Patricia Schoenlein, Ph.D.           
pschoenl@mail.mcg.edu

Phone: (706) 721-6281

Room: CB2906

• Associate Professor: Department of Cellular Biology and Anatomy

• Associate Professor, Department of Radiology

• Associate Professor, Department of Surgery

Research Emphasis - The focus of research in my laboratory is to identify mechanisms underlying the induction of apoptosis in cancer cells and to understand the relationship between apoptosis, micronucleation and the increased chemosensitivity of cancer cells. There are two main projects in my laboratory. One dealing with how certain genes which are amplified in cancer can be preferentially eliminated by radiation treatment allowing tumor cells resistant to chemotherapy to now become vulnerable to chemotherapeutic drugs. The second project addresses the molecular mechanisms underlying the increased efficacy of combinatorial drug treatments for breast cancer.

Project One - Molecular mechanisms underlying chemosensitization of cancer cells by radiation treatment

The first project in my laboratory is quite unique and relevant to my interest in developing strategies to circumvent the multidrug resistant phenotype of tumor cells. We have made the novel observation that fractionated radiation therapy, at doses commonly administered to cancer patients, induces the loss of extrachromosomally amplified multidrug resistant genes from cancer cells. Concomitant with the loss of extrachromosomally amplified genes is a reduction in the multidrug resistance of the cancer cells (increased chemosensitivity). This result may have direct clinical relevance because gene amplification is very common in cancer cells. In > 50% of tumor biopsies analyzed, gene amplification structures have been documented. In particular, the most common carrier of amplified oncogenes and drug resistance genes in human cancer cells in vivo are extrachromosomal circular DNA molecules, referred to as double minute chromosomes (dmin) and episomes. The presence of amplified oncogenes has been shown to impart a multidrug resistant phenotype to cancer cells. Thus, any treatment modality that specifically reduces the copy number of extrachromosomally amplified genes in tumor cells has the potential to increase chemosensitivity in the clinical setting.

The model system for our radiation studies has been multidrug resistant (MDR) cancer cells that harbor amplified multidrug resistance (MDR1) genes exclusively on extrachromosomal circular DNA molecules ranging from 750 – 1890 kb. These circular DNA molecules can be readily visualized using fluorescence in situ hybridization studies in which metaphase chromosome spreads of MDR cells are hybridized to an MDR1-specific cosmid probe (Fig. 1). During our analysis of irradiated cells, we have observed that radiation treatment results in the “selective” entrapment of extrachromosomally amplified genes in radiation-induced micronuclei (Fig. 2 ). Micronuclei are small, membrane-bound nucleus-like particles that are localized to the cytoplasm of a cell after mitotic division. It has been well established in published studies that either acentric DNA fragments or whole chromosomes or chromatids displaced from the mitotic spindle lead to radiation-induced micronucleation. However, we were the first research group to observe that fractionated radiation therapy leads to the entrapment of extrachromosomally amplified genes and their subsequent loss. This novel observation was recently published in Cancer Research (Sanchez et al., 1998).

It is our immediate goal to determine if radiation-induced loss of extrachromosomally amplified drug resistance genes and oncogenes is a universal end point of fractionated radiation therapy that results in increased chemosensitivity and/or death (apoptosis) of tumor cells . To this end, we are currently expanding our studies in the next year to include the analysis of the COLO320 DM cell line and two Neuroblastoma cell lines harboring amplified MYCC and MYCN genes, respectively, on double minute chromosomes. We also have recently begun mechanistic studies to understand the process of radiation-induced micronucleation in tumor cells, the role of p53 in this process, and the relationship, if any, between micronucleation and apoptosis (summarized in Fig. 3).

Our collaborators in this project at MCG include Dr. John T. Barrett - a radiation oncologist, Dr. Xinbin Chen - a p53 expert, Dr. Anita Kulharya – a cytogeneticist, and Dr. Dave Welter – an expert on nuclear and chromosomal morphology. Additionally, we have initiated a collaboration with Dr. Geoff Wahl at the Salk Institute who has studied hydroxyurea-induced loss of extrachromosomally amplified genes via micronucleation for the past decade.

