- Email: [email protected]
Nucleolar RNA synthesis in heterokaryons derived from senescent and pre-senescent human fibroblasts
Copyright Q 1980 by Academic Press. Inc. All rights of reproduction in any form reserved ,“,11-4827/80!100?97-0~$0?.~in
Experimental
NUCLEOLAR
Cell Research 129 (1980) 297-301
RNA SYNTHESIS
DERIVED
IN HETEROKARYONS
FROM SENESCENT
PRE-SENESCENT
HUMAN
AND
FIBROBLASTS
JOAN RENER’ and ROLAND M. NARDONE Deportment
ofBio/og_v. The Cntholic Uni~vrsi~y qfAmerictr.
W’uvhington. D.C. 20064.
crnd ‘Lnborator~ of Pnrnsitic Disensas. Notioncrl Institute oj’illlergy md lqfecriotts Disetrses. Notiontrl Institutes oj’Hea/th.
Bethesdn,
MD -70014. USA
SUMMARk Normal human fibroblasts, serially passaged in vitro. demonstrated decreasing synthesis of ribosomal RNA (rRNA). Senescent and pre-senescent WI38 cells were fused with one another in order to study age-related factors affecting the production of nucleolar RNA. Autoradiograms revealed that the young nucleus of a dichronic heterokaryon (2 nuclei of different ages) had an impaired ability to produce nucleolar RNA, while the young nucleus of a monochronic heterokaryon (2 nuclei of the same age) was not affected. Old nuclei of dichronic heterokaryons, and old monochronic heterokaryons displayed the same nucleolar RNA synthesis rate as did their single, unfused counterparts
Normal human diploid fibroblasts, grown in vitro, have finite replicative lifespans [ 1, 21. While the seminal events which cause in vitro aging are not agreed upon at this time, metabolic and cellular parameters which change as fibroblast populations lose their replicative capacity have been extensively characterized [3, 41. It has been demonstrated that DNA synthesis [4], and RNA synthetic activity [4, 8, 9, lo] decreases as diploid fibroblasts age. Bowman et al. [ 1l] demonstrated that the decline in RNA synthesis is due mainly to decreased synthesis of nucleolar RNA, nucleoplasmic synthesis being relatively unchanged. Elucidation of the mechanism of template shutdown remains to be resolved. This study was undertaken to determine whether or not a diffusible repressor of ribosomal DNA (rDNA) is associated with the nuclei of late passage (old) cells and whether or not the repressed 20-801806
rDNA template of old nuclei could be derepressed by factors present in nuclei of early passage (young) cells. Cell hybridization has been a useful tool in probing the factors responsible for the shut down of template activity in old cells. Initial experiments by Goldstein & Lin [5], followed by extensive work by Norwood et al. [6, 71, indicated that DNA replication in old fibroblasts can be derepressed after fusion with transformed cells, but that the senescent phenotype is dominant in a heterokaryon formed by fusion with a presenescent cell. Consideration is given here to the effects of fusion between cells of different ages upon the transcription of rDNA. Because it is localized within the nucleolus and codes specifically for a single 45 S precursor molecule of rRNA [ 12, 131. rDNA is uniquely suited to an autoradiographic study of regulation of transcription in a Exp Cc/l Rrs 129 11980~
defined
segment
of the human
In these experiments. thesis was examined
genome
nucleolar RNA synin both single. unfused
WI38 cells and in heterokaryons from cells of different
MATERIALS Cells and culture
[ 161.
produced
ages.
AND
METHODS
conditions
Human diploid fibroblasts (W138~. at passage 15. were obtained from the American Type Culture Collection. The cells were grown in 75 or 150 cm* plastic tissue culture flasks (Corning). They were fed with Eagle’s Minimum Essential Medium- (MEM) supplemeoted with 10% fetal bovine serum (FBS). 2 mM L-glutamine. 100 U/ml penicillin and 100 pg/ml streptomycin and buffered at pH 7.3 with NaHCO,. The cells were incubated at 37°C; a SC; C0,-95qii air mixture served as the gas phase. Confluent cultures were subcultured every 3 or 4 days by a I : ? split. A 0.25% solution of trypsin (activity I : 250) in calcium and magnesium-free ohosphate-buffered saline (CMF-PBS) was used to free ;he cklls for harvesting. Culture age was verified by the method of Cristofalo & Scharf[ 161. Senescent populations were defined as those which had completed X0% of their lifespans (passage 40 or above). Pre-senescent populations were defined as those which had completeh (45% of their lifespans and had been passaged less than 28 times. To maintain an adequate supply of old cells, confluent cultures p40 or above. were held for up to one month in I50 cm* flasks with intermittent feeding every 7 days. To insure an adequate stock of young cells. near confluent cultures in 75 cm? flasks were harvested between ~18 and ~22. The cells were transferred to amooules containing I ml MEM. IO% DMSO and ISc’c FB’S. They were left at 4°C for 4 h to overnight and then frozen and stored in liquid nitrogen. Throughout the studies cells and spent media were checked for the presence of mycoplasma. Aliquots were tested with mycoplasma enrichment media (BioQuest) under aerobic and anaerobic conditions. All cultures were negative for mycoplasma.
