Landry Gayet

Transcriptional regulators of the human multidrug resistance 1 gene: recent views.

The multidrug resistance (MDR) phenotype is the major cause of failure of cancer chemotherapy. This phenotype is mainly due to the overexpression of the human MDR1 (hMDR1) gene. Several studies have shown that transcriptional regulation of this gene is unexpectedly complex and is far from being completely understood. Current work is aimed mainly at defining unclear and new control regions in the hMDR1 gene promoter as well as clarifying corresponding signaling pathways. Such studies provide new insights into the mechanisms by which xenobiotic molecules might modify the physiological hMDR1 expression as well as the possible role of oncogenes in the pathological dysregulation of the gene. Here we report recent findings on the regulation of hMDR1 which may help define specific targets aimed at modulating its transcription.

Multidrug-resistant cancer cells contain two populations of P-glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic force microscopy and biochemical analysis

Considerable interest exists about the localization of P-gp (P-glycoprotein) in DRMs (detergent-resistant membranes) of multidrug resistant cancer cells, in particular concerning the potential modulating role of the closely related lipids and proteins on P-gp activity. Our observation of the opposite effect of verapamil on P-gp ATPase activity from DRM and solubilized-membrane fractions of CEM-resistant leukaemia cells, and results from Langmuir experiments on membrane monolayers from resistant CEM cells, strongly suggest that two functional populations of P-gp exist. The first is located in DRM regions: it displays its optimal P-gp ATPase activity, which is almost completely inhibited by orthovanadate and activated by verapamil. The second is located elsewhere in the membrane; it displays a lower P-gp ATPase activity that is less sensitive to orthovanadate and is inhibited by verapamil. A 40% cholesterol depletion of DRM caused the loss of 52% of the P-gp ATPase activity. Cholesterol repletion allowed recovery of the initial P-gp ATPase activity. In contrast, in the solubilized-membrane-containing fractions, cholesterol depletion and repletion had no effect on the P-gp ATPase activity whereas up to 100% saturation with cholesterol induced a 58% increased P-gp ATPase activity, while no significant modification was observed for the DRM-enriched fraction. DRMs were analysed by atomic force microscopy: 40-60% cholesterol depletion was necessary to remove P-gp from DRMs. In conclusion, P-gp in DRMs appears to contain closely surrounding cholesterol that can stimulate P-gp ATPase activity to its optimal value, whereas cholesterol in the second population seems deprived of this function.

Characterization of the typical multidrug resistance profile in human uveal melanoma cell lines and in mouse liver metastasis derivatives.

Uveal melanoma is the most common intraocular malignancy. To study its biology, stable cell lines provide a useful tool, but these are very difficult to obtain. A stable and rapidly growing human choroidal melanoma cell line composed of pure epithelioid cells was established and maintained for at least 4 years. In vivo transplantation into BALB/cByJ nude mice induced vascularized tumours at the injection sites. Interestingly, two of three cases produced a liver metastasis. Other uveal melanoma cell lines displaying different morphological aspects were also obtained. To avoid the bias due to uncertain immunologically based staining approaches, several methods were juxtaposed to establish the multidrug resistance (MDR) profile. All the uveal melanomas studied expressed significant levels of the MDR-related MDR1, MRP1 (MDR-related protein 1) and LRP/MVP (lung resistance protein/major vault protein) messenger RNAs (mRNAs), produced their corresponding proteins and were able to functionally extrude daunomycin. When compared with the established MEWO skin melanoma cell line, our data showed that both primary and metastatic uveal melanomas intrinsically expressed the typical MDR phenotype, which precludes the use of any anticancer drugs known to be substrates of MDR-related proteins to treat the disease. Moreover, it appears that the metastasizing process does not change the status of the MDR phenotype.

Données cytogénétiques actuelles : vers la monosomie du chromosme 3 comme principal facteur pronostique du mélanome uvéal

Le melanome uveal est la tumeur intra-oculaire la plus frequente. Les progres technologiques recents (l’analyse des microsatellites et l’hybridation comparative genomique notamment) ont permis de preciser la cytogenetique et l’anamnese de cette tumeur. La monosomie du chromosome 3 (50 a 60 % des melanomes uveaux) semble un evenement precoce et determinant dans la progression tumorale. Les melanomes portant cette anomalie ont un pronostic extremement pejoratif. Des travaux recents suggerent que ces derniers constituent une entite pathologique distincte des melanomes qui ont conserve leurs deux chromosomes. Les aberrations du chromosome 6 temoignent probablement d’une seconde voie d’entree dans le processus de cancerisation. A contrario , les gains de 8q paraissent de survenue plus tardive dans l’histoire naturelle de cette tumeur, comme le suggere leur frequence dans les tumeurs les plus grosses. Ces progres ont permis de converger vers les regions des chromosomes 3, 6 et 8 les plus probablement impliquees dans la tumorogenese et font esperer la decouverte des genes responsables dans les prochaines annees.

Multichimiorésistance du mélanome uvéal

In spite of important progress in the local treatment of uveal melanoma, the most frequent primitive intraocular tumor, 15%-30% of patients still die because of tumor metastasis. This tumor is characterized by constitutive chemoresistance, thwarting any attempt to control it using the usual chemotherapy protocols. The chemoresistance of uveal melanoma is mainly due to the typical multidrug resistance phenotype (MDR), which is linked to overexpression of membrane proteins that actively extrude anticancer drugs from the cell. Typical MDR is particularly complex in this tumor since several chemoresistance-related proteins are simultaneously produced. The negative prognostic significance of the overexpression of P-glycoprotein, the main representative among the typical MDR-related proteins, was shown in uveal melanoma. The atypical MDR phenotype, which refers to other chemoresistance mechanisms such as resistance to apoptosis also contributes to the chemoresistance of uveal melanoma. Thanks to the recent progress in molecular biology, the chemosensitization strategies of gene therapy approaches, which aim at weakening the pathological activity of MDR genes in cancer cells, are currently on the rise. This approach will disrupt current therapeutic strategies and necessarily improve and standardize the methods used to characterize the chemoresistance profile of this cancer. Indeed, we will have to know the genes to be targeted for each melanoma in order to induce cell chemosensitivity.