Nuclear protein content and DNA-histone interaction

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1972 by Academic Press, Inc. in any form reserved

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Experimental Cell Research 7.5(1972) 231-236

NUCLEAR

PROTEIN

CONTENT

AND

DNA-HISTONE

INTERACTION

G. AUER Institute

for Medical Cell Research & Genetics, Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, Sweden

SUMMARY Epithelial kidney cells newly seeded in primary slide cultures show a marked increase in nuclear protein content which always precedes initiation of DNA synthesis. The accumulation of proteins in the nucleus is accompanied by a decreased stainability of DNA-bound residues in histones, indicating a decreased interaction between DNA and histones. This is further supported by the increased acridine orange binding to DNA phosphate groups in the DNA-histone complex in cells with increased nuclear protein content. The results are interpreted to reflect a masking of DNA-bound residues in histones due to the accumulation of non-histone proteins in the cell nucleus.

Previous work has shown that the vast majority of epithelial kidney cells from 14day-old mice, newly seeded in primary slide cultures, consist of cells with small, compact nuclei, condensed chromatin and relatively low nuclear protein content [5, 61. Prior to initiation of DNA synthesis and cell proliferation the nuclei in these cells undergo a sequence of events of which two steps have been identified by cytochemical methods. The first involves an initial change of the DNP complex, characterized by an increased dye binding capacity of DNA phosphate groups [2, 3, 12, 24-281 and by a decreased stainability of DNA-bound residues in histones [1,2]. This initial DNP change has been found to be non-enzymatic [3] and is sensitive to changes of the environmental ion composition [4]. The second step involves a marked increase of proteins in the cell nucleus

associated with a dispersion of the chromatin and an increased rate of RNA synthesis [5,6]. The mechanism by which protein accumulation in the nucleus is involved in the stimulated RNA synthesis is not known. One possibility may be that protein accumulation in the cell nucleus affects the interaction between DNA and histones increasing the template activity of DNA for RNA synthesis. The aim of the present investigation was to study the cytochemical properties of the DNP complex in intact cells in relationship to the accumulation of proteins in the nucleus. For this purpose epithelial kidney cells in newly seeded primary cultures were used as the biological system. The interaction between DNA and histones was studied by means of the alkaline bromphenol blue dye binding method combined with quantitative microspectrophotometry. Exptl Cell Res 75 (1972)

232 G. Auer Cytochemical techniques Nuclear dry mass determinations in individual cells were oerformed in a microinterferometer with a diaphragrn for limiting of the field of measurement as described elsewhere [8]. For this reason ethanol/ acetone-fixed slide cultures were mounted in glycerol (n = 1.455). The microinterferometer records the surface integral of the optical path difference, ,,J”@da, expressed in ems. This is related to the dry mass, DM, as follows:

T

I

0

I

I

/

1

100

200

300

1. Abscissa: projected nuclear area Cum”); ordinate: nuclear dry mass (N-DM). Relative amounts of nuclear dry mass (mean and S.E.M.) plotted against the projected nuclear area, (n =25). (From [5].)

Fig.

MATERIALS AND METHODS Cell cultures Kidneys from 14-day-old mice were cut into small pieces-and treated for 12 h at +4”C in a 0.25 % solution of trypsin in phosphate buffer. Subsequently trypsinization-was allowed to continue for ] h at 37°C with mechanical stirring. The tissue fragments were then separated by centrifugation and dissociated into single cells and small cell complexes by pressing the cell susoension back and forth through a fine needle. Eagie’s minima1 essential medium -(EMEM) containing 10 % heat-inactivated calf serum (GIBCo) was used as the culture medium. The cells were inoculated into 55 mm plastic Petri dishes (Nunc) containing 5 ml culture medium and a glass slide on the bottom. With inoculations of lo4 to lo6 cells/ml of culture medium and an inoculation time of 1 h, slide cultures with initial population densities of 50 to 3 000 cells/cm* were obtained. Large variations in local population density were observed within each slide culture. The Petri dishes were incubated at 37°C in an atmosnhere of 10% CO, in air. After 1 and 12 h of inoculation, the slide cultures were rinsed in 0.9 % saline and fixed for 24 h either in 10 % neutral formalin or in a mixture of absolute ethanol and acetone (1: 1 v/v). Exptl Cell Res 75 (1972)

