David G. Kaufman, MD, PhD
Office: 919-966-1396 or 919-966-5952
There are two areas of research in our laboratory.
One is a basic research study that is investigating a general characteristic of cancer development. It is largely a genomics study using molecular biology methodology. It concerns DNA replication origins and regions of DNA replicated at the beginning of S phase.
The second is a translational study concerned with the mechanisms of development of a specific type of cancer: endometrial cancer. We reconstruct human endometrium in culture from its constituent cells and study the interaction between endometrial epithelial and stromal cells that determine normal tissue structure and function. In this system we are attempting to reproduce the progressive steps of endometrial cancer development.
These projects are described in greater detail below.
A Search for Targets for Malignant Transformation among DNA Sequences Replicated Early in S Phase
One research goal is to discover molecular mechanisms that relate cell proliferation and chemical carcinogenesis. Previous studies showed that cells are most susceptible to malignant transformation when treated with chemical carcinogens during the earliest part of the S phase. We also found that DNA is preferentially damaged when it replicates, with elevated carcinogen binding to DNA at replication forks. We are now trying to determine whether susceptibility in early S phase occurs because critical DNA target sites for malignant transformation are replicated in the earliest part of the S phase. We evaluated the time sequence of replication of genes during the S phase and found DNA to replicate in a specific temporal order in normal human fibroblast cultures (NHF1 cells). We labeled DNA replicated early in S phase and in the next metaphase identified the highest labeling in six chromosomal bands. We also prepared libraries from genomic DNA that replicated in the earliest part of the S phase. Subsequently the nearly 10,000 clones were sequenced and mapped to the human genome, revealing the distribution of early S phase DNA replication and the presence of clones in each of the early replicating chromosomal bands. Starting with a clone form one of the earliest replicating chromosomal bands (1p36.1) we found a region that begins replication early in the first hour of S phase at one end of the clone and a region that replicates one hour later at the other end. We then found origins of DNA replication that correspond to the two regions separated by about 20 kB, a length that could be replicated in far less than an hour. From this we deduce that DNA replication either ceases or progresses very slowly at the boundary between these two regions. Continuing studies are using new technology that allows us to stretch DNA fibers in parallel orientation permitting the examination of DNA replication through specific genomic regions. We are examining the process of replication at different intervals of the S phase and comparing replication in normal and malignant cells. Since our goal was to identifying the earliest replicated regions in an effort to identify target sequences with a role in malignant transformation, it is interesting to note that the majority of the genes in the apoptotic pathway, despite the fact that they are located on several chromosomes, are the genes most strongly linked to replication at the start of S phase.
Reconstruction and Transformation of Human Endometrium in Cell Culture
In another line of research, we have been studying biologic and molecular features of malignant transformation in human endometrium using human endometrial cells in culture. Using cultures of epithelial and stromal cells of human endometrium we have examined interactions between endometrial stromal and epithelial cells in defining normal differentiated structure and function of endometrial tissue. Functional endometrial glands form when these cells are combined in a basement membrane-like matrix. The epithelial cells in these glands have polarized structure, they are interconnected by functional gap junctions, they express estrogen and progesterone receptors, they respond to estrogens and progestins, and they produce hormone-dependent products. When preneoplastic or malignant human endometrial epithelial cells are substituted for normal epithelial cells, glandular structures form that resemble the abnormal structures seen in hyperplasias and cancers of the human endometrium in vivo. For many of these functions, endometrial stromal cells can be removed and replaced with media conditioned by endometrial stromal cells, which indicates that paracrine factors mediate some of these stromal cell functions. Recently we have generated or obtained versions of normal stromal and epithelial cells immortalized by constitutively-expressing telomerase reverse transcriptase and use these cells to substitute for normal cells with limited life-span and proliferative capacity. Current studies are focused on the separate functions of estrogen receptors alpha and beta, and signaling through the IGF-I pathway. We are also evaluating the genes that are abnormally regulated in endometrial intraepithelial neoplasia (EIN). Eventually our goal is to reproduce in vitro the progressive steps of human endometrial carcinogenesis in this model of endometrial tissue in culture. We hope to relate morphologic and functional alterations in endometrial tissue growth and differentiation during endometrial cancer development, to alterations of gene function and cellular interactions as reflected in these cultures.
Cohen, S.M., Chastain P.D., Cordeiro-Stone, M, and Kaufman, D.G.: DNA Replication and the GINS Complex: Localization on Extended Chromatin Fibers. BMC Epigenetics and Chromatin 2: 6, 2009.
Frum, R.A., Khondker, Z.S., and Kaufman, D.G.: Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells. Cell Cycle 8: 1-16, 2009. PMCID: PMC2829940
Asagoshi, K., Tano, K., Chastain II, P.D., Adachi, N., Sonoda, E., Kikuchi, K., Koyama, H., Nagata, K., Kaufman, D.G., Takeda, S., Wilson, S.H., Watanabe, M., Swenberg, JA., and Nakamura, J., FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells. Mol Cancer Res. 8: 204-215, 2010. PMCID: PMC2824787
Chastain II, P.D., Nakamura, J., Rao, S., Chu, H., Ibrahim, J., Swenberg, J.A.., and Kaufman, D.G. Abasic Sites Preferentially Form at Sites of Replication. FASEB J. 24: 3674-3680, 2010. PMID: 20511393; PMCID: PMC2996904
Cohen, S.M., Chastain II, P.D., Rosson, G.B., Groh, B.S., Weissman, B.E., Kaufman, D. G., and Bultman, S.J. BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression. Nucleic Acids Res. 38: 6906-6919, 2010. PMCID: PMC2978342
Luke, A.M., Chastain II, P.D., Pachkowski, B.F., Afonin, V., Takeda, S., Kaufman, D.G., Swenberg, J.A.., and Nakamura, J. Accumulation of True Single Strand Breaks and AP sites in Base Excision Repair Deficient Cells. Mutat Res. 694: 65-71, 2010. PMCID: PMC2996904.
Kaufman, D.G., Cohen, S.M., and Chastain, P.D. Temporal and Functional Analysis of DNA Replicated in Early S Phase. Adv. Enzyme Regul. 51:257-271, 2011. PMID: 21093474.
Wang, Y., Chastain, P.D., Yap, P.-T., Kaufman, D.G., Guo, L., and Shen, D. Automated DNA Fiber Tracking and Measurement. Proc. IEEE ISBI 2011, pp. 1349-1352, Chicago, Illinois, U.S.A, Mar 30-Apr 2, 2011.
Sampey, B.P., Lewis, T.D., Barbier, C.S., Makowski, L, and Kaufman, D.G. Genistein Effects on Stromal Cells Determines Epithelial Proliferation in Endometrial Co-Cultures. Exp. Mol. Path. 90: 257-263, 2011. PMID: 21281625.