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.
Shen
D-W
DW
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892-4254, USA.
Su
A
A
Liang
X-J
XJ
Pai-Panandiker
A
A
Gottesman
M M
MM
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016190:Carboplatin / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D019175:DNA Methylation; D000077209:Decitabine; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004705:Endocytosis / Q000502:physiology; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D008727:Methotrexate / Q000494:pharmacology; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D000077209:Decitabine; D005492:Folic Acid; D016190:Carboplatin; D020559:Monomeric GTP-Binding Proteins; D001374:Azacitidine; D002945:Cisplatin; D008727:Methotrexate