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Since the discovery of the structure of DNA in 1953 by Watson and Crick, our understanding of the genetic causes and the regulations involved in tumor development have hugely increased. The important amount of research developed since then has led to the development of gene therapy, which specifically targets and treats cancer cells by interacting with, and correcting their genetic material. This study is a review of the most accomplished research using gene therapy aimed at treating malignant ophthalmologic diseases, and focuses more specifically on uveal melanoma and retinoblastoma. Such approaches are remarkable regarding the efficiency and the cellular targeting specificity. However, gene therapy-based treatments are so recent that many long-term interrogations subsist. The majority of the reviewed studies are conducted in vitro or in murine models, thereby requiring several years before the resulting therapies become part of the daily ophthalmologists' arsenal. However, the recent spectacular developments based on advanced scientific knowledge justify an up-to-date review that would benefit the ophthalmologist community., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

LRP130 is a ubiquitous protein involved in cellular homeostasis, microtubule alteration, and transactivation of a few multidrug resistance genes. Its role in resistance to apoptosis in HepG2 and HUH7 hepatocarcinoma cells was investigated. Using shRNA-producing lentiviruses to down-regulate the LRP130 gene, we showed that i) LRP130 did not affect the capacity of hepatocarcinoma cells to extrude drugs since LRP130 down-regulation was insufficient to significantly reduce P-glycoprotein production in these cells, and ii) the expression of 11 apoptosis-related genes measured by PCR-array was significantly reduced. Interestingly, six of these genes encode extrinsic pathway proapoptotic proteins whose expression was higher in LRP130-non producing than in LRP130-producing HepG2 cells. Fluorescence microscopy confirmed this new anti-apoptotic role of LRP130, which is strengthened by a significantly reduced cytochrome c oxidase activity in LRP130-down-regulated hepatocarcinoma cells.

We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.

Background: Most uveal melanomas are currently treated by eye-preserving radiotherapies. However, for melanomas of the largest size or with initial complications, enucleation remains the reference treatment. Enucleation is called primary when it is proposed as the only local treatment option for a melanoma. There is very little literature on the use of primary enucleation after the introduction of conservative treatments. Our main goal was to evaluate the survival of melanoma patients treated by primary enucleation since the introduction of proton-beam therapy in France in 1991., Methods: All melanoma patients undergoing primary enucleation in our department between 1991 and 2002 were included in this retrospective study. The 5-year melanoma-specific survival rate was calculated using the Kaplan-Meier method. The multivariate prognostic analysis was performed using the Cox proportional hazards model., Results: Forty patients, representing 8% of all patients with choroidal uveal melanoma diagnosed and followed up in our department during an 11-year period, were included in the study. No patient was lost to follow-up. The 5-year melanoma-specific survival rate was 31.45% (SE: 7.8) after primary enucleation. Significant prognosis factors in the multivariate analysis were: tumor thickness > 12 mm (p = 0.03), anterior margin of the tumor involving the iris (p = 0.018), and presence of epithelioid cells (p = 0.02)., Conclusions: The very low survival rate reported reflects the evolution of primary enucleation, which is currently indicated only for melanomas with the worst prognosis. The knowledge of current post-enucleation survival rates represents an essential achievement for both correct assessment of conservative therapies and patient counseling.

p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood-brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood-brain barrier.

The multidrug resistance (MDR) phenotype of cancers has generated a large amount of research, owing to its constant fatal clinical outcome. Many studies have focused on the discovery of chemomodulators; however, in spite of this huge effort, the side effects that these products induce, and their additive toxicity when used in the presence of anticancer drugs, have led to the disaffection of the pharmaceutical industry and possibly slowed down research in pharmacological modulation. New tools developed using molecular biology techniques have opened the way for gene therapy and given birth to new therapeutic hopes. However, these discoveries and especially their clinical applications have slowed due to a lack of knowledge of the systems that finely regulate the MDR genes. This weakness explains why, to date, no general review has focused on the possibilities of gene therapy of MDR derived form the strategic options now available. Based on molecular foundations and recent fundamental discoveries, we seek to inform clinicians of the therapeutic hopes for chemoresistant tumors brought about by potent and specific new tools such as transcriptional decoys, interfering RNAs, etc. After describing the causes and mechanisms of MDR, we critically review these new strategies and their corresponding clinical trials.