where J, is the specific optical path difference [8], expressed in cmS/g. Knowing JI the recorded integral of the optical path difference can be converted into grams. The value for mouse liver cells embedded in glycerol (0.077 cm3/g [9]) was used in the present investigation. Previous quantitative cytophotometrical analyses of cell nuclei indicate that the nuclear dry mass increase reported herein is largely the result of an increase in protein content of the cell nucleus. [lo, 111. The interferometric nuclear dry mass analysis in whole cells is subject to a measuring error because of the cytoplasmic sheet over-and underlying the cell nucleus. This error can in genera1 be neglected in flat cells with comparable geometrical shapes. In the present study the cells in the newly seeded slide cultures changed from a spherical to a flat shape. The nuclear dry mass values of newly attached but not fully flattened cells might thus be somewhat too high as-compared with the mass values of flattened cells. In flattened cells from early cultures a nearly linear relationship has been found to exist between the increase of the projected nuclear area and the increase of the nuclear dry mass (fig. 1). In some experiments the determination of the nuclear dry mass was replaced by the determination of the projected nuclear area. AO-staining of ethanol/acetone-fixed cells was carried out according to the procedure described previously [12]. AO-stained cells were excited individually in UV light (1 365 M-I) and the fluorescence intensities at 530 mn (reflecting the amount of AObinding sites in the DNP complex [12]) were measured in a microspectrofluorimeter described by Caspersson et al. 1131. Bromphenol blue (BPB) staining was performed as described previously [14, 151. Slide cultures, fixed in 10 % neutral formalin were washed 3 x 5 min in distilled water and hydrolysed either in 5 % freshly prepared trichloracetic acid (TCA) at 90°C for 15 min or in saturated picric acid (PA) at 60°C for 3 h. After hvdrolvsis- the slide cultures were stained in 0.01 % BPB, at pH 8.2 for 1 h, followed by differentiation for 60 set in 0.0035 M borate buffer (pH 8.2). The slide cultures were then quickly dehydrated and mounted in DePeX. Absorption measurements were performed in a rapid scanning microspectrophotometer [16] at 370 nm (absorption maximum of PA) and 592 nm (absorution maximum of BPB). The amounts of BPB‘and PA bound in individual epi-

Nuclear protein content and DNA-histone

interaction

thelial cells were calculated from a two-wavelength equation [l, 21 and expressed in relative units. It has been shown previously that TCA hydrolysis exposes both arginine and lysine residues in the DNAbound histones for the subsequent BPB binding, whereas PA binds strongly to the arginine residues and thereby mainly exposes the lysine residues [l, 21. The amount of BPB (M nPB) bound to the cell following TCA hydrolysis thus reflects the total number of arginine and lysine residues bound to DNA and the amount of BPB bound in the cell following PA hydrolysis mainly reflects the DNA-bound lysine residues in histones [I, 21. The arginine residues in histones bound to DNA are reflected by the PA binding in the cell as well as by the relative decrease in BPB binding when the extraction of DNA is carried out in PA instead of TCA [1, 21. The population density was determined by counting the number of cells within microscopic fields of 0.95 mm2 (sparse cultures) and 13 000 pm2 (dense cultures). The projected nuclear area was determined by measuring the slide surface area occupied per nucleus.

cl

0

RESULTS

0

The interaction between DNA and histones has been investigated by means of the alkaline bromphenol blue (BPB) binding method, (a) in cells exhibiting initial DNP changes (judged from the increased capacity to bind the basic dye acridine orange (AO) [12, 271); (b) during the accumulation of proteins in the cell nucleus preceding initiation of DNA synthesis (judged from the progressive increase of the nuclear dry mass [5]). Recent work has shown that in epithelial kidney cells an increase of the initial population density induced a rapid increase of the

233

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100

200

35

25

50

300

63

Fig. 2. Abscissa: (upper) pm2; (lower) x lo-r2 g; ordinate: Mn,, (rel. units). Relative amounts of BPB (M,,,) of individual epithelial cells BPB-stained after either 0, TCAor l , PA- hydrolysis, plotted against the projected nuclear area, respectively the approximate nuclear dry mass. (In flattened epithelial kidney cells a good correlation exists between the projected nuclear area and the nuclear dry mass, cf fig. 1).

