Changes in enzymic activity during cultivation of human cells in vitro

Printed in Sweden Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form resewed

Experimental Cell Research 80 (1973) 305-312

CHANGES

IN ENZYMIC OF HUMAN

ACTIVITY CELLS

DURING

CULTIVATION

IN VITRO

B. I. SAHAI SRIVASTAVA Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, N. Y. 14203, USA

SUMMARY The composition of chromatin, its template activity and the activity of certain chromatin-associated enzymes, including DNA polymerase (DP) and soluble RNase, DNase, DP and seryl tRNA synthetase, were examined in early and late passage of WI-38 cells and of WI-38VA13 cells. No significant changes in soluble RNase, DNase, seryl tRNA synthetase or soluble and chromatin-associated DP were found with increasing passage of WI-38 cells. The activity of seryl tRNA synthetase and DP in WI38VA13 cells was, however, significantly higher than WI-38 cells in all passages. A decline in RNA synthesizing activity of chromatin, an increase in the proportion of RNA and histone in chromatin, as well as an increase in the activities of ‘chromatinassociated enzymes’ (RNase, DNase, protease, nucleoside triphosphatase, DPN pyrophosphorylase) were noted in WI-38 cells with increasing passages. Although RNA synthesizing activity of chromatin from WI38VAl3 cells was lower than that from WI-38 cells, the former also were much lower in ‘chromatin-associated enzymes’. An increase of chromatin-associated enzymes responsible for RNA, DNA and protein degradation in WI-38 cells in successive passages, and a much lower activity of these enzymes in WI-38VA13 cells (which have an indefinite doubling potential in vitro) suggests that an elevation in the activity of these enzymes, which would seriously interfere with the chromatin function, could result in ‘aging’ of WI-38 cells.

Hayflick [12] has shown that cultures of the human diploid cell line WI-38, which originated from fetal lung tissue, have an in vitro lifespan of 50+ 10 passages. Hayflick & Moorhead [13] have suggestedthat these cells ‘age’ in culture, that is, with an increasing number of passages the culture generation time increases, the cells begin to deteriorate [phase III] and eventually die. However, a heteroploid cell line [WI-38VA13] derived from WI-38 cells by SV40 virus transformation [ll] grows like a ‘permanent’ cell line. These two cell lines which may offer an excellent system for the study of human aging at the cellular level, have been used by several

investigators in the study of various enzymes [6, 7, 14, 17, 321. Increases of chromatin-associated nuclease, protease and nucleoside triphosphatase [26, 271 and unpublished data) were observed in this laboratory during senescencein a plant model system. It was considered desirable, therefore, to determine whether analogous changes also occur in human cells during ‘aging’. This study primarily concerns chromatin-associated enzymes and the template activity of the chromatin in WI-38 cells at various stages of aging as compared with WI-38VA13 cells. DNA polymerase (DP), RNase, DNase, and seryl tRNA synthetase Exptl Cell Res 80 (1973)

306

B. I. Sahai Srivastava

activity of the soluble fraction have also been protein determinations. Any remaining preparation was stored frozen at -20°C for subsequent assay of examined. RNase and DNase activity. Alternate

procedure for the preparation

of cell extract.

The cells of a diploid human fibroblastcellline(WI-38) were grown and supplied to us by Dr L. Hayflick. These cells were cultured in Eagle basal medium (GIBCo) supplemented with 10 % fetal calf serum and

About 1 g (fresh wt) of cells was homogenized by 40-50 strokes in buffer C (25 mM Tris-sulfate. nH 8.3; 1 mM MgSO,; 6 mM NaCl; 4 mM dithiothreitol and 0.1 mM EDTA) in a glass homogenizer. Glycerol was added to the homogenate to a final volume of 10% and the contents centrifuged at 37000 g for 20 min. A part (ca 5 ml) of the supernatant was saved

50 /-%/ml

for DNA

MATERIALS AND METHODS

of aureomYcin.