Uveal melanoma is the most frequent intra-ocular cancer. The recent development of new chromosome-related technologies have permitted the elucidation of both the cytogenetics and the natural history of this disease. Fifty to 60% of uveal melanomas are linked to a monosomy 3, which appears as an early and determinant event in tumor progression. Tumors with this anomaly have a very poor prognosis. Recent work suggests that this category of uveal melanoma represents a distinct pathologic entity from that associated with normal disomy 3. Chromosome 6 aberrations probably constitute a second entry point into the process of cancerogenesis, while gains in 8q seem to appear later in the natural history of uveal melanomas due to their higher frequency in larger tumors. Other anomalies will be reviewed. In spite of significant improvements in the local treatment of uveal melanoma, many patients die due to tumor metastasis. This disease is characterized by a constitutive chemoresistance whose typical multidrug resistance phenotype (MDR) is particularly complex since different combinations of several resistance proteins are simultaneously produced. Regulation of the expression of these proteins is a research priority, increasingly so as gene therapy-dependent chemosensitization strategies expand. Therefore, the development and improvement of methods to determine the chemoresistance profile become a crucial objective today in the therapeutic strategies against uveal melanoma.

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.

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.

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.

P-glycoprotein (P-gp) is the most well-known ATP-binding cassette (ABC) transporter involved in unidirectional substrate translocation across the membrane lipid bilayer, thereby causing the typical multidrug resistance (MDR) phenotype expressed in many cancers. We observed that in human CEM acute lymphoblastic leukemia cells expressing various degrees of chemoresistance and where P-gp was the sole MDR-related ABC transporter detected, the amount of esterified cholesterol increased linearly with the level of resistance to vinblastine while the amounts of total and free cholesterol increased in a nonlinear way. Membrane cholesterol controlled the ATPase activity of P-gp in a linear manner, whereas the P-gp-induced daunomycin efflux decreased nonlinearly with the depletion of membrane cholesterol. All these elements suggest that cholesterol controls both the ATPase and the drug efflux activities of P-gp. In addition, in CEM cell lines that expressed increasing levels of elevated chemoresistance, the amount of P-gp increases to a plateau value of 40% of the total membrane proteins and remained unvaried while the amount of membrane cholesterol increased with the elevation of the MDR level, strongly suggesting that cholesterol may be directly involved in the typical MDR phenotype. Finally, we showed that the decreased daunomycin efflux by P-gp due to the partial depletion of membrane cholesterol was responsible for the efficient chemosensitization of resistant CEM cells, which could be totally reversed after cholesterol repletion.

Uveal melanoma is the most frequent intraocular cancer. The recent development of new technologies such as microsatellite analysis and comparative genomic hybridization have elucidated both the cytogenetics and the natural history of this disease. Fifty to 60% of uveal melanomas are linked to monosomy 3, which appears as an early and determinant event in tumor progression. Tumors with this anomaly have a very poor prognosis. Recent work suggests that this category of uveal melanomas represents a distinct pathological entity from that associated with normal disomy 3. Chromosome 6 aberrations probably make up a second entry point into the process of carcinogenesis, while gains in 8q seem to appear later in the natural history of uveal melanoma because of their higher frequency in larger tumors. Progress in genome analysis has identified regions in chromosomes 3, 6, and 8 as those most probably involved in tumorigenesis. It is to be hoped that this will soon lead to the discovery of the genes responsible.

The MDR1 gene is a key component of the cytotoxic defense network and its overexpression results in the multidrug resistance (MDR) phenotype. However, the molecular mechanisms that regulate the MDR1 gene and coordinate multiple MDR-related genes expression are poorly understood. In a previous study, we identified a new 12 bp cis-activating region in the 5'-flanking region of the human MDR1 gene, which we called inverted MED1. In the present study, we characterized the precise binding element, which we named invMED1, and revealed the presence of the LRP130 protein as the nuclear factor. Its binding intensity increases with the endogenous MDR1 geneexpression and with the MDR level of CEM leukemia cells. Interestingly, the LRP130 level did not vary with the chemoresistance level. We observed the involvement of LRP130 in the transcriptional activity of the MDR1 gene promoter, and moreover, in that of the MDR-related, invMED1-containing, MVP gene promoter. We used siRNAs and transcriptional decoys in two unrelated human cancer cell lines to show the role of the invMED1/LRP130 couple in both MDR1 and MVP endogenous genes activities. We showed that invMED1 was localized in the -105/-100 and -148/-143 regions of the MDR1 and MVP gene promoters, respectively. In addition, since the invMED1 sequence is primarily located in the -160/-100 bp region of mammalian MDR-related genes, our results present the invMED1/LRP130 couple as a potential central regulator of the transcription of these genes.

