Reduced expression of small GTPases and hypermethylation of the folate binding protein gene in cisplatin-resistant cells.

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 [(14)C]-carboplatin, [(3)H]MTX, [(3)H]folic acid (FA), [(125)I]epidermal growth factor, (59)Fe, [(3)H]glucose, and [(3)H]proline, as well as (73)As(5+) and (73)As(3+), 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 [(14)C]carboplatin, [(3)H]FA, and [(3)H]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.