Cancer is a disease characterized by alterations in both cell structure and DNA organization. Pathologists have long realized that cancer cells have markedly altered nuclear shapes and patterns of chromatin. The causes of these alterations in nuclear and DNA organization have not been well characterized.
The aim of this research effort is to characterize and elucidate the mechanisms involved in determining the three-- dimensional DNA organization within the cancer cell. The long range goal of the sponsor's laboratory program has been to understand how alterations in cell structure within the cancer cell may contribute to alterations of DNA organization . Utilizing prostate cancer as a model, the laboratory has demonstrated that alterations in cancer cell nuclear morphology correlate with changes in the composition of the nuclear matrix. The nuclear matrix is the structural element which determines in part the three-dimensional organization of the nucleus and is involved in DNA organization and function. The applicant will investigate whether alterations in the nuclear matrix contribute to alterations of DNA organization and therefore, alterations of gene expression observed within cancer cells. Transfection of normal cells with specific oncogenes and putative tumor suppressor genes will be used to better understand their respective roles in altering DNA organization and nuclear matrix composition. Specifically, the three-dimensional organization of DNA within normal prostate cells as well as transformed prostate cancer cells will be investigated utilizing in situ hybridization and confocal microscopy. By utilizing cDNA probes for genes that are located on and off the nuclear matrix, alterations in three-dimensional structural DNA organization will be correlated with changes in nuclear matrix-gene interactions. In addition, alterations in nuclear matrix composition will be investigated utilizing high resolution two-dimensional gel electrophoresis. The second objective of this application is to support the training of a physician as a medical scientist. The applicant aspires to take a clinical education from his medical residency at the University of Chicago and fellowship in medical oncology at Johns Hopkins into a career of independent scholarship in oncology investigation. Toward this goal, Dr. Pienta will train in the Laboratory of Cell Structure and Function at the Johns Hopkins Oncology Center under the guidance of Dr. Donald S. Coffey, the award sponsor. In addition to the rigorous laboratory experience in cellular and molecular biology, Phase I of the award will also encompass a didactic program in cellular and molecular biology. A faculty advisory committee will review Dr. Pienta's progress every four months. After successful completion of Phase I, Dr. Pienta will assume a faculty position to further pursue these studies in Phase II.
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