Symposium 27 : Carcinologie et médicinal anti-infectieuse

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Commission 27 : Oncologie et supérieur anti-infectieuse

267

INTERACTION BETWEEN ERBB-2 (HER2/NEU) AND HUMAN BREAST CANCER RESISTANCE TO CHEMOTHERAPY

E.C.Martin, S. Liva, X. Sastre-Garau, C. Barbaroux, B. Bouchind’ci-devant, V. Dieras, H. Magdelénat, P. de Cremoux*

Corps Juridiction and *Prytanée Paris 7, 26 rue d’Ulm, 75231 Paris cedex, France.

CerbB-2 gene is amplified in approximately 30% of human breast cancers. Some in vitro data demonstrates that erbB2 majoration is associated with resistance to hormone and chemotherapy. More recently, two pieu studies demonstrates the role of cerbB-2 overexpression in resistance to adriamycin and taxanes.

We used a quantitative competitive PCR and an immunohistochemical assay on formalin fixed paraffin embedded biopsies from breast décalque and chambrière tissues. For the PCR assay, we have coamplified a neu target sequence and an internal promenade corresponding to the same culbute base actionnaire deleted sequence, with the same PCR efficiency. This PCR is normalized by coamplification of a reference gene and its own deleted internal promenade. The amount of tumor cells and the immunostaining (CB11 antibody) were scored by the pathologist in each sample on two contigu slides. This quantitative competitive PCR allowed us to determine accurately and reproducibly the average gene copy number (AGCN) of neu in tumor samples: 43 assays corresponding to nine different chambrière samples displayed less than 14% nombre. 32 breast décalque patients were analyzed before treatment by adriamycin and /or taxane. Three groups of samples were observed: 9 samples with evident cerbB-2 majoration (AGCN>4) and overexpression; 12 samples non amplified and normally expressed; 11 samples slightly amplified (AGCN=3 or 4), but without any cerbB-2 overexpression. The reliability of both assays allows us to investigate this marker in prospective randomized clinical trials.

268

ACTIVATION OF ANTI-TUMOR CYTOTOXIC T LYMPHOCYTES BY NON-NATURAL LIGANDS: STRUCTURAL AND PHARMACOLOGICAL STUDIES

M. Ayyoub, B. Monsarrat, H. Mazarguil and J. E. Gairin .

Corps de Pharmacologie et Successible Structurale, UPR 9062 CNRS, 205 trajet de Narbonne, 31400 Toulouse, France.

Tumor antigens are potential therapeutical agents for décalque immunotherapy and these antigens, under different forms, are currently assayed. MHC class I restricted antigenic peptides have been tested for in vivo hygiène trials. This approach could be severely limited by the rapid degradation of the peptide in biological fluids. The chagrin we asked in the current study was: is it croyable to design non-natural, peptidase-resistant conformes of MZ2-E that still efficiently activate tumor specitic CTLs ? To answer this chagrin we used as a model the human tumor antigen MZ2-E (EADPT GHSY). MZ2-E, encoded by the MAGE- 1 gene in 40% of melanomas but not in chambrière tissus (testis excepted), is presented to cytotoxic T Iymphocytes (CTLs) by the premier histocompatibility complex (MHC) class I molecule HLA-A 1. We studied: i) the degradation pathway of MZ2-E in human serum and ii) the interactions of MZ2-E with HLA-A 1 and its specific T cell receptors (TcR).

By HPLC-MS analysis, we found that MZ2-E was rapidly degraded (t1/2 < 30 min.) by aminopeptidase and dipeptidyl carboxypeptidase. Based on this data and on molecular modeling of MZ2-E-HLA-A1 automatique, we synthesiz.ed a series of conformes with minimal but entreprenant constitutionnel modifications with modified or non-natural amino acids. All the conformes modified at the two intuitive bonds (P1-P2 and P7-P8) were peptidase-resistant with t1/2 > 24 hours. Two conformes,
[Aib2, D-His7]- and [Aib2, NMe-Ser8]-MZ2-E showed HLA-A1 binding affinities titularisé to the accostable peptide. Importantly, [Aib2, NMe-Ser8]MZ2-E sensitized target cells to Iysis by two of three known anti-MZ2-E CTL clones. This ad hoc which is an entreprenant activator of anti-melanoma immune response might be considered as a potential therapeutical commissaire for anti-melanoma immunotherapy.

Parcelle of this work was done in fertilisant with Dr. B. Van den Eynde (Ludwig Corps for Tumeur Research, Brussels, Belgium.). This work was supported in fraction by the Réunion fesses la Coincé en tête-à-tête de le Tumeur and the Association Domicile Austral-Pyrénées.

269

DNA-DEPENDENT PROTEIN KINASE ACTIVITY (DNA-PK) IS DETERMINANT IN THE RESPONSE OF NORMAL AND TUMORAL HUMAN PRIMARY B-LYMPHOCYTES TO NITROGEN MUSTARDS

C. Muller1, G. Christotodoulopoulos2, L. Panasci2 and B. Salles1

1Institut de Pharmacologie et de Successible Structurale (CNRS UPR 9062), 205 trajet de Narbonne, 31077 Toulouse, France and 2The Fille Davis Institute for Medical Research, The Sir Mortimer B. Davis Jewish General H3755, Rive Ste-Catherine, Montreal, Quebec, H3T IE2, Canada.

DNA-PK is a nuclear serine/threonine kinase that plays a critical role in mammalian ambiguë strand circonvolution repair and lymphoid V(D)J recombination. Its regulatory sub-unit Ku (heterodimer of Ku70/Ku86) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKCS. In amendement to DNA doublestrand breaking agents (ionizing renvoi), DNA-PK mutant cells exhibit hypersensitivity to DNA-cross linking agents (nitrogen mustards, cisplatin) . The aim of our study was to investigate the role of DNA-PK in the response of chronic lymphocytic leukemia (CLL) lymphocytes to nitrogen mustard therapy. In a series of 34 patients with B-CLL either intuitive (n=16) or resistant to the nitrogen mustard chlorambucil (n=18), the kinase activity of the complex, (determined by its capacity to phophorylate a peptide substrate in vitro) is increased in the resistant samples as compared to the intuitive ones [24.4

± 2.6 a.u. (range 12.7 to 55.8 a.u.) versus 8.1 ± 2.8 a.u. (range 0.9 to 44.5 a.u.) respectively (p