Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

Puked in Sweden Copyright Q 1974 hy Acndrmic Press, Inc. All rights of rrprodocrion in my.fonn r~wrwd

Experimental Cell Research 83 (1973) 271-280

SYNTHESIS

OF DNA-BINDING

PROTEINS

DURING

THE CELL CYCLE OF WI-38 CELLS B.-K. CHOEl and N. R. ROSE1 The Center for Immunology and Department of Microbiolog,y, School of Medicine, State University of New York at Buffalo, Buffalo, N. Y. 14214, USA

SUMMARY The DNA-binding proteins synthesized by WI-38 cells at specific periods during the cell cycle were chromatographed on calf thymus DNA-cellulose and analyzed by SDS-polyacrylamide gel electrophoresis. The DNA-binding proteins which were predominantly labeled in Cl had approximate mol wts of 20 000, 30 000 and 68 000. During the S phase, in addition to the G 1 uroteins. some large - _proteins and histones were labeled. Proteins of auurox. 45 000 D seemed to be unique to the S period. The profile of DNA-binding proteins synthesized in G2 is very similar to that of G 1. The labeling of DNA-binding proteins in the early G 1 was markedlv reduced in the presence of 5 ,ttg/ml of actinomycin D indicating that synthesisof these G I proteins is dependent on new transcription. In order to determine the relationship between the DNA-binding proteins prepared from the whole cell sonicates and nuclear proteins, DNA-binding proteins of cytoplasm and nuclear fractions were examined. DNA-binding proteins are found to exist both in nuclear and cytoplasmic fractions of WI-38 cells. Nucleocytoplasmic migration of DNA-binding proteins was also studied by pulse labeling G 1 cells with 3H-1eucine and determining the amount of radioactivity incorporated into cytoplasmic and nuclear DNA-binding protein fractions after chasing for various periods of time. Furthermore, incubation of unlabeled isolated nuclei with the 3H-DNA-binding proteins in vitro revealed that fractions of these cytoplasmic DNA-bindingproteins are transported in nuclei. Present experiments suggest that DNA-binding proteins synthesized in the late G I are correlated with the onset of S and that a fraction of DNA-binding proteins synthesized in the cytoplasm migrates into the nuclei.

Cell kinetic studies have established the existence of a biochemical cycle during the growth of a mammalian cell [IO]. However, the underlying mechanism for the progression of these sequential events is poorly understood [3, 20, 21, 231. The molecular processes occurring in the early G 1 and during the period of G I-S conversion are prime determinants of accumulative growth. The requirements for RNA and protein syntheses in the early Gl and G 1-S periods have been established and these findings have been the subject of several recent reviews [3, 23, 311. Particularly, rapid

1 Present address: Department of Immunology and Microbiology, Wayne State University, School of Medicine, Detroit, Mich. 48201, USA. 18-731810

synthesis of non-histone chromosomal (NHC) proteins and the control of RNA-transcription by these newly synthesized proteins has been observed in G 1 cells. Therefore, a correlation of NHC proteins with the onset of DNA synthesis in Gl-S period has been postulated [12, 301. Many proteins which function in association with DNA replication and transcription show DNA-binding properties at certain ionic strengths. DNA-binding protein synthesis has been investigated in 3T6 cells stimulated to divide after addition of serum [26] and in synchronous cultures of Chinese hamster ovary cells [ 131.These results indicate that certain DNA-binding proteins appear at specific stages. Using similar techniques, we have investigated the accumulaExptl Cell Res 83 (1974)

212

B.-K. Choe & N. R. Rose

tion of DNA-binding proteins in the serumstimulated WI-38 human diploid fibroblast cultures [9]. Our observation confirmed that the accumulation of a certain mol. wt group of DNA-binding protein is associated with the onset of DNA synthesis. It is, however, not clear whether this unique pattern of synthesis occurs only in the GO cells stimulated to divide or if it also occur in the Gl period of continuously growing cells. It is not clear what classes of proteins were screened by the DNA-cellulose chromatography of whole cell sonicates as described in the nreceding paper [9]. In the present paper we describe the characterization of the DNAbinding proteins by examining their synthesis and transport in synchronized Gl cell cultures. 1

