Influence of melanosome dynamics on melanoma drug sensitivity.

BACKGROUND: Malignant melanomas are intrinsically resistant to many conventional treatments, such as radiation and chemotherapy, for reasons that are poorly understood. Here we propose and test a model that explains drug resistance or sensitivity in terms of melanosome dynamics. METHODS: The growth and sensitivity to cisplatin of MNT-1 cells, which are melanotic and enriched with mature stage III and IV melanosomes, and SK-MEL-28 cells, which have only immature stage I and II melanosomes, were compared using clonogenic assays. Differences in pigmentation, melanosome stages, melanosome number, and cellular structures in different cell lines in response to various treatments were examined by electron microscopy. The relative numbers of melanosomes of different stages were compared after treatment with 1-phenyl-2-thiourea. The relationship between drug transporter function and endogenous melanogenic toxicity was assessed by treating cells with the cyclosporin analog PSC-833 and by assessing vacuole formation and cell growth inhibition. All statistical tests were two-sided. RESULTS: Endogenous melanogenic cytotoxicity, produced by damaged melanosomes, resulted in pronounced cell growth inhibition in MNT-1 cells compared with amelanotic SK-MEL-28 cells. The sensitivity to CDDP of MNT-1 cells was 3.8-fold higher than that of SK-MEL-28 cells (mean IC(50) for SK-MEL-28 and MNT-1 = 2.13 microM and 0.56 microM, respectively; difference = 1.57 microM, 95% confidence interval = 1.45 to 1.69; P = .0017). After treatment with 6.7 microM CDDP for 72 hours, the number of stage II-III melanosomes in surviving MNT-1 cells was 6.8-fold that of untreated cells. Modulation of MNT-1 cells to earlier-stage (II, II-III, III) melanosomes by treatment with the tyrosinase inhibitor 1-phenyl-2-thiourea dramatically increased CDDP resistance. Furthermore, PSC-833 principally suppressed MNT-1 melanotic cell growth via an elevation of autophagosome-like vacuolar structures, possibly by inhibiting melanosome membrane transporters. CONCLUSIONS: Melanosome dynamics (including their biogenesis, density, status, and structural integrity) regulate the drug resistance of melanoma cells. Manipulation of melanosome functions may be an effective way to enhance the therapeutic activity of anticancer drugs against melanoma.

Differential phosphorylation patterns of P-glycoprotein reconstituted into a proteoliposome system: insight into additional unconventional phosphorylation sites.

Membrane vesicles from the multidrug-resistant KB-V1 and KB-C1 cell lines overexpressing P-glycoprotein (Pgp), responsible for pleiotropic chemotherapeutic agents resistance, were solubilized with octyl-glucoside (OG-EX) and further fractionated on DEAE-sepharose column with increased concentrations of NaCl. The fraction containing Pgp (F3) was reconstituted into proteoliposomes (F3-PLP). Comparisons of the phosphorylation levels of Pgp achieved throughout the purification and reconstitution steps were addressed in this study. The [delta32 P] ATP-driven phosphorylation of Pgp was strongly increased in OG-EX, decreased in F3 and not detected in F3-PLP, when compared to Pgp phosphorylation in native plasma membrane vesicles. [delta32 P]ATP-phosphorylation of Pgp in F3-PLP could be restored by exogenously added PKC or by the catalytic sub-unit of PKA. The vanadate-induced hyperphosphorylation effect on Pgp by [delta32 P]ATP observed with plasma membrane vesicles was maintained in OG-EX, but was lost in F3 and did not enable labelling in F3-PLP. Enhancement of [delta32 P]-labelling of native Pgp via [delta32 P]ATP combined with GTP was maintained and also triggered phosphorylation of purified/reconstituted Pgp in F3-PLP as well. Altogether, our data suggest differential phosphorylation patterns of the transporter linked to environmental molecular composition (lipids, presence of detergent) and structure (unfolded versus embedded). In addition, restoration by GTP of Pgp phosphorylation by [delta32 P]ATP in the frame of F3-PLP suggests intra-molecular modulations and hints that other phosphorylation sites and processes, different from the classic ones involving PKC and/or PKA, may participate in the transporter's mechanism.

Modulation by the ATP/GTP ratio of the phosphorylation level of P-glycoprotein and of various plasma membrane proteins of KB-V1 multidrug resistant cells.

The level of protein phosphorylation is known to affect the properties of various membrane proteins. We have previously shown that GTP is capable of greatly enhancing the phosphorylation by [gamma-32P]ATP of P-glycoprotein (Pgp) from KB-V1 cells (3). Investigating the possibility of a general modulation of [gamma-32P]ATP plasma membrane protein phosphorylation, we found that phosphorylation of other membrane proteins are also modulated by various combinations of [ATP + GTP]. The ATP/GTP ratio giving the highest phosphorylation level depended on the protein studied. Modulation of the [gamma-32P]ATP-mediated phosphorylation of numerous membrane proteins requires hydrolysis of both ATP and GTP. ADP and GDP also increased [gamma-32P]ATP-driven phosphorylation but to a lesser extent than GTP. This plasma membrane endogenous phosphorylation activity was neither inhibited by specific inhibitors of protein kinase C, nor by inhibitors of cAMP- or cGMP-dependent protein kinases or of casein kinase II, respectively. Mastoparan, a G-protein regulator, increased the phosphorylation of some proteins that were already enhanced by the presence of [ATP + GTP] mixtures, especially proteins migrating in gels at the same position as P-glycoprotein.

P-glycoprotein, expressed in multidrug resistant cells, is not responsible for alterations in membrane fluidity or membrane potential.

Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. However, the physiological role of P-gp in normal tissues is not well understood. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity and membrane potential associated with P-gp expression, but interpretation of these studies is difficult, because most experimental cells have been selected for long periods in the presence of cytotoxic drugs and may have other host alterations. Therefore, we created two cell lines in which a transfected human MDR1 cDNA is repressed by tetracycline and induced in the absence of tetracycline. One cell line was derived from a mouse embryonic fibroblast cultured from a double (mdr1a/1b) knockout mouse, and the other was from a human HeLa cell line. Analysis of the kinetics of expression of P-gp showed that the mRNA had a half-life of approximately 4 h, and the protein had a half-life of approximately 16 h. P-gp cell surface expression (measured with monoclonal antibody MRK-16) and P-gp function (measured with a fluorescent substrate, rhodamine 123) was characterized by using fluorescence-activated cell sorting. No differences in membrane potential using the fluorescent probe oxonol or in membrane "fluidity" using fluorescent anisotropy probe or electron spin resonance probe were observed in the tet-repressible P-gp-expressing cells. In contrast, several drug-selected cells that express P-gp showed an increase in membrane fluidity and membrane potential. These results suggest that expression of P-gp per se has little effect on membrane fluidity or membrane potential, and it does not have H(+) pump activity. The changes in these parameters observed in drug-selected cells must reflect other host adaptations to drug selection.