When

confluent

(approach-

ing stationary phase), the cells were harvested by scraping with a Bellco silicone rubber scraper in serum free medium, washed 4 times with phosphatebuffered saline (pH 7.2) and shipped on dry ice. SV40-transformed WI-38 cells (cell he wI-38vA13) obtained from Dr Hayflick were grown, scraped and washed according to the above procedure by Dr J. Minowada at Roswell Park Memorial Institute.

polymeras

assay and

proteindetermination.

Th e remainder (ca 20 ml) was dialysed (3 h at 1°C) against buffer C containing 10% glycerol (buffer D) and was used for DEAE-cellulose chromatography. DEAE-cell&se

chromatography.

The dialysed

super-

natant was clarified by centrifugation (37 000 g, 20 min) and loaded on a column (1.3 x 20 cm) of DEAEcellulose equilibrated with buffer D. After initial washing with buffer D, the column was eluted with a Isolation of chromatin. Chromatin was isolated essen- linear gradient formed with 100 ml of buffer D and tially by the method of Bekhor et al. [3]. This involved 100 ml of buffer D containing 1 M NaCl; 60-drop washing the cells by pelleting (1 500 g for 15 min) in f ractions were collected and the A,,,,, of each fracbuffer A (0.05 M Tris-HCI, pH 7.6, 0.13 M NaCl, tion was determined against buffer D. Aliquots (0.1 0.025 M KCI, 0.0025 M MgCI,) followed by single ml) from each fraction were assayed for DP using pelleting from 4 x diluted buffer A. The cells were native calf thymus DNA as the template. Fractions &hen lysed in deionized water by 15-20 strokes in a containing DP activity were pooled; the protein from glass homogenizer and the nuclei pelleted at 1 500 g. this pool was precipitated at between 30-50 % saturaThe nuclear pellet was homogenized by hand with tion with ammonium sulfate. This precipitate, which 40-60 strokes in 0.01 M Tris, pH 8.0. Chromatin contained most of the DP activity, was dissolved in was pelleted and washed by repelleting (1 500 g for buffer D; the preparationwas clarified by centrifu15 min) 2 times from 0.05 M Tris buffer pH 8.0. The gation and used for the assay of DP using various resulting crude chromatin was purified by layering templates. on 1.7 M sucrose and pelleting 2 h at 60 000 g. The . estimation on extracts. Aliquots from the chromatin pellet was washed by suspending in 0.15 M p ro tem NaCl and centrifuging at 37000 g for 30 min. The enzyme preparations were precipitated with 5 % washed chromatin pellet was dispersed in 0.01 M trichloroacetic acid (TCA). The precipitates, after Tris-HCl, pH 8.0, and dialysed overnight against washing with ethanol and ethanol/ether (1 : l), were the same buffer. All operations were carried out at solubilized in 0.3 N NaOH and the protein was esti1°C and the purification of nuclear and chromatin mated by the procedure of Lowry et al. [16]. preparations was followed microscopically after staining with methylene blue. All the washings and extracts Chemical analysis of chromatin. The histones from obtained prior to ultracentrifugation of the chromatin chromatin were extracted with 0.5 N HCI and estiwere pooled and saved for the preparation of nucleic mated by the procedure of Lowry et al. [l6]. The acid-free extract. residue left after HCI extraction was treated with 5 % TCA (I5 min at 90°C) and the contents centrifuged. DNA and RNA were estimated in the supernatant Preparation of nucleic acid free extract. The pooled cell extract (ca 100 ml from 0.5-I g cells) was made by the diphenylamine [4] and orcinol [I91 procedures, 0.01 M with respect to p-mercaptoethanol and centri- respectively. fuged at 37000 g for 20 min. Ammonium sulfate (and ammonium carbonate lj5Oth that of ammonium sulfate) was added to the supernatant to 80 % satura- Enzyme assays tion and the precipitate obtained was dissolved in 9 ml of buffer B (0.01 M Tris-HCI, pH 8.0, 0.01 M MgCl$, DP. The ‘soluble’ extract equivalent to 5O-300,~g of 0.01 M/3-mercaptoethanol, 5 % glycerol). Strepto- protein, or chromatin equivalent to 1640 ,ug DNA mycin sulfate (1 ml of a 10% solution) was added to were used in each assay. Several different concenrrathis preparation and, after the removal of the preci- tions of the enzyme preparations in the linear range and incubation periods of 30-60 min (during which pitate by centrifugation, the protein in the supematant was precipitated by adding ammonium sulfate to 80 % the reaction was found to be linear with time) were saturation. The protein pellet obtained by centrifuga- used. The complete reaction mixture for the assay tion was dissolved in about 4 ml of buffer B and dia- of DP contained in 0.5 ml final volume: Tris-HCl lysed overnight against the same buffer. The dialysed buffer, pH 8.3, 25 pmoles; MgAc, 3 pmoles; dithiopreparation was clarified by centrifugation and used threitol, 10 pmoles; NaCI, 30pmoles; dATP, dCTP, for DNA polymerase, seryl tRNA synthetase and and dGTP, each, 0.4 pmoles; [3H-methyl]TTP, 5 yCi Exptl Cell Res 80 (1973)

