"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

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, derepressed 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):
 

Contrasts between Euchromatin and Heterochromatin in Animal Cells. (a)

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),
(Figure 1), 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 JH, 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). 

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