MATERIALS

AND METHODS

Synchronized cell cultures. WI-38 human diploid fibroblast cultures [16] were provided by Professor L. Hayflick of Stanford University. The cultures used in the present investigation were between 20th and 30th passages.Exponentially growing cells were maintained in Eagle’s minimum essential medium (MEM; GIBCo) supplemented with 10% fetal calf serum (North American Biologicals) and 50 pg/ml of chlortetracycline. HEPES (10 mM) was included to maintain the pH at 7.4. The cells were periodically tested for mycoplasma contamination in our laboratory. Cells in G I, S and G2 phases of the cell cycle were obtained by blocking randomly growing cultures with 2 mM thymidine for 12 h, releasing the cells into fresh, thymidine-free deoxycytidine containing medium, and plating the cells in roller bottles (Bellco Glass Inc.). Six to 8 h after plating, the monolayers were washed and overlayered with warm fresh medium. One to 2 h later, mitotic cells were selectively detached into fresh medium from the monolayers by vigorous shaking [24, 361. The initial mitotic synchrony was usually determined by visual inspection of cells under the microscope. After harvesting, the mitotic cells were plated in 75 cm2 (Falcon) plastic flasks and allowed to proceed through G 1, S and G 2 as shown in fig. 1 A. Cells in G2 and G 1 phases of the cell cycle were also obtained by blocking random cultures with either 2 mM thy&dine or-l mM hydroxyurea for 12 h, releasing the cells into thymidine or hydroxyurea-free deoxycytidine (0.01 mM) containing fresh medium for 9 h and blocking with 2 mM thymidine for an additional 12 h [6]. After release from the second thymidine block, the cells progressed through S and into G 2 as shown in fig. 1B. Exptl Cell Res 83 (1974)

Labeling

of proteins

with radioactice

amino

acids.

Labeling medium was prepared with leucine-omitted, tryptophan-omitted or lysine-omitted amino acid mixtures and 2 26 dialysed calf serum. The following radioactive amino acids (New England Nuclear, Boston) and amounts were added to the labeling medium: 3H-4,5-L-leucine (100 &i/ml, 67 ,’ IO-” M leucine), “C-U-L-leucine (10 &i/ml, 3.5-4.5 ’ IO--” M leucine), 3H-G-L-tryptophan (100 &i/ml, 8 ’ 10--fiM tryptophan) and 14C-U-L-lysine (lO$Zi/ml, 1.5 \’ 10m5 M lysine). For each T-75 culture flask 10 ml of labeling medium was used. of cell extracts. Labeled cultures were washed with 200 ml of ice-cold saline, frozen at ~70°C and thawed, repeating the freeze-thaw cycle 3 times. Cells were collected in a small volume (usually 445 ml) of Tris-HCl pH 7.4 containing 2mercaptoethanol (lOm3M), sodium bisulfite (10m3M) and NaCl (0.15 M), and sonicated 40 set at 60 W in After~~centrifueine an ice-bath with Branson ~.~~~~ Sonifier. ..~~~~~~ ~~~ .~~~~~~-.~o the lysed cells for 30 min at I2 800 g at 4°C 200 pg/ml of DNAse (Sigma) was added to the supernatant and left at room temperature for 15 min. After DNAse digestion, the extract was centrifuged 1 h at 100 000 g in a Spinco 40 rotor at 4°C and the supernatant was dialyzed overnight at 4°C against 2 :c lo-’ M Tris-HCl (pH 8.2) -1m 10e3 EDTA tm10m3M 2 mercaptoethanol and 0.05 M NaCl to remove Mg ion. The dialysed samples were further centrifuged for 30 min at 100000 g in a Spinco 40 rotor before loading onto the DNA-cellulose columns. Preparation

Calf thymus DNA-cellulose chromatography. Details of DNA-cellulose chromatography have been described in the preceding paper [9]. gel electrophoresis. Procedures of electrophoresis on SDS-polyacrylamide gel are detailed in the preceding paper [9].

