Inhibitory effects of acid polysaccharides from sea urchin embryos in RNA synthesis in vitro

ARY

NOTES

Inhibitory effects of acid polysaccharides from sea urchin embryos in RNA synthesis in v&o Y. AOKI

and H. KOSHIHARA,

Zoology, Tokyo Kyoiku ku, Tokyo, Japan

University,

Department of Ootuka,

Bunkyo-

Summary The supernatant fraction from cytoplasm in sea urchin embryos exhibited a remarkable inhibitory effect on RNA synthesis in vitro and an inhibitory substance present was mostly derived from intercellular substances. This inhibitor was attributed to the acid polysaccharides. In the course of development, the inhibitory effects of the acid polysaccharides altered as indicated in the elution profiles of Ecteola column chromatography.

It is generally accepted that nuclear RNA synthesis, in vivo or in vitro, is controlled to a great extent by various cytoplasmic factors. This has been demonstrated by many investigators using various procedures [6, 7, 13, 14, 151. Our preliminary experiment showed that a cytoplasmic fraction prepared from sea urchin eggs or embryos exhibited a remarkable inhibitory effect on RNA synthesis in vitro [I]. However, this fraction showed no significant activity of nucleotide phospholysis or RNA hydrolysis. Furthermore, the inhibitory activity of this cytoplasmic fraction could not be affected by dialysis, heat treatment or pronase digestion. In the present experiments it was demonstrated that the inhibitory effect of the cytoplasmic supernatant was almost entirely attributable to the acid polysaccharides of intercellular substances. The inhibitory profiles of fractions obtained by Ecteola cellulose column chromatography altered gradually in the course of development.

Materials

and methods

Cell dissociation and intercellular substances: Embryos used in these experiments were obtained from the sea urchin, Anthocidaris cvassispina, at hatching blastula, gastrula or prism stage. Loosely packed embryos were suspended in 4 vol of the cell-dissociation medium (composed of 0.44 M sucrose. 0.01 sodium citrate,’ 0.061 M EDTA and 0.01 M ?risbuffer. DH 8.1) and left standing for several minutes. Whilst check&g with the optical microscope, this suspension was shaken by hand until completely dissociated. Dissociated cells and intercellular substances were separated by centrifugation at IO 000 g for 10 min. Each fraction was quickly frozea and stored at - 20°C until used. Preparation of acid polysaccharides: Four volumes of acetone were added direct to the intercellular substances and from the resulting precipitate, lipids were completely removed by extraction with ethanol and ether-ethanol (I :3) in order. Then, in order to remove proteins, pronase equivalent to 20 9/oof total protein &as addkd to the above residue suspended in 0.02 M Tris-HCl buffer adjusted to oH 8.0. After complete digestion, acid pol;saccharidks in solution were exclusively sedimented by addition of 2 vol of 0.2 % cetyl trimethyl ammonium bromide (CTA For removal of CTAB, the resulting sediment was solubilized with 0.2 M KSCN and precipitated with 4 vol of ethanol. The sediment was washed with 4 vol of ethanol in order to remove KSCN. The finally washed sediment was designated as the acid polysaccharides. If necessary, a small amount of contaminant RNA was digested with pzncreatic RNase. Acid polysaccharides were determined by the cetylpyridinium chloride method 1121, using heparin (167 units/mg, Daiichi Pure Chemicals Co., Tokyo) as an internal standard. ‘kid Fractionation of acid polysaccharides: polysaccharides were fractionated by Ecteola cellulose column chromatography. All procedure was according to the method of Ringertz & Reichard [I 11. Before the fractionation. acid Dolvsaccharides were treated with RNase, whi& was -5O-pg per OD,,, = 3: at 37°C for 15 min. Neoarin (from Daiichi) and chondroitin sulfate (Zelia Chemicals Co.) were used as markers for chromatography.

