"Single-Cell Analysis of DNase I-Sensitive Sites during Neoplastic and Normal Cell Differentiation within Human Bone Marrow."
John H. Frenster
Departments of Medicine, Stanford University and Santa Clara Valley
Medical Center,
San Jose, California 95128
E-mail: frenster@euchromatin.net
DNase I-sensitive sites in chromatin correspond to points of active gene transcription (1), and can be visualized by high-resolution electron microscopy of intact single cells (2). These methods were applied to leukemic and normal human bone marrow spicules, and quantitative analysis revealed an inverse correlation between the advancing stages of granulocytic or erythrocytic cell differentiation and the number and size of DNase I-sensitive sites per cell nucleus. The sites range in size from 25 to 700 nm in length, corresponding to 70-2000 base pairs in length of DNA helix. These DNase I-sensitive sites, which represent localized DNA helix openings (3), are found exclusively within extended, de-repressed euchromatin and never within condensed, repressed heterochromatin.
Detailed studies of the molecular biophysics, biochemistry, and ultrastructure of euchromatin and heterochromatin within animal cells (4) have revealed a striking partition of function (5) between the two states of chromatin (Table 1):
| Euchromatin | Heterochromatin |
| Extended microfibrils | Condensed masses |
| Active RNA synthesis | No RNA synthesis |
| Early DNA replication | Late DNA replication |
| Many DNA helix openings | No DNA helix openings |
| DNase I-sensitive sites | No DNase I-sensitive sites |
| Many nuclear polyanions | Few nuclear polyanions |
| Loose binding of histones to DNA | Tight binding of histones to DNA |
| Reduced number of nucleosomes | Full number of nucleosomes |
| Increased binding of steroid hormone | Decreased binding of steroid hormone |
| Binding of oncogenic viral DNA | No binding of oncogenic viral DNA |
| Binding of chemical carcinogens | Little binding of chemical carcinogens |
| Little binding of PHA mitogen (b) | Much binding of PHA mitogen (b) |
| Decrease during cell differentiation | Increase during cell differentiation |
| Decrease during cell division | Increase during cell division |
| Increase during cell neoplasia | Decrease during cell neoplasia |
| Increase during lymphocyte activation | Decrease during lymphocyte activation |
| Resistance to added RNA | Response to added RNA |
| (a) Individual References in Ref. (5) | |
| (b) PHA: Phytohemagglutinin
|
Since oncogenic viral DNA requires DNA helix openings within complementary
host DNA sequences for effective viral oncogenesis (6),
these sequences in euchromatin (7) also allow gene transcription
characteristic of the neoplastic state (8), often involving
derepression of previously inactive embryonic genes (9);
loss of viral DNA from these integration sites correlates with reversion
of the transformed cells to the normal state (10).
Figure 1. Comparison of viral oncogenesis and cell differentiation in animal cells.
If normal cells are susceptible, they may be infected by an oncogenic virus (step 1). If the normal cells contain DNA sequences complementary to sequences in the viral DNA, the viral DNA may be bound and integrated (step 2). Bound viral DNA may then induce synthesis of RNA species usually repressed in a normal cell (step 3). The resultant transformed cell may enter the G1 and S phases of the proliferation cycle (step 4). An excess of cell proliferation over cell destruction results in a growing neoplasm (step 5). Cell differentiation processes pass through similar steps (9). Steps 3, 2, and 1 may be reversible, as steps 6, 7, and 8, in both systems (10).
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.
References:
1. Frenster JH, "Electron microscopic localization of acridine orange binding to DNA within human leukemic bone marrow cells." Cancer Res. 31: 1128-1133 (1971).
2. 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. 63: 331-335 (1979).
3. Frenster JA, "Selective control of DNA helix openings during gene regulation." Cancer Res. 36: 3394-3398 (1976).
4. Frenster JH, "Ultrastructure and function of heterochromatin and euchromatin." In: "The Cell Nucleus." Busch H, editor. Vol. 1: 565-580, Academic Press, New York. (1974).
5. Frenster JH, "Selective gene de-repression by de-repressor RNA." In: "Eukaryotic Gene Regulation." Kolodny GM, editor. Vol. 1: 131-143, CRC Press, Boca Raton, FL. (1980).
6. Tereba AL, Skoog PK, Vogt PK. "RNA tumor virus specific sequences in nuclear DNA of several avian species." Virology 65: 524-534 (1975).
7. De La Maza LM, Faras MA, Varmus H, Vogt PK, Yunis JJ, "Integration of avian sarcoma specific DNA in mammalian chromatin." Exp. Cell Res. 93: 484-489, (1975).
8. Van Dyke MW, Roeder RG, Sawadogo M, "Physical analysis of transcription preinitiation complex assembly on a class II gene promoter." Science 241: 1335-1338 (1988).
9. Frenster JH, Herstein PR. "Gene De-Repression." New Eng. J. Med. 288: 1224-1229 (1973).
10. Nomura S, Fischinger PJ, Mattern CFT, Gerwin BI, Dunn KJ. "Revertants of mouse cells transformed by murine sarcoma virus: Flat variants induced by FUDR and colcemide." Virology 56: 152-163 (1973).
Additional References:
1. "Electron Microscopy of Human Lymphocytes before and after Activation by PHA" (Busch H, 1974).
2. "Oncogenes as Molecular Targets within Active Chromatin", (Frenster JH, 1999).
3. "Activation of DNA Transcription within Repressed Chromatin",
(Frenster JH, 2001).
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