Polyacrylamide

Isolation of nuclear proteins. The following procedures

were carried out in an ice-bath or in the cold room. The cultures were washed 3 times in 40 vol of phosphate buffered saline (PBS), pH 7.2 and the harvested cell pellets were washed in 20 volumes of hypotonic buffer, RSB [22]. The pellet was resuspended in 5 vol of RSB, swollen for 10 min, and Triton X-100 was added to a final concentration of 0.7 96 and mixed carefully [15]. After another 5 min the suspension was homogenized gently (5 strokes) in a Dounce homogenizer and the nuclei were pelleted by centrifugation for 3 min at 600 g. Recoverv of the nuclei was &ally 85 to 90% and- the nuclei prepared in this fashion had a protein:DNA ratio of 3 and an RNA: DNA ratio of 0.3. The nuclei were then extracted 3 times with 2 vol of 0.1 M Tris-HCI (pH 6.5). 3 times with 2 vol of 0.15 M NaCI. and 3 times with 2 vol of 0.35 M NaCl. The extracted nuclei were dissociated in 3 M NaCl containing lO-3 M sodium bisulfite. 30 % guanidine-HCl, 0.01 MTris-HCl (pH 8.0) for 24 h to reduce NaCI. The protein solution was loaded on QAE Sephadex A-50, previously equilibrated with 30 0; guanidine-HCI, 0.01 M Tris-HCI (pH 8.0). Histone was eluted with the same buffer. Non-histone chromosomal (NHC) proteins were eluted with 3 bed

DNA-binding protein during cell cycle volumes of 0.01 M Tris-HCl (pH 8.0) + 3 M NaCl i 30 ^b guanidine-HCI [14]). After reducing the concentrations of NaCl and guanidine-HCI by dialysing against 0.02 M Tris-HCI (pH 8.0) overnight, histone and NHC proteins were concentrated to an appropriate volume by ultrafiltration in molecular membranes (Amicon). The proteins were finally lyophilized and stored at --70°C. Purities of preparations were examined by the SDS-polyacrylamide gel electrophoresis.

060M

2 OOM Na Cl

NaCl

4

25

ZO-

273

i

Other methods. DNA, RNA and proteins were determined quantitiatively by the procedures described by Shatkin [28]. :

RESULTS

lm ~::::~~:!!~;-ll-;-?~t& !( ‘\” IO

DNA-binding cell cycle

proteins labeled during

Synchronous cultures of WI-38 cells were obtained either by the detachment of mitotic cells or by the double thymidine block. Fig. 1 shows the progression of such cells through the cycle. Synchronized cells were grown with radioactive amino acids during the intervals

Fig. 1. Abscissa: time after (A) mitosis; (B) release; ordinate: (left) cpm s 1O-3(O-O); (right) fig protein

(A-A). Incorporation of 3H-thymidine into WI-38 cell DNA (A) at various times after detachment of mitotic cells and (B) after release from a double 2 mM thymidine block. IO6 cells were labeled for 30 min with 10 ml of 3H-thymidine (0.5 mCi/ml medium) and the incorporation of radioactivity into cold 20~6 TCA-precipitable material was measured. Average of duplicate cultures was plotted. G 1 lasted for 6 h and S for 10 h. G2 was estimated from generation time of the culture and the length of G I + S to be 4 to 6 h. Arrows indicate the onset of each phase of growth cycle.

20

--..L i” . . . . ..-.

,p ,).. --. 30

40

50

60

J 70

Fig. 2. Abscissa: fraction no.; ordinnfe: labeled protein

bound ( % total cellular protein). Chromatography of extracts of WI-38 cells on calf thymus DNA-cellulose. Ordinate gives cpm eluted in each fraction, expressed as %, of the total applied to the column. Proteins were loaded onto the columns equilibrated with 0.05 M NaCl buffer and were eluted with 0.15 M, 0.6 M and 2 M NaCl buffers. Fractions of 1 ml were collected and 50 ~1 of each fraction was counted in 5 ml of scintillation fluid. Each 8 x 10fi cells were labeled during G 1 (O--O) and G 2 ( :<) with 3H-leucine and S cells (a--a) were labeled with ‘*C-lysine. Labeled G 1 and S cells or labeled G2 and S cells were mixed and the extracts were chromatographed.