In a reprebentative esperimrnr. ~27 .mu p-12 cellr iterr grown in 75 and I50 cm’ flask\. re3pectivel>. md were prelabeled as debcribed above. Four d,l!\ hrfor-e fusion. 5x IO young cells/ml or I * IO’ old ~rll\/ml were nlated into Leighton tube3 containing fl\ine glass coverslips. Tw&tv-four hour3 after -pla;inf. these cells were synchron;ed for3648 h bv iholeucine deprivation and dlocked in Cl [IS]. RN.4 iynthesis i, continuous but not uniform in rate during the cell cycle: hence synchronization zerved to reduce normal variations. The cells were fed with complete medium. Fusion was accomplished after I8 h h) a modification of the technique described by Norwood et ai. [7]. Prelabeled cells were rinsed twice the CMF-PBS. harvested. pelleted and resuspended in I ml cold CMFPBS. Tube5 containing the cell5 uerc placed on crushed ice while cell count5 were ohtained with a Coulter Counter Model B. During thi\ period the >ynchronized cells were also placed on cl-ushed ice. These Here then refed with 0.3-0.6 ml of serum-free MEM containing IO00 HAU of inactivated Sendai vim\ (Connaught Labs). These tubej remained on ice for- an additional I5 min. ~-IX IOF pre-labeled young cells. 01 6x IO5 pre-labeled old cells suspended in 0.1 ml serumfree MEM were added to each Leighton tube. After 15 additional minutes on ice. the tubes were incubated for 90 min at 37°C. The virus was removed by gentlq rinsing the cultures twice with warm MEM containing serum. The heterokaryons were refed Lbith fresh medium and incubated for I8 h. .4t the beginning of the 18th h they were refed with MEM containing [“HIuridine (5 &i/ml; spec. act. 25 Cilmmol: Nem England Nuclear) and 2.5~ IOM deoxycytidine and 2.5~ IO-’ M thymidine. The cultures were incubated for 3 h to insure saturation labeling conditions. Young and old control cultures consisted of synchronized populations exposed to fusion conditions without cocultivation and ‘mock’ fusion in which no Sendai virus was present in the serum-free MEM. Monochronic were formed between pre-labeled heterokaryons young cells and synchronized cells of the same age. ot pre-labeled old cells and synchronized old cells. Dichronic heterokaryons were formed between prelabeled young cells and synchronized old ones or prelabeled old cells and synchronized young ones. Autoradiograms for each class were prepared in triplicate.
Autoradiograph> Identification
of parent
populations
In order to recognize rhe respective nuclei in heterokarvons. one of the parent populations used for fusion was heavily pre-labiled wit-h [jH]thymidine (spec. act. 28 Cilmmol, New England Nuclear Corp.). Old cells were labeled with 0.03 uCi/ml for l&2/ davs. Thev were refed with the same concentration oi labeleh thvmidine in fresh MEM on the 4th and 8th davs. Ybung cells were labeled with 0.2 &i/ml for 24-46 h prior to a fusion experiment. Control studies (unpublished) showed that these concentrations of radioisotope administered for the stated times were not toxic to the cells in terms of plating efficiency or population doubling times. Erp Cd
Res 129 I 19801
At the end of the final incubation period, the cells were rinsed twice with CMF-PBS, fixed with methanol and air-dried. The finished slides were dipped in fresh emulsion (Kodak NTB-2) which had been diluted I : I with 0.05 % sodium lauryl sulphate. The backs of the slides were wioed free of emulsion and thev were allowed to dry ior at least 2 h before they were-stored in light-tight boxes for 54 days at 4°C. The slides were developed over crushed ice in Kodak D-19 developer for 2 min. rinsed for 30 set in distilled water and fixed for 7 min in Kodak Rapid Fixer. After a I5 min rinse in running tap water, they were stained for 4 min with Giemsa stock diluted 1: 25 with distilled water. rinsed. dehydrated in isopropanol for 2 min and cleared in fwo changes of xylene.
