Shirley L. Nakatsu, Marilyn A. Masek, Sharon Landrum, and John H. Frenster

Division of Medical Oncology, Stanford University School of Medicine, Stanford, California 94305 

Introduction:
Materials and Methods:
Results:
Discussion and Conclusions:
Support:
References:
Links to Related Sites: 

Bone marrow spicules (5) and peripheral blood cells (6) were isolated under sterile conditions from patients as previously described, and were subjected to the analysis of of active templates of active DNA templates while in the living state (4). Acridine orange was used as previously described (4) to probe for the location and number of active DNA templates within each cell (5). Control experiments omitting either the acridine orange or the DNase steps from the reaction sequence did not reveal any reaction product (5). Positive cells revealed large (>0.1 um) or small (0.025-0.1 um) reaction products localised exclusively within the nuclear euchromatin complexes (5). These probe sites were counted within each cell by one observer, and another observer independently classified the stage of cell division or of cell differentiation, using the ultrastructural criteria previously reported for dividing cells (7), differentiating granulocytes (8), and differentiating erythrocytes (9).

A total of fifty dividing normal bone marrow cells were analysed (Table 1).
 

Table 1. Probe counts in dividing bone marrow cells.

Stage of cell division	Mean probe count per cell
Early prophase	35.3
Late prophase	14.8
Metaphase, anaphase, early telophase	0
Late telophase	85.0
Interphase	35.1

These analyses revealed a progressive decrease in the number of probe sites per cell through the early and late stages of prophase, to an absence of any probe sites in metaphase, anaphase and early telophase, with a subsequent rapid increase in the number of probe sites per cell cell in late telophase, returning to a basal level in interphase.

Similarly a total of 123 normal differentiating granulocyte precursor cells and 176 normal differentiating erythrocyte precursor cells (Table 2) were analyzed.
 

Table 2. Probe counts within differentiating marrow cells.

Stage of Cell  Differentiation:	Percent Cells Containing:		Mean Probe Count per Positive Cell:	Ratio:
	(Large Probes)/  (Small Probes)		(Large Probes)	Large Probes/ Total Probes
Granulocytes:
Promyelocytes	91.5 / 100		9.55	0.19
Myelocytes	85.9 / 96.4		8.75	0.18
Metamyelocytes	47.6 / 85.7		3.9	0.05
Band  Granulocytes	0 / 20.0		0	0
Segmented  Granulocytes	0 / 12.2		0	0
Erythrocytes:
Proerythroblasts	100 / 100		16.0	0.65
Early  Erythroblasts	84.7 / 91.6		8.5	0.32
Late  Erythroblasts	14.8 / 17.2		3.4	0.20
Nucleated  Erythrocytes	0 / 8.3		0	0

These analyses revealed a progressive decrease during the course of normal cell differentiation in the percentage of cells containing either large or small probe sites. In addition, these analyses revealed a progressive decrease during cell differentiation in the number of large probe sites per positive cell, as well as a progressive decrease in the fraction of all probe sites represented by large probe sites (Table 2). These observed decreases in DNA template activity during the course of normal bone marrow cell differentiation are statistically highly significant (P< 0.01) by Mann's test for trend significance (10).

Independent analyses have previously revealed a direct correlation between DNA template activity as measured by probe sites within isolated chromatin, and the rate of RNA synthesis by such isolated chromatin when supplied with excess amounts of exogenous RNA polymerase and monoribonucleotides (11). The studies reported here measuring probe sites within intact living cells similarly correlate closely with previous independent studies demonstrating a progressive decrease in RNA and DNA synthesis and cell division during normal bone marrow cell differentiation (2). These quantitative data suggest that a progressive restriction of DNA templates is an important mechanism during normal cell division and bone marrow cell differentiation (12). We are currently studying the molecular species (13) mediating such restriction and release of DNA template activity during normal (6) and leukemic cell division and differentiation (14).

This work was supported in part by grants from the National Cancer Institute and the American Cancer Society, and by a Research Scholar Award from the Leukemia Society to J.H.F to whom all correspondence should be sent.

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4. Frenster JH, Nature New Biol. 236, 175 (1972).

5. Frenster JH, Cancer Res. 31, 1128 (1971).

6. Frenster JH, Best WR, and Winzler RJ, Proc. Soc. Exp. Biol. Med. 98, 887 (1958).

7. Robbins E, and Gonatas NK, J. Cell Biol. 21, 429 (1964).

8. Anderson DR, J. Ultrastruc. Res. Suppl. 9, 5 (1966).

9. Bessis MC, and Breton-Gorius J, Blood 19, 635 (1962).

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11. Seligy VL, and Lurquin PF, Nature New Biol. 243, 20 (1973).

12. Frenster JH, and Herstein PR, New Eng. J. Med. 228, 1224 (1973).

13. Frenster JH, Nature 208, 1093 (1965).

14. Frenster JH, in "The Cell Nucleus" (edit. Busch H), 1, 565-580 (Academic Press, New York, 1974).

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