The typical multidrug resistance phenotype (MDR), the major cause of failure of cancer chemotherapy, is the result of the overexpression of the human MDR1 gene, the regulation of which is still incompletely understood. Using several EMSA experiments, we have identified a new regulatory sequence located from -103 to -98 bp relative to the +1 start site in the MDR1 promoter region. This sequence, which we called inverted MED-1, acts as a cis-activator for this gene. In transient transfection experiments of highly resistant human lymphoblastic CEM/VLB5 cells, its deletion from the promoter region is responsible for 60% inhibition of the MDR1 transcriptional activity. This sequence specifically binds a nuclear protein of about 150-160 kDa. We showed that its binding capacity is related to the chemoresistance level of the studied cell lines and may reflect the increased transcriptional activity of the MDR1 gene in multidrug-resistant cells.

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.

Several recent studies have shown that proteins of the cadherin-catenin complex are not only involved in cell-cell adhesion but also in the proliferation and differentiation processes. For the first time, we investigated the effect of the quantity of cytoplasmic beta-catenin on dermal fibroblast proliferation by overexpressing human beta-catenin in human dermal fibroblasts. Our results show that dermal fibroblasts overexpressing normal beta-catenin or a stabilized beta-catenin mutant have a higher growth rate than control fibroblasts. Moreover, when confluence is reached, the number of fibroblasts is increased when the cells overexpress beta-catenin suggesting a role for beta-catenin in the regulation of contact growth arrest. Finally, by comparing proliferation in normal dermal fibroblasts and dermal fibroblasts expressing E-cadherin we observed a negative regulatory effect of E-cadherin expression on fibroblast proliferation. These data demonstrate the involvement of beta-catenin and cadherin in the dermal fibroblast proliferation process and in contact growth arrest.

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.

P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.

Up to now, removal of sodium dodecyl sulfate (SDS) from proteins in terms of restoration of their activity was an unsolved problem. A general procedure using ceramic hydroxyapatite (HAP) chromatography was developed for the complete removal of SDS bound to soluble or membrane proteins. This procedure involves (i) the binding of the SDS-protein complexes onto the ceramic hydroxyapatite column, (ii) extensive washing of bound proteins with phosphate buffer containing a mild detergent to exchange SDS, (iii) elution of the retained protein by increasing the phosphate concentration. Using this approach, complete exchange of [35S]SDS into a nonionic detergent such as dodecyl maltoside was achieved with a 90-100% protein recovery. The efficiency of protein-bound SDS removal is very likely due to the combined effect of phosphate ions and the hydrophobic tail of nonionic detergent: acting together, they are able to displace SDS molecules from their protein-binding sites. The advantages of this HAP-mediated SDS removal method include high efficiency, rapidity, simplicity and general applicability to a wide variety of detergents and soluble or membrane proteins. Of utmost importance, SDS-treated P-glycoprotein, glutamate dehydrogenase, and lysozyme fully recovered their enzymatic activities after HAP chromatography, including lysozyme electroeluted from SDS-polyacrylamide gel electrophoresis. This demonstrates that reactivation of SDS-treated protein can be achieved, provided that SDS is completely removed under mild conditions.

Multidrug resistance of cancer cells is a serious problem in the treatment of tumors and is the leading cause of the frequent failure of chemotherapy. Cancer cell chemoresistance is based on the development of several mechanisms among which one of the most important concerns the overexpression of membrane proteins to remove cytotoxic compounds from the cytoplasm. The leading archetype of these proteins is the P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, or traffic ATPases. In the recent past years, new non P-glycoprotein membrane proteins, several of which being members of the ABC superfamily of transporters, and new genes, have been discovered in cancer cells with a multiple drug resistance phenotype. In this article, we briefly review these newly discovered entities.

