Jeannette A. Hovsepian ^{1, @} and John H. Frenster ^{2, @}

Departments of  ¹Radiology and of  ² Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

^@ Present Address:  Physicians' Educational Series, Atherton, California, 94027-5446 USA
Phone:  +1 650 367 6483;   Fax:   +1 650 364 1773
e-mail:   frenster@euchromatin.net

Supported in part by a USPHS Research Career Development Award (CA-17857) from the National Cancer Institute to J.H.F., and by USPHS Research Grants from the National Cancer Institute.

High-resolution electron microscopy of mammalian cell nuclei reveals interphase chromatin as partitioned between condensed heterochromatin and extended euchromatin. DNase I-sensitive ultrastructural probes of intact cells reveals active DNA sites that are confined exclusively to euchromatin. Active DNA sites within intact cells can be counted, measured, and analyzed for shape and for correlation to cell type, cell differentiation, and/or cell neoplasia. Within the cell nucleus, euchromatin is found as extended 10 nm microfibrils, that tend to occupy the nuclear center, with displacement of condensed heterochromatin masses to the nuclear margin. In the conversion of heterochromatin to euchromatin during mitogen-induced blastogenesis, euchromatin microfibrils are furthered extended, with subsequent enlargement of the cell nucleus and extreme displacement of condensed heterochromatin to the nuclear margins. Euchromatin fractions contain an excess of nuclear polyanions compared to heterochromatin fractions, while polycationic histones are more evenly distributed between euchromatin and heterochromatin. This distribution gives euchromatin microfibrils a net negative charge, while heterochromatin condensed masses are near electro-neutrality. When heterochromatin predominates, as in mature erythrocytes, lymphocytes and sperm, the cell nucleus is reduced in diameter. The most likely mechanism for euchromatin microfibril extension is the mutual repulsion generated by excess polyanions on the euchromatin microfibrils. Such mutual repulsion expands the size of the cell nucleus, increases the diffusible space between the microfibrils, and allows the free circulation and diffusion of enzymes, proteins, RNA and oligonucleotides among the microfibrils and to the nuclear pores on the nuclear membrane. Small RNA molecules are particularly important in this environment as both activators and products of active DNA sites. 

Nuclear size varies during cell differentiation within mammalian cells, and may relate to the extension or compaction of individual euchromatin 10 nm microfibrils. Extremes of chromatin compaction and small nuclear size are often found within cells with a high degree of heterochromatin and reduced nuclear activity, such as mature erythrocytes, granulocytes, lymphocytes, and spermatids [1].

Most of these cells have reduced activity of RNA synthesis, DNA synthesis, and protein synthesis, with predominant masses of compacted heterochromatin and reduced or missing nucleoli, and with diameters of cell nuclei reduced from those cells earlier in the cell differentiation pathway [2].

Certain mammalian cells are capable of a reversible nuclear enlargement and contraction in vitro, such as in T-lymphocytes during and after exposure to a mitogen such as phytohemagglutinin (PHA). The effects of PHA on T-lymphocytes are known as lymphoblastoid transformation, and include increases in euchromatin microfibrils, nuclear size, RNA synthesis, nucleolar size, DNA synthesis, and mitotic cell division, all within 72 hours of first exposure to PHA. In the absence of PHA, none of these effects occurs in the culture [3].

If PHA is withdrawn from the T-lymphocyte culture, all of these effects are reversed, with a cessation of mitosis and of DNA synthesis, a decrease in RNA synthesis, a decrease in nucleolar size, a decrease in nuclear size, and a decrease in the amount of euchromatin, with a full conversion of the extended euchromatin 10 nm microfibrils to a compacted heterochromatic state [4].

The availability of the PHA-lymphoblastoid transformation system in vitro enables a closer look at the extensile properties of euchromatin 10 nm microfibrils, and the role such extensile forces play in the dimensions and activity of the mammalian cell nucleus [5, 6].