AO-binding capacity. This occurred a few minutes after the cells had attached to the slide surface and before any increase of the nuclear protein content could be detected

Table 1. Relative amounts of BPB (MBPB) and PA (MPA) of epithelial cells with small nuclei (25 x IO-12g) and large nuclei (65 x lo-l2 g), BPB-stained after either TCA- or PA-hydrolysis and seeded at population densities between 50 and 3 000 cells/cm2 slide surface area Small nuclei (25 x lo-l3 g) 50-75 cells/cm2

2 500-3 000 cells/cm2

Large nuclei (65 x IO-r2 g) 50-3 000 cells/cm2

Hydrolysis

TCA

PA

TCA

PA

TCA

PA

M BPB

8.0621.25 n=30 0.8210.09 n=50

5.7lzhO.87 n=30 3.71kO.95 n=50

5.91 kl.32 n=30 0.61 kO.07 n=50

5.37kO.78 n=30 1.02kO.21 n=so

2.21 iO.41 n=50 -

2.04 kO.37 n=50 -

M PA

Exptl Cell Res 7.5(1972)

234

G. Auer

/

50

1000

2000

I

3000

Fig. 3. Abscissa: number of cells/cm2; ordinate: AO-binding sites in DNP (rel. units). Fluorescense intensity at 530 nm (mean and S.E.M.) of AO-stained epithelial cells with average nuclear dry mass values around O---O, 25 x lo-l2 g and o-o, around 65 x 10V2 g, plotted against the local population density (number of cells per cm2 slide surface area) (n = 50).

by means of microinterferometrical methods [5]. These initial DNP-changes were then followed by a progressive accumulation of proteins in the nucleus preceding initiation of DNA synthesis and cell proliferation [5]. In the present study initial DNP-changes (reflected by an increase of the cellular AObinding capacity from 1.35 kO.21 to 3.74& 0.32, n=50) were observed 1 h after inoculation by increasing the initial population density from 50-75 to 2 500-3 000 cells/cm2 slide surface area. Cells with increased nuclear protein content (reflected by increased nuclear dry mass values from 25 x IO-l2 to 65 x lo-l2 g in average) were obtained by culturing the cells at population densities between 50-75 and 2 500-3 000 cells/cm2 slide surface area during 12 h. Exptl Cell Res 75 (1972)

It is clear from table 1 that initial DNP changes were connected with a decrease of the BPB-binding capacity after TCA hydrolysis (reflecting arginine and lysine residues in histones bound to DNA [l, 21) (from 8.06* 1.25 to 5.71& 1.32 rel. units), whereas after PA-hydrolysis (reflecting mainly lysine residues in histones bound to DNA [I, 21) the BPB-binding capacity remained more or less unchanged (5.71 i0.87 and 5.37 F 0.78 rel. units respectively). Initial DNP changes were also connected with a decreased PAbinding capacity (reflecting arginine residues in histones bound to DNA) (from 3.71+0.95 to 0.61 iO.07 rel. units). Table 1 shows furthermore that in cells with increased nuclear dry mass (65 x lo-l2 g in average) the BPB-binding capacity was as low as 2.21 iO.41 relative units after TCA hydrolysis and 2.04iO.37 relative units after PA hydrolysis. Thus, increase of the nuclear dry mass seemed to be connected with a marked decrease of the stainability of basic residues in histones bound to DNA and liberated both after TCA and PA hydrolysis. The relationship between projected nuclear areas (reflecting the nuclear protein contents, cf p. 232) and the relative amounts of BPB bound in individual cells after either TCA or PA hydrolysis is illustrated in fig. 2. It is clear from fig. 2 that a progressive increase of the projected nuclear area is connected with a progressive decrease of the cellular BPB-binding capacity. The decreased BPB stainability of histones in cells with increased nuclear protein content is paralleled by an increased A0 binding to DNA phosphate groups. Fig. 3 illustrates that at comparable population densities cells with increased nuclear dry mass (65 x lo-l2 g in average) bound significantly more A0 per unit amount of DNA than cells with low nuclear dry mass (25 X lo-l2 g in average).

Nuclear protein content and DNA-histone DISCUSSION In the present study the relationship between protein accumulation in the nucleus and the interaction between DNA and histones was analysed in non-proliferating cells responding to growth stimuli. In connection with a progressive increase of the nuclear protein content a progressive decrease in the stainability of DNA-bound histones was observed, as judged from the alkaline bromphenol blue binding reaction. This decreased stainability of DNA-bound histones could clearly be distinguished from the decreased stainability of DNA-bound histones observed in connection with the initial DNP changes which have been found always to precede the accumulation of proteins in the nucleus [5]. The results can be interpreted to reflect either (a) a quantitative decrease in histone content; (b) an increased loss of histones during the staining procedure, in particular during acid hydrolysis; (c) a masking of basic residues in DNA-bound histones. Evidence has been obtained [17] indicating that growth stimulation does not result in a quantitative alteration of the histone content. However, an increased loss of histones from cells with increased nuclear protein content as compared to cells with low nuclear protein content could be a reasonable explanation for the results reported here. It is known that some histones are lost during acid hydrolysis, especially during TCA hydrolysis. This loss has however been shown to be minimized if hydrolysis was performed in PA, which forms insoluble precipitates with basic proteins [14, 151. Thus, if the herein reported decrease in histone stainability would mainly be due to a loss of histones, a less pronounced decrease should be expected in PA-hydrolysed cells as compared with TCAhydrolysed cells. The same decrease in histone stainability in nuclei with increased

