Last Updated: 3/23/2010

Clinical Interests
Pathology of gynecologic cancers
General anatomic pathology

Research Interests
One of our research goals is to discover molecular mechanisms that relate cell proliferation and chemical carcinogenesis. In previous studies, we have found that cells are most susceptible to malignant transformation when they are 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 the S phase and identified six labeled sites in chromosomal bands in the next metaphase. We also prepared libraries from genomic DNA that replicated in the earliest part of the S phase. The libraries were screened for the presence of DNA markers corresponding to the chromosomal bands labeled early in the S phase. Starting with library clones we mapped the chromosomal bands and determined the time of replication in S phase of small fragments of these clones. Using this and complementary approaches, we have found very early replicating regions in the four bands we have studied. In one of the earliest replicating chromosomal bands (1p36.1) we found a region that replicates very early with adjacent regions on each side that replicate one to two hours later. DNA replication either ceases or progresses very slowly at the boundaries of this early replicating region. We have found an origin of DNA replication within this early replicating region, thus this bounded early replicating region may represent a replicon. Continuing studies are seeking similar early replicating regions in the other five chromosomal bands previously identified. We wish to determine whether all of these early replicating regions are bounded by later replicating regions with transition zones of paused on slow progressing replication. We also wish to determine whether these other sites are equally gene-poor. Since we use origins of DNA replication as a parameter of functional genomic analysis, we will continue to identify origins and characterize their properties both in terms of DNA sequence and protein binding. By identifying the earliest replicated regions, we hope to screen these regions in tumor cells to identify possible new target sequences with a role in malignant transformation.

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. Our culture methods allow both the epithelial and stromal cells of human endometrium to undergo a wide range of differentiation in vitro. In recent studies with human endometrial cells in culture 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, and they respond to these hormones by producing hormone-dependent products when induced with hormones. When preneoplastic or malignant human endometrial epithelial cells are substituted for normal epithelial cells, the glandular structures that are formed 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 indicating that paracrine factors mediate some of these stromal cell functions. The normal cells used in these studies to date have limited life-span and proliferative capacity. Ongoing studies are seeking to develop human endometrial stromal and epithelial cells that have extended life-span by transferring into them constitutively-expressed telomerase reverse transcriptase and/or other genes or gene suppressors. Further studies will explore the effects of targeted inactivation of genes who's activity are lost in endometrial cancers or by introducing mutant forms of genes found to be mutated in endometrial cancers. Our goal in these studies is to reproduce in vitro the progressive steps of endometrial carcinogenesis in this model of endometrial tissue in culture. In this process we will 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.

Recent Accomplishments and Honors
a. We demonstrated that interaction between normal endometrial epithelial cells and stromal cells are essential for normal functions of endometrial epithelial cells including growth in response to estrogen, formation of intercellular gap junctions and production of secretory proteins. Knowledge of these normal interrelationships is important because these functions are lost in endometrial cancers.

b. We have developed human endometrial stromal cells that have extended life-spans in cell culture by transferring into them constitutively expressed telomerase reverse transcriptase. Initial characterization of these cells indicates that they have few differences in gene expression from primary cultures of endometrial stromal cells from which they were derived. Comparable studies are in progress to generate human endometrial epithelial cells that have extended life-spans in cell culture. The availability of these long-live cells will enable us to better study epithelial stromal interaction and endometrial carcinogenesis in this experimental model system.

c. We have demonstrated six chromosomal bands in which DNA replication begins at the onset of S phase in normal human fibroblasts. We have begun mapping these six chromosomal bands and in the four bands studied to date we found regions within the bands that replicate very early in S phase.

d. We have characterized one segment of DNA in chromosome band 1p36.1 that is replicated extremely early in S phase. This region is 200kb in length and iis bounded by regions that start to replicate one to two hours later is S phase. We have identified one origin of DNA replication within this region and we are evaluating whether there are additional origins. These results identify a replicon (or a small bank of replicons) that replicate very early in S phase.

e. We have found that DNA replication pauses for about 10 minutes soon after initiation during the first hour of S phase in normal human fibroblasts; replication resumes thereafter. Pausing does not occur later in S phase in these normal cells and is absent at any time during S phase in a cancer cell line. Pausing of replication, like the S checkpoint, can be inhibited with caffeine which suggests that pausing may represent a previously unrecognized DNA damage surveillence process operative specifically on the earliest replicating DNA sites.

Training

M.D. 1968. Washington University (St.Louis)

Ph.D. 1973. Washington University (St.Louis), Experimental Pathology

Internship and Residency in Anatomic Pathology, 1968-70. Barnes Hospital and Washington University
Hospitals, St. Louis, MO

Research Associate (Postdoctoral Fellowship) in Cancer Research with Michael B. Sporn at the National Cancer Institute, 1970-1975.

Publications
Cohen, S.M., Cobb, E.R., Cordeiro-Stone, M., and Kaufman, D.G.: Chromosomal Localization of the Earliest Replicating DNA Regions in Normal Human Fibroblasts. Experimental Cell Research 245: 321-329, 1998.

Schlemmer, S.R., and Kaufman, D.G.: Endometrial Stromal Cells Regulate Gap-Junction Function in Normal Human Endometrial Epithelial Cells but not in Endometrial Carcinoma Cells. Molecular Carcinogenesis, 28: 70-75, 2000.