Methods:

Tritiation of Phytohemagglutinin:
Twenty-four mg. of highly purified PHA in the form of the glycoprotein octamer (Rigas, [7]) was exposed for 2 weeks to 15.0 curies of tritium gas at a pressure of 660 mm. Hg and a temperature of 26^oC. [3]. The resultant ³H-PHA was lyophylized from 20 ml. of distilled water, and possessed a specific activity of 1.4 millicuries/milligrams. When assayed within 2 weeks following the completion of tritiation, the mitogenic activity was only slightly reduced compared to the pre-tritiation mitogenic activity.

Incubation Conditions:
A modification of the method of Moorehead, et al was employed [8]. Normal human blood was aspirated during the fasted state under sterile conditions into heparinized syringes, was thoroughly mixed with heparin, 4 units/ml, was centrifuged at 10 g. for 10 minutes, and the resultant leukocyte-rich supernate was collected and diluted 1:5 with medium 199. Additional heparin was added to a concentration of 4 units/ml, penicillin and streptomycin were each added to 0.060 mg/ml, and ³H-PHA (described above) was to 0.010 mg/ml, 5 ml aliquots of the resultant cell suspension, containing approximately 10⁶ lymphocytes/aliquot, were transferred to 15 ml screw-cap plastic flasks. These were tightly sealed, placed flat on their sides, and incubated at 37^oC for periods of time ranging from 15 minutes to 48 hours.

Electron Microscopy:
At the conclusion of incubation, the contents of each flask were centrifuged at 200 g for 10 minutes, and 5 mg of 5 percent glutaraldehyde dissolved in medium 199 was added to the resultant centrifuge pellet. These were allowed to stand at 4^oC for 48 hours, and were then post-fixed in 1 percent OsO₄, dehydrated in progressive ethanol concentrations, embedded in epon, uniformly sectioned at 1000 A^o thickness, stained with uranyl acetate, and prepared for high resolution radioautography by the method of Caro and Van Tubergen [9]. After 2 months exposure to the Ilford 4 emulsion, the grids were examined without NaOH removal of the developed emulsion on a Siemens Elmiskope 1A at 80 KV. The resultant micrographs were printed on a low-contrast photographic paper to enhance radioactive grain contrast.

Results:

Fig. 1 (left, above). Electron micrograph of a normal human blood lymphocyte incubated in vitro in the absence of phytohemagglutinin for 48 hours. The cytoplasm is scanty and composed largely of monosomal ribosomes. The nucleus is compact with a major part of the DNA contained within condensed heterochromatin masses arranged directly underneath the nuclear membrane. Only a small minority of nuclear chromatin is arrayed as euchromatin 10 nm microfibrils. The nucleolus is small and surrounded by additional condensed heterochromatin. (X 16,000).

Fig. 2 (right, above). Electron micrograph of normal human blood lymphocyte incubated in vitro in the presence of phytohemagglutinin for 48 hr (see Fig. 1). The lymphocyte has undergone lymphoblastic transformation [6] with an increased cytoplasm composed largely of polysomal ribosomes. The nucleus has enlarged with a major part of the DNA contained within extended euchromatin 10 nm microfibrils dispersed throughout the nucleus. The nucleolus has enlarged and is free of any surrounding heterochromatin. The plasma membrane has increased its number of microvillus projections and phagocytic activity. (X 9,000).

1. Mammalian cell nuclei increase their dimensions after cell activation.

2. Human T-lymphocytes can be activated in-vitro by PHA mitogen.

3. After 48 hours in PHA culture, T-lymphocytes have undergone the following:

     a. Conversion of condensed heterochromatin to extended 10 nm euchromatin microfibrils.
     b. Onset of nuclear RNA synthesis throughout euchromatin.
     c. Onset of conversion of monoribosomes to polyribosomes throughout cytoplasm.
     d. Tripling in size of nuclear diameters.
     e. Doubling in size of nucleolar diameters.
     f. Doubling in size of cell diameters.
     g. Onset of nuclear DNA synthesis throughout euchromatin.