Plasma membrane P-glycoprotein is known as an ATP-dependent drug efflux pump that confers multidrug resistance to tumor cells. None of the reported purification procedures worked properly for our P-glycoprotein-overproducing cell lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10, and human lymphoblastic leukemia CEM/VLB5 cells. We have thus developed a general procedure for efficient purification of P-glycoprotein by combining solubilization with sodium dodecyl sulfate and chromatography on ceramic hydroxyapatite. This procedure was successful for the three cell lines and yielded 70% of the P-glycoprotein present in the starting plasma membranes with more than 99% purity. After exchanging sodium dodecyl sulfate into dodecyl maltoside and reconstitution into liposomes, purified P-glycoprotein exhibited a specific ATPase activity of about 200 nmol/min/mg, which was very similar to that obtained for P-glycoprotein solubilized and purified with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. This ATPase activity was sensitive to orthovanadate inhibition and stimulated by verapamil and other drugs. More importantly, drug transport properties of the reconstituted P-glycoprotein were comparable with those of P-glycoprotein embedded in plasma membranes. Since it is virtually devoid of lipids, this preparation is suitable for both functional and structural investigations.

The cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction. NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage. Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity. TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations. A similar affinity binding was monitored by quenching of intrinsic fluorescence. The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides. TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%). The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment. A high quenching was observed upon nucleotide interaction with similar affinity. The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.

The central glycolytic and oxidative pathways and the ATP-producing mechanisms differ in sane and malignant cells by their regulation and dynamics. Fast-growing, poorly-differentiated cancer cells characteristically show high aerobic glycolysis. In the same way, cholesterol biosynthesis, which occurs by normal pathways in tumors, is deficient in feed-back regulation and in sterol-transport mechanisms. Other metabolic ways are deficient, as for example, intramitochondrial aldehyde catabolism, at the origin of a possible acetaldehyde toxicity, which can be circumvented by the synthesis of an unusual and neutral product for mammalian cells acetoin, through tumoral pyruvate dehydrogenase. If most of the glycolytic pyruvate is deviated to lactate production, little of the remaining carbons enter a truncated Krebs cycle where citrate is preferentially extruded to the cytosol where it feeds sterol synthesis. Glutamine is the major oxidizable substrate by tumor cells. Inside the mitochondrion, it is deaminated to glutamate through a phosphate-dependent glutaminase. Glutamate is then preferentially transaminated to alpha-ketoglutarate that enters the Krebs cycle. Glutamine may be completely oxidized through the abnormal Krebs cycle only if a way of forming acetyl CoA is present: cytosolic malate entering mitochondria is preferentially oxidized to pyruvate + CO2 through an intramitochondrial NAD(P)(+)-malic enzyme, whereas intramitochondrial malate is preferentially oxidized to oxaloacetate through malate dehydrogenase, thus providing a high level of intramitochondrial substrate compartmentation. These and other regulatory aberrations in tumor cells appear to be reflections of a complex set of non-random phenotypic changes, initiated by expression of oncogenes.

Isolated mitochondria from highly glycolytic Ehrlich and AS30-D tumor cells have a 12.4- and a 2.3-fold higher cholesterol level, respectively, than that of rat liver mitochondria. The passive proton permeability of Ehrlich and AS30-D tumor inner membrane mitochondria is, respectively, 4- and 1.4-fold lower than that of rat liver mitochondrial membrane. This feature is accompanied by a lower proton leak current in tumor mitochondria. A 3.5-fold cholesterol enrichment of rat liver mitochondria decreases their passive proton permeability by a factor of 2, thus establishing a direct relationship between the cholesterol contents of mitochondrial membranes and the passive proton permeability. Creatine kinase activity is present in the cytosol of these cells and is mostly represented by the BB isoform. Since AS30-D tumor cells' treatment with the creatine analogue beta-guanidinopropionic acid decreases their life span and viability, creatinine kinase is an indispensable enzyme entering a main energy distribution pathway starting from mitochondrial ATP, through glycolysis and creatine phosphorylation, to satisfy the large energy demands of tumor cell division.