0
18
3 Hoursafter release
Fig.
I.
release
from
21
synchrony
Frequency distribution of cells in S phase after from an isoleucine-deficiency induced block.
Analysis of slides The slides were scanned for heterokaryons containing a nucleus with a heavy deposit of silver grains over its whole surface (resulting from [“Hlthymidine incorporation) and a second nucleus in which silver grains (due to [3H]uridine uptake) were displayed in a typical fashion over and around its nucleoli, while the nucleoplasm was relatively grain-free. Nucleolar RNA synthesis in these cells was compared with that in control cultures in order to verify that the fusion event did not cause an effect. The following criteria were rigorously observed: (I) All slides were examined ‘blind’; (2) heterokaryons were chosen from fields showing maximum [“Hluridine incorporation into unfused cells: (3) to be considered as a heterokaryon. a cell had to have a clear outline containing two distinguishable nuclei; (4) if the cell membrane was at all indistinct, the nuclei had to be in juxtaposition with the same axial orientation: (5) ‘edging grains’ were counted only if the interior nucleolar region was clearly labeled, or if the grains made a clear outline of a nucleolus.
greater than those which characterized old cultures. This is not unexpected. By definition, senescent cultures contain a very small fraction of actively dividing cells. Thus the increased nucleolar RNA synthesis which is apparent in young cultures as they escape from Cl and enter S phase [ 17. 181 is absent from the old cultures as a whole. Cells of like ages were fused and [3H]uridine incorporation into the nuclei which had been blocked in Cl was scored. Fig. 3a. b summarizes uridine incorporation by these monochronic heterokaryons. While the mean values for young and old monochronic heterokaryons are significantly different from one another. they are similar to those of their single, unfused counterparts (fig. ?I. The amount of r3H]uridine incorporation into the young and old nucleoli of dichronic heterokaryons is presented in fig. 40, b. It is evident that the mean number of silver grains over the nucleoli of the young nucleus is considerably lower than is depicted in figs 2n and 3~1. Rather, it approaches the value associated
35 30 25 20
RESULTS Fig. 1 is a frequency distribution of the per cent of young and old cells in S phase after release from isoleucine deficiencyinduced synchrony. A maximum number of cells of both ages (70 9%of young cells, 20 CT of old cells) were in S phase 18 h after release. The frequency distribution of silver grains localized over the nucleoli of unfused ~28 and ~45 cells during this period is presented in fig. Za, 6. It is apparent that both the mean and the range of the number of silver grains from the young population are
7) '5 0" s
10
-m
5
5 0
z
30 25 20 15 10 5
Naofsilvergrains Fig.
2. Frequency distribution of silver grains over the nucleoli of (a) young; (b) old unfused cells, labeled with [3H]uridine.
EXP CellRes 129f/980)
a
I
o
z
4
6
8
10
Fig. 3. Frequency distribution of silver grains over the nucleoli of (a) young and (6) old monochronic heterokaryons labeled with [“Hluridine IS h after fusion.
with the grain counts over the nucleoli of old cells (fig. 2h). The grain counts associated with the old nucleoli of both types of heterokaryons were virtually the same. Fusion with young cells did not increase their ability to synthesize nucleolar RNA. On the contrary, it appears that the presence of regulatory factors associated with senescent cells can, at least temporarily. repress rDNA template activity in a young cell. These results are tabulated in table I.
0
2
4
No. of silver
6
8
10
grains
Fig. 4. Frequency distribution of silvrr grains over the nucleoli of (n) young; (b) old nuclei in dichronic heterokaryons.
