Analysis of the Mechanisms of Drug Resistance in Cancer by DNA Microarray
Analysis of the Mechanisms of Drug Resistance in Cancer by DNA Microarray
By Roald Ravatn, MSc, PhD and Khew-Voon Chin, PhD
Drug resistance in cancer is a major obstacle to successful chemotherapy. Emergence of resistance implies that tumor cells are able to survive chemotherapy and eventually metastasize. Cancers may be primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur to form a multi-drug resistant tumor (acquired resistance).1 Certain mechanisms of drug resistance in cancer are well studied, e.g., overexpression of the multidrug resistance protein (MDR1), overexpression of the multidrug resistance-associated protein (MRP), and increased DNA repair.2-4
Mechanism of Development of Intrinsic Drug Resistance
The mechanisms of development of intrinsic drug resistance are not understood thoroughly and may be associated with tumor progression. It is known that various regulatory genes in the cell targeted for genetic alterations during tumorigenesis also may influence cellular sensitivity to chemotherapeutics.5 These genetic alterations involve a diverse group of gene products that include tumor suppressors, oncogenes, cell cycle regulators, transcription factors, growth factor receptors, and cell death regulators. Therefore, a single mechanistic pathway cannot explain the genesis of intrinsic resistance in cancer. Intrinsic drug resistance likely involves the altered expression of a diverse group of genetic factors influencing various biochemical pathways, thus giving rise to the commonly observed multidrug resistance phenotype in cancer. The emergence of acquired resistance, on the other hand, may be associated with either drug induction or drug selection of tumor cells during chemotherapy, and results in relapses that are refractory to treatment. The mechanisms of acquired resistance also are likely to involve the expression of multiple genes that contribute to the resistance phenotype.
The recent advent of high-density DNA microarray technology and its capacity for simultaneous probing of the genome has enabled the analysis of the expression profiles for thousands of genes in yeast and man.6-14 In view of the complex array of genetic factors contributing to drug resistance, DNA microarray should be extremely useful for examining the development of drug resistance in cancer. The resulting analyses ultimately may enable the use of the signature expression profiles of drug-resistant tumors to predict tumor response to specific drugs and to design better therapeutic regimens to circumvent drug resistance.
Gene Expression Patterns After Drug Induction and Drug Selection
In the case of acquired drug resistance, we have asked whether there are differences in the genetic factors that confer resistance, when derived either by drug induction or drug selection during chemotherapy. To address this issue, we utilized DNA microarray to monitor the expression profiles of MCF-7 breast cancer cells in response to doxorubicin treatment, as well as MCF-7 cells that had previously been selected for resistance to doxorubicin (MCF-7/D40).15 The expression profiles for the induced cells subsequently were compared with the expression profiles for the doxorubicin-resistant MCF-7/D40 cells (see Table). The drug doxorubicin is a topoisomerase II inhibitor that causes DNA damage, followed by induction of programmed cell death or apoptosis.16
| Table-Expression Profiles and Resistance | |||
| Name | Accession number |
Fold changes in gene expression 15 hours after exposure of MCF-7 to doxorubicin |
Fold changes in gene expression in MCF/D40 cells (constitutive) |
| Cell cycle genes | |||
| CDC28 kinase 1 | L29222 | -7.3 | -2.3 |
| XRCC1 | M36089 | 9.8 | 7.6 |
| Protein CDC27 | AA489098 | 5.9 | 5.5 |
| Neuronal genes | |||
| Ataxin 2 | U70323 | -7.7 | -2.8 |
| Human neuronal apoptosis inhibitory protein | U19251 | 4.6 | 3.7 |
| Synaptotagmin | M5504/J05710 | 14.0 | 10.4 |
| Signal transduction genes | |||
| Fms-related protein tyrosine kinase | D00133 | 23.0 | 9.5 |
| Granulin | M75161 | 17.1 | 11.6 |
| Recoverin | AB001838 | 5.1 | 7.6 |
| Transferrin receptor protein | M11507 | 7.4 | 4.6 |
| Collapsin response mediator protein 1 | U17278 | 20.7 | 13.5 |
| Transcription factors | |||
| RNA polymerase II | L37127 | -7.8 | -2.0 |
| Sigma 3B | X99459 | 27.5 | 16.4 |
| Metabolic genes | |||
| Cellular retinoic acid-binding protein | M68867 | 29.4 | 17.0 |
| Epoxide hydrolase | J03518 | 15.8 | 11.3 |
| Ubiquitin-proteasome | |||
| 26S proteasome regulatory subunit 4 | AA622905 | 18.6 | 11.6 |
| Protein secretion | |||
| Pescadillo | U78310 | 11.9 | 8.8 |
| A subset of genes transiently induced or repressed by doxorubicin in MCF-7 cells, which intersects with genes altered in the doxorubicin-resistant MCF-7/D40 cells. All changes in expression levels are calculated relative to the untreated MCF-7 cells. | |||
In one set of experiments, the MCF-7 cell line was treated transiently with doxorubicin (1 mcg/mL) for various time periods, ranging from one to 15 hours. In another experiment, the doxorubicin-resistant cell line MCF-7/D40 was cultured for 15 hours without doxorubicin treatment. Total RNA prepared from these cultured cells was used to synthesize 33P-labeled cDNAs by reverse transcription, followed by hybridization to the human cDNA microarrays from Research Genetics, Inc. (http://www.resgen.com, Huntsville, AL). These arrays are 5 x 7 cm nylon filters with approximately 5700 spots corresponding to known genes, ESTs, and reference points. The clones selected contain the 3’ untranslated region, with an average size of 1 kb. After hybridization and washing, the microarrays were exposed to phosphor- imaging screens and then scanned on a Molecular Dynamics Storm Imager. The resulting images were processed and analyzed with Pathways software (Research Genetics, Inc.) and the output data were further analyzed using MS Excel and Cluster/Treeview (a software package from Stanford University).