4. 72 hours after transfer to a PHA-free culture, all of these changes are reversed.

5. T-lymphocytes can undergo several such cycles of activation and de-activation.

Conclusions:

1. Changes in cell nucleus dimensions reflect changes in the cell nucleus chemistry  [10].

2. An early change during T-lymphocyte activation by PHA is tunneling of heterochromatin masses.

3. Tunneling results in conversion of heterochromatin masses to euchromatin 10 nm microfibrils.

4. Euchromatin 10 nm microfibrils contain an excess of polyanions compared to heterochromatin [10].

5. Such euchromatin 10 nm microfibrils are tethered to remaining original heterochromatin masses [11].

6. Mutual repulsion between tethered negatively-charged euchromatin 10 nm microfibrils results in:

     a. Linear uni-dimensional extension of increasing numbers of euchromatin 10 nm microfibrils.
     b. Promoted acceleration of conversion of heterochromatin masses to euchromatin 10 nm microfibrils.
     c. Displacement of remaining heterochromatin masses to nuclear margins.
     d. Concentration of extended euchromatin 10 nm microfibrils to the nuclear center.
     e. Increase in diameters of cell nuclei after cell activation.

7.  Euchromatin 10 nm microfibrils are a dynamic extensile force within the mammalian cell nucleus  [11].

1. Frenster JH, "Ultrastructure and Function of Heterochromatin and Euchromatin", in:  "The Cell Nucleus", vol. 1, pp. 565-580, (1974), (Busch H, ed.), New York, Academic Press.

2. Frenster JH, Nakatsu SL, and Masek MA, "Ultrastructural Probes of DNA Templates within Human Bone Marrow and Lymph Node Cells", "Advances in Cell and Molecular Biology", vol. 3, pp. 1-19 (1974), ed. DuPraw EJ, New York: Academic Press.

3. Stanley DA, Frenster JH, and Rigas DA, "Localization of ³H-Phytohemagglutinin within Human Lymphocytes and Monocytes", "Proceedings of the Fourth Annual Leukocyte Culture Conference, 1969", (McIntyre OR, ed.), pp. 1-11, (1971), New York: Appleton-Century-Crofts.

4. Polgar PR, Kibrick S, and Foster JM, "Reversal of PHA-Induced Blastogenesis in Human Lymphocyte Cultures", Nature 218, 596 (1968).

5. Rubin AD, and Cooper HL, "Evolving Patterns of RNA Metabolism during Transition from Resting State to Active Growth in Lymphocytes Stimulated by Phytohemagglutinin", Proc. Natl. Acad. Sci. U.S. 54, 469 (1965).

6. Tokuyasu K, Madden SC, and Zeldis LJ, "Fine Structural Alterations of Interphase Nuclei of Lymphocytes Stimulated to Growth Activity In Vitro", J. Cell Biol. 39, 630 (1968).

7. Rigas DA, and Head C, "The Dissociation of Phytohemagglutinin of Phaseolus Vulgaris by 8.0 M Urea and the Separation of the Mitogenic from the Erythroaggluntinating Activity", Biochem. Biophys. Res. Comm. 34, 633 (1968).

8. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, and Hungerford DA, "Chromosome Preparations of Leukocytes Cultured from Human Peripheral Blood", Exp. Cell Res. 20, 613 (1960).

9. Caro LG, and Van Tubergen RP, "High-Resolution Autoradiography. I. Methods", J. Cell Biol. 15, 173 (1962).

10. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature 206, 680-683 (May 15, 1965).

11. Frenster JH, "Ultrastructural Continuity Between Active and Repressed Chromatin",
Nature 205, 1341-1342 (March 27, 1965).

12. Hovsepian JH, and Frenster JH, "Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription", Molec. Biol. Cell, vol. 14, supp. p. 242a (November, 2003).

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E-mail: frenster@euchromatin.net