DISCUSSION Our observation of decreased rRNA synthesis in aging WI38 cells is consistent with the findings of Macieira-Coelho et al. [8], Ryan & Cristofalo [9] and of Bowman et al. [I I]. We have initiated an investigation of the regulation governing this phenomenon by fusing young and old cells and looking at nucleolar RNA synthesis in the respective nuclei of the heterokaryon. Since 70% of
the pre-senescent cells were in S phase at the time of the experiment, it is believed that the differences in nucleolar RNA synthesis reported herein are due not merely to random position in the cell cycle. but must be attributed to the inhibitory influence of the senescent phenotype. Preliminary experiments in which old cells were treated with actinomycin D or cycloheximide prior
Nucleolar RNA s_vnthesis in heterokaryons Table 1. The autoradiographic determination of rDNA template activity in the nucleoli of heterokayons 18-21 h after fusion Population labeled with r3H]uridine
Mean no. of silver grains +S.E.M.”
Population labeled ?
Unfused ~27 Unfused p42
6.250.24 3.0&O. 14
93 58
Monochronic heterokaryons p27 p42
6.61tO.286 2.2rtO.28
93 36
Dichronic heterokaryons p27 p41
2.4to. I2b 2.6kO.27
25 40
fusion have yielded no clues as to the nature of the inhibitory factor(s). The mechanism of age-related regulation of template activity is unknown. Regulatory molecules can interact with RNA polymerases or on the level of chromatin itself. Several studies indicate that the activities of RNA polymerases do not change with increasing age [ 19, 201. Chromatin, however, does exhibit temporal changes. It has been demonstrated that the F 1 histone subfraction in rat liver and spleen chromatin is more tightly bound to DNA in aging rats [2 11, than in young ones concomitant with a decline in acetylation [9, 221. A specific decline in rDNA transcription may be due to the inability of RNA polymerase I to interact with the chromatin template. It is known that regulatory molecules pass freely between the nuclei of a heterokaryon [23]; perhaps senescent regulatory factors influence the accessibility of the DNA template by influencing histone binding. Since an obligatory increase in rRNA synthesis precedes S phase in quiescent
Printed in Sweden
cells which are stimulated to divide [13], it is suggested that an initial impairment of an aging cell’s ability to express its rDNA template cannot be ruled out as a causative factor in the loss of proliferative capacity. Future experiments in which nucleolar RNA synthesis is scored at intervals beginning immediately after fusion may illuminate this question.
REFERENCES
U Corrected background. h p
to
301
I. Hayflick. L. Exp cell res 37 (1965) 614. 1. Martin. G, Sprague, C & Epstein. C. Lab invest 23
(1970) 86. 3. Hayflick, I, The handbook of the biology of aging (ed C Finch & L Hayflick) chap. 7. Van Nostrand Reinhold. New York (1976). 4. Cristofalo, V, Adv gerontol res 4 (1972) 45. 5. Goldstein, S & Lin. C. Exo cell res 70 (1972) 436. 6. Norwood, T. Pendergrass: W & Martin. G,‘J cell biol 64 (1975) 55 I. 7. Norwood. T, Pendergrass. W. Sprague. C & Martin. G. Proc natl acad xi US 7 1 (1974) 223 I. 8. Macieira-Coelho, .4. Ponten, J & Philipson, L, Exp cell res 42 (I%61 673. 9. Ryan. J & Cristofalo, V, Exp cell res 90 (1975)456. 10. Castle, T. Katz, A & Richardson, A. Mech aging dev 8 (1975) 383. I I. Bowman. P. Meek. R & Daniel, C, Exp cell res 101 (1975) 434. I?. Busch, H & Smetana, K, The nucleolus. Academic Press, New York (1970). 13. Baserga, R. Huang. C, Rossini, M, Chang. H & Ming. P, Cancer res 36 (1976). 14. Wellaur. P & David. T. Proc natl acad sci US 70 (1975) 2827. 15. Tobev, R, Methods in cell bioloav (ed D Prescott) p. 67:Academic Press, New Yo;k (1973). 16. Cristofalo. V & Scharf. B. Exe cell res 76 (1973) 419. 17. Parmeley, R, Dow. L & Mauer, A. Cancer res 37 (1977)4313. 18. Nicolini. G. Kendall, F. Baserga, R. Dessaive, C. Clarkson. B & Fried. J, Exp cell res 106 (1977) III. 19. Gibas. M & Harman. A, J gerontol 25 (1970) 105. 20. Harker, C & Benzon, R, Fed proc 36 (1977) 294. 21. Medvedev, Z. Medvedev, M & Huschtscha. L. Gerontology 23 (1977) 334. 22. Petricevic. C, Denko, C & Messined. L. Mech agingdev 8 (1978) 231. 23. Gordon, S &Cohn. Z. Exp med 133 (1971) 321. Received December 27, 1978 Revised version received April 15, 1980 Accepted April 18, 1980
Experimental
NUCLEOLAR
Cell Research 129 (1980) 297-301
RNA SYNTHESIS
DERIVED
IN HETEROKARYONS
FROM SENESCENT
PRE-SENESCENT
HUMAN
AND
FIBROBLASTS
JOAN RENER’ and ROLAND M. NARDONE Deportment
ofBio/og_v. The Cntholic Uni~vrsi~y qfAmerictr.
