Rationale: The tendency of tumor cells to gain resistance to multiple forms of therapy concurrently has long been a consistent barrier to effective treatment of disease. This effect significantly reduces the treatment options available to the clinician, and makes disease-free recovery difficult for the patient. The ability to discern particular mechanisms governing multi-modality resistance and exploit them, therefore, could result in more efficacious treatment strategies. We have previously shown that stress-induced transcription factor activation (e.g., AP-1) results in decreased response to therapeutic modalities. Furthermore, others have established a link between epigenetic regulation of gene expression (such as hypermethylation of gene promoters) and the malignant phenotype. Interestingly, there also appears to be a correlation between AP-1 and an epigenetic regulator of transcription, DNA methyltransferase. We attempted to delineate a mechanistic pathway involving stress-induced transcriptional events and methylation to identify potential molecular targets for reversing multi-modality resistance in tumor cells. Research Synopsis: Through a collaboration with Dr. Douglas Spitz (previously at Washington University and recently at the University of Iowa), we obtained an oxidative stress-resistant fibroblast cell line with morphological and physiological characteristics of transformed cells. Among other treatments, these cells were significantly more resistant to hydrogen peroxide, cisplatin, etoposide, and heat-induced radiosensitization modalities. Exposure of the oxidative stress-resistant cells to the quintessential redox stressor, hydrogen peroxide, and chemotherapeutic agents concurrently with indomethacin resulted in a loss of the resistant phenotype. Interestingly, we also found that these stress-resistant cells also overexpressed AP-1 constitutively;indomethacin also inhibited the basal activity of this transcription factor (Cancer Res 61: 3486-92, 2001). In pursuing a mechanism for this action, we found that additional cell lines made by Dr. Tom Curran (St. Jude Children's Hospital) to genetically overexpress the c-Fos constituent of AP-1 also exhibit resistance to several chemotherapeutic agents (e.g., cisplatin, etoposide) and show increased expression levels of the epigenetic regulator DNMT-1 (Kaushal et al., in preparation). Microarray analysis and subsequent Western blotting confirmed that Bag-1, an anti-apoptotic protein, was upregulated in c-Fos and DNMT-1 overexpressing cell lines, identifying the Bag family as a potential marker and prospective target for reversing the resistant phenotype. An ongoing collaboration with Drs. Andrew Feinberg and Bert Vogelstein (Johns Hopkins University), making use of microarray technology in DNMT-deficient cells, has yielded several prospective avenues for continuing this investigation (Gius et al., submitted). Project Roles: Work on this project has been headquartered in our laboratory, but has been a largely multi-institutional effort. Dr. Gius and his colleagues worked with principal investigators to coordinate research activities and acquire the necessary materials, in addition to performing much of the intellectual and experimental work necessary for progress on the project. Our laboratory's experiments with the stress-resistant cell lines resulted in publication of a primary author manuscript. A second manuscript, which links DNMT1 with stress resistance, is in preparation, and we have just submitted one of several manuscripts that will hopefully arise from our work with the knockout cell lines in a microarray platform.Rationale: The tendency of tumor cells to gain resistance to multiple forms of therapy concurrently has long been a consistent barrier to effective treatment of disease. This effect significantly reduces the treatment options available to the clinician, and makes disease-free recovery difficult for the patient. The ability to discern particular mechanisms governing multi-modality resistance and exploit them, therefore, could result in more efficacious treatment strategies. We have previously shown that stress-induced transcription factor activation (e.g., AP-1) results in decreased response to therapeutic modalities. Furthermore, others have established a link between epigenetic regulation of gene expression (such as hypermethylation of gene promoters) and the malignant phenotype. Interestingly, there also appears to be a correlation between AP-1 and an epigenetic regulator of transcription, DNA methyltransferase. We attempted to delineate a mechanistic pathway involving stress-induced transcriptional events and methylation to identify potential molecular targets for reversing multi-modality resistance in tumor cells. Research Synopsis: Through a collaboration with Dr. Douglas Spitz (previously at Washington University and recently at the University of Iowa), we obtained an oxidative stress-resistant fibroblast cell line with morphological and physiological characteristics of transformed cells. Among other treatments, these cells were significantly more resistant to hydrogen peroxide, cisplatin, etoposide, and heat-induced radiosensitization modalities. Exposure of the oxidative stress-resistant cells to the quintessential redox stressor, hydrogen peroxide, and chemotherapeutic agents concurrently with indomethacin resulted in a loss of the resistant phenotype. Interestingly, we also found that these stress-resistant cells also overexpressed AP-1 constitutively;indomethacin also inhibited the basal activity of this transcription factor (Cancer Res 61: 3486-92, 2001). In pursuing a mechanism for this action, we found that additional cell lines made by Dr. Tom Curran (St. Jude Children's Hospital) to genetically overexpress the c-Fos constituent of AP-1 also exhibit resistance to several chemotherapeutic agents (e.g., cisplatin, etoposide) and show increased expression levels of the epigenetic regulator DNMT-1 (Kaushal et al., in preparation). Microarray analysis and subsequent Western blotting confirmed that Bag-1, an anti-apoptotic protein, was upregulated in c-Fos and DNMT-1 overexpressing cell lines, identifying the Bag family as a potential marker and prospective target for reversing the resistant phenotype. An ongoing collaboration with Drs. Andrew Feinberg and Bert Vogelstein (Johns Hopkins University), making use of microarray technology in DNMT-deficient cells, has yielded several prospective avenues for continuing this investigation (Gius et al., submitted). Project Roles: Work on this project has been headquartered in our laboratory, but has been a largely multi-institutional effort. Dr. Gius and his colleagues worked with principal investigators to coordinate research activities and acquire the necessary materials, in addition to performing much of the intellectual and experimental work necessary for progress on the project. Our laboratory's experiments with the stress-resistant cell lines resulted in publication of a primary author manuscript. A second manuscript, which links DNMT1 with stress resistance, is in preparation, and we have just submitted one of several manuscripts that will hopefully arise from our work with the knockout cell lines in a microarray platform.
