There are two areas of research in our laboratory: 1.) 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 to study DNA replication origins and regions of DNA replicated at the beginning of S phase. 2.) 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
Our goal is to discover molecular mechanisms that relate cell proliferation and chemical carcinogenesis. We showed that cells are most susceptible to malignant transformation when treated with chemical carcinogens during the earliest part of the S phase. DNA is preferentially damaged when it replicates; elevated carcinogen binding to DNA occurred at replication forks. This implies that susceptibility in early S phase occurs because DNA targets critical for malignant transformation are replicated early in 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). DNA replicated early in S phase was labeled and the highest labeling was identified in six chromosomal bands in the metaphase. Libraries of genomic DNA replicated in the earliest part of the S phase yielded nearly 10,000 clones which were sequenced and mapped to the human genome; clones were found in each of the early replicating chromosomal bands. Ontogeny analysis showed that 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. Somewhat less strongly linked were genes in the Wnt pathway and genes for base excision repair. Starting with a clone from one of the earliest replicating chromosomal bands (1p36.1) we mapped the timing of replication through this region early in S phase. Two origins of replication separated by about 20 kB were found in this region; they are activated one hour apart. DNA replication either ceased or progressed very slowly at a boundary between these two origins where palindromic DNA was present. Further studies used stretched DNA fibers to examine the timing of DNA replication through specific genomic regions. This technique was used for analysis of DNA damage and repair. We found DNA damage using antibodies and measured the length of DNA examined to determine the intensity of DNA damage as the number of damage sites per 106 base pairs. This technique proved to yield accurate measurements with low variability using as few as 5-10 cells. We are now testing whether we can detect DNA damage in small numbers of circulating tumor cells (CTCs) recovered from patients treated with cancer chemotherapeutic drugs. If successful this analysis may serve as a means of determining the efficacy of the drug for a specific patient’s cancer.
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. 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. We studied the different functions of estrogen receptors alpha and beta, and the effect of signaling through the IGF-I pathway on epithelial cell proliferation. We are also evaluating genes expressed abnormally in endometrial intraepithelial neoplasia (EIN) to seek an immunohistochemical test to identify EIN.
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.
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.
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.
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.
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.
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.
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.
Schlemmer, S.R., and Kaufman, D.G.: Re-establishment of Gap-Junction Intercellular Communication in Human Endometrial Carcinoma Cells by Prostaglandin E2. Experimental and Molecular Pathology 93: 441-448, 2012.
Smith-Roe, S.L., Nakamura, J., Holley, D., Chastain, P.D., Rosson, G.B., Simpson, D.A., Ridpath, J.R., Kaufman, D.G., Kaufmann, W.K., and Bultman, S.J. SWI/SNF Complexes are Required for Full Activation of the DNA-Damage Response. Oncotarget. 6: 732–745, 2015.