W’uvhington. D.C. 20064.
crnd ‘Lnborator~ of Pnrnsitic Disensas. Notioncrl Institute oj’illlergy md lqfecriotts Disetrses. Notiontrl Institutes oj’Hea/th.
Bethesdn,
MD -70014. USA
SUMMARk Normal human fibroblasts, serially passaged in vitro. demonstrated decreasing synthesis of ribosomal RNA (rRNA). Senescent and pre-senescent WI38 cells were fused with one another in order to study age-related factors affecting the production of nucleolar RNA. Autoradiograms revealed that the young nucleus of a dichronic heterokaryon (2 nuclei of different ages) had an impaired ability to produce nucleolar RNA, while the young nucleus of a monochronic heterokaryon (2 nuclei of the same age) was not affected. Old nuclei of dichronic heterokaryons, and old monochronic heterokaryons displayed the same nucleolar RNA synthesis rate as did their single, unfused counterparts
Normal human diploid fibroblasts, grown in vitro, have finite replicative lifespans [ 1, 21. While the seminal events which cause in vitro aging are not agreed upon at this time, metabolic and cellular parameters which change as fibroblast populations lose their replicative capacity have been extensively characterized [3, 41. It has been demonstrated that DNA synthesis [4], and RNA synthetic activity [4, 8, 9, lo] decreases as diploid fibroblasts age. Bowman et al. [ 1l] demonstrated that the decline in RNA synthesis is due mainly to decreased synthesis of nucleolar RNA, nucleoplasmic synthesis being relatively unchanged. Elucidation of the mechanism of template shutdown remains to be resolved. This study was undertaken to determine whether or not a diffusible repressor of ribosomal DNA (rDNA) is associated with the nuclei of late passage (old) cells and whether or not the repressed 20-801806
rDNA template of old nuclei could be derepressed by factors present in nuclei of early passage (young) cells. Cell hybridization has been a useful tool in probing the factors responsible for the shut down of template activity in old cells. Initial experiments by Goldstein & Lin [5], followed by extensive work by Norwood et al. [6, 71, indicated that DNA replication in old fibroblasts can be derepressed after fusion with transformed cells, but that the senescent phenotype is dominant in a heterokaryon formed by fusion with a presenescent cell. Consideration is given here to the effects of fusion between cells of different ages upon the transcription of rDNA. Because it is localized within the nucleolus and codes specifically for a single 45 S precursor molecule of rRNA [ 12, 131. rDNA is uniquely suited to an autoradiographic study of regulation of transcription in a Exp Cc/l Rrs 129 11980~
defined
segment
of the human
In these experiments. thesis was examined
genome
nucleolar RNA synin both single. unfused
WI38 cells and in heterokaryons from cells of different
MATERIALS Cells and culture
[ 161.
produced
ages.
AND
METHODS
conditions
Human diploid fibroblasts (W138~. at passage 15. were obtained from the American Type Culture Collection. The cells were grown in 75 or 150 cm* plastic tissue culture flasks (Corning). They were fed with Eagle’s Minimum Essential Medium- (MEM) supplemeoted with 10% fetal bovine serum (FBS). 2 mM L-glutamine. 100 U/ml penicillin and 100 pg/ml streptomycin and buffered at pH 7.3 with NaHCO,. The cells were incubated at 37°C; a SC; C0,-95qii air mixture served as the gas phase. Confluent cultures were subcultured every 3 or 4 days by a I : ? split. A 0.25% solution of trypsin (activity I : 250) in calcium and magnesium-free ohosphate-buffered saline (CMF-PBS) was used to free ;he cklls for harvesting. Culture age was verified by the method of Cristofalo & Scharf[ 161. Senescent populations were defined as those which had completed X0% of their lifespans (passage 40 or above). Pre-senescent populations were defined as those which had completeh (45% of their lifespans and had been passaged less than 28 times. To maintain an adequate supply of old cells, confluent cultures p40 or above. were held for up to one month in I50 cm* flasks with intermittent feeding every 7 days. To insure an adequate stock of young cells. near confluent cultures in 75 cm? flasks were harvested between ~18 and ~22. The cells were transferred to amooules containing I ml MEM. IO% DMSO and ISc’c FB’S. They were left at 4°C for 4 h to overnight and then frozen and stored in liquid nitrogen. Throughout the studies cells and spent media were checked for the presence of mycoplasma. Aliquots were tested with mycoplasma enrichment media (BioQuest) under aerobic and anaerobic conditions. All cultures were negative for mycoplasma.
