Evaluation of drug transporters' significance for multidrug resistance in head and neck squamous cell carcinoma.

BACKGROUND: Multidrug resistance (MDR) hampers chemotherapy in head and neck squamous cell carcinoma (HNSCC). There is little information about MDR mediating drug transporters in HNSCC. METHODS: Nine HNSCC cell lines were characterized concerning drug transporter expression and susceptibility to cisplatin, paclitaxel, and 5-fluorouracil (5-FU) using a DNA microarray and proliferation assays. Three cells lines were precisely investigated for transporter expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and drug resistance before and after simulated chemotherapies. RESULTS: The cell lines differed in drug resistance and transporter expression. Cisplatin and paclitaxel resistances were inversely associated. Drugs rendered HNSCC cells resistant, cross-resistant, or cross-sensitive. Sensitivity changes were accompanied by transporter mRNA expression changes. CONCLUSION: Paclitaxel was identified as a potent inducer of numerous drug transporters and phenotypic MDR in HNSCC. Thus, cytostatic susceptibility of HNSCC cannot exclusively be deduced from the expression of single transporters but more likely of the entire transportome and non-transporter modulators.

Expression profiling of ATP-binding cassette transporters in childhood T-cell acute lymphoblastic leukemia.

A major issue in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters. The majority of these proteins have not yet been examined in T-ALL. Using a newly developed microarray for the simultaneous quantification of 38 ABC transporter genes, we observed a consistent overexpression of ABCA2/ABCA3 in clinical samples of ALL. Therefore, we analyzed the association of these two genes with drug resistance. Treatment of CCRF-CEM and Jurkat cells with methotrexate, vinblastine, or doxorubicin led to an induction of ABCA3 expression, whereas a significant increase of ABCA2 expression was only observed in Jurkat cells. To study the causal relationship of ABCA2/A3 overexpression with drug resistance, we applied RNA interference (RNAi) technology. RNAi specific for ABCA2 or ABCA3 led to a partial decrease of expression in these two ABC transporters. Upon cotreatment of RNAi for ABCA2 with methotrexate and vinblastine, a partial decrease of ABCA2 expression as well as a simultaneous increase of ABCA3 expression was observed. Vice versa, ABCA3 RNAi plus drugs decreased ABCA3 and increased ABCA2 expression. This indicates that down-regulation of one ABC transporter was compensated by the up-regulation of the other. Application of RNAi for both ABCA2 and ABCA3 resulted in a more efficient reduction of the expression of both transporters. As a consequence, a significant sensitization of cells to cytostatic drugs was achieved. In conclusion, ABCA2 and ABCA3 are expressed in many T-ALL and contribute to drug resistance.

ABCA3 as a possible cause of drug resistance in childhood acute myeloid leukemia.

BACKGROUND: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters that function as drug efflux pumps. The majority of these proteins have not yet been examined in malignant diseases. EXPERIMENTAL DESIGN: A newly developed microarray for the simultaneous quantification of 38 ABC transporter genes and Taqman real-time PCR was used to analyze the expression of ABC transporters in pediatric AML and healthy bone marrow. Small interfering RNA was used to verify the role of ABCA3 in drug resistance. RESULTS: Using the microarray, we identified four new ABC transporters, which were overexpressed in many AML samples compared with healthy bone marrow: ABCA2, ABCA3, ABCB2, and ABCC10. The overexpression of these four genes was verified by real-time PCR in 42 samples from children with AML and 18 samples of healthy bone marrow. The median expression of ABCA3 was three times higher in 21 patients who had failed to achieve remission after the first course of chemotherapy than in a well-matched group of 21 patients who had achieved remission at this stage (P = 0.023). Incubation of cell lines with a number of different cytostatic drugs induced an up-regulation of ABCA3. Down-regulation of ABCA3 by small interfering RNA sensitized cells to doxorubicin. CONCLUSION: Our results show that ABCA2, ABCA3, ABCB2, and ABCC10 are overexpressed in childhood AML compared with healthy bone marrow. ABCA3 is the most likely transporter to cause drug resistance.

Microarray Expression Profiling of ABC Transporters in Human Breast Cancer.

The ATP-binding cassette (ABC) transporters are highly conserved genes involved in the translocation of molecules through biological membranes. Several of them are involved in tumor drug resistance, and it is thought that many others may contribute to the development of the tumor phenotype in a still unknown way. A low-density DNA microarray was recently developed for the analysis of 38 ABC-transporter genes and 3 other transporters. In the present pilot study, clinical samples from 16 breast cancer patients were tested. Of the 41 transporters analyzed, 10 were not or very seldom expressed, while 23 were found to be expressed, sometimes at very high levels, in the majority of the tumors. Comparison of the treated and untreated tumors showed an unexpected similarity of results. The signal obtained on the capture probes for ABCC6/8/9 was, however, found to be higher in the treated samples. The microarray data were validated on 15 ABC-transporter genes by real-time PCR. The present results showed that the expression of the majority of the ABC transporters was a clear feature of breast tumors, whether treated or not.

Molecular characterization of breast cancer cell lines by a low-density microarray.

We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors.

Microarray-based detection of multidrug resistance in human tumor cells by expression profiling of ATP-binding cassette transporter genes.

Different mechanisms of drug resistance, including ATP-binding cassette (ABC) transporters, are responsible for treatment failure of tumors. We developed a low-density DNA microarray which contains 38 genes of the ABC transporter gene family. This tool has been validated with three different multidrug-resistant sublines (CEM/ADR5000, HL60/AR, and MCF7/CH1000) known to overexpress either the ABCB1 (MDR1), ABCC1 (MRP1), or ABCG2 (MXR and BCRP) genes. When compared with their drug-sensitive parental lines, we observed not only the overexpression of these genes in the multidrug-resistant cell lines but also of other ABC transporter genes pointing to their possible role in multidrug resistance. These results were corroborated by quantitative real-time reverse transcription-PCR. As the microarray allows the determination of the expression profile of many ABC transporters in a single hybridization experiment, it may be useful as a diagnostic tool to detect drug resistance in clinical samples.