¹Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892-4254

Correspondence: M Gottesman, E-mail: mgottesman@nih.gov

²Su's current address: Pritzker School of Medicine, University of Chicago, 924 East 57th Street, Chicago, IL 60637-5415, USA

³Pai-Panandiker's current address: National Capital Consortium, National Cancer Institute, Radiation Oncology Sciences Program, Radiation Oncology Branch, NIH, Building 10, Room B3B69, Bethesda, MD 20892

Received 19 January 2004; Revised 24 March 2004; Accepted 25 March 2004; Published online 15 June 2004.

Abstract

Reduced accumulation of cisplatin is the most consistent feature seen in cisplatin-resistant (CP-r) cells that are cross-resistant to other cytotoxic compounds, such as methotrexate. In this report, defective uptake of a broad range of compounds, including [¹⁴C]-carboplatin, [³H]MTX, [³H]folic acid (FA), [¹²⁵I]epidermal growth factor, ⁵⁹Fe, [³H]glucose, and [³H]proline, as well as ⁷³As⁵⁺ and ⁷³As³⁺, was detected in CP-r human hepatoma and epidermal carcinoma cells that we have previously shown are defective in fluid-phase endocytosis. Downregulation of several small GTPases, such as rab5, rac1, and rhoA, which regulate endocytosis, was found in CP-r cells. However, expression of an early endosomal protein and clathrin heavy chain was not changed, suggesting that the defective endocytic pathway is clathrin independent. Reduced expression of the cell surface protein, folate-binding protein (FBP), which is a carrier for the uptake of MTX, was also observed in the CP-r cells by confocal immunofluorescence microscopy and Real-Time PCR. Reactivation of the silenced FBP gene in the CP-r cells by a DNA demethylation agent, 2-deoxy-5-aza-cytidine (DAC) demonstrates that hypermethylation occurred in the CP-r cells. The uptake of [¹⁴C]carboplatin, [³H]FA, and [³H]MTX increased in an early stage CP-r cell line (KB-CP1) after treatment with DAC. Both a defective endocytic pathway and DNA hypermethylation resulting in the downregulation of small regulatory GTPases and cell surface receptors contribute to the reduced accumulation of a broad range of compounds in CP-r cells.