In a reprebentative esperimrnr. ~27 .mu p-12 cellr iterr grown in 75 and I50 cm’ flask\. re3pectivel>. md were prelabeled as debcribed above. Four d,l!\ hrfor-e fusion. 5x IO young cells/ml or I * IO’ old ~rll\/ml were nlated into Leighton tube3 containing fl\ine glass coverslips. Tw&tv-four hour3 after -pla;inf. these cells were synchron;ed for3648 h bv iholeucine deprivation and dlocked in Cl [IS]. RN.4 iynthesis i, continuous but not uniform in rate during the cell cycle: hence synchronization zerved to reduce normal variations. The cells were fed with complete medium. Fusion was accomplished after I8 h h) a modification of the technique described by Norwood et ai. [7]. Prelabeled cells were rinsed twice the CMF-PBS. harvested. pelleted and resuspended in I ml cold CMFPBS. Tube5 containing the cell5 uerc placed on crushed ice while cell count5 were ohtained with a Coulter Counter Model B. During thi\ period the >ynchronized cells were also placed on cl-ushed ice. These Here then refed with 0.3-0.6 ml of serum-free MEM containing IO00 HAU of inactivated Sendai vim\ (Connaught Labs). These tubej remained on ice for- an additional I5 min. ~-IX IOF pre-labeled young cells. 01 6x IO5 pre-labeled old cells suspended in 0.1 ml serumfree MEM were added to each Leighton tube. After 15 additional minutes on ice. the tubes were incubated for 90 min at 37°C. The virus was removed by gentlq rinsing the cultures twice with warm MEM containing serum. The heterokaryons were refed Lbith fresh medium and incubated for I8 h. .4t the beginning of the 18th h they were refed with MEM containing [“HIuridine (5 &i/ml; spec. act. 25 Cilmmol: Nem England Nuclear) and 2.5~ IOM deoxycytidine and 2.5~ IO-’ M thymidine. The cultures were incubated for 3 h to insure saturation labeling conditions. Young and old control cultures consisted of synchronized populations exposed to fusion conditions without cocultivation and ‘mock’ fusion in which no Sendai virus was present in the serum-free MEM. Monochronic were formed between pre-labeled heterokaryons young cells and synchronized cells of the same age. ot pre-labeled old cells and synchronized old cells. Dichronic heterokaryons were formed between prelabeled young cells and synchronized old ones or prelabeled old cells and synchronized young ones. Autoradiograms for each class were prepared in triplicate.
Autoradiograph> Identification
of parent
populations
In order to recognize rhe respective nuclei in heterokarvons. one of the parent populations used for fusion was heavily pre-labiled wit-h [jH]thymidine (spec. act. 28 Cilmmol, New England Nuclear Corp.). Old cells were labeled with 0.03 uCi/ml for l&2/ davs. Thev were refed with the same concentration oi labeleh thvmidine in fresh MEM on the 4th and 8th davs. Ybung cells were labeled with 0.2 &i/ml for 24-46 h prior to a fusion experiment. Control studies (unpublished) showed that these concentrations of radioisotope administered for the stated times were not toxic to the cells in terms of plating efficiency or population doubling times. Erp Cd
Res 129 I 19801
At the end of the final incubation period, the cells were rinsed twice with CMF-PBS, fixed with methanol and air-dried. The finished slides were dipped in fresh emulsion (Kodak NTB-2) which had been diluted I : I with 0.05 % sodium lauryl sulphate. The backs of the slides were wioed free of emulsion and thev were allowed to dry ior at least 2 h before they were-stored in light-tight boxes for 54 days at 4°C. The slides were developed over crushed ice in Kodak D-19 developer for 2 min. rinsed for 30 set in distilled water and fixed for 7 min in Kodak Rapid Fixer. After a I5 min rinse in running tap water, they were stained for 4 min with Giemsa stock diluted 1: 25 with distilled water. rinsed. dehydrated in isopropanol for 2 min and cleared in fwo changes of xylene.
