Chromatin structure, DNA synthesis and transcription through the lifespan of human embryonic lung fibroblasts

Experimental

Cell Research

151 (1984) 283-298

Chromatin Structure, DNA Synthesis and Transcription through the Lifespan of Human Embryonic Lung Fibroblasts FRANCINE

PUVION-DUTILLEUL,’ EDMOND PUVION,’ CHRISTINE ICARD-LIEPKALNS2 and ALVARO MACIEIRA-COELH02

‘Znstitut de Recherches Scienti$ques SW le Cancer, CNRS, 94802 Villejuif Cedex, and 2Znstitut de CancCrologie et d’Zmmunogt!nCtique, ZNSERM, 94804 Viifejuif Cedex, France

The presence in terminal embryonic tibroblasts of small molecular weight (MW) DNA independent of bulk DNA could be ascertained by three different techniques performed in parallel. This alteration was not artifactualiy induced, either by high pH and the detergent used or by the release of cellular enzymes. An increased thermolability of old chromatin was also observed. Cells with altered chromatin synthesized DNA and RNA according to a pattern similar to young type nuclei. Long-term treatment with hydrocortisone significantly increased the cell yield but did not prevent, in the late passages, the occurrence of oldtype chromatin; the nucleolar filamentous masses, however, maintained a ‘young’ pattern. Short-term treatment induced only a moderate reversion in the appearance of chromatin lesions. Direct evidence was obtained of increased gene expression in the presence of hydrocortisone.

Several studies have confirmed that there is a limit to the proliferative potential of human fibroblasts, corresponding to 50+ 10 population doublings (PD) for cells derived from embryonic lung [I]. Extensions of this limit can be obtained by modifying the nutrient medium. Maintenance of cells in an arrested state for instance can increase their growth potential [2, 31. On the other hand, certain batches of serum [4] and some steroid hormones [5,6] can also extend the limit of PD; the effect, however, delays rather than prevents the ageing process. The treatment with steroid hormones is associated with increased RNA and protein synthesis [7-l 11. During the terminal stages of the fibroblast population lifespan there is a dramatic fall in the proportion of cells synthesizing DNA per unit of time [12, 131, which is associated with two nuclear alterations at the ultrastructural level [14, 151, suggesting profound changes in RNA synthesis, as well as in chromatin organization. Even in the final stages there are cells with a division cycle similar to that of young cells [13, 161; the remaining population corresponds to cells with a low likelihood of entering DNA synthesis and some authors have suggested that they are terminally differentiated cells [17]. RNA synthesis activity has been a controversial subject, since some authors claim a decline [12, 18-211, others an increase [22], while still others claim no variation [23]. Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved aol4-4827184 $03.00

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Biochemical studies have certain limitations, since they analyse populations having considerable heterogeneity. Electron microscopy has the advantage of allowing the study of individual cells. In the present work we used biochemical as well as electron microscopy techniques to analyse chromosomal DNA and DNA and RNA synthesis. The effect of hydrocortisone, which is known to increase the growth potential of these cells, was also studied with regard to the changes induced in the same molecular structures. MATERIAL

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METHODS

Cell Culture Cells from the fetal lung flbroblast line ICIG-7 [ 131were grown in MEM medium supplemented with 10% fetal calf serum (FCS) and 16 &ml gentamycin. The cells were found to be free of Mycoplasma by the orcein [24] and uridine-uracyl ratio [25] methods. They were serially propagated and examined in resting phase from the 1lth to 56th population doubling level (PDL), which was the last doubling. The population lifespan is divided into four phases (I-IV) according to previously defined criteria [l , 261. Phase I corresponds to the first 4-5 doublings during which cells adapt to the in vitro conditions and is expressed by the elimination of cells with gross chromosome abnormalities and an increase in growth rate. Phase II is characterized by a stable fraction of cycling cells which is close to 100%. The transition to phase III is defined by the failure to respond to low inocula through an increase in the rate of entrance into the cell cycle; during phase III there is a slight decline in the number of cells which enter DNA synthesis during a 24-h period. Finally, phase IV is characterized by a dramatic fall in the maximum cell densities and in the number of cells entering DNA synthesis during a 24-h period, an unpredictability of the entrance into the cell cycle, a pronounced rise in the number of cells with chromatin changes [15] and marked metabolic events. Identical cultures were incubated in the same nutritive medium containing 5 ug/ml of hydrocortisone-21.phosphate either during one doubling prior to fixation or from the 19th PD on to the end of the culture.

