Facteurs de résistance cellulaire aux agents xénobiotiques : contribution des gènes MDR-1, BCL-2 et P53
(1997)
Files
247632_Gala_These.pdf
Restricted Access - Adobe PDF
- 40.01 MB
Details
- Authors
- Supervisors
- Abstract
- This work is an overview if the current knowledge and personal data relating to three mechanisms of tumor resistance to chemo- and/or radiotherapy: P-glycoprotein (P-gp), bcl-2 and p53. P-gp is the gene product of the multidrug resistance gene mdr1. Bcl-2 and p53 are the gene products of the protooncogene bcl-2 and the tumor suppressor gene p53, respectively. Our first goal was to optimize the immonudetection of these proteins and to study the prognostic value of their expression in leukemias : P-gp and myeloblastic leukemia (B-CLL). We identified two forms of P-pg expression in malignant cells (membrane and intracutoplasmic localization) and confirmed the negative prognostic value of both form of P-gp expression in AML. On the contrary, bcl-2 expression was constitutively expressed in ALL and had no prognostic value. Mutation of p53 was associated with primary resistance to 2-chlorodeoxyadenosine (2-CDA) in B-CLL patients. <BR> We also paid attention on functional assays for P-gp (measure of daunorunicin or rhodamine incorporation by flow cytometry) and p53 (FASAY or Fonctional Analysis of Separated Allele in Yeast). These functional aspects are indeed key factors determining chemoresistance. Concomitantly, we designed and used a therapeutical protocol based on the combination of cyclosporine-quinine, for the pharmacological modulation of P-gp related MDR. This protocol was assessed in patients presenting with resistant or refractory disease such as multiple myeloma (15 cases) and thymoma (1 case). Partial and transient responses were observed in these patients. <BR> Using immunocytochemical methods, we focused on the detection of residual disease (RD) in B-CLL. We first assessed the feasibility of antigenic detection using Bouin’s fixed bone marrow trephine biopsies. We then validated the use of bcl-2 expression as a “malignant marker” in B-CLL on this material. We showed that RD is the rule in B-CLL patients achieving an haematological complete response (CR) at the end of the treatment by 2-CDA. We also demonstrated that the pattern of RD is clearly distinct from benign or reactive lymphoid aggregates, which are frequently found in physiological and non malignant diseases. In a next step, we set up a method for the quantification of RD using computarized image analysis -. We showed the interstitial progression of RD at distant follow-up (one year or more) in all the patients assessed. <BR> As drug resistance is a multifactiorial disease, unravelling its mechanisms requires a multiparametric analysis. Flow cytometry is particularly suitable for this analysis. However, detection of the intracellular antigens requires effective fixation and permeabilization of the cell membrane, a procedure which is not yet standardized. Accordingly, we assessed a new method of permeofixation based on the microwave heating method. We compared the results with two commercial chemical reagents (Orthopermeafix and Fix&Perm). The three methods were applied to the detection of intracellular epitopes of P-gp, bcl-2 and p53 on positive and negative control cell lines. An excellent correlation between the three methods was achieved. The microwaves heating based method appeared to be a valuable, versatile and cheap alternative compared to the two other reagents, allowing further clinical evaluation in resistant or refractory diseases
- Affiliations
Citations
Gala, J.-L. (1997). Facteurs de résistance cellulaire aux agents xénobiotiques : contribution des gènes MDR-1, BCL-2 et P53. https://hdl.handle.net/2078.5/111307