0
18
3 Hoursafter release
Fig.
I.
release
from
21
synchrony
Frequency distribution of cells in S phase after from an isoleucine-deficiency induced block.
Analysis of slides The slides were scanned for heterokaryons containing a nucleus with a heavy deposit of silver grains over its whole surface (resulting from [“Hlthymidine incorporation) and a second nucleus in which silver grains (due to [3H]uridine uptake) were displayed in a typical fashion over and around its nucleoli, while the nucleoplasm was relatively grain-free. Nucleolar RNA synthesis in these cells was compared with that in control cultures in order to verify that the fusion event did not cause an effect. The following criteria were rigorously observed: (I) All slides were examined ‘blind’; (2) heterokaryons were chosen from fields showing maximum [“Hluridine incorporation into unfused cells: (3) to be considered as a heterokaryon. a cell had to have a clear outline containing two distinguishable nuclei; (4) if the cell membrane was at all indistinct, the nuclei had to be in juxtaposition with the same axial orientation: (5) ‘edging grains’ were counted only if the interior nucleolar region was clearly labeled, or if the grains made a clear outline of a nucleolus.
greater than those which characterized old cultures. This is not unexpected. By definition, senescent cultures contain a very small fraction of actively dividing cells. Thus the increased nucleolar RNA synthesis which is apparent in young cultures as they escape from Cl and enter S phase [ 17. 181 is absent from the old cultures as a whole. Cells of like ages were fused and [3H]uridine incorporation into the nuclei which had been blocked in Cl was scored. Fig. 3a. b summarizes uridine incorporation by these monochronic heterokaryons. While the mean values for young and old monochronic heterokaryons are significantly different from one another. they are similar to those of their single, unfused counterparts (fig. ?I. The amount of r3H]uridine incorporation into the young and old nucleoli of dichronic heterokaryons is presented in fig. 40, b. It is evident that the mean number of silver grains over the nucleoli of the young nucleus is considerably lower than is depicted in figs 2n and 3~1. Rather, it approaches the value associated
35 30 25 20
RESULTS Fig. 1 is a frequency distribution of the per cent of young and old cells in S phase after release from isoleucine deficiencyinduced synchrony. A maximum number of cells of both ages (70 9%of young cells, 20 CT of old cells) were in S phase 18 h after release. The frequency distribution of silver grains localized over the nucleoli of unfused ~28 and ~45 cells during this period is presented in fig. Za, 6. It is apparent that both the mean and the range of the number of silver grains from the young population are
7) '5 0" s
10
-m
5
5 0
z
30 25 20 15 10 5
Naofsilvergrains Fig.
2. Frequency distribution of silver grains over the nucleoli of (a) young; (b) old unfused cells, labeled with [3H]uridine.
EXP CellRes 129f/980)
a
I
o
z
4
6
8
10
Fig. 3. Frequency distribution of silver grains over the nucleoli of (a) young and (6) old monochronic heterokaryons labeled with [“Hluridine IS h after fusion.
with the grain counts over the nucleoli of old cells (fig. 2h). The grain counts associated with the old nucleoli of both types of heterokaryons were virtually the same. Fusion with young cells did not increase their ability to synthesize nucleolar RNA. On the contrary, it appears that the presence of regulatory factors associated with senescent cells can, at least temporarily. repress rDNA template activity in a young cell. These results are tabulated in table I.
0
2
4
No. of silver
6
8
10
grains
Fig. 4. Frequency distribution of silvrr grains over the nucleoli of (n) young; (b) old nuclei in dichronic heterokaryons.
DISCUSSION Our observation of decreased rRNA synthesis in aging WI38 cells is consistent with the findings of Macieira-Coelho et al. [8], Ryan & Cristofalo [9] and of Bowman et al. [I I]. We have initiated an investigation of the regulation governing this phenomenon by fusing young and old cells and looking at nucleolar RNA synthesis in the respective nuclei of the heterokaryon. Since 70% of
the pre-senescent cells were in S phase at the time of the experiment, it is believed that the differences in nucleolar RNA synthesis reported herein are due not merely to random position in the cell cycle. but must be attributed to the inhibitory influence of the senescent phenotype. Preliminary experiments in which old cells were treated with actinomycin D or cycloheximide prior
Nucleolar RNA s_vnthesis in heterokaryons Table 1. The autoradiographic determination of rDNA template activity in the nucleoli of heterokayons 18-21 h after fusion Population labeled with r3H]uridine
Mean no. of silver grains +S.E.M.”