Changes in enzymic activity during cell cultivation (spec. radioact. 7.7 mCi/pmole:); 20 pg of native or denatured calf thymus DNA where indicated; and the enzyme preparation. After 30-60 min incubation at 37°C 100 pg of yeast RNA and 1 ml of cold 25 % TCA were added. The precipitates were collected on presoaked (overnight in saturated pyrophosphate solution) B, membrane filters, washed with 5 % TCA, dried and counted using toluene based scintillation fluid 1261. Srryl tR NA synthetase. The complete reaction mixture for the assay of seryl tRNA synthetase activity contained in 0.5 ml final volume: Tris-HCl buffer, pH 8.3, 50 pmoles; MgAc, 20 pmoles; ATP, 2 pmoles; KCI, 2.5 pmoles; B-mercaptoethanol, 2 pmoles; W-Lserine, 0.3 ,Li (spec. radioact. 103 mCi/mmole); rat liver tRNA 15 ,ug; and nucleic acid free extract equivalent to 200-300 fig protein. After 0, 5, 10 and 15 min incubation of duplicate samples at 37°C the 5 “h TCA-precipitable radioactivity was collected on BB membrane filters and counted as described above. The values for the first 10 min during which the reaction was linear, after subtraction of blank (zero time) counts, were used for calculation of enzyme activity. RNuse and DNuse. RNase and DNase activities were determined by incubating (3 and 6 h at 37°C) the enzyme preparation with Torula RNA or highly polymerized calf thymus DNA (in buffer plus 0.001 M MgCI,), and measuring the increase in the A,,, of the acid-soluble products over a zero time control [26]. Since pH optima for chromatin and soluble nucleases were around 7.5 and 6.5 respectively, 0.05 M Tris-HCl buffer, pH 7.5, and 0.05 M sodium acetate buffer, pH 6.5, were used in the assays. Protease. For the determination of protease activity, 0.7 ml of the chromatin preparation was incubated with 2 ml of 1 % bovine serum albumin (in 0.05 M Tris-HCI, pH 7.5) at 37°C for 0, 3 and 6 h. The reaction was terminated by adding 1 ml of 20% (v/v) HCIO,. After chilling for 5-6 h, the contents were centrifuged at 30000 g for 30 min. Aliquots (I ml) from the supernatant, following neutralization with NaOH, were reacted with ninhydrin reagent [20]. The increase in Aj,0 of the ninhydrin color over that obtained with the supernatant from zero time incubation gave the amount of protease activity.

