SODIUM-INDUCED CHANGES IN THE NUCLEI OF MONOLAYER HeLa CULTURES

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1 J. Cell Sci. II, (1972) 669 Printed in Great Britain SODIUM-INDUCED CHANGES IN THE NUCLEI OF MONOLAYER HeLa CULTURES K. JOBST AND N. KELLERMAYER Department of Clinical Chemistry, Medical University of Pics, Pics, Hungary SUMMARY In HeLa cells cultured in a medium containing 142 mequiv./l. sodium ions only the mitotic forms are birefringent (anisotropic index, 27/1000). In a culture medium containing mequiv./l. sodium ions, large numbers of nuclear forms reminiscent of the prophase were found. In such a medium the anisotropic index was 86/1000. At low sodium ion concentration ( mequiv./l.) these anomalous, prophase-like forms were not seen and the anisotropic index was 18/1000. The appearance of the birefringent, prophase-like forms is related to a structural rearrangement and condensation of DNP in a hypertonic medium. INTRODUCTION In an earlier study (Kellermayer & Jobst, 1970) we related the ion-dependent anisotropy of interphase nuclei isolated in sucrose to the dissociation of DNP structures. We also described an anisotropic nuclear effect in the condensed mitotic chromosomes of tissue culture cells, simultaneously with isotropic nuclei in the intact interphase cells (Kellermayer, Jobst & Angyal, 1970). On the basis of the 2 similar morphological and polarization optical patterns it could be assumed that perhaps even in the living state there existed a relationship between the development of birefringence in mitotic chromosomes and the actual cationic concentration of the nuclei. According to Siebert, Langendorf & Hannover (1965), a rapid equilibration of sodium and potassium ions takes place between extracellular and intranuclear media. Therefore in order to clarify the above relationship we tried to produce, in tissue cultures, the anisotropic nuclear effect characteristic of mitotic nuclei by changing the sodium ion concentration of the culture medium. A possible change in numbers of the mitotic forms, i.e. in the anisotropic index (= the ratio of anisotropic to isotropic nuclei), would support the role of sodium and potassium cations in the development of mitotic chromosomes as well as in the ultrastructural changes (condensation) of DNP (Matsuura & Iwabuchi, 1962; Whitfield, Broh^e & Youdale, 1966; Brasch, Seligy & Setterfield, 1971). MATERIAL AND METHODS The examinations were carried out on HeLa coverglass cultures. Our strain had previously been adapted to a culture medium consisting of 85 % Hanks's solution and 15 % bull serum (standard medium = SM). We tried to influence the cationic concentration of the cells and/or that of the nuclei by changing the sodium ion concentration of the medium. Therefore we either added 5 % sodium chloride to the isotonic Hanks's solution containing 142 mequiv./l. sodium ions or decreased the sodium ion concentration of the Hanks's solution (in the latter case we