Gl, S, and G2 for 2 to 3 h, as indicated by arrows in fig. 1. Fig. 1 indicates that synchrony of WI-38 cells under our experimental conditions diminishes and becomes poor during G2. Therefore, stage-specific proteins could be expected to be detected only because of their relative abundance. Proteins labeled in G 1 cells (1 to 3 h post-mitosis) with 3H-leucine and S cells (6 to 9 h post-mitosis) with 14C-lysine were co-chromatographed on calf thymus DNA +cellulose columns (fig. 2). The fractions of the total extract which bound to calf thymus DNA were 3 to 6 % respectively. The DNA-binding protein fraction of G2 cells was considerably smaller than that of Gl and S cells. The reason for this reduction of DNA-binding protein fraction is at present unclear. Proteins eluted at 0.15 M NaCl represented proteins binding DNA weakly and those eluted at 0.6 M NaCl could Exvil

Cell Res 83 (1974)

214

B.-K. Ckoe & N. R. Rose

0025

Fig. 3. Abscissa: (rap) approx. mol. wt; (bottom) fractio no.; ordinate: cpm/fraction/total cpm in cell protein ( %). Co-electrophoresis of DNA-binding proteins of 0.15 M NaCl and 0.6 M NaCl elutes obtained from Gl, S and G2 cells. Labeling of the cultures and chromatography on DNA-cellulose was the same as described in fig. 1: (A) the 0.15 M NaCl eluates of the mixture of 3H-labeled G 1 proteins (O--O) and Y-labeled S proteins ( x - ::); (B) the 0.15 M NaCl eluates of the mixture of 3H-labeled G2 proteins (O--O) and ‘JC-labeled S proteins ( x - x ); (C)the 0.6 M NaCl eluates of the 3H-labeled G 1 (O--O) and W-labeled S proteins ( i -- ‘. ); (D) the 0.6 M NaCl eluates of the 3H-labeled G2 (o--a) proteins. Counts are expressed as percent of the total in the soluble extracts on the ordinates.

be regarded as moderately firmly bound proteins. Eluted protein fractions were further analysed on 7.5 “/b SDS-polyacrylamide gels. The electrophoretic profile of 0.15 M NaCl eluates showed 10 to 15 different size classes of proteins (fig. 3). In Gl cells, however, 0.15 NaCl eluates revealed accumulation of three proteins of approximate molecular weights of 20 000-22 000, 30 000 and 90 000. These proteins were detected as the major proteins in the 0.15 M NaCl fraction of density-inhibited WI-38 cells stimulated to synthesize DNA after the change of medium Expil CeN Res 83 (1974)

[9]. During the S phase 0.15 M NaCl eluates showed nearly equal amounts of large and small mass proteins as in G 1 cells. However, protein(s) of mol. wt 40 000-43 000 seems to be unique to the S period. This protein fraction was, in fact, detected in density-inhibited cells only at the late stage (S period) of serum stimulation [9]. Patterns of DNA-binding proteins of 0.6 M NaCl (fig. 3c, d) show proteins having mol. wt of 40 000-45 000. However, correlation between these proteins and the cell cycle is still difficult to evaluate. Proteins which elute with 2 M NaCl (results

DNA-binding protein during cell cycle Table 1. Effect of actinomycin D on the synthesis of DNA-binding

275

protein

Culture bottles each containing approx. 4 j: IOfi cells were synchronized by the double thymidine bloc. Sixteen h after release from the block cells were pulse-labeled for 5 min in 50 ml of Earle’s balanced salt solution (BSS) containing 20 &i/ml of 3H-leucine. After washing with each 500 ml of ice-cold BSS, 200 ml of prewarmed MEM supplemented with IO % fetal calf serum and 5 pgjml of actinomycin D were added (start of ‘chase’). At 0, 30 and 120 min after the addition of actinomycin the cultures were sampled and DNA-binding proteins were isolated as described in the Materials and Methods. In a parallel culture, cells were incubated in absence of actinomycin for 2 h as a control Radioactivity contained in Chase periodb (min)

Initial cell extract (A) (cpm _ 106)

0. I5 M NaCl eluate (B) (cpm 103 (B/AU;)”

0.6 M NaCl eluate (C) (cpm IO?) (C/A “c,)a

0 30 120 Control’

5.25 5.3 6.23 1.45

2.26 2.09 3.38 16.8

0.8 1.35 2.33 9.55

(0.43) (0.395) (0.54) (1.15)

(0.15) (0.255) (0.375) (0.66)

a Percent of initial soluble cell extract. b Five minute pulse-labeling and 0 to 20 min incubation in presence of actinomycin D. (’ Continuous labeling for 120 min in absence of actinomycin D.

