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Cholesterol increases in a nonlinear way with the level of chemoresistance of the human CEM acute lymphoblastic leukemia and the membrane cholesterol can modulate the P-glycoprotein ATPase activity. The analysis highlights the complex effect of cholesterol on the multidrug resistance (MDR) phenotype and also the possibility of chemosensitizing resistant cells by the modulation of their membrane cholesterol content.

A study was conducted to analyze the overexpression and purification of the C-terminal nucleotide-binding domain of a parasite Pgp-like multidrug transporter that is encoded by the ltrmdr I of Leishmania tropica. Direction interaction with various classes of flavonoids and reversal of the multidrug resistance was also examined. Results indicated that flavonoids that bind with high affinity to the cytosolic domain of the transporter increases daunomycin accumulation in a L. tropica line overexpressing the transporter.

A hexahistidine-tagged C-terminal nucleotide-binding domain ([H.sub.6]-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2[prime](3[prime])-N-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated to fluorescence resonance-energy transfer. [H.sub.6]-NBD2 also bound all flavonoids known to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells, with similar affinities and relative efficiencies. Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4[prime]-methoxy 3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of [H.sub.6]-NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by preincubation with ATP, or partly displaced upon ATP addition. Interestingly, kaempferide was also able to partly prevent the binding of the antiprogestin RU 486 to a hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, which occurred at the ATP site and extended to the vicinal steroid-interacting hydrophobic region, was completely prevented or displaced by kaempferide. All results indicate that flavonoids constitute a new class of modulators with bifunctional interactions at vicinal ATP-binding site and steroid-interacting region within a cytosolic domain of P-glycoprotein.

Experiment was conducted to prove the hypothesis that a longer 371-705 domain, including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacts with steroids that have been proven to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Results prove that both ATP and steroid-binding regions are present on the cytosolic extended-NBD1 domain of P-glycoprotein which constitutes a promising tool for the identification of amino acids involved in both types of binding.

summary: 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. [Copyright &y& Elsevier]

Epithelialization of normal wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the barrier function. Keratinocyte migration is one of the most earliest and crucial event determining the efficiency of the overall wound repair process. Laminin 332, composed by the association of α3, β3 and γ2 chains, is a major adhesion substrate for keratinocytes and is known for its role in supporting cell adhesion and migration during wound repair. The α3 chain comprises a large globular region in its carboxyl-terminal end, which consists of five homologous globular domains (LG1-LG5), known to be involved in cellular interactions. Recent findings have suggested that the α3 chain C-terminal domains LG45 may have a role to play during the epithelialization phase in wound repair. In the present study, we have analyzed whether a peptide mimicking the major heparin binding sequence KKLRIKSKEK in α3LG45 may interact with keratinocytes to promote cell adhesion and migration. In vitro experiments supported this hypothesis and revealed that the KKLRIKSKEK peptide induces human primary keratinocyte adhesion and has the ability to promote keratinocyte migration when added in the culture medium. To examine the peptide efficacy in vivo, the KKLRIKSKEK peptide was applied over partial-thickness cutaneous wounds in pigs. Compared with vehicle-treated cutaneous wounds, the peptide application significantly promoted early-stage wound healing by accelerating re-epithelialization. Additional beneficial effects such as reduced inflammatory response and decreased granulation tissue formation were also noticed in the peptide-treated wounds.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-alpha (ERalpha) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERalpha in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERalpha remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERalpha ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERalpha monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERalpha ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

The bulky side chains of antiestrogens hinder folding of the ligand binding domain (LBD) of estrogen receptors (ERs) into a transcriptionally active conformation. The presence of a tertiary amine in the side chain of raloxifene, which interacts with a negatively charged residue in helix H3 of the ER LBD [Asp351 in human (h)ERalpha], is important for antiestrogenicity in animal and cellular models. To better understand the molecular basis of the differential activity of tamoxifen and raloxifene, we have examined the influence of tertiary amine substituents and of mutations at position 351 in hERalpha on the activity profiles of tamoxifen derivatives. Results obtained in several cellular model systems suggest that the degree of antagonist activity of tamoxifen derivatives does not strictly correlate with the basicity of the side chain but depends on an optimal spatial relationship between the tertiary amine of these antiestrogens and the negative charge at position 351. Although altering the position of the negative charge at residue 351 (mutation D351E) had little effect on transcriptional activity in the presence of tamoxifen, it drastically increased the partial agonist activity of a tamoxifen derivative with improved antagonist activity as well as that of raloxifene. Our results suggest that contrary to raloxifene, tamoxifen and most of its derivatives do not interact with Asp351 in an optimal manner, although this can be improved by modifying tertiary amine substituents.