The ATP-binding cassette (ABC) transporter proteins are a large superfamily of membrane proteins comprising 48 members (plus one pseudogene) divided into seven different families based on sequence similarities (4
). The nomenclature for human ABC transporter genes is provided at: http://nutrigene.4t.com/humanabc.htm
. There is a high sequence homology among all the members, especially those within a particular family. ABC transporters have a wide array of cellular roles including regulation of lipid homeostasis (25
) and protection of the organism by effluxing toxins out of the cells (26
). Similarly, they are involved in MDR by protecting cancer cells from the toxicity of chemotherapeutic agents. In the last three decades, approximately 25 ABC transporters have been shown to be involved in MDR in in vitro
studies; however, numerous studies investigating ABC transporter gene expression in clinical samples have failed to directly link these transporters to drug resistance (5
). Inappropriately designed studies and poorly chosen cohorts are some of the main reasons for this failure. The limited sensitivity and/or probe specificity of platforms that have been used to assess the expression profiles of highly homologous genes such as ABC transporters is another shortcoming in these studies. The establishment of a standard analytical platform that would allow the precise discrimination of highly homologous genes using a small amount of sample would help to produce a more unified picture of MDR. It could lead to progress not only in understanding the mechanisms governing multidrug resistance but also in the translation of this knowledge to clinical practice, especially in personalized medicine (31
DNA microarrays have been used to explore the relationship between gene expression patterns and drug resistance in cancer cells; however, pinpointing individual genes in gene families possessing high homology represents a major shortcoming of that technology. Although quantitating ABC transporter expression in routine clinical applications is challenging, our previous study indicated that real-time PCR has the ability to discriminate among genes in a complex multigene family, thereby allowing meaningful correlations to be drawn between gene expression and subtle differences in drug sensitivity phenotype (16
). We wanted to evaluate the accuracy and sensitivity of TLDA and BioMark 48.48, two currently available high-throughput platforms based on qRT-PCR using TaqMan chemistry to discriminate highly homologous ABC transporter genes. Our previously established database (16
) was thus chosen as a model to appraise the accuracy and sensitivity of these micro- and nanofluidic high-throughput TaqMan-based qRT-PCR platforms to analyze the expression profiles of ABC transporters. The NCI-60 panel includes a diverse set of human cancer cell lines derived from nine tissues of origin which have been extensively studied using microarrays, rendering this panel of cancer cell lines ideal for further analysis (20
). The present data indicate that the microfluidic TaqMan-based qRT-PCR platform (TLDA) provides the greatest sensitivity, accuracy, and precision for ABC transporter gene expression profiling when compared to SYBR Green-based qRT-PCR. These advantages vis-a-vis current technologies make TLDAs more applicable to clinical use. Merging nanotechnology and biological profiling could enable personalized medicine to advance to the next stage in its development (36
). Here we assessed the ability of a nanofluidic TaqMan-based qRT-PCR platform, the BioMark 48.48 Dynamic Array, to precisely detect ABC transporter genes in the NCI-60 panel. Although pre-amplification is a requirement for gene-expression analysis with this platform, it demonstrates reliability and accuracy similar to the TLDA platform.
The National Cancer Institute’s Developmental Therapeutics Program has extensively screened over 100,000 anti-cancer compounds using the NCI-60 cancer cell line panel since 1990 (17
). We correlated the gene expression profiles obtained from the microfluidic TaqMan-based qRT-PCR platform (TLDA) and the growth inhibitory profiles of a subset of 1,429 candidate anticancer drugs tested against the panel to establish a database identifying compounds as substrates of one or more ABC transporter(s). Our improved database confirms several predictions made by the previous study, and also highlights formerly unidentified anticancer and yet unexplored compounds that are substrates of ABC transporters. This was demonstrated for three extensively studied ABC transporters: ABCB1, C1 and G2. Also the ability of this database to reveal compounds whose activity is potentiated by ABC transporters was confirmed experimentally with an increased sensitivity of ABCB1 overexpressing cells to NSC693871.
Dramatic advances in gene expression profiling have occurred in the past few years. In this study, two TaqMan qRT-PCR platforms, based on micro- and nano-fluidic systems, TLDA and BioMark 48.48 Dynamic Arrays, were singled out with the potential to be further developed for individualized cancer management. The superiority of these platforms was clearly demonstrated over established technologies in assessing ABC transporter expression profiles. Our investigations led to the refinement of a previously established database with the capability to more precisely identify compounds whose resistance is mediated by ABC transporters as well as ascertain which compounds are responsible for collateral sensitivity. The challenge, now, is to apply these platforms to elucidate the gene signatures for MDR in a well-designed clinical study. That could also lead to progress not only in understanding the mechanisms governing multidrug resistance but also in the translation of this knowledge to clinical practice, especially in personalized medicine.