Validation of an in vitro screen for phospholipidosis using a high-content biology platform.

Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to 100 micro mol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, non-phospholipidogenic controls were correctly called.

Interaction of an estramustine photoaffinity analogue with cytoskeletal proteins in prostate carcinoma cells.

To identify specific drug targets of the antimitotic drug estramustine, a photoaffinity analogue, 17-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]estradiol-3-N-bis(2- chloroethyl)carbamate, was synthesized and reacted in competition assays with cytoskeletal protein preparations. By attaching the photoaffinity ligand to the 17 beta-position of the steroid D-ring, the cytotoxic properties of the drug were maintained. In cytoskeletal protein preparations from human prostate carcinoma cells (DU 145) or a clonally selected, estramustine-resistant cell line (E4), the major microtubule-associated protein (MAP) present was MAP4. In both cytoskeletal fractions and reconstituted microtubules, 17-O-[[2-[3-(4-azido-3-[125I]iodophenyl)propionamido] ethyl]carbamyl]estradiol-3-N-bis(2-chloroethyl)carbamate bound to both MAP4 and tubulin. From competition assays, the apparent binding constant for MAP4 from DU 145 cells was 15 microM. Similar calculations for tubulin gave values of 13 microM (bovine brain), 19 microM (DU 145 wild-type cells), and 25 microM (E4 cells). The identification of these cytoskeletal proteins as specific drug targets provides a direct explanation for the antimicrotubule and antimitotic effects of estramustine.

P-glycoprotein binding and modulation of the multidrug-resistant phenotype by estramustine.

BACKGROUND: Previous preclinical studies of combinations of estramustine and vinblastine or paclitaxel (Taxol) have shown that it is possible to achieve a greater than additive cytotoxicity with these antimicrotubule drug combinations. Phase II studies in hormone-refractory prostate cancer have demonstrated clinical antitumor activity of sufficient magnitude to stimulate further laboratory and clinical studies of these drugs combinations. PURPOSE: Our purpose was to characterize the interactions of estramustine with P-glycoprotein and to determine its effects. METHODS: Standard laboratory techniques were used to study the effects of estramustine on intracellular drug concentrations, cytotoxicity, and induction of messenger RNA (mRNA) for the MDR1 (also known as PGY1) gene. Using a photoaffinity analogue of estramustine 17-0-[[2-[3-(4-azido-3-[125I]-iodophenyl) propionamido]ethyl]-carbamyl]estradiol-3-N-bis(2-chloroethyl)ca rba mate ([125I]AIPP-estramustine), binding to the membrane proteins of human ovarian (SKOV3) and their multidrug-resistant counterpart SKVLB1 cells was studied. Southern-blot analysis was performed on DNA extracted from human prostate carcinoma wild-type DU145, estramustine-resistant cell line (E4), and SKVLB1 cells. RESULTS: Membrane fractions from SKOV3 and SKVLB1 cells were analyzed for proteins that could be photoaffinity labeled with [125I]AIPP-estramustine. Competitive inhibition of this binding was achieved with excess concentrations of (in order of efficacy) estramustine, vinblastine, verapamil, progesterone, and to a lesser degree, by paclitaxel but not with estramustine phosphate, estradiol, and estriol. SKVLB1 cells accumulated much less [3H]vinblastine and [3H]paclitaxel than did SKOV3 cells. Estramustine caused a concentration-dependent enhancement of drug accumulation in the SKVLB1 cells to a maximum of approximately 12-fold. No effect of estramustine was apparent for the wild-type SKOV3 cells. In comparison with verapamil, estramustine was less effective as a modulator; however estramustine demonstrated good chemosensitizing activity in combination with actinomycin D and vinblastine. Neither short-term, low-dose no longer-term, higher concentration were found to produce measurable transcript (mRNA for the MDR1 gene levels. Such data suggest that, at least levels. Such data suggest that, at least for two distinct human cell line (SKOV3 and DU145), estramustine does not induce the overexpression of the MDR1 gene. CONCLUSION: It is apparent from the P-glycoprotein data that estramustine interacts with this efflux pump, altering intracellular drug accumulation. Overall, the nonempiric basis for including estramustine in clinical protocols that contain other multidrug-resistant drugs is strengthened by the present data.