NA was RNA synthesis: Sea urchin spermal prepared by the method of Marmur [9]. ~~rifica~~o~ of RNA polymerase from E. coli was perfo according to the procedure of Chamberlin & [Z] up to their fraction 3 stage. The standard reaction mixture for assay of RNA synthesis in chromatinor DNA-template system contained in final volume

432

Y. Aoki & H. Koshihara

of 0.55 ml: 40 rcmoles of Tris-HCl buffer (uH 8.0). 4 pmoles of M&l,, 1 pmole of MnCl, (TMM), 1.2 pmoles of p-mercaptoethanol, 0.4pmoles each of ATP, CTP and GTP, 20/&g DNA or pluteus chromatin equivalent to 80 pg DNA, 30-1OOflg of RNA polymerase (160-900 units/mg protein for 30 min at 3O”C), 0.4 &i of 3H-UTP (spec. act. 11.0 Ci or 25.0 Ci mmole) and 0.1 ml of each cytoplasmic fraction or acid nolvsaccharides. DNA was determined bv- dinhenvl_ amine method [4]. For RNA synthesis in nuclei, which were prepared by homogenizing embryos at pluteus stage in isotonic medium and centrifuging at 1 500 g for 10 min, the reaction mixture contained nuclei equivalent to 5.5 mg protein (suspended in 0.2 ml of the medium composed of 0.05 M Tris-HCl buffer pH 7.6, 0.003 M MgC& and 0.25 M sucrose) and the-same mixture as described above except TMM. The reaction was carried out at 30°C for 10 min and stopped by addition of 3 ml of 5 % TCA solution. After washing twice, the acidinsoluble material was counted in a liquid scintillation counter. Alternatively, an aliquot of synthesized RNA was placed on a Whatman 3 MM filter which was prepared by washing with 0.1 M sodium pyrophosphate, and the filter was then washed three times with 5 % TCA and once with ethanol, and finallv was dried and counted. Protein was determined by the method of Lowry et al. [S].

Results and Discussion Inhibitory effects of cytoplasmic supernatant or intercellular substances from sea urchin embryos on RNA synthesis in vitro: In the preliminary studies, the supernatant fraction from sea urchin embryos exhibited a remarkable inhibitory effect on RNA synthesis in vitro. For a more precise localization and characterization of the cytoplasmic inhibitor, we first compared the inhibitory effects of different cytoplasmic fractions prepared from whole embryos or their dissociated cells and intercellular substances. Data shown in table 1 were obtained with fractions prepared from gastrulae of A. crassispina, using the chromatin-template RNA synthesizing system described in Methods. It was seen that only the 105 000 g supernatant of embryos and the intercellular substances

exclusively

showed

intense

activity,

while the supernatant of dissociated cells did not show the inhibitory effect so significantly. These results suggest that the whole embryos supernatant contains the interExptl Cell Res 70

cellular substances which exhibit the intense inhibitory effect. Both the precipitable and non-precipitable fractions in intercellular substances acted as inhibitory factors so effectively that the RNA synthesis was almost completely arrested. The non-precipitable fraction in the substances proved to be especially effective as an inhibitor, as estimated by their protein contents. On the other hand, an appreciable inhibitory effect of the microsomal fraction prepared from whole embryos may be due to its being identical with the precipitable fraction in the intercellular substances. The mitochondrial fraction had a slight stimulation activity, but it seems to be due to additive RNA synthesis in a few contaminating nuclei. From these results, it is demonstrated that the inhibitory effect of cytoplasm is attributable to the intercellular substances. On the other hand, it was found in our preliminary experiment that the above cytoplasmic effects on RNA synthesis in vitro could not be due to concomitant RNase activity or nucleotide phosphatases activity [l]. Consequently, we attempted to isolate the cytoplasmic inhibitor from the intercellu1a.r substances since these acted as a powerful inhibitor in situ. Inhibitory effects of acid polysaccharides extracted from the intercellular substances: From preliminary observations it is clear that the inhibitor of cytoplasmic supernatant is non-dialysable, heat-stable and pronaseresistant, suggesting that it is not a protein. Therefore, in analysing the inhibitory effect of the intercellular substances, we extracted acid polysaccharides from them, and observed if any inhibitory effect was exhibited by addition of these to the three different RNA synthesizing systems-RNAtemplate or chromatin-template system, and isolated nuclei. Extraction of acid poly-