Brylawski, B.P., Cohen, S.M., Longmire, J.L., Doggett, N.A., Cordeiro-Stone, M., and Kaufman, D.G.: Construction and Characterization of Cosmid Libraries of DNA Replicated Very Early in the S Phase of Normal Human Fibroblasts. J. Cellular Biochemistry, 78: 509-517, 2000.

Albright, C.D., and Kaufman, D.G.: Lactoferrin: A Tamoxifen-Responsive Protein in Normal and Malignant Endometrial Cells in Culture. Experimental and Molecular Pathology 70: 71-76, 2001.

Arnold, J.T., Kaufman, D.G., Seppl, M., and Lessey, B.A.: Endometrial Stromal Cells Regulate Epithelial Cell Growth In Vitro: A New Coculture Model. Human Reproduction 16: 836-845, 2001.

Arnold, J.T., Lessey, B.A., Seppl, M., and Kaufman, D.G.: Effect of Normal Endometrial Stroma on Growth and Differentiation in Ishikawa Endometrial Adenocarcinoma Cells. Cancer Res. 62: 79-88, 2002.

Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., and Kaufman, D.G.: Mapping of an Origin of DNA Replication Near the Transcriptional Promoter of the Human HPRT Gene. J. Cellular Biochemistry 85: 346-356, 2002.

Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., and Kaufman, D.G.: The Same Origins of DNA Replication Function on the Active and Inactive Human X Chromosomes. J. Cellular Biochemistry 88: 923-931, 2003.

Brylawski, B.P., Cohen, S.M., Horne, H., Irani, N., Cordeiro-Stone, M, and. Kaufman, D.G: Transitions in Replication Timing in a 340-kb Region of Human Chromosome R-Band 1p36.1. J. Cellular Biochemistry 92: 755-769, 2004.

Cohen, S.M., Hatada, S., Brylawski, B.P., Smithies, O., Kaufman, D.G, and. Cordeiro-Stone, M: Complementation of Replication Origin Function in Mouse Embryonic Stem Cells by Human DNA Sequences. Genomics 84: 475-484, 2004.

Barbier, C.S., Becker, K.A., Troester, M., and Kaufman, D.G.: Expression of Exogenous Human Telomerase in Cultures of Endometrial Stromal Cells Does Not Alter Their Hormone Responsiveness. Biol. of Reproduction 73: 106-114, 2005.

Tang, M., Mikhailik, A., Pauli, I., Giudice, L,C., Fazelabas, A.T., Tulac, S., Carson, D.D., Kaufman, D.G., Barbier, B., Creemers, J.W.M., and Tabibzadeh, S.: Decidual Differentiation of Stromal Cells Promotes Proprotein Convertase 5/6 Expression and Lefty Processing. Endocrinology 146: 5313-5320, 2005.

Chastain, P.D., Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M, and. Kaufman, D.G: A Late Origin of DNA Replication in the Trinucleotide Repeat Region of the Human FMR2 Gene. Cell Cycle 5: 869-872, 2006.

Chastain II, P.D., Heffernan T., Nevis, K.R., Lin, L., Kaufmann, W., Kaufman, D.G., and Cordeiro-Stone M. Checkpoint Regulation of Replication Dynamics in UV-Irradiated Human Cells. Cell Cycle 5: 2160-2167, 2006.

Cohen, S.M., Furey, T.S., Doggett, N.A., and Kaufman, D.G: Genome-wide Sequence and Functional Analysis of Early Replicating DNA in Normal Human Fibroblasts. BMC Genomics 7:301, 2006.

Chastain II, P.D., Nakamura, J, Swenberg. J., and Kaufman, W. Non-random Distribution of Endogenous Apurinic/Apyrimidinic Sites in Genomic DNA of Mammalian Cells. FASEB J. 20: 2612-2614, 2006.

Brylawski, B.P., Chastain, P.D., Cohen, S.M., Cordeiro-Stone, M, and. Kaufman, D.G.: Mapping of an Origin of DNA Replication in the Promoter of Fragile X Gene FMR1. Exp Mol Pathol. 82: 190-196, 2007

Kaufman, D.G., Cordeiro-Stone M., Brylawski, B.P., Cohen, S.M., and Chastain II, P.D.: Early S Phase DNA Replication: A Search for Targets of Carcinogenesis. Adv Enzyme Regul. 47: 127-138, 2007.

Cohen, S.M., Cordeiro-Stone, M, and Kaufman, D.G: Early Replication and the Apoptotic Pathway. J. Cell. Physiol. 213: 434-439, 2007.

Barbier, C.S., Kloosterboer, H.J., and Kaufman, D.G.: Effects of Tibolone on Human Endometrial Cells in Co-Culture. Reproductive Sciences 15: 75-82, 2008.

Frum, R.A., Chastain P.D., Qu, P., Cohen, S.M., and Kaufman, D.G.: DNA replication in early S phase pauses temporarily near newly activated origins. In press, Cell Cycle, 2008.

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.

Tano, K., Chastain II, P.D., Asagoshi, K., 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., Essential Role of Fen1 on the Elongation of DNA Replication Fork under Oxidative Stress, Mol Cancer Res. 2010 Feb 9. [Epub ahead of print].

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 Journal, 2010, in press.

E-mail: uncdgk@med.unc.edu
Telephone: (919) 966-1396
Address: 620 Brinkhous-Bullitt, CB# 7525 Chapel Hill, NC 27599-7525

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