The effect of acetyl-L-carnitine on succinate oxidation, adenine nucleotide pool and lipid composition of synaptic and 'free', non-synaptic, mitochondria in cerebral hemispheres of senescent rats has been studied. Fisher rats (24- or 28-month-old) were treated with acetyl-L-carnitine (300 mg/kg body wt., intraperitoneally (i.p.)) 3 h before being killed. Oxygen consumption was measured using succinate as a substrate; adenine nucleotides and lipids were analyzed by high performance liquid chromatography (HPLC). Acetyl-L-carnitine reverses, in synaptic mitochondria, the age-related decrease in the respiratory control ratio due to a higher state 4 respiration rate. Administration of acetyl-L-carnitine to senescent rats does not affect the total adenine nucleotide pool of synaptic and non-synaptic mitochondria which was unchanged with age. Finally, pretreatment of senescent rats with acetyl-L-carnitine brings the cholesterol and phospholipid contents of synaptic mitochondria, reduced in senescent rats, to the adult level; pretreatment of adult rats has no such effect. Altogether these results suggest that acetyl-L-carnitine is able to reverse age-related deficits of brain mitochondria.

In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d.

Acetoin, an unusual metabolite of highly glycolytic mammalian tumor cells, is synthesized from decarboxylated pyruvate and active acetaldehyde in mitochondria. It plays important roles in the regulation and detoxification of pyruvate metabolism through pyruvate dehydrogenase. We show in this report the inhibitory effect of acetoin on succinate oxidation by Ehrlich tumor cell mitochondria, and thus its regulatory role on intermediate metabolism. Acetoin utilization by Ehrlich mitochondria may lead to small quantities of citrate formation which increase the already increased cholesterol synthesis of cancer cells. Membranes, in particular the inner mitochondrial membrane, flooded with cholesterol, show a proton passive permeability twice as low as that of control mitochondrial membranes, a feature that may be related to drastic changes in membrane potential-dependent metabolism of cancer cells.

The N-terminal amino acid residues of the beta-subunit in the rat liver mitochondrial ATPase - ATPsynthase have been identified by direct microsequencing after electrophoresis of either purified F1 or F0F1. The mature rat liver beta-subunit begins by two alanine residues that precede the glutamine recently proposed as the first amino acid of the sequence (Boulet, D., Poirier, J. and Côté, C., 1989, Biochem. Biophys. Res. Commun. 159, 1184-1190). This result indicates that the proteolytic cleavage of the beta-subunit precursor may occur at the level of this first alanine. This may be important in the understanding of proteolytic processing events which lead to the assembly of the ATPase-ATPsynthase subunits during mitochondrial biogenesis.

[14C]Pyruvate was rapidly non-oxidatively decarboxylated by Ehrlich tumor mitochondria at a rate of 40 nmol/min/mg of protein in the presence or absence of ADP. A search for decarboxylation products led to significant amounts of acetoin formed when Ehrlich tumor mitochondria were incubated with 1 mM [14C] pyruvate in the presence of ATP. Added acetoin to aerobic tumor mitochondria was rapidly utilized in the presence of ATP at a rate of 65 nmol/min/mg of protein. Citrate has been found as a product of acetoin utilization and was exported from the tumor mitochondria. Acetoin has been found in the ascitic liquid of Ehrlich and AS30-D tumor-bearing animals. These unusual reactions were not observed in control rat liver mitochondria.

Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

A glucose injection given immediately after birth delays the maturation which normally occurs in rat liver mitochondria and which increases the rate of ATP synthesis coupled to succinate oxidation from a low value at birth to the adult value a few hours after birth [R. Meister, J. Comte, L. Baggetto, C. Godinot and D. C. Gautheron, Biochim. biophys. Acta 722, 36 (1983)]. Alkylxanthine (pentoxifylline, HWA 285) administration at birth has no effect on the maturation of mitochondria prepared from 2-hr-old rat livers while DBcAMP administration increases their RCR and their rate of ATP synthesis. On the contrary, both alkylxanthines and DBcAMP reverse the glucose-induced inhibition of mitochondrial maturation. This DBcAMP effect cannot be mimicked by butyrate and is therefore related to cAMP. The cAMP content of rat liver increases during this postnatal period in both control and glucose-treated rats, although glucose administration tends to decrease the level of cAMP. Alkylxanthine administration restores after 2 hr the cAMP level in glucose-treated animals. The variations of RCR could not be completely correlated with the level of cAMP. The possible involvement of other factors in the mitochondrial maturation and the glucose effect is discussed.