Director: Susan O. McGuire, PhD

The cell culture lab is complementary to all of the sections of the core facility. The laboratory is equipped with state-of-the-art tissue and cell culture facilities along with additional analytical instrumentation for the processing of cells and tissues.

The development of specific human and animal cell lines and dedicated tissue culture methods have aided in the understanding of the pathogenesis of cancer, thrombotic disorders, inflammatory processes and the immunomodulatory effects of cytokines and other mediators. The culturing of skin, vascular, and neuronal cells has played a major role in the development of newer diagnostic methods for the treatment of such diseases as thrombotic stroke, myocardial infarction, Parkinson’s disease, and inflammatory disorders. Because of the high throughput and batch analysis, these methods are very attractive for large-scale screening studies.

This laboratory is available to Pathology faculty free of charge. Other departments’ faculty members and external scientists may request permission to use the facility.

Ongoing projects:

• Folate-directed drugs may increase specificity of cancer therapy.
  In conventional cancer chemotherapy, a linear increase in the fraction of cells killed often requires an exponential increase in drug dose, but the lack of tumor specificity produces dose-limiting toxicity that has impeded improvement in survival. In an attempt to develop a new technique that can deliver the maximum dose of drug specifically and selectively, a covalent complex of folate, fluorescein and hemoglobin was synthesized and characterized. Using cell culture, the specificity and selectivity uptake of this reagent by cancer cells is being examined. Human neuroblastoma SH-SY5Y cells were incubated with various FITC labeled hemoglobin analogues. Preliminary data suggests that the folate-XLHb-FITC complex was readily taken up by the cells via endocytosis. Conversely, the XLHb-FITC complex was unable to permeate the cells. As a further proof of principle, the presence of 100 mM folic acid blocked the folate receptors, allowing only minute amounts of the folate-XLHb-FITC complex to permeate the cells. To our knowledge, this is the first study to demonstrate that human neuroblastoma SH-SY5Y cells contain folate receptors. The hemoglobin-based, folate-directed PDT agent model appears to be a viable approach, and may open up new potential avenues for cancer therapy.
• Studying urinary HB-EGF/APF levels in adults may contribute to diagnosis and treatment of painful bladder syndrome.
  The level of HB-EGF (Heparin-binding epidermal growth factor-like growth factor) and APF (Antiproliferative factor) in the urine of women with painful bladder syndrome is being measured (1) to determine if the HB-EGF/APF levels differ from volunteers who do not have painful bladder syndrome, (2) to determine whether levels of HB-EGF/APF vary with the menstrual cycle in normal volunteers and in those with painful bladder syndrome, (3) to determine if the HB-EGF results from our laboratory agree with those from the University of Maryland laboratory that performs the same test, and (4) to determine whether levels of HB-EGF/APF vary when bladder symptoms change. These urinary chemicals may eventually be used to diagnose and treat painful bladder syndrome.
• Culturing human bronchial epithelial cells to aid in the understanding of the etiology and pathogenesis of Kawasaki Disease.
  Kawasaki Disease (KD) is the leading cause of acquired heart disease in children in developed nations, and can result in coronary artery aneurysm formation, myocardial infarction, and sudden death. The etiology of KD is unknown, although clinical and epidemiological data support infection with a ubiquitous microbial agent. Recently, in tissue from children with acute fatal KD, we discovered spheroids detected by synthetic KD antibodies and visualized by electron microscopy as homogenous, perinuclear, electron-dense inclusions. The data indicate that the inclusions appear consistent with aggregates of viral proteins and associated nucleic acid, and are likely to derive from the etiologic agent of KD. December 18, 2006try and that it replicates in the bronchial epithelium. Our specific aim is to determine if clinical respiratory samples from acute KD patients infect in vitro human ciliated bronchial epithelial cell cultures. For this project, human epithelial cells are isolated from primary tissue and grown for 28 days on an air-liquid interface, where they differentiate and grow cilia. Cells are treated with clinical samples from KD patients, and are later subjected to immunohistochemistry and treatment with the synthetic KD antibody.

• Simian virus 40 (SV40) appears to cause malignant mesothelioma, a fatal cancer of the lungs previously associated only with asbestos inhalation in humans.
  

During the past 8 years we have had the exciting experience of seeing our research move from tissue culture, to animal models, to humans, and, soon, to clinical trials.

Our initial finding that SV40 small t antigen was required to induce DNA replication and to transform resting cells (J. Virology 1992 and 1994) and to inactivate p300 (EMBO J. 1996) led to our discovery that SV40 induced mesotheliomas and osteosarcomas in hamsters (Am. J. Pathol 1993, Virology 1992). These results prompted experiments in which we demonstrated the presence of SV40 in human mesotheliomas (Oncogene 1994) and osteosarcomas (Oncogene 1996). We demonstrated that SV40 inhibited p53 (Nature Medicine 1997) and Rb (Nature Medicine 1997) in human mesothelioma, and that the same strains of SV40 found in mesothelioma were present in the poliovaccines used in the 50s (Cancer Res. 1999, Virology 2001). We discovered the mechanisms that make mesothelial cells susceptible to SV40, and we found that asbestos and SV40 may be co-carcinogens (PNAS 2000). We investigated the molecular pathways induced by SV40 that cause a unusual high rate of human mesothelial cell transformation. We found that SV40 induces telomerase activity (Oncogene 2002), and Notch-1 (Oncogene 2003), and in collaboration with Adi Gazdar (University of Texas), that SV40 inactivates RASSF1A (Oncogene 2002). More recently we uncovered the viral and cellular mechanisms that make mesothelial cells susceptible only to SV40 and not to other polyomaviruses (Carbone et al., Cancer Res. Adv. In brief, In press) and a chemical inhibitor of the SV40Tag-p53 interaction (Carbone et al., Virology in press). Our findings have now been reviewed positively by 3 independent panels: George Klein et al., Oncogene 2002; Institute of Medicine (IOM), Vaccine Safety Review Committee, October 2002; May Wong et al., JNCI 2002. Presently, there are more than 70 papers from 60 different laboratories that have confirmed the association of SV40 with human mesothelioma, bone and brain tumors (reviewed in Adi Gazdar et al., Nature Cancer Review, 2002). This project (SV40 and human mesothelioma) is funded by the NIH/NCI and by a generous donation of the Riviera Country Club in Illinois.

We also discovered that mesothelioma is genetically transmitted in some families in Turkey (The Lancet 2001). We hope that by studying these families we will be able to identify a gene/s that when altered causes a high incidence of mesothelioma in individuals exposed to carcinogenic fibers or tumor viruses. This project (genetic predisposition to mesothelioma) is funded by the American Cancer Society and by the Cancer Research Foundation of America.

Our studies suggest that viruses, environmental carcinogens, and genetics can interact to cause human cancer. Our research is most relevant to the fields of SV40 and mesothelioma. However, our work has general relevance to cancer research. In the long term, we hope that our work will help to elucidate the biological effects of SV40 when present in human tissues, and the pathogenesis of mesothelioma. We want to understand how viruses, environmental carcinogens, and genetics interact in causing human cancer. Our hope, of course, is that our research will help to prevent and treat mesothelioma and other cancers. The diagnostic pathology expertise of Dr. Carbone in mesothelioma has proven a great resource for our research by providing new ideas and specimens to study.