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Gene-expression profiling reveals down-regulation of equilibrative nucleoside transporter 1 (ENT1) in Ara-C-resistant
Kazuchika Takagaki1, Susumu Katsuma, Yoshinori Kaminishi
1Discovery Research Laboratories, Nippon Shinyaku Co., Ltd, 3-14-1 Sakura, Tsukuba, Ibaraki 305-0003, Japan. k.takagaki@nippon-shinyaku.co.jp
Abstract:
We have investigated the mechanism of resistance of leukemia cells to Ara-C using an in-house cDNA microarray designed for the analysis of leukemia cells. We produced Ara-C-resistant cells from the CCRF-CEM (acute lymphoblastic leukemia) cell line and compared their gene-expression profile with that of wild-type cells. The adenosine deaminase (ADA) gene was highly up-regulated in Ara-C-resistant cells, while equilibrative nucleoside transporter 1 (ENT1) and several cell-cycle-related genes were down-regulated. Of all these genes, ENT1 seemed the most likely to be relevant to Ara-C resistance. To investigate the role of ENT1 in Ara-C-resistant cells, we transfected the cells with the gene. ENT1-transfected Ara-C-resistant cells resembled wild-type CCRF-CEM cells more closely than untransfected Ara-C-resistant cells in terms of growth rate, Ara-C-uptake characteristics, and ADA expression levels. The down-regulation of the ENT1 gene is expected to result in nucleotide deficiency in addition to blockage of Ara-C influx. Accordingly, Ara-C-resistant cells showed low growth rates, which were restored by transfection with ENT1. These low growth rates were also correlated with the phosphorylation level of cell-cycle checkpoint kinase 2. In this study we identified down-regulation of ENT1 as the factor responsible for Ara-C resistance, and this knowledge may be used to devise a clinical regimen that will overcome the resistance.
Insights
Equilibrative nucleoside transporter 1 (ENT1) down-regulation causes leukemia cell resistance to Ara-C chemotherapy. Restoring ENT1 levels in resistant cells re-sensitizes them to Ara-C treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Leukemia treatment often involves chemotherapy agents like Ara-C.
- Mechanisms of drug resistance can limit treatment efficacy.
- Understanding resistance pathways is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the molecular mechanisms behind leukemia cell resistance to Ara-C.
- To identify specific genes and pathways involved in Ara-C resistance.
- To explore potential therapeutic strategies to overcome Ara-C resistance.
Main Methods:
- Utilized an in-house cDNA microarray for gene expression profiling of leukemia cells.
- Generated Ara-C resistant cell lines from CCRF-CEM (acute lymphoblastic leukemia) cells.
- Performed gene transfection experiments to validate the role of identified genes.
Main Results:
- Identified up-regulation of adenosine deaminase (ADA) and down-regulation of equilibrative nucleoside transporter 1 (ENT1) in Ara-C resistant cells.
- Demonstrated that ENT1 down-regulation is a key factor in Ara-C resistance.
- Restored Ara-C sensitivity and normalized growth rates in resistant cells by transfecting ENT1.
Conclusions:
- Down-regulation of ENT1 is a primary mechanism conferring Ara-C resistance in leukemia cells.
- ENT1 plays a critical role in Ara-C uptake and cellular response.
- Targeting ENT1 may offer a novel strategy to overcome Ara-C resistance in clinical settings.
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