not shown) are very complex, thus it is difficult to correlate them with the cell cycle. It is relevant here to mention that most of the proteins having a mol. wt of 20 000-22 000 which were eluted in 0.15 M NaCl or 0.6 M NaCl fraction are not histones. Since it is known that histone does not contain tryptophan but is rich in lysine [19], cells in the S period were grown for 3 h in the presence of 3H-tryptophan and r”C-lysine. Then the cells were extracted and chromatographed on the DNA-cellulose using purified calf thymus histone as the marker and the 0. I5 and 0.6 M NaCl eluates were electrophoresed on the SDS-polyacrylamide gels. Comparison of staining profiles and the distribution of radioactivity revealed that under our extraction condition only small amounts of histones were extracted. Thus it is established that the protein(s) of 20 000-22 000 D synthesized during G I and G2 are not histones. Profiles of DNA-binding proteins in G2 cells are essentially the same as G I cells. It is not clear whether this similarity is real or due to the poor synchrony of the cells at G2 period. Synthesis of proteins eluted by 0.6 M NaCl

seems to be continuous during GI, S and G2, although small quantitative differences are noticeable. Effect of actinomycin D and the DNAbillding protein synthesis Experimental evidence has established the requirements for RNA and protein syntheses in the early Gl before the onset of DNA synthesis in the subsequent S phase [3,23, 311. As we have observed continuous synthesis of DNA-binding proteins during G2, it was of interest to determine whether the continued synthesis of these proteins in G 1 is necessary for the onset of DNA synthesis. G 1 cells were pulse-labeled with 3H-leucine for 5 min and the label was chased for 30 and 120 min in the presence of 5 ,ug/ml of actinomycin D which blocks both the transcription and the subsequent DNA synthesis. As shown in table 1, DNA-binding protein synthesis in Gl is reduced regardless of the possible presence of messenger RNA for these proteins during the preceding G2 in the continuously growing cultures. These results suggest that new transcription Exptl Cell Res 83 (1974)

276 B.-K. Choe & N. R. Rose Table 2. Amount of cellular proteins and DNA-binding proteins in WI-38 cells /cg proteins per 3.5 x 108cells

Percent of initially loaded protein eluted at 0.15 M NaCl 0.6 M NaCl

2 M NaCl

Sum

7.29 x IO’

Total proteins Cytoplasmic proteins (crude soluble proteins) Nuclear proteins RNP (buffer soluble) 0.15 M NaCl soluble 0.35 M NaCl soluble histone NHC protein (2 M NaCl soluble) Residual NHC proteins

5.76 x IO” 1.65

2.05

0.3

4.0”

5.3 x 102

2.2

5.6

1.39 ” 103 4.58 r 103

0.11

7.ga

0.1

90.0

2.0

90.0b

3.31 x 103

17.0

4.8

0.1

22.0b

1.8 x lo3

4.55 “: 103

Amounts of nuclear proteins were corrected on the basis of nuclear counts. ’ Fractions of DNA-binding proteins in soluble cytoplasmic proteins and (RNP -t 0.15 M NaCl soluble :m0.35 M NaCl soluble) in nuclear proteins are determined by the radioactivity counting of these labeled proteins. b Unlabeled purified histone and NHC (2 M NaCl soluble) proteins were employed, see also fig. 4.

of RNA and assembly of new polysomes are required for the accumulation of DNA-binding proteins in G 1. The synthesis of the DNA-binding proteins is sensitive to actino-

: 05M 015M

06LC

4

i 04t 02

! c

1 i

: : t..m...... ..-............ ..~....-....?.A..

B I.i 05M

06

O4

+ -,

1

015M

0.6M

2M No Cl

+

4

4

I ‘\i Ii\

IO20

30

40

50

60

70

Fiw. 4. Abscissa: fraction no.: ordinate: absorbance at 233 nm.

Binding of WI-38 nuclear proteins to calf thymus DNA-cellulose. (A) Histones (8 mg) are applied to approx. 15 mg calf thymus DNA immobilized on 10 g of cellulose and fractions of 2 ml were collected. (B) NHC proteins (8 mg) are applied on a column containing approx. I6 mg of calf thymus DNA in 10 g of cellulose and fractions of 0.5 ml were collected. Arrows indicate the applications of NaCl buffers. Exptl Cell Res 83 (1974)

mycin D just as the NHC protein synthesis is observed in WI-38 cells [25]. DNA-binding and nucleus