Publications from my laboratory relevant to extrachromosomal gene amplification, multidrug resistance and radiation induced loss of extrachromsomally amplified genes in tumor cells include the following:

Schoenlein, P.V., Shen, D-w., Barrett, J. T., Pastan, I., and Gottesman, M. M.: Double minute chromosomes carrying the human MDR1 and MDR2 genes generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells. Mol. Biol. Cell. 3: 507-520, 1992.

Schoenlein, P.V.: Molecular cytogenetics of multiple drug resistance. Cytotechnology 12: 63-89, 1993.

Schoenlein, P.V., Van Devanter, D. R., and M. M. Gottesman: Extrachromosomal elements in mammalian cells. In Adolph K.W. (Ed.): Gene and Chromosomes Analysis part B. vol. 2. Academic Press, Inc. 78-103, 1993.

Schoenlein, P.V.: The role of gene amplification in drug resistance. In Ozols, R. F. and Goldstein, L. J. (Eds.): Anticancer Drug Resistance: Advances in Molecular and Clinical Research. Pp. 167-200 Norwell, MA, Kluwer Academic Publishers, 1995.

Sanchez, A.M., Barrett, J. T., and Schoenlein, P.V. Fractionated ionizing radiation accelerates loss of amplified genes harbored by extrachromosomal circular DNA in tumor cells. Cancer Res. 58: 3845-3854, 1998.

Schoenlein, P.V., Welter, D., and Barrett, J. T. The degradation profile of extrachromosomal circular DNA during cisplatin-induced apoptosis is consistent with preferential cleavage at matrix attachment regions. Chromosoma 108: 121-131, 1999.

Additional references from other laboratories that are relevant to micronucleation:

Project Two - Hormonal Treatment of Breast Cancer to induce apoptosis of tumor cells.

The second project ongoing in my laboratory is specifically focused on breast cancer. We are studying the underlying mechanism(s) that mediate active cell death (induction of apoptosis) following the hormonal treatment of breast cancer cells. Specifically we are studying the efficacy of combined therapy with the antiprogestin mifepristone and the antiestrogen tamoxifen in growth inhibition and/or induction of apoptosis of breast cancer cells. Both in vitro (cell culture) and in vivo studies (human xenografts in nude mice) are being pursued with several breast cancer model systems, including MCF7 cells which express estrogen receptor (ER+) and progesterone receptor (PR+) and MDA-231 cells which lack both estrogen and progesterone receptors. We have demonstrated an additive inhibitory effect on the growth of human breast cancer cells in vitro. This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of the anti-apoptotic gene bcl2, and induction of TGFB1 protein as compared to monotherapy with either tamoxifen or mifepristone. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive and significantly different from the effect of monotherapy (P<0.05). These data are summarized in the following publication: Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells. M. Fathy El Etreby, Yayun Liang, Robert W. Wrenn, and Patricia V. Schoenlein. Breast Cancer Research and Treatment 51: 149-168, 1998. Thus, our current data has led to our overall hypothesis that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of breast cancer. We are currently funded to test our hypothesis (NIHR01CA70897-01A1) and to delineate the molecular pathways involved in the breast cancer cell's response to combined hormonal therapy . We are most interested in the underlying signal transduction pathways affected by tamoxifen and mifepristone binding to the ER and PR, respectively, which in turn promote active cell death of breast cancer cells (summarized in Fig. 4 ). Collaborators on this project include Dr. Robert Wrenn – a PKC expert, Dr. Tom Ogle – an ER and PR expert, Dr. Jill Lewis – a molecular biologist, Dr. Fathy El Etreby – an expert in hormonal therapeutic applications, and Dr. Rory Dalton – a surgical oncologist.

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