307

cuvettes, each of which contained 2 ml of the reaction mixture which was composed of 0.6 M glycine, pH 9.6, 0.3 M nicotinamide and 3 % ethyl alcohol; 1 ml of water was added to the second cuvette which served as the blank. After initial reading at 340 nm, 50 ,ug (in 0.05 ml of 0.1 M potassium phosphate buffer, pH 7.5) of alcohol dehydrogenase was added to each tube and the increase in A840(which reached maximum within 15 min) against blank cuvette was read everv 5 min. This max?mum reading for a 2 h incubated sample (which was used for the calculation of activity) was twice compared to the 1 h sample. DNA-dependent RNA synthesizing activity of chromatin. The complete incubation mixture for this assay

contained: Tris-HCI buffer, pH 8.0, 40,r~moles; MgCl, 4 @moles; MnCI,, 1 pmole; CTP, GTP and UTP, each, 0.2 pmole; [VCIATP, 0.2 pCi (spec. radioact. 25 @X/,umole); /I-mercaptoethanol, 12pmoles; E. coli RNA polymerase, 3 units; and chromatin equivalent to 0.5-3 pg DNA in a final volume of 1 ml. The reaction was started by adding chromatin last. After 15 min incubation at 37°C 40 /Lmoles of unlabelled ATP, 100 ,ug of yeast RNA and 1 ml of cold 25 % TCA were added. The precipitates were collected on B, membrane filters, washed with 5 % TCA and counted in a scintillation counter. Usually chromatin at three different concentrations was assayed to assure linearity.

RESULTS AND DISCUSSION

The data presented in table 1 show that RNase and DNase activity associated with the chromatin from WI-38 cells increased with increasing passages. Furthermore, in WI-38VA13 cells, which have apparently indefinite doubling potential, the activity of these enzymes was barely detectable regardless of whether the determinations were made at neutral, alkaline or acid pH. The activity of other chromatin-associated enzymes preATPase and CTPase, GTPase, UTPase activity. ATP (1 mg) or CTP, GTP and UTP (1 mg each) in sented in table 1 also increased with increasing 1 ml of 0.05 M Tris-HCI, pH 7.5, were incubated with 0.5 ml of chromatin for 3 and 6 h at 37°C and the passages in WI-38 cells and again the activity amount of inorganic phosphorus released over the of these enzymes was low in WI-38VA13 zero time control value was determined colorimetricells. Protease activity has been previously tally [I]. DPNpyrophosphorylase. The assay of this enzyme found to be associated with chromatin from was done according to the procedure of Traub [31] WI-38 cells [8], whereas the other enzymes which involved incubation of 0.6 ml of chromatin preparation with 0.6 ml of a mixture containing 54 listed have been found to be associated with /imoles of glycyl-glycine buffer, pH 7.4, 2.4 @moles chromatin from various mammalian sources of ATP, 2.4 pmoles of nicotinamide mononucleotide (NMN), 6 pmoles of MgCl, and 180 pmoles of nico30,311. The changes in tinamide. After 1 or 2 h at 37°C the reaction was [2,9, 10,21,22,25,28, stopped by chilling and adding 15 pl 75 % TCA. The enzyme activities during ‘aging’ in WI-38 precipitate formed was removed by centrifugation (30 000 g, 15 min) and the supernatant saved. One ml cells are essentially in agreement with changes of supernatant was added to one of the two matched occurring in plant tissues [26, 271 and unExptl Cell Res 80 (1973)