proteins in cytoplasm

Many nuclear proteins are known to bind to DNA with various affinities [2, 18,35, 38, 391. However, it has never been closely determined what types of proteins are found by the calf thymus DNA-cellulose chromatography of whole cell sonicates. Therefore, a reconstruction experiment was attempted. First, we examined the DNA-binding protein fractions in nuclei and cytoplasm of WI-38 cells (table 2). Nuclei were prepared as described in Materials and Methods and the purities of each protein fraction were examined on the SDS-polyacrylamide gel electrophoresis. Soluble nuclear proteins (combined fraction of 0.1 M Tris-HCI +O. 15 M NaCl+O.35 M NaCl-soluble proteins) showed 7 to 8 % binding to calf thymus DNA. Histones are the most tightly binding proteins to DNA since they could not be eluted by 0.15 M NaCI. Histones eluted almost completely (97 %) at 0.6 M NaCI, with a very small fraction eluting at 2 M NaCl (~2 “/o) (fig. 4). NHC proteins

DNA-binding pro rein during cell cycle Table 3. Transport of cycloheximide

of cytoplasmic

DNA-binding

277

proteins into nuclear fraction in the presence

Migration of 3H-leucine labeled DNA-binding proteins at various times following a 5 min pulse. Culture bottles each containing approx. 5 x lo6 cells were synchronized by the 2 mM thymidine and 1 mM hydroxyurea block. Sixteen hours after release from hydroxyurea block, cells were pulse labeled for 5 min in 50 ml of Earle’s balanced salt solution (BSS) to which were included 20 &i/ml of 3H-leucine. After washing with each 500 ml of ice-cold BSS, 200 ml of prewarmed MEM containing 2 % fetal calf serum and 0.5 pug/ml of cycloheximide were added to each culture (start of ‘chase’). At 0,30 and 60 min after start of chase, the cultures were removed, washed harvested and swollen in ice-cold hypotonic buffer (RSB). Then cells were further fractionated into nuclear and cytoplasmic fractions by I “/ATriton X-100. In parallel experiments exact duplicate cultures were prepared and labeled with 14C-leucine 2 pCi/ml in 50 ml of MEM supplemented with 2 % fetal calf serum for 2 h. Each 3Hlabeled nuclear or cytoplasmic fraction was mixed with W-labeled whole cells and sonicated. These cell extracts were chromatographed on the calf thymus DNA cellulose and 0.15 M NaCl eluates and 0.6 M NaCl eluates were taken. These eluates were further electrophoresed on SDS-polyacrylamide gels. W-cell extracts served as references in analytical procedures. Only 3H-leucine incorporation among various fractions are presented aRadioactivity contained in Chase period (min) 0 30 60

Cellular fraction

Initial cell fraction (cpm %105) ( :;)b

Cytoplasmic Nuclear Cytoplasmic Nuclear Cytoplasmic Nuclear

8.0 8.8 9.45 7.8 6.5 16.2

47.5 52.5 54.75 45.25 28.60 71.40

0.15 M NaCl eluate (cpm x lOa) ( :O)b 8.0 4.5 5.6 9.1 II.0 20.7

4.75 2.68 3.25 5.21 4.85 9.12

0.6 M NaCl eluate (cpm y 10”)

( ?“h)(l

2.5 2.15 2.16 3.2 3.2 7.35

1.49 1.30 1.25 1.86 1.41 3.27

a “H-leucine incorporation. * :‘:, of the sum of the radioactivities (H) in cytoplasmic and nuclear fractions.

(3 M NaCl soluble) consisted of weakly and moderately DNA-binding proteins and nearly 28 96 of them did not bind to calf thymus DNA. Elution with 0.15 M NaCl eluted approx. 17 Sb, 0.6 M NaCl eluted 4.8 % and the 2 M NaCl step eluted 0.1 % of NHC proteins under present experimental conditions (fig. 4). Binding of NHC proteins to calf thymus DNA seems to be strongly dependent on the ionic strength of buffers employed for the column equilibration and the elution of the proteins. Difficulties were encountered with NHC proteins because of their precipitation at low ionic strength. Therefore, it is possible that some of the NHC proteins formed aggregates on the column and thereby increased the binding fraction at the 0.15 M NaCl elution step.