Electron Microscopic

Techniques

Triplicate cultures were taken for conventional electron microscopy, loosening’s procedure and Miller’s technique. Cultures were fixed conventionally with 1.6 % glutaraldehyde in 0.1 M Sorensen phosphate buffer, pH 7.3, dehydrated in ethanol and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined on a Siemens Elmiskop 101 electron microscope. For the loosening procedure, cultures were covered with an aqueous solution of 1% formaldehyde containing 0.1 M sucrose and 0.4% Photo flo (Kodak-Patht, Chalon, France), pH 8.5-9.0, as previous described [14]. Cells were scraped from the plastic substratum and embedded as described above. In some cases, additional cultures at high PDL (52nd) were treated with a loosening solution adjusted to pH 7.0-7.3 or with a detergent-free loosening medium, pH 8.5-9.0. In addition, to find out whether the nuclear changes that occurred were the effect of the release of cellular enzymes during loosening, we have mixed lysed Werner’s syndrome cells (purchased from the Human Mutant Cell Repository, Camden, N.J.) which are known to have profound chromatin changes [27], with ICIG-7 monolayer cells with young type chromatin. For this experiment, Werner’s cells were first suspended in the complete loosening medium and then added to ICIG-7 monolayer cells. The mixed populations were examined in order to detect possible nuclear alterations in ICIG-7 cells. Monolayer cultures were submitted to Miller’s spreading technique [28] according to our usual experimental conditions [29]. The grids were rotary shadow-cast with platinum.

High-resolution Autoradiography RNA synthesis. Confluent phases II and IV monolayers were incubated for 5 min in 2 ml culture medium containing 100 @X/ml [5-3H]uridine (SA 27 Ci/mmol, CEA). They were rinsed for 1 min in a Exp Cell

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culture medium containing 0.1% non-radioactive uridine and exposed either to glutaraldehyde tixative or loosening medium. Some cultures were maintained in a non-radioactive medium for 10 min, 30 min, 1 or 2 h. DNA synthesis. Confluent cells in phases II, III and IV were stimulated with a medium change and 24 h later were incubated for 10 min in the presence of 100 @i/ml [methy13H]thymidine (SA 51 Ci/mmol). One group of terminal cultures was incubated after stimulation during 10 and 66 h in the presence of 100 @i/ml tritiated thymidine (r3H]TdR) (SA 5 Ci/mmol). In addition, some cultures were maintained after a 10 min labelling in a non-radioactive medium for 10 h. After the pulse and chase treatments, cultures were exposed either to glutaraldehyde solution or to loosening medium. After fixation, cells were embedded in Epon as described above. Ilford L4 emulsion was applied on Formvar-coated copper grids by the loop technique. Four months later, the autoradiographs were developed with the gold-latensification technique [30]. The grids were stained with uranyl acetate alone (glutaraldehyde-fixed material) or followed by lead citrate (loosened material). To determine the percentage of labelled interphases by optical microscopy 48 h after a 1 : 2 split, 0.1 @i/ml [3H]thymidine (SA 2 Ci/mM) was added to the nutrient medium and duplicate cultures were fixed and processed for autoradiography, as described previously [12]. Three thousand cells were counted on each sample.

Cytophotometry Feulgen-pararosaniline (SO,) staining was done by the method of Mares and Van Der Ploeg [31]. Absorbance measurements were made using a stage scanner and the HID ACSY3 computer-assisted program for scanning cytophotometry [32]; sample and line separation were 0.25 pm.

Sucrose Gradient Centrifugations Cells were grown on 13-mm diameter plastic coverslips until they became confluent. When no mitosis could be seen the supematant was replaced by fresh medium supplemented with 20% FCS. Eight hours later, 0.5 @i/ml [14C]TdR was added for 16 h and then the supematant was again replaced with nutrient medium without serum, supplemented this time with 10 Kg/ml of unlabelled thymidine. Half an hour later, the coverslips were layered on top of a lysing solution (0.4 ml) (0.5 M NaCI, lo-* M EDTA, 0.45 M NaOH, pH 12.0) containing 0.1% lauryl sulfate, which was layered on 11 ml of a 5-20% alkaline sucrose gradient (0.9 M NaCI, 0.3~ lo-’ M EDTA, 0.2-0.4 M NaOH, pH 12.0). Lysis was done during 4 h in the dark. When ultrastructural analysis with the loosening procedure was to be compared with the results obtained with sucrose gradient centrifugation, the cells grown on the coverslips were treated exactly as described above for the loosening procedure, scraped and layed directly on the top of the gradient. The gradients were centrifuged for 2 h at 40000 rpm in an SW41 Beckman rotor at 12°C. Lambda phage (~40s) was used as a reference and centrifuged simultaneously with the cell samples. Sedimentation coefficients were calculated from the formula [33] &a, w=/ID/(rpm)zxt. The molecular weights were calculated according to the method of Studier [34] from the formula SzO,[email protected].