308 B. I. Sahai Srivastava Table 1. Enzyme activity” associated with chromatin from WI-38 and WI-38 VA13 cells

Cell type WI-38 p-19/53c p-22150 p-31153 p-40150 p-45150 WI-38VA13

RNase

DNase

1.4&0.2b ::z 2.4 2.5 0.1

0.3 kO.1 0.3 0.6 1.2 1.0 co.1

Protease

ATPase

CTP GTP ase UTP i

0.7kO.l 1.0 1.2 1.2 3.3 0.2

0.8 ) 0.2 0.7 1.2 2.1 2.8 0.4

1.2io.2 1.0 1.7 2.2 2.6 0.4

DPN pyrophosphorylase

1.250.2 ::: 2.7 2.7 0.5

’ Expressed as enzyme units/mg of DNA in chromatin. One enzyme unit represents an increase of 1.0 A,,, (for RNase and DNase) or 1.0 A,,, (for protease) or 1.0 A,,, (for triphosphatases) or 1.0 Az4,, (for DPN-pyrophorylase) in 1 ml of solution for an incubation period of 1 h. ’ Mean F S.D. (n = 3). ’ The P-numbers in this and other tables refer to the passage at which the cells were assayed and the passage at which the cells phased out. Differences between passages 19-22 and passages 3145 groups in RNase, DNase, ATPase, CTPase, GTPase, UTPase, and DPN pyrophosphorylase activity were significant atp < O.OOl,p < 0.01,p c 0.01,p < 0.01 andp c 0.05 level respectively. Although differences between these two groups in protease activity were not significant, passage 45 cells had distinctly high protease activity. Activities of the above enzymes in WI-38 VA1 3 cells were significantly different (p ~0.01) from WI-38 cells.

published data); in plant tissue the DPNpyrophosphorylase activity declined with’age. DPN-pyrophosphorylase which is always associated with chromatin has been found to be Table 2. The ratios of DNA, histone and RNA in chromatin from WI-38 and WI-38VA13 Cdl.9

Cell type

DNA

Histone

RNA

WI-38 p-19/53 p-22150 p-29150 p-31153 p-40150 p-44145 p-45150 WI-38VA13

1 1 1 1 1 1 1 1

1.12+0.09a 1.16 I .05 1.20 1.38 1.30 1.34 1.03

0.39 10.05 0.37 0.35 0.44 0.61 0.64 0.55 0.18

a Mean & S.D. (n = 4). Differences between passages 19-31 and passages 40-45 groups in the proportion of histone and RNA in chromatin were significant at p ~0.01 level. Differences between WI-38 and WI-38VA13 cells in the proportion of RNA and that between passages 40-45 WI-38 cells and WI-38VA13 cells in the proportion of histone were also significant (p < 0.001). Exptl Cell Res 80 (1973)

low in dividing and high in non-dividing [25, 281 mammalian cells. The chromatin in this study was prepared by conventional procedures which included ultracentrifugation through 1.7 M sucrose and washing with 50 vol of 0.15 M NaCl. Furthermore, the chromatin enzymes examined here were those which have been found to be associated with chromatin [2, 9, 10, 21, 22, 24, 25, 28, 30, 31, 331 and distinct from cytoplasmic or lysosoma1 enzymes. Nevertheless, the possibility that some of these enzymesin WI-38 cells become associated with chromatin during its preparation can not be excluded until the enzymes in question have been purified and characterized. The ratios of DNA, histone, and RNA in chromatin from early passage WI-38 cells presented in table 2 are similar to those found by Rovera & Baserga [23] in these cells. It is interesting to note that the proportion of both histone and RNA in chromatin increased with increasing passages in WI-38 cells. The chromatin from WI38VA13 cells, however, had nearly the same

Changesin enzymic acti ity during cell cultivation proportion of histone as early passageWI-38 cells but a lower proportion of RNA. The DNA dependent RNA synthesizing activity of chromatin in table 3 indicates that this activity was highest between passage 22150and 31153but it declined between passages 40150 and 45150 in WI-38 cells, and that this activity in WI-38VA13 cells was lower than in WI-38 cells. This activity included inhibition by actinomycin D, DNase and RNase and dependenceon Mg2+, Mn2+, all four triphosphates and E. coli RNA polymerase. To determine whether the lower activity in the later passageswas due to chromatin-associated RNase, 20 ,ug actinomycin D was added to parallel samples after 15 min of incubation, and incubation continued for an additional 15 min before precipitation with TCA. The loss of counts for both early (p-19/50) and late (p-45/50) passage cells in this experiment was less than 10%, indicating that the low RNA synthesizing activity of chromatin from late passage cells was not due to a higher level of chromatin-associated RNase. Although the possible effects of chromatin-associated RNase, DNase, pro-

