"Single-Cell Analysis of DNase I-Sensitive Sites During Neoplastic Cell Differentiation within Hodgkin's Disease Lymph Nodes."
John H. Frenster, M.D.
Department of Medicine, Stanford University, and Institute for Medical Research, Santa Clara Valley Medical Center, San Jose, California 95128, USA.
Neoplastic cells can undergo a form of cell differentiation and maturation both in-vitro and in-vivo, mediated by a progressive reduction in gene transcription and RNA synthesis (1). DNA base sequences which are actively transcribed to RNA within intact chromatin are characterized by the presence of DNA helix openings (2) and by a selective sensitivity to digestion by DNase I (Proc Natl Acad Sci US, Vol 78, 2669 (1981). An electron microscopic technique was developed to localize sites of DNase I-sensitivity within intact single cells (3,4). The discrete electron-dense reaction products formed were confined to the transcription-active extended euchromatin portion of the cell nucleus. Omission of DNase-I resulted in complete absence of any reaction product. The DNase I-sensitive sites range in size from 25-700 nm, equivalent to a minimum of 70-2000 base pairs in DNA helix length. Intact lymph nodes positive for the nodular sclerosis type of Hodgkin's Disease were biopsied from untreated patients at the time of original laparotomy staging of their disease, and each cell, normal or neoplastic, within each biopsied lymph node was analyzed for the location, size, and number of DNase I-sensitive sites per single cell. Neoplastic Hodgkin's cells and Reed-Sternberg cells were identified in their separate stages of differentiation and maturation by ultrastructural criteria (5). Neoplastic cells earliest in the cell maturation sequence (Hodgkin's cells) contained the largest number of DNase I-sensitive sites per cell nucleus, and these sites were also of the largest size per site. At each morphologic step in neoplastic cell maturation, from mononuclear Reed-Sternberg cell, to binuclear Reed-Sternberg cell, to multinuclear Reed-Sternberg cell, the number and size of individual DNase I-sensitive sites decreased with increasing cell maturation, correlating with a similar decrease in RNA synthesis, DNA synthesis, and mitotic index within these same neoplastic cells during such in-vivo cell differentiation and maturation. Neoplastic cell differentiation within the involved lymph nodes of untreated patients with Hodgkin's Disease may thus share some important biological and molecular features with normal cell differentiation in other human cell systems (6,7).
References:
1. Frenster JH, Herstein PR, "Gene De-Repression", New
Eng J Med vol. 288, 1224-1229 (1973).
2. Frenster JH, "Selective Control of DNA Helix Openings during Gene Regulation", Cancer Research, vol. 36, 3394-3398 (1976).
3. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bone Marrow Cells", Cancer Research vol. 31, 1128-1133 (1971).
4. Frenster JH, Papalian MM, Masek MA and Frenster JA, "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease", J Natl Cancer Inst vol. 63, 331-335, (1979).
5. Archibald RB, Frenster JH, "Quantitative Ultrastructural Analysis of Lymphocyte-Reed Sternberg Cell Interactions in Hodgkin's Disease", Natl Cancer Inst Monogr, vol. 36, 239-245(1973).
6. Nakatsu SL, Masek MA, Landrum S and Frenster JH, "Activity of DNA Templates during Cell Division and Cell Differentiation", Nature vol. 248, 334-335 (1974).
7. Frenster JH, "Electron Microscopic Analysis of Asymmetry within the Cell Nucleus of DNA Helix Openings during Cell Activation and Cell Differentiation", Proc 38th Annual Meeting Electron Microscopy Society of America, 544-545, edit. by Bailey GW (Claitor's Publ Div, Baton Rouge, LA/1980).
Supported in part by Research Grants CA-10174 and CA-13524 from the National Cancer Institute, by Research Grant IC-45 from the American Cancer Society, and by a Research Scholar Award from the Leukemia Society of America.
Additional references:
0. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).
1. Hodgkin's Disease Abstracts Published November 1998.
2. Hummel M, Marafioti T, and Stein H, "Clonality of Reed-Sternberg Cells in Hodgkin's Disease", New Eng. J. Med. 340: 394-395 (1999).