Successful transfer of drug resistance genes into hematopoietic cells might allow the administration of higher doses of chemotherapy and, therefore, increase regression of chemosensitive tumors. In addition, this approach can be used to select in vivo transduced cells by their enrichment after administration of cytotoxic drugs. Our group has studied the potential value of MRP1 to protect hematopoietic cells. The interest in the use of MRP1 as an alternative to MDR1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in clinic to decrease MDR1 tumor resistance, have little or no effect on MRP1 function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by MRP1. We constructed two different MRP1-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or phosphoglycerate kinase (PGK) as promoters and generated ecotropic producer cells. MRP1 transduced fibroblasts were more resistant to doxorubicin, vincristine, and etoposide and their chemoprotection was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP1 function. Lethally irradiated mice were engrafted with bone marrow (BM) cells transduced with MRP1 vectors (PGK promoter). We demonstrated that high expression of MRP1 in murine hematopoietic cells reduces doxorubicin-induced leukopenia and mortality. In addition, in vivo selection of MRP1-transduced BM cells was achieved following doxorubicin administration and allowed a better chemoprotection after the second chemotherapy cycle

Unreliability of carcinoembryonic antigen (CEA) reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting contaminating breast cancer cells in peripheral blood stem cells due to induction of CEA by growth factors.

RT-PCR is increasingly used for the detection of minimal residual disease in solid tumors. Carcinoembryonic antigen (CEA) RT-PCR seemed to be highly specific for detection of tumor cells when tested on PBMC. A very high frequency of RT-PCR amplification product for CEA in PBSC from breast cancer patients mobilized with G-CSF was found. However, this result contrasted with tumor cell detection by immunocytochemistry (ICC) which showed no correlation with RT-PCR results. In addition, CEA mRNA was amplified in most G-CSF-mobilized PBSC samples derived from patients with hematological malignancies and from healthy donors of allogeneic stem cells, although no circulating epithelial cells could be demonstrated by ICC. CEA RT-PCR expression was observed in PBMC from healthy individuals incubated in vitro with G-CSF. These data suggest that CEA transcription can be induced by G-CSF, resulting in a loss of specificity of CEA RT-PCR for tumor cell detection in PBMC. We conclude, CEA RT-PCR may not be recommended to detect tumor cell contamination in peripheral blood from patients treated with G-CSF. This may have implications on tumor cell detection by RT-PCR in tissues where endogenous or exogenous growth factors may induce the transcription of CEA or other genes