Acid polysaccharides

able 1. Effects

of cytoplasmic NA synthesis in vitro

fractions

from sea urchin embryos and from

sea urchin

embryos

NA synthesis

433

on ~l~rornat~~ template

Embryos were collected at gastrula stage and homogenized in medium composed of 0.25 M sucrose, 0.15 M KCI, 0.15 M NaCI, 0.003 M MgCl, and 0.01 M Tris-HCl buffer pH 7.2. Mitochondria fraction was sedimented by centrifugation at 12 000 g for 10 min, and its supernatant was then separated into microsome and supernalant fractions by centrifugation at 105 000 g for 90 min. This centrifugation also separated the intercellular substances into precipitate and supernatant. Each fraction equivalent to 100 pg protein was added to each tube containing chromatin equivalent to 80 pg DNA. Chromatin was isolated from frozen plutei as follows: After homogenizing in the homogenization medium and centrifuging at 10 000 g for 30 min, the sediment was rehomogenized with 0.05 M Tris-HCl buffer (pH 8.0) and centrifuged at 10 000 g. This washing procedure was repeated twice and the finally washed sediment rehomogenized with I .9 M sucrose and centrifuged for 2 h at 22 000 rpm in a Spinco SW 25.1 rotor. The resulting gelatinous pellet was suspended in 0.05 M Tris-NC1 buffer and used as chromatin Addition of the homogenization medium (a), the cell dissociation medium (b) in place of a cytoplasmic fraction. The reaction mixture and condition of the experiment were as described in Methods. -pmoles 3H-UMP incorporation per 100 pg DNA Addition

of fraction ( %)

Source of cytoplasm

Nonea

mitochondria

microsome

Embryos Dissociated cells

110 (100) 110 (100)

102 (93) 131 (119)

90 (82) 108 (98)

Noneb

Total

precipitate

supernatant

16 (26)

11 (18)

Intercellular substances

105000

62

(100)

9 (15)

105 000 g supernantant

-

42 (38) 99 (90)

g

105000g

-

Figures in parentheses are percentages.

saccharides was carried out according to a slight modification of Scott’s method [12] and assays with three RNA synthesizing system.s were done respectively, as described in Methods. At the same time, authentic heparin used as an internal standard was tested for its inhibitory effect and compared with that of the acid polysaccharides. As shown in table 2, it was found that acid polysaccharides from hatching blastulae and gastrulae exhibited a remarkable inhibitory effect on the DNA- or chromatin-template RNA synthesizing system, whereas they were less inhibitory in isolated nuclei when added in similar amount. Furthermore, the same amount of heparin exhibited an inhibitory effect to a comparable degree with that of the acid polysaccharides from the above embryos at both stages. Concerning the effect of heparin on RNA synthesis in vitro, there remains the slightly

puzzling fact that isolated nuclei or ‘aggregating enzymes’ were stimulatively affected NA- or in contrast with the results in chromatin-template E. coli systems [3, lo]. The foreg that the polyanion, heparin or chondroitin sulfate, derepresses RNA synthesis mainly by binding to the poiycation, histones, and then by the resulting partial removal of histones from chromatin [5]. Thus, RNA polymerases in nuclei or ‘aggregating enzymes’ already bind to chromatin or continue to synthesize RNA (most merase engaging in RNA synthesis). Therefore, the enzymes would not be inhibited by the polyanion, i.e. heparin. Our L~~~u~lisbed data suggest that acid polysaccharides containing heparin and chondroitin sitfate could interact only with free and nonengaging RNA polymerase and have no effect in the middle of RNA synthesis fmanw-