309

Table 4. DNA polymerase activity of chromatin from WI-38 and WI-38VA13 cells assayed in the presence and in the absence of exogenousDNA cpm incorporated into DNA/mg chromatin DNA/hour Cell type

Chromatin alone

Chromatin plus native calf thymus DNA

WI-38 p-19153 p-19150 p-40150 p-44145 p-45150 WI38VA13 WI38VA13

19 170 12000 11500 13 150 10 600 11900 18 270

45 510 56 200 47 610 60200 49 ooo 90 700 109 250

DNA polymerase activity of the chromatin assayed in the presence of DNA was significantly different (p
tease and nucleoside triphosphatase on the in vitro measurements of RNA synthesizing activity of chromatin cannot be excluded, the activities of these enzymeswere probably not Table 3. Activity of chromatin from WI-38 high enough to be serious during the short and WI-38VA13 cells in DNA-dependent (15 min) period of incubation. Decline in RNA synthesis RNA synthesizing activity of chromatin during aging in mouse tissue [18] and loss of cpm incorporated into certain fibroblast functions with increasing RNA/pg chromatin DNA on 15 min passagesin WI-38 cells [14] have been reCell type incubation ported. Slower rates of RNA synthesis in phase III WI-38 cells, compared with phase I WI-38 cells, have also been reported [17] by autop-19153 555 p-19/50 531 radiographic techniques. p-22150 652 Since DNA polymerases represent key enp-31153 620 p-40150 340 zymes involved in the regulation of cell divip-45150 185 sion, the changes in these enzymes, which W138VA13 138 occur both in cytoplasm and chromatin [5, RNA synthesizing activity of the chromatin from pas- 24, 331, were examined. The data shown in sages 19-31 WI-38 cells was significantly different table 4 indicate that chromatin from WI-38 or (~‘0.01) from that of passages 4&45 WI-38 or WI-38VA13 cells. from WI-38VA13 cells, like that from other Exptl Cell Res 80 (1973

3 10 B. I. Sahai Srivastava

Table 5. DNA polymerase activitya of nucleic

sources [15], was a poor template for endogenous DP and addition of exogenous DNA was required for full expression of DP activity. The DP activity of the chromatin assayed with or without exogenous DNA did not change significantly with increasing passagesin WI-38 cells. In contrast, the DP activity of chromatin from WI-38VA13 cells assayed in the presence of exogenous DNA was twice that of WI-38 cells. The DP activity of the “nucleic acid-free extract” or of the “extract prepared by the alternate procedure” again did not change (table 5) significantly with increasing passage in WI-38 cells, although the DP activity in these preparations from WI-38VA13 cells was 13-20 times higher compared with WI-38 cells. The lower DP activity of “nucleic acid free extracts” from WI-38 cells compared with the “extracts prepared by the alternate procedure” resulted from higher protein content of the former extracts due to pooling of all extracts and washings. On the other hand, the difference in the ratio of WI-38 to WI38VAl3 DP activity between the two extrac-

acid-free extracts and of extracts prepared by the alternate procedure from WI-38 and WI38VA13 cells

Cell type

cpm incorporated into DNA/mg protein/hour

Nucleic acid free extract WI-38 6 252 p-19153 5 695 p-20145 p-29150 5 855 6 112 p-40150 4 443 p-45150 WI-38VA13

124 500

Extracts prepared by alternate procedure WI-38 10 500 p-i s/so 9000 p-44145 WI-38VA13

131 492

a Heat-denatured calf thymus DNA was used as template. DNA polymerase activity of WI-38VA13 cells was significantly different (p
Table 6. Response of DNA polymerases from WI-38 (p-19/50) cells to different templates cpma incorporated/O.1 ml enzyme/hour System No template added Plus calf thymus native DNA (20 pup) Plus calf thymus denatured DNA (20 ,ug) Plus calf thymus “activated” DNA (20 pg) Plus calf thymus denatured DNA (20 pg) but minus dATP, dCTP, dGTP Poly(rA). poly(rU) Plus human ribosomal RNA (10 ,ug)

