Acidification and Recovery Results in Nuclear Accumulation of Supravital Dyes during Interphase

Abstract

Recent studies using real time imaging demonstrated relative nuclear insulation for ion-size particles. We show here that acidification and recovery converted the insulated interphase nuclei of KB carcinoma and nontumorigenic Chang cells into intense nuclear accumulating states marked by sequestration of the exogenous supravital dyes neutral red, methylene blue, and brilliant cresyl blue. The phenomenon was not affected by Na⁺-free and HCO₃-free conditions nor by the presence of cationic and anionic antiport regulators of cytosolic pH. Cytological, microspectrophotometric, and flow cytometric evaluation of whole cell populations showed that the nuclear influx was abolished by omitting the pH recovery response, and by modulating the recovery response. The abolition of nuclear influx in the presence of the P-ATPase and F_o-ATPase inhibitors, vanadyl(IV) ions and oligomycin, respectively, suggest that H⁺-translocating ATPase pumps are involved in regulating cytosolic acidification in Na⁺-free and HCO₃-conditions vanadyl(IV) inhibited nuclear uptake of supravital dyes in a dose dependent manner. Nuclear uptake of dyes, however, was not affected by up to 1 mM of genistein even though tyrosine-specific phosphorylation and DNA synthesis were abolished. Upgradient nuclear influx involving proton pump is novel. KB cancer cells and nontumorigenic Chang cells had differential dye accumulations induced by acidification and recovery.

Key Words:

• interphase nuclear uptake
• supravital dyes
• proton modulation
• flow cytometry
• microspectrophotometry
• vanadyl(IV) sulfate
• oligomycin
• Chang cells
• KB carcinoma cells