A most unexpected observation was the cytoplasmic DNA-binding protein. It reached nearly 3 ‘X0of total soluble proteins. Reconstructed DNA-binding proteins from the cytoplasmic fraction and the nuclear proteins did not show the electrophoretic profile of whole cell sonicates. However, judging from our extraction conditions, it is likely that proteins from whole cell sonicates eluted with 0.15 M NaCl contained predominantly soluble nuclear proteins and cytoplasmic DNA-binding proteins, whereas proteins from whole cell sonicates eluted with 0.6 M NaCl would contain NHC proteins and small amounts of histones. It is relevant to mention that one of the cytoplasmic DNA-binding proteins has been isolated from 3T6 and SB cells prior to our study [37]. Exptl Cell Res 83 (1974)

218 B.-K. Choe & N. R. Rose investigate their nucleo-cytoplasmic relationship to obtain further information on their biological roles. If the cytoplasmic DNA-binding proteins represented newly synthesized nuclear proteins and certain cytoplasmic proteins which can migrate into nuclei only under stimulated conditions, one would expect to observe the transport of such proteins into the nucleus. G 1 cells were pulse labeled for 5 min with 3H-leucine and then the labels were chased for 30 and 60 min in the presence of 0.5 pg/ml of cycloheximide IO 20 (CalBiochem) and 0.1 mM L-leucine (regular 30 40 50 60 70 MEM). After fractionating the cells into Fig. 5. Abscissa: (top) approx. mol. wt; (botron2) nuclei and cytoplasm, each fraction was fraction no.; ovdinafe: cpm . IO-“. (A) 0.15 M; (B) 0.6 M NaCl fraction. In vitro mixed with whole cell sonicates of G 1 cells incorporation of DNA-binding proteins into isolated Wl-38 cell nuclei. Exponentially growing WI-38 cells which have been prepared and labeled for 2 h were labeled for 12 h with 3H-leucine (2 pCi/ml), with llC-leucine as in the parallel cultures as harvested, cytoplasmic extracts were fractionated a reference. The mixed extracts were chromaon calf thymus DNA-cellulose and 0.15 M NaCl fraction and 0.6 M NaCl fraction were pooled, diatographed on the calf thymus DNA-cellulose lysed against 0.15 M NaCl-Tris buffer (pH 7.4). These dialysed DNA-binding proteins were further con- columns 0.15 M NaCl and 0.6 M NaCl eluates centrated by Sephadex G-200 powder to obtain 800 were obtained and resolved by the SDSpug/ml of proteins. WI-38 cell nuclei were isolated polyacrylamide gel electrophoresis (table’3). from approx. 4 ‘. 10’ cells by 1 “b Triton X-100. Isolated nuclei were divided into 2 parts incubated A comparison of 3H-leucine incorporation with (1) 800 /ng DNA-binding proteins (0.15 M NaCl profiles (fig. not shown) and the redistribufraction); (2) 400 /-tg DNA-binding proteins (0.6 M NaCl fraction). Incubations were carried out in open tion of radioactivity (table 3) shows that up test tubes in a water bath at 37’C without shaking for 30 min. The reactions were run in a final volume of to 50°d of the cytoplasmic DNA-binding 1.0 ml containing 0.15 M NaCI, 0.05 M Tris-HCI proteins are transported into nuclei within (pH 7.6), 0.01 M MgCI,, 5 mM 2-mercaptoethanol, 0.5/tmoles ATP, 0.1 /moles GTP, 5 jlmoles phos- 60 min in the absence of continued protein phoenolpyruvate and 2 1/g of pyruvate kinase. After synthesis. the incubation, the nuclei were washed twice with iceWhen isolated nuclei were incubated with cold 30 ml of 0.15 M NaCI-Tris-HCI buffer, treated with 1 o/, Triton X-100 for 5 min in the ice-bath and “H-leucine labeled cytoplasmic DNA-binding washed once with NaCl buffer. The nuclei were sonicated for 1 min and digested with DNAse (100 proteins in vitro (fig. 5), 4(;,, of 0.6 M NaCl /(g/ml) for 20 min at room temperature. The digested proteins and 0.02 “/o of 0.15 M NaCl proteins nuclei were centrifuged at 100000 g for 1 h and the appeared in the nuclei. We have prepared supernatants were concentrated to an appropriate volume. Polyacrylamide gel electrophoresis were carDNA-binding proteins on calf thymus DNA ried out as described in Materials and Methods. Arinstead of on WI-38, which makes it imposrows indicate the approximate position of staining calf thymus histones in the reference gels.