“Soluble” DNA polymeraseb

Solubilized chromatin DNA polymeraseC

0 600 466 882

0 555 252 838

30 0 0

12 0 0

a Blank counts (without enzyme) of about 80-100 cpm have been subtracted. b Protein precipitated between 30-50 % saturation with ammonium sulfate from DP peak fractions obtained by DEAE-cellulose chromatography. c Chromatin was stirred (2 h) with 1 M NaCl (dissolved in 0.01 M Tris-HCl, pH 8.0, containing 0.01 M/Imercaptoethanol) and the 1 M NaCl extract recovered by centrifugation (37 000 g 20 min) was dialysed (4 h) against 6 vol of water. The orecipitate formed was removed by centrifugation and the supernatant containing D’P activity was used in the-assays. Exptl Cell Res 80 (1973)

Changes in enzymic activity during cell cultivation tion procedures resulted from greater variation in DP activity of WI-38VA13 cells. An examination of the extract (prepared by the “alternate procedure”) from early (p-19/50) and late (p-44/45) passage WI-38 cells by DEAE-cellulose column chromatography revealed only one DNA directed DNA polymerase (DDDP) activity peak. This DP activity peak corresponds with the soluble DP reported by Weissbach et al. 1331 in WI-38 cells. Similarly the properties of partially purified DP from the ‘soluble’ and the chromatin fractions presented in table 6 are similar to soluble and nuclear enzymes reported by Schlabach et al. [24] in HeLa and WI-38 cells. The A,,, protein profile of ‘soluble’ extracts from early and late (phase III) WI-38 cells as obtained by DEAE-cellulose chromatography showed quantitative differences in the proportion of different protein fractions from these cells. Unfortunately, the limitation of the material precluded further examination along these lines. Nevertheless, the activities of seryl tRNA synthetase, RNase and DNase in unfractionated “nucleic acid free extract” were determined because translational changes [29] or changes in soluble nucleases could occur during aging. Aminoacyl tRNA synthetases, other than for serine, were not assayed due to the practical limitations imposed by the simultaneous assay of many other enzymes within a day. Estimation of seryl tRNA synthetase activity (cpm bound to tRNA/mg protein/min) of p- 19/53, p-20145, p-29150, p-40150 and p-45150 WI-38 cells gave 230, 277, 279, 249 and 228 cpm, indicating no significant change with increasing passages. This activity in WI38VA13 cells was twice that of WI-38 cells. It should be recognized that we measured only seryl tRNA synthetase; the activity of other aminoacyl tRNA synthetases may change during aging in WI-38 cells. The data in table 7 show that the activity

3 11

Table 7. RNase and DNase activity of nucleic acid-free extractsa from WI-38 cells Enzyme unitsb/mg of protein Cell type

RNase

DNase

p-19/53 p-20145 p-40150 p-45150

5.9 5.1 4.8 6.4

1.2 1.0 0.9

a Samples were stored several months at -20°C before assay of RNase and DNase activity at pH 6.5. b One enzyme unit represents an increase of 1.0 AZeO in 1 ml solution for an incubation period of 1 h. Differences between passages 19-20 and passages 40-45 were not significant.

of soluble RNase and DNase (assayed at pH 6.5) did not change significantly with increasing passage in WI-38 cells. The results presented in this paper show that cessation of cell division in phase III WI-38 cells is not paralleled by changes in DNA polymerase activity of these cells. On the other hand, aging in WI-38 cells appears to be associated with increase in those chromatin-associated enzymes which may degrade DNA, RNA, protein and precursors of nucleic acids. Even one of these changes could seriously impair chromatin function and precipitate aging of WI-38 cells. This hypothesis is supported by the finding of extremely low activities of these chromatinassociated enzymes in WI-38VA13 cells which have indefinite doubling potential in vitro. Since suppression of the activities of these chromatin-associated enzymes by cytokinin plant hormones retards senescence in plant tissues [27], it is reasonable to assume that substances which may suppress the increase in the activity of these enzymes in WI-38 cells may also retard aging in these cells. The author wishes to thank Dr L. Hayflick and Dr J. Minowada for their assistance. Dr Hayflick was Exptl Cell Res 80 (1973)

312 B. 1. Sahai Srivastava assisted by contract 69-2091 from the National Institute of Child Health and Human Development. This work was supported by NSF Grant GB-27554 and by USPHS grant (CA-13038).