Population labeled ?
Unfused ~27 Unfused p42
6.250.24 3.0&O. 14
93 58
Monochronic heterokaryons p27 p42
6.61tO.286 2.2rtO.28
93 36
Dichronic heterokaryons p27 p41
2.4to. I2b 2.6kO.27
25 40
fusion have yielded no clues as to the nature of the inhibitory factor(s). The mechanism of age-related regulation of template activity is unknown. Regulatory molecules can interact with RNA polymerases or on the level of chromatin itself. Several studies indicate that the activities of RNA polymerases do not change with increasing age [ 19, 201. Chromatin, however, does exhibit temporal changes. It has been demonstrated that the F 1 histone subfraction in rat liver and spleen chromatin is more tightly bound to DNA in aging rats [2 11, than in young ones concomitant with a decline in acetylation [9, 221. A specific decline in rDNA transcription may be due to the inability of RNA polymerase I to interact with the chromatin template. It is known that regulatory molecules pass freely between the nuclei of a heterokaryon [23]; perhaps senescent regulatory factors influence the accessibility of the DNA template by influencing histone binding. Since an obligatory increase in rRNA synthesis precedes S phase in quiescent
Printed in Sweden
cells which are stimulated to divide [13], it is suggested that an initial impairment of an aging cell’s ability to express its rDNA template cannot be ruled out as a causative factor in the loss of proliferative capacity. Future experiments in which nucleolar RNA synthesis is scored at intervals beginning immediately after fusion may illuminate this question.
REFERENCES
U Corrected background. h p
to
301
I. Hayflick. L. Exp cell res 37 (1965) 614. 1. Martin. G, Sprague, C & Epstein. C. Lab invest 23
(1970) 86. 3. Hayflick, I, The handbook of the biology of aging (ed C Finch & L Hayflick) chap. 7. Van Nostrand Reinhold. New York (1976). 4. Cristofalo, V, Adv gerontol res 4 (1972) 45. 5. Goldstein, S & Lin. C. Exo cell res 70 (1972) 436. 6. Norwood, T. Pendergrass: W & Martin. G,‘J cell biol 64 (1975) 55 I. 7. Norwood. T, Pendergrass. W. Sprague. C & Martin. G. Proc natl acad xi US 7 1 (1974) 223 I. 8. Macieira-Coelho, .4. Ponten, J & Philipson, L, Exp cell res 42 (I%61 673. 9. Ryan. J & Cristofalo, V, Exp cell res 90 (1975)456. 10. Castle, T. Katz, A & Richardson, A. Mech aging dev 8 (1975) 383. I I. Bowman. P. Meek. R & Daniel, C, Exp cell res 101 (1975) 434. I?. Busch, H & Smetana, K, The nucleolus. Academic Press, New York (1970). 13. Baserga, R. Huang. C, Rossini, M, Chang. H & Ming. P, Cancer res 36 (1976). 14. Wellaur. P & David. T. Proc natl acad sci US 70 (1975) 2827. 15. Tobev, R, Methods in cell bioloav (ed D Prescott) p. 67:Academic Press, New Yo;k (1973). 16. Cristofalo. V & Scharf. B. Exe cell res 76 (1973) 419. 17. Parmeley, R, Dow. L & Mauer, A. Cancer res 37 (1977)4313. 18. Nicolini. G. Kendall, F. Baserga, R. Dessaive, C. Clarkson. B & Fried. J, Exp cell res 106 (1977) III. 19. Gibas. M & Harman. A, J gerontol 25 (1970) 105. 20. Harker, C & Benzon, R, Fed proc 36 (1977) 294. 21. Medvedev, Z. Medvedev, M & Huschtscha. L. Gerontology 23 (1977) 334. 22. Petricevic. C, Denko, C & Messined. L. Mech agingdev 8 (1978) 231. 23. Gordon, S &Cohn. Z. Exp med 133 (1971) 321. Received December 27, 1978 Revised version received April 15, 1980 Accepted April 18, 1980