interaction

235

protein content observed after DNA extraction in either TCA or PA hydrolysis may therefore be interpreted to reflect mainly a masking of basic residues in histone. The mechanism responsible for such a masking of DNA-bound basic residues in histones is unclear. The high correlation between the accumulation of non-histone proteins in the nucleus and the decreased stainability of histones suggests that nonhistone nuclear proteins are involved in such a masking process, thereby affecting the interaction between DNA and histones. This suggestion is supported by findings demonstrating that phosphoproteins are able to form complexes with histones which are thereby inhibited from binding to the DNA [18-211. It has also been shown that in connection with growth stimulation acetylation of the basic residues in histone occurs [22-241. Such a process is consistent with the herein reported decreased stainability of DNA-bound histone residues. Recent studies on epithelial cells have also suggested that structural changes of the DNP complex are involved in the regulation of RNA synthesis [6]. Thus it is possible that the accumulation of non-histone proteins in the cell nucleus induces a decreased binding between DNA and histones which is of importance for the dispersion of nuclear chromatin and its increased RNA synthetic activity. These studies were supported by grants from the Swedish Cancer Society and Deutsche Forschungsgemeinschaft. The development of the biophysical instruments used in this work was supported by grants from the Swedish Natural Science Research Council to Professor T. Caspersson.

REFERENCES 1. Zetterberg, A & Auer, G, Exptl cell res 56 (1969) 122. 2. Auer, G, Zetterberg, A & Killander, D, Exptl cell res 62 (1970) 32. 3. Dariynkiewicz, Z, Bolund, L & Ringertz, N R, Exptl cell res 56 (1969) 418. Exptl Cell Res 75 (1972)

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4. Mazia, D, Proc natl acad sci US 4 (1954) 521. 5. Auer, G & Zetterberg, A, Exptl cell res 75 (1972) 245. 6. Auer. G. Moore, G P M, Ringertz, N R & Zetterberg, A, Exptl cell res 76 (1973). .In press. 7. Caspersson, T & Lomaka, G, Instrumentation in biochemistry. Biochem Sot Symp no 26 (ed T W Goodwin) p, 25. Academic Press, New York (1966). 8. Carl&n, L, Acta histochem, suppl. 6, (1965) 397. 9. - Thesis. Balder AB. Stockholm (1970). 10. Zetterberg, A, Thesis. Almqvist &. Wiksell, Uppsala, 1966. 11. - Exptl cell res 43 (1966) 517. 12. Rigler, R, Acta physiol Stand, suppl. 67 (1966) 267. 13. Caspersson, T, Lomakka, G & Rigler, R, Acta histochem, suppl. V 1 (1965) 123. 14. Bloch. D P & Hew. H Y C. J bionhvs biochem cytol ir (1960) 69. ’ ’ 1 15. Ringertz, N R & Zetterberg, A, Exptl cell res 42 (1966) 243. 16. Lomakka, G, Acta histochem, suppl. 6 (1965) 47.

Exptl Cell Res 7.5 (1972)

17. Black, M M & Ansley, H R, J cell biol 26 (1965) 797. 18. Langan, T A & Smith, L, Fed proc 25 (1966) 778. 19. Frenster, J H, Nature 206 (1965) 680 20. - Ibid 206 (1965) 1269. 21. Frenster, J H, C’J Dawe & Wilkenns Co, 78. 22. Poao. B G T. Allfrev. V G & Mirskv. A E. Proc natl acah’sci US’55 (1966) 805. _ 23. Dariynkiewicz, Z, Bolund, L & Ringertz, N R, Exptl cell res 56 (1969) 418. Bolund, L, Thesis. Balder AB, Stockholm, 1971. E: Killander, D & Rigler, R, Exptl cell res 39 (1965) 701. 26. - Ibid 54 (1969) 163. 27. Rigler, R & Killander, D, Exptl cell res 54 (1969) 171. 28. Bolund, L, Ringertz, N R & Harris, H, J cell sci 5 (1969) 71.

Received June 20, 1972