Characterization of the intracellular distribution and binding in human adenocarcinoma cells of N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), a photoaffinity analogue of the antitumor diarylsulfonylurea sulofenur.
A photoactivatable diarylsulfonylurea, N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), has been examined as a potential probe to elucidate the intracellular distribution and binding of antitumor diarylsulfonylureas. Our results demonstrated that against the human colon adenocarcinoma cell line GC3/c1, LY219703 is a more potent cytotoxic agent than N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (Sulofenur; ISCU), whereas a subline selected for resistance to ISCU was cross-resistant to LY219703, suggesting a similar mechanism of action or resistance. Cellular pharmacology studies showed that [3H]LY219703 concentrated in cells, and that its concentrative accumulation could be inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that it was similar to other antitumor diarylsulfonylurea (DSU) drugs examined. Accumulation of [3H]LY219703 in cells was progressively decreased by co-incubation with increasing concentrations of ISCU, and in cells incubated to steady state with 1 microM [3H]LY219703, ISCU (500 microM) rapidly displaced the photoaffinity analogue. Photoactivation of [3H]LY219703 by UV light (5-30 min) prevented efflux of radiolabeled drug during a 20-min wash in drug-free medium. Subsequent distribution studies showed that 89% of the radiolabel was associated with particulate components, and that approximately 20% of the radiolabel in the 320,000 g pellet could be extracted with acetone. Subcellular distribution showed approximately 6% associated with nuclei, 52% with mitochondria and 26% in the microsomal fraction. The effect of UV photoactivation on the distribution of [3H]LY219703 in soluble and particulate fractions was also examined in GC3/c1 cell preparations sonicated prior to being incubated with [3H]LY219703. A high proportion (83%) of radiolabel associated with the 100,000 g pellet, and distribution between soluble and particulate fractions was not altered by UV irradiation. Specific activities of protein in the 100,000 g supernatant and pellet were 0.186 and 0.537 nmol/mg, respectively. Putative binding species were analyzed by SDS-PAGE. Using SDS-PAGE, ten major binding proteins were identified in 320,000 g pellets from GC3/c1 cells: M(r) 110, 88, 76, 70, 64, 58, 48, 36, 26, and 24 kDa, and at least four of these (88, 70, 64, and 36 kDa) were also detected in mitochondria isolated from cells after photoactivation, or in mitochondrial preparations that were incubated with [3H]LY219703 and photoactivated after isolation from cells. Results suggested that under conditions of SDS-PAGE some dissociation of radiolabel from proteins also occurred. Binding of [3H]LY219703 to a model substrate, bovine serum albumin, and the effect of denaturing conditions used for sample preparation prior to SDS-PAGE, showed that relatively mild denaturing conditions (23 degrees, 2 hr) caused significant dissociation of radiolabel from BSA.(ABSTRACT TRUNCATED AT 400 WORDS)
Houghton
P J
PJ
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101-0318.
Sosinski
J
J
Thakar
J H
JH
Boder
G B
GB
Grindey
G B
GB
D000230:Adenocarcinoma / Q000378:metabolism*; D000345:Affinity Labels; D000970:Antineoplastic Agents / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D004351:Drug Resistance; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D010777:Photochemistry; D013347:Subcellular Fractions / Q000378:metabolism; D013453:Sulfonylurea Compounds / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D000970:Antineoplastic Agents; C092475:LY 219703; D013453:Sulfonylurea Compounds; C058179:sulofenur