Nuclear-cytoplasmic migration of DNAbinding proteins In view of a large DNA-binding protein pool in the cytoplasm we felt it was necessary to Exptl Cell Res 83 (1974)

sible to claim a rigorously

specific interaction

of these proteins with chromatin. The presence of proteins in the cell that can be transported and bind with homologous chromatin, however, suggest their possible functions in genetic regulatory

processes.

DNA-binding

DISCUSSION Weakly DNA-binding proteins (0.15 M NaCl elutates) which accumulated predominantly during the G 1 or the G2 period had mol. wts of 20000, 30000 and 90000. During the S period there is a unique accumulation of proteins of 45 000 D in addition to the proteins synthesized during G 1. In the preceding communication we have described the accumulation of small mol wt DNA-binding proteins in the density-inhibited WI-38 cells which were stimulated to synthesize DNA after a medium change [9]. One of the proteins mol. wt 45 000) was synthesized in association with the onset of DNA synthesis. Therefore, there is a conformity of the pattern in DNAbinding (0.15 M NaCl eluates) protein synthesis in stimulated cells and continuously growing cells. Protein binding to DNA with moderate affinity (0.6 M NaCl eluates) which were synthesized by Gl and S cells were similar, but accumulation of one or more class of proteins was markedly reduced in the G2 cells. The profile of the accumulation of DNA-binding proteins of 0.6 M NaCl eluates in WI-38 cells confirms similar observations of Fox & Pardee [13] with the CHO cells. In growing cells the DNA-binding proteins seem to be continuously synthesized, as it is evident from the profile of the proteins synthesized during G 1, S and G2. However, there are stage-specific variations at least in their accumulation and their syntheses, suggesting continuous new transcriptions. In the present experiments, the nature of DNA-binding proteins in whole cell sonicates was not fully examined, but DNA-binding properties of cellular proteins in different compartments were investigated. DNA-binding properties of nuclear proteins have been investigated in other laboratories [18, 19, 25, 35, 38, 391. However, the finding of cyto-

protein during cell cycle

279

plasmic DNA-binding protein was largely unexpected. Previously, Stein & Baserga [33] have demonstrated the cytoplasmic synthesis of acidic chromosomal proteins, and the kinetics of nuclear transport have been investigated by Stein & Borun [7, 321, and Carlsson et al. [8]. Therefore, large fractions of cytoplasmic DNA-binding proteins could be newly synthesized nuclear proteins. In our experiments, we have demonstrated that the bulk of cytoplasmic DNA-binding proteins is actually transported into the nuclei. The cytoplasms of mammalian cells have been reported to contain proteins which bind readily to exogenous RNA [1, 27, 341. One of these RNA-binding proteins (mol. wt 43 000) has been further demonstrated by Schweiger & Spitzauer [27] to bind to DNA as well. Therefore, they have speculated that the cytoplasmic RNA-binding proteins might be the newly synthesized one of NHC proteins. Nevertheless, a certain fraction of cytoplasmic DNA-binding proteins could be of predominantly cytoplasmic location [37]. In the ionic environment of the cell, which includes more than 0.15 M potassium ion, the binding of 0.15 M NaCI-eluate proteins to nucleic acids in vivo must be rather weak. However, it is possible that freely reversible interactions of these proteins and specific effector molecules may lead the complexes in a state that increases their affinity for specific sites on the DNA. The effector molecules can be hormones or quasi-hormonal molecules. In fact, the implication of cytoplasmiceffector binding proteins and NHC-proteins in the steroid hormone vs target cell systems have been investigated in many laboratories [4, 11, 17, 29, 401. There is, at present, no direct evidence that these DNA-binding proteins described in the present paper are NHC proteins or hormone-binding proteins. Purification and immunochemical investigations of the DNAbinding proteins are in progress in our Exptl Cell Res 83 (1974)

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laboratory for the purpose of comparing these proteins with NHC proteins. We thank Professor L. Hayflick of Stanford University for the supply of WI-38 cell cultures and acknowledge the suggestions from Professor T. Y. Wang at the State University of New York at Buffalo. This work was supported in part by NIH Research Grant CA 05203 and a Henry C. and Bertha Buswell Fellowship. This is publication No. 65 from The:Center for Immunology.

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