REFERENCES 1. Allen, R J L, Biochem j 34 (1940) 858. 2. y;;ley, J & Chalkley, R, J biol them 245 (1970) 3. Bekdor, I, Bonner, J & Dahmus, G K, Proc natl acad sci US 62 (1969) 271. 4. Burton, K, Biochem j 62 (1956) 315. 5. Chang, L M S & Bollum F J, Biochemistry 11 (1972) 1264. 6. Cristofalo, V J, Aging in cell and tissue culture (ed E Holeckova & V J Cristofalo) p. 83. Plenum Press, New York (1970). 7. Cristofalo, V J, Parris, N & Kritchevsky, D, J cell physol 69 (1967) 263. 8. Farber, J, Rovera, G & Baserga, R, Biochem j 122 (1971) 189. 9. Furlan, M & Jericigo, M, Biochim biophys acta 147 (1967) 135. 10. Garrels, J I, Elgin, S C R & Bonner, J, Biochem biophys res commun 46 (1972) 545. 11. Girardi, A J, Jensen, F C & Koprowski, H, J cell phys 65 (1965) 69. 12. Hayflick, L, Exptl cell res 37 (1965) 614. 13. Hayflick, L & Moorhead, P S, Exptl cell res 25 (1961) 585. 14. Houck, J C, Sharma, V K & Hayflick, L, Proc sot exptl biol med 137 (1971) 331. 15. Lindsay, J G, Berryman, S & Adams, R L P, Biochem j 119 (1970) 839. 16. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265.

Exptl

Cell Res 80 (1973)

17. Macieira-Coelho, A, Ponten, J & Philipson, L, Exptl cell res 42 (1966) 673. 18. Mainwaring, W I P, Biochem j 110 (1968) 79. 19. Markham, R, Modern methods of plant analysis (ed K Paech & M V Tracey) p. 246. SpringerVerlag, Berlin (1955). 20. Moore, S & Stein, W H, J biol them 211 (1954) 907. 21. Morgan, C R & Banner, J, Proc natl acad sci US 65 (1970) 1077. 22. O’Connor, P J, Biochem biophys res commun 35 (1969) 805. 23. Rovera, G & Baserga, R, J cell phys 77 (1971) 201. 24. Schlabach. A. Fridlender. B. Bolden, A & Weissbath, A, B&hem biophys res corn&n 44 (1971) 879. 25. Siebert, G & Bennet-Humphrey, G, Adv in enzymol 27 (1965) 239. 26. Srivastava, B I S, Biochem j 110 (1968) 683. 27. - Biochem biophys res commun 32 (1968) 533. 28. Stirpe, F & Aldridge, W N, Biochem j 80 (1961) 481. 29. Strehler, B, Hirsch, G, Gusseck, D, Johnson, R & Bick, M, J theor biol 33 (1971) 429. 30. Swingle, K F, Cole L J & Bailey, J S, Biochim bionhvs acta 149 (1967) 467. 31. Traub,- A, Methods in. cancer research (ed H Busch) p. 353. Academic Press, New York (1970). 32. Wang, K-M, Rose, N R, Bartholomew, E A, Balzar, M, Berde, K & Foldvary, M, Exptl cell res 61 (1970) 357. 33. Weissbach, A, Schlabach, A, Fridlender, B & Bolden, A, Nature new biol 231 (1971) 167.

Received December 18, 1972 Revised version received February 20, 1973