434

Y. Aoki d H. Koshihara

Table 2. Effects of the acid polysaccharides of the intercellular embryos on RNA synthesis in vitro

substances from sea urchin

Acid polysaccharides of intercellular substances at hatching blastula and gastrula stages, equivalent to 25 ,ug heparin, were added to each tube. The reaction mixture and condition of the experiment were as described in Methods. pmoles 3H-UMP incorporation

per mg DNA or proteina Acid polysaccharides ( %) intercellular substances

System DNAtemplate Chromatin template Isolated crude nucleia

None

Addition heparin

8 515 (100)

12 (0)

593 (100) 3.03 (100)

69 (12) 2.55 (84)

of

h. blastula

gastrula

162 ( 2)

93 ( 1)

123 (21) 2.18 (72)

72 (12) 1.65 (55)

Figures in parentheses are percentages.

script in preparation). The above explanation might account for these contradictory results. When comparing hatching blastula with gastrula, it is noteworthy that the acid polysaccharides at the more advanced stages are, to a certain extent, more effective than those at hatching blastula stage. This tendency of their inhibitory effect in the course of development was ascertained by fractionation and subsequent assay. Fractionation of acid polysaccharides through Ecteola cellulose column and the inhibitory activities of their fractions in RNA synthesis in vitro: The acid polysaccharides from embryos at hatching blastula, gastrula and prism stages, respectively, were charged through an Ecteola cellulose column. Elution patterns were obtained according to the method of Ringertz & Reichard [l 11. In order to remove contaminant RNA in these acid polysaccharides preparations, they were treated with RNase under the same condition as described in Methods and then charged to the columns. As shown in fig. 1 a, b, c, each of the three acid polysaccharide fractions was similarly divided into mainly two major peaks, which began to elute at 0.6 M and 1.0 M of Cl-ions Exptl Cell Res 70

through the column, respectively. Thus, the first is a relatively sharp peak and the second appears as a complicated and wide-spread form. Comparison of the acid polysaccharides from embryos of three different developmental stages revealed that the ratios of acid polysaccharides of the first peak to those of the second gradually decrease: 1.23, 0.93 and 0.8 in hatching blastula, gastrula and prism. Identification of the two peaks suggests that the former corresponds to chondroitin sulfate, the latter to heparin fraction in elution patterns of the authentic chemicals through the same column. At this time, the run-off fraction through the column contains RNase and nucleotides digested with it as noncharged substances. Prior to assay of RNA synthesis in presence of each eluate, it was dialysed against distilled water to remove HCl-NaCl, and 0.1 ml aliquot of the each eluate (12.0 ml) was added to the reaction mixture containing DNA from sea urchin sperm, and E. coli RNA polymerase. In fig. 1 a, b, c, the relative values of the inhibitory effects of each eluate are plotted against the number of each tube, and superimposed on the elution patterns. The in-

FZg. 1. Abscissa: fraction number; ordinate: (Zeff)*ASSQ; (right) inhibition ( X). E&ion profiles of the acid polysacc~a~d~ of intercellular substances through Ecteala ~~~u~~ and the rate of inhibitory effect of each fraction on DNA-template RNA synthesis. Linear gradient c~omato~a~hy. Resesvoir: 400 ml of 3.0 M NaCl/WCl (1: l), mixing vessel: 400 ml of 0.1 M NaCljHCl (1: I). Each fraction was collected at 12 ml. Acid polysaccharides fractions of intercellular substances were obtained from sea urchin embryos and charged through the column in the following amounts: (a) 1.1 mg in batching blastuiae; (b> 2 mg in gastrulae; (c) 2 mg in prism. These were pretreated with RNase for removal of concomitant RNAs. This RNase and rmcleotides digested with it appeared as an eluate of run-off. Acid po~ysaccha~~d~ were determined at 530 rnp by turbidity with 0.1 % of cetyl-pyridiniurn chloride. After each fraction was dialysed against pure water, an ahquot (0.1 ml) was added to DNA-template RNA synthesizing system as described in Methods. ----, Am; - -> Asa; -, A,,,; m- -3 inhibition of RNA synthesis ( %). The value at AZsa or Azso was negligible except at run-off.

