Abstract
Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae L-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human L-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, L-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients.
Highlights
Cell Lines and Culture Conditions—NT2/D1 cells were obtained from the American Type Culture Collection (ATCC) and the HCT116 human colorectal carcinoma cell line was kindly provided by Dr Bert Vogelstein (Johns Hopkins, Baltimore, MD)
HCT2 Expression Levels Correlate with Sensitivity to BLM— As outlined in the Introduction, we recently identified the carnitine permease Agp2 as conferring high affinity polyamine and BLM-A5 transport activity in yeast [25, 27]
We reasoned that permeases having dual carnitine and polyamine transport activities might be responsible in mammals for the transport of BLM-A5, a BLM species characterized by the presence of a spermidinyl moiety on the bithiazole group
Summary
Agp overexpression greatly sensitized cells to BLM-A5, which correlated with enhanced damage to DNA [25]. Agp transports only one species of BLM, namely BLM-A5, which contains a spermidine moiety [27] This observation led to the remarkable discovery that Agp expression is responsible for high affinity polyamine transport [27]. Our genome-wide screen has established for the first time that altered transport is a critical mechanism leading to BLM resistance in yeast. This raised the possibility that mammalian cells might use related genes to regulate BLM influx. We propose that a high hCT2 activity level in a tumor cell sample would be prognostic of sensitivity to BLM treatment, whereas hCT2 deficiency would correlate with drug resistance
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