RESULTS Chromatin Changes during In Vitro Aging Analysis of chromatin. Chromatin from cells in phases II, III and IV was compared by three different methodologies. Cells treated for mild decondensation, as described under Material and Methods, were either centrifuged in alkaline-sucrose gradients or further processed to be examined by electron microscopy. Simultaneously cells lysed above the sucrose gradient by conventional methods were also centrifuged in parallel. Identical cultures were also prepared for Miller’s spreads. Fig. 1 a illustrates one of the experiments performed with cells in phase III. Exp Cell

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The radioactivity found in the different fractions collected from both gradients formed a single peak. The chromosomal DNA prepared by the two methods had the same sedimentation velocity. Mild decondensation of identical cultures showed (fig. 1 b) that chromatin threads were homogeneously distributed within the extra-nucleolar area and oriented perpendicularly to the lamina. The spacing of the threads was always lower than 0.1 urn. Miller’s spreads of identical cultures (fig. 1 c) showed that most of the DNA fibres displayed a nucleosomal organization (about 30 nucleosomes per urn of DNA fibre). The same results were found in several experiments performed with cells at different PDL during phase II and III. With phase IV cells, however, the radioactivity collected from the gradients formed two peaks with different MW (fig. 1 d). The curves corresponding to chromosomal DNA prepared by the two different methods overlapped, the smaller MW DNA had the same sedimentation velocity as 1 phage. Identical results were obtained in four separate experiments. At the ultrastructural level after mild decondensation, the chromatin threads were widely spaced, especially at the nuclear periphery, where the threads were shorter and infrequent along the lamina, which was sometimes devoid of chromatin threads (fig. I e). With Miller’s spreading technique (fig. lj), most of the DNA fibres were punctuated by highly spaced nucleosomes (less than six particles per urn of DNA fibre) or were entirely extended. Effects of experimental conditions on chromatin changes. The chromatin changes observed after the decondensation procedure, usually performed at I. Phase III (U-C) and phase IV (d-f) fibroblasts prepared in parallel for electron microscopy and sucrose gradient centrifugation. (n, d) Sucrose gradient centrifugation; (b, e) loosening procedure; (c, fi Miller’s spreads. The curves represent the radioactivity found in the different fractions collected from the gradients. -, Cells treated the same way as for loosening procedure: ---, cells prepared by conventional methods. Vertical dashed line indicates the fraction where A-phage was found. For further description, see text. Fig.

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5 Fig. 3. Distribution of DNA content in arbitrary units found on anaphases of fibroblasts at different PDL. The mean DNA content with the 95 % confidence limits is indicated. f=80+3.20: (a) 54th PD; (b) 45th PD; (c) t=83+3.32,31st PD. AMOUNTOF

DNA

alkaline pH, were also found when the pH was adjusted to 7.0-7.3. Indeed, at neutral pH, typical ‘old’ type nuclei were observed at high PDL of ICIG-7 cells (not shown). On the other hand, when Photo flo was omitted during the preparation of the loosening medium, old-type chromatin-as well as nuclear peripheral areas free of chromatin-were also seen (not shown), although the nuclear swelling and the nucleolar disruption did not occur. These data demonstrated that old-type nuclei were not artificially induced by alkaline pH or the detergent during loosening. In addition, when Werner’s syndrome cells suspended in the complete loosening medium were mixed with ICIG-7 cells at low PDL, no rarefaction of the chromatin threads was induced in ICIG-7 cells. The latter were identified by their regular shape, whereas Werner’s syndrome nuclei were multilobed, as previously shown [27]. These data indicated that released enzymes were not responsible for the nuclear changes observed in old cultures. In previous experiments [35] we have found a higher frequency of small molecules and a wider range of MW after centrifugation of DNA from phase IV cells. We have now found that this occurs when cells are lysed above 3O”C, indicating increased thermolability of phase IV chromatin (fig. 2). Fig. 2a shows an experiment where phase III cells were lysed at room temperature and at 37°C. At both temperatures, the radioactivity of the different fractions formed single peaks, although the peak obtained from the cells lysed at 37°C had a smaller MW. Identical results were obtained in six different experiments performed with phases II and III cells. With phase IV cells, however (fig. 2b) DNA from cells lysed at 37°C was polydispersed; DNA from cells lysed at room temperature sedimented with the same pattern as in the experiment shown in fig. 1 d, i.e., one main peak with high MW and another peak with the same sedimentation velocity as A-phage. Identical data were obtained in four separate experiments. DNA Content