436

S. Goldstein h C. C. Lin

hibitory effect appeared from the first to the second peak in rough parallel with the elution profiles by turbidity. A remarkable change in the patterns representing inhibitory activity occurred between hatching blastula and gastrula stages. The inhibitory activity of the first eluting fraction of hatching blastula diminished drastically at the more advanced stages and shifted considerably toward the second peak (corresponding to heparin fractions) which appeared to be composed of several heterogeneous peaks in a wide-spread range on the gradient. From the profiles, two separate inhibitory activities appear at concentrations corresponding to the optical density peaks in blastula, whereas at the other more advanced stages the former disappears or shifts to the latter activity, indicating that the former changes or transfers to the latter, additively. Although this is plausible, it is equally possible that the first fraction, identical with chondroitin sulfate, diminished, and the second fraction corresponding to heparin reversely increased, in proportion to the amount in each fraction. From these observations, acid polysaccharides in intercellular spaces may become more polymerized or sulfated forms at the later than the earlier stage of hatching blastula. Further investigations are needed to clarify this point. References 1. Aoki, Y & Koshihara, H, Jap j develop biol 23 (1969) 32. 2. Chamberlin, M & Berg, P, Proc natl acad sci US 48 (1962) 81. 3. Chambon, P, Ramuz, M, Mandel, P & Doly, J, Biochim biophys acta 157 (1968) 504. 4. Dische, Z & Schwarz, K, Microchem acta 2 (1937) 13. 5. Frenster, J H, Nature 206 (1965) 680. 6. Gurdon, J B & Brown, D D, J mol biol 12 (1965) 27. 7. Harris, H, J cell sci 2 (1967) 23. Exptl Cell Res 70

8. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 9. Marmur, J, J mol biol 3 (1961) 208. 10. Novello, F & Stirpe, F, Biochem j 112 (1969) 721. 11. Ringer& N R & Reichard, P, Acta them Stand 14 (1960) 303. 12. Scott, J E, Methods of biochemical analysis, vol. 8, p. 162. Interscience, New York (1960): 13. Thompson, L R & McCarthy B J, Biochem biophys res comm 30 (1968) 166. 14. Unsprung, H & Markert C L, Develop biol 8 (1963) 309. 15. Yamana, K & Shiokawa, K, Exptl cell res 44 (1966) 283. Received August 9, 1971 Revised version received October 4, 1971

Rescue of senescent human fibroblasts by hybridization with hamster cells in vitro S. GOLDSTEIN’ and C. C. LIN,2 lDepurtment of Medicine, and 2Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada

Summary Cell hybridization studies were carried out to explore the nature of senescence of diploid human fibroblasts in vitro. A heteroploid line of golden hamster cells was fused with four different strains of senescent human fibroblasts with uroduction of viable hybrids in each case. This suggests that senescence in vitro is a quasidifferentiated state which is amenable to genetic reprogramming.

The human diploid fibroblast has a limited proliferative capacity when cultured in vitro [l] and thus has been used increasingly as a model for the study of aging [2]. Although substantial evidence now supports the validity of this system [2], the molecular basis for the loss of mitotic potential and eventual death of these cells remains unknown. Two general mechanisms could be responsible for initiating the cellular decline: accumulation of faults in informational macromolecules, and/ or a genetic program related to differentiation [2]. Recent work has shown that the ability to perform DNA repair, normally present in all cells, diminishes after serial subculture of human fibroblasts. However, defects in this