Simultaneous altera-tions in the expression of approximately 500 genes were observed, with gradual changes (range: two- to 30-fold) in a time-dependent manner following treatment with doxorubi-cin.15 The biochemical functions of the altered genes are diverse and in-clude transcription factors, protein kinases and phosphatases, cell cycle regulators, proteases, and apoptotic and anti-apoptotic factors, as well as a large number of metabolic genes. Altered expression of the transcription factors included down-regulation of the general transcription factor RNA polymerase II, the transcription corepressor Dr1-associated protein, and the enhancer binding proteins AP-3 and AP-4. Decreased expression of these genes may trigger a general repression of transcription in response to the cytotoxic effects of doxorubicin. In addition, increased expression of cytochrome c, which triggers apoptosis by activating the caspases and down-regulation of Bcl-2, an anti-apoptotic factor, was consistent with the induction of apoptosis by doxorubicin.17,18 We also observed changes in the expression of a group of previously unknown putative zinc finger transcription factors.
Another cluster of genes that showed striking changes after doxorubicin treatment were the genes involved in proteolysis. Protein degradation is recognized to be critical in the regulation of cell cycle, transcription, and signal transduction.19 We found that some ubiquitin-associated factors and subunits of the 26S proteasome, including Poh1, were up-regulated after doxorubicin treatment. It has been shown previously that overexpression of Poh1 confers multidrug resistance.20,21 Interestingly, the regulatory subunit 4 of the 26S proteasome, which is induced upon doxorubicin treatment, also is overexpressed in the doxorubicin-resistant cell line, suggesting a role for the regulatory subunit 4 of the 26S proteasome in the development of resistance to doxorubicin.
An interesting pattern of expression in the cell cycle genes following exposure to doxorubicin also was observed. Cyclin D2 and its catalytic partner, cyclin-dependent kinase 6, were induced by doxorubicin, indicating that these cells would proceed through the G1 phase of the cell cycle. In fact, it has been observed that cells exposed to a lethal, but not excessive concentration of doxorubicin will proceed through the G1-S phase and die in G2.22 Consistent with this observation, we found that the levels of cyclin A and cyclin E remained unchanged, whereas the CDC28 protein kinases 1 and 2 (CKS1 and CKS2) were down-regulated. These two kinases normally inhibit the activity of cyclin A/CDK2 kinase, which is involved in the G1-S transition of the cell cycle.
Expression Profile of Doxorubicin-Resistant Cells
Next we examined the expression profile of the doxorubicin-resistant MCF-7/D40 cells. A subset of genes with altered expression in MCF-7 after exposure to doxorubicin also was constitutively altered in the doxorubicin-resistant cell line MCF-7/D40 (see table). In view of their normal functions, overexpression of some of these genes may inadvertently confer drug resistance in cancer. For example, epoxide hydrolase, a drug-metabolizing enzyme, which has been found to be overexpressed in breast cancer and hepatocellular carcinoma, may enhance the metabolism of doxorubicin, thereby giving rise to an apparent increase in resistance in these cancers.23,24
Our results also showed the 26S proteasome regula-tory subunit 4 gene to be overexpressed both in the doxorubicin-induced and the doxorubicin-resistant MCF-7 cells. Overexpression of another proteasome gene, Poh1, has been shown to confer multidrug resistance.20,21 Therefore, increased expression of the 26S proteasome regulatory subunit 4 gene may contribute to increased resistance to doxorubicin. The precise role of the ubiquitin-proteasome pathway in drug resistance remains to be determined.
The single-stranded DNA repair protein XRCC1 also is overexpressed after doxorubicin induction, and in the drug resistant MCF-7/D40 cell line. This protein plays a role in the repair of single-stranded DNA breaks in mammalian cells and forms a repair complex with b-polymerase, ligase III, and poly (ADP-ribose) polymerase.25 In addition, XRCC1 specifically binds single-stranded DNA breaks (gaps and nicks), also in a gap DNA-b-polymerase complex. It is conceivable that XRCC1 also could bind to and repair double-stranded DNA gap lesions produced by topoisomerase II. Thus, elevated expression of XRCC1 may increase the efficiency of the repair of topoisomerase II-generated lesions, and contribute to increased resistance.
The altered expression levels observed for the other members of this subset of overlapping genes and their functional significance in drug resistance are not immediately apparent, and need to be investigated further. Nevertheless, their coordinated expression may represent a distinct signature profile for doxorubicin resistance in cancer. Clearly, further comparisons of expression profiles will be necessary to determine the prevalence of the expression of this subset of genes in various other doxorubicin-resistant cell lines. Whether by induction or selection, our results suggest that there is a convergence in the genes and pathways that are critical for the emergence of drug resistance in cancer.
Conclusion
In our analysis, the use of cDNA microarrays containing about 5000 gene elements has provided a genomic view of the mechanisms of acquired resistance in breast cancer cells by drug induction and selection. Now that the human genome sequence nearly has been completed, and when all the genes are identified, DNA microarrays containing EST elements corresponding to every single gene will provide a true global genomic analysis of gene expression. In the future, such analysis of drug resistance will yield unambiguous insights into the mechanisms of drug resistance, and ultimately will lead to more effective treatment strategies to circumvent tumor resistance to chemotherapy. (Dr. Ravatn is a Postdoctoral Fellow at The Cancer Institute of New Jersey in New Brunswick, and Dr. Chin is an Assistant Professor of Medicine and Pharmacology at The Cancer Institute of New Jersey and in the Department of Pharmacology, Robert Wood Johnson Medical School, Piscataway, NJ.) v
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