To ascertain that DNA was not lost during mild decondensation, and since it is difftcult to make DNA measurements after treatment with fixative containing Fig. 4. Autoradiograms of phase IV fibroblasts incubated with [3H]TdR for (a) 10 min; (b) 66 h prior to the loosening procedure. Exp Cell

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1. Frequency of the ICIG-7

et al. of altered nuclei at different cells revealed by the loosening

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Frequency

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Exceptional (< 1%) Rare (-5 %) Frequent (-98 %)

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aldehyde groups, cells were labelled with [i4C]thymidine during 15 h and then processed as described above for conventional fixation or mild decondensation; before reaching the inclusion step they were washed with cold 5 % perchloric acid (PCA) and the precipitable radioactivity was measured in a liquid scintillation counter. The same radioactivity was found in both groups. To ascertain if age-dependent changes in DNA content might be responsible for the sparse chromatin fibres after mild decondensation, cytophotometric analysis was done in cells at different PDL. When one measures the DNA above interphases one cannot be sure of the real spread of Gl values, because some of the cells included in the Gl peak are already engaged in DNA synthesis. Hence we measured the DNA contents above each half of anaphases and telophases when one is sure of finding the true Gl value. The results show (fig. 3) that there was no difference even during the final stages (phase IV), either in the spread or the mean of DNA content. DNA Synthesis

Incorporation of [methyl-3H]thymidine in nuclei of ICIG-7 cells at different PDL displayed the same patterns. In conventionally fixed young and old cultures, two types of localization of silver grains occurred, with an increased number of grains after longer labelling periods. Silver grains were in the main either scattered within the non-nucleolar area or were located at the nuclear periphery over the dense chromatin (not shown); this must correspond to cells at different time points in the S period. Curiously, these two patterns of the distribution of the silver grains were not modified whatever the duration of the labelling of the tritiated precursor and the presence or absence of a long chase period; they were also found after the loosening procedure. With the latter technique the peripheral labelling was not located at the level of the lamina densa, but rather at a short distance (fig. 4a). After a 10 min pulse, 31% of the nuclei were labelled in young cultures and after a 10-h chase, 47%. In phase IV cultures, the number of labelled nuclei after a lo-min pulse (23 %) was not much modified as compared with low PDL cells; after a 10-h chase it was 36 %. It is interesting to observe that old-type nuclei with both altered chromatin threads and nucleolar filamentous masses were able to incorporate [3H]TdR, Exp

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Growth curve of O-O, control; W-0, hydrocortisonetreated cells; (6) percent labelled interphases found after autoradiography (optical microscopy) in cells grown in the presence of [3H]TdR. -, Control; ---, hydrocortisone. Bars represent the extreme values between samples. Fig. 5. (a)

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although often at a lower level, during short-term incubation (fig. 4a). The labelling was markedly increased after long-term incorporation (fig. 4 b). RNA Synthesis In addition to the disappearance of dense chromatin, nuclear changes occurring during the in vitro lifespan of ICIG-7 cells also included a decrease in the amount of non-nucleolar RNP fibrils and nucleolar hypertrophy, as revealed in ultrathin sections of glutaraldehyde-fixed material (not shown). Following the loosening procedure, this nucleolar modification was related to the unusually coarse appearance of the nucleolar filamentous masses resulting from the dissociation of the fibrillar component (fig. 6b). The time of appearance of the age-related morphological changes is shown in table 1. Following a short pulse with [5-3H]uridine, the distribution of silver grains in the nucleolar and extranucleolar zones of the nuclei revealed no significant qualitative differences between cells with young-type and old-type chromatin, although about half of the old-type nuclei were unlabelled or exhibited only a few silver grains (not shown). These results obtained after both conventional fixation and the loosening procedure suggested that, although nucleolar and non-nucleolar RNA synthesis persists in old cultures, there might be a decrease. Over