2 670 K. Jobst and N. Kellermayer used sucrose to make the solution isotonic). Thus we prepared culture media of sodium ion concentrations between 40 and 160 mequiv./l. and checked their sodium content namephotometrically. Some of the coverglass cultures pre-cultured for 72 h in SM, were transferred under sterile conditions to a medium of higher ionic concentration for 2 h, and others to a solution of lower ionic concentration for 24 h. For polarization- and light-microscopical study the coverglass preparations were rinsed with Hanks's solution for 10 s, dried in air at 22 C for 30 min and, omitting the conventional fixatives, stained for 10 min with 005 % toluidine blue dissolved in ph 3-5 veronal-sodium acetate buffer. Then the preparations were quickly washed with 09 % NaCl solution. In order to stabilize the dye on the structure, it was precipitated by treatment of the slides with 2 % potassium ferricyanide for 2 min. After the potassium ferricyanide had been made to run off the slides, they were covered with gum arabic containing 2 % glycerine and 2 % potassium ferricyanide (= toluidine blue staining followed by precipitation = TBP staining) (Romhanyi, 1963). Some preparations were mounted in pure gum arabic without previous staining. The gum arabic-covered preparations were dried in air and then studied under a Zeiss-Opton polarization microscope equipped with rotating 20- and 57-/tm compensators and a continuous interference filter. TBP staining was also used for light-microscopic study of the preparations. Switching on the analyser of the microscope enabled us to examine in polarized light the forms classified by light microscopy as mitotic or mitosis-like. Thus on the basis of an analysis of the same nuclei the number of dividing forms observed under the light microscope and characterized by the mitotic index could be compared with the number of birefringent nuclei characterized by the anisotropic index. The mitotic and anisotropic indices of the preparations were determined on the basis of studying a total of 3000 nuclei per coverglass. After incubation in the media of lower or higher ionic concentration, the preparations were postcultured in SM for h and thereafter stained and studied in several experiments. It should be mentioned that in the unstained preparations mounted in gum arabic, the mitotic nuclei showed only weak intrinsic birefringence. This birefringence was, however, markedly enhanced by the above-mentioned TBP staining, which results in an oriented alignment of the dye molecules on the DNA of the chromosomes. RESULTS Light microscopy showed that the mitotic index of the HeLa cells cultured in SM averaged 27/1000 (range, 25-29/1000). In a medium of low sodium ion concentration (40-80 mequiv./l.) the cells did not divide and became detached from the coverglass. In such a medium the cells are not viable. In a medium containing mequiv./l. sodium ions the cells are attached, and the mitotic index was 18/1000. The index rose to 23/1000 after a 24-h period of postculture but failed to reach the value of 27/1000 obtained in the SM. Thus a culture medium of decreased sodium ion concentration hinders the development of mitotic, birefringent forms. At the same time the morphological appearance of the mitotic forms seen here did not differ from those observed in SM, and these mitotic forms were birefringent. In a medium of higher sodium ion concentration (over 142 mequiv./l.) we found large numbers of condensed, prophase-like nuclei, which were invariably anisotropic (see Fig. 1 c, D). Since according to our previous studies it is only the chromatin of mitotic forms that TBP staining shows to be birefringent (Kellermayer et al. 1970), we believe that the large number of condensed nuclei appearing in a medium containing between 145 and 160 mequiv./l. sodium ions can, on the basis of their anisotropy,

3 Sodium-induced changes in nuclei 671 Table 1. Effect of sodium ion concentration of medium on anisotropic index and mitotic index Sodium ion concentration of medium, mequiv./l I5O-I Anisotropic index, / 00 (polarization microscopy) 27 ±3 34 ±3 54 ±3 86 ±5 Mitotic index, / m (light microscopy) 28 ±2 33 ±3 50 ±5 80 ±7 be regarded as mitotic. This observation seems to indicate a relation (see Table 1) between the existing sodium ion concentration of the medium and the number of anisotropic nuclei (anisotropic index), and a similar relation between the said ion concentration and the number of nuclei classified as mitotic by light microscopy (mitotic index). That is, with increasing sodium ion concentration of the medium both the anisotropic and the mitotic indices increased. When preparations cultured at a sodium ion concentration of mequiv./l. were post-cultured in an isotonic medium for 48 h, then their mitotic index decreased to 33-35/1000. At this index almost all mitotic forms are typical. DISCUSSION The investigations presented here indicate that in a medium of high sodium ion concentration a morphological transformation of HeLa cell nuclei takes place (Robbins, Pederson & Klein, 1970; Schachtschabel & Foley, 1972). This manifests itself mostly in nuclear forms reminiscent of the late prophase of untreated cells, which are birefringent just like the mitotic forms in SM (Fig. 1 A, B) and the isolated nuclei incubated in vitro with 142 mequiv./l. sodium chloride (Jobst & Kellermayer, 1967). This observation indicates that the sodium ion plays an important role in mitosis: it may control and/or block the mitotic activity of the cell (Cone & Tongier, 1971). As an explanation it may be assumed that the local increase in sodium ion concentration (Siebert & Langendorf, 1970) results in DNP dissociation, chromatin aggregation and condensation of the nucleus, which is reflected in optical polarization in the birefringence of such nuclei (Jobst & Kellermayer, 1967). Thus the appearance of nuclear anisotropy is indicative of a structural rearrangement of DNP. The anomalous mitotic cell forms produced in vivo under the effect of hypertonic salt confirm, on the one hand, the role played by inorganic salts in the intracellular metabolic processes of the nucleus, and on the other hand, they show that optical anisotropy is a sensitive indicator of the intranuclear changes of chromatin structure.

4 672 K. Jobst and N. Kellermayer REFERENCES BRASCH, K., SELIGY, V. L. & SETTERFIELD, G. (1971). Effects of low salt concentration on structural organization and template activity of chromatin in chicken erythrocyte nuclei. Expl Cell Res. 65, CONE, C. D. & TONGIER, M. (1971). Control of somatic cell mitosis by simulated changes in the transmembrane potential level. Oncology zs, JOBST, K. & KELLERMAYER, M. (1967). Submicroscopic structure and dry weight of isolated thymus nuclei following trypsin and salt treatment. Polarization optical, interference microscopic and cytophotometric studies. Acta morph. Acad. Sci. hung. 15, KELLERMAYER, N. & JOBST, K. (1970). Ion-dependent anisotropy of deoxyribonucleoprotein structures in tissue cultures. Expl Cell Res. 63, KELLERMAYER, M., JOBST, K. & ANGYAL, T. (1970). Polarization-optical study of the ultrastructure of cell nuclei in tissue cultures. Acta morph. Acad. Sci. hung. 18, MATSUURA, H. & IWABUCHI, M. (1962). Effect of inorganic salts on cell division II. J. Fac. Sci. Hokk. Univ. 8, ROBBINS, E., PEDERSON, T. & KLEIN, P. (1970). Comparison of mitotic phenomena and effects induced by hypertonic solutions in HeLa cells. J. Cell Biol. 44, ROMHANYI, GY. (1963). t)ber die submikroskopische strukturelle Grundlage der metachromatischen Reaction. Acta histochem. (jfena) 15, SCHACHTSCHABEL, D. O. & FOLEY, G. E. (1972). Serial cultivation of Ehrlich ascites tumor cells in hypertonic media. Expl Cell Res. 70, SIEBERT, G., LANGENDORF, H. & HANNOVER, R. (1965). Untersuchungen zur Rolle des Natrium- Stoffwechsels im Zellkern der Rattenleber. Hoppe-Seyler's Z. physiol. Client. 343, SIEBERT, G. & LANGENDORF, H. (1970). Ionenhaushalt im Zellkern. Natnnvissenscliaften 57, WHITFIELD, J. F., BROHEE, H. & YOUDALE, T. (1966). Mitotic stimulation in normal and irradiated suspension cultures of rat bone marrow by an elevated salt concentration. Expl Cell Res. 41, (Received 27 March 1972)

5 Sodium-induced changes in nuclei 673 Fig. 1. A, B, a HeLa culture grown for 72 h in so-called standard medium containing 142 mequiv./l- sodium ions, and stained with the TBP method. A, light microscope; B, polarization optical picture, x 550. As well as several isotropic interphase nuclei there is a birefringent nucleus in prophase, with loosened chromatin structure. C, D, HeLa culture grown in standard medium for 72 h, then incubated at 37 C for 2 h in a medium containing 155 mequiv./l. sodium ions and stained by the TBP method, c, light microscope; D, polarization optical picture, x 550. The light microscopy image shows 2 condensed, basophilic nuclei, which, unlike the interphase forms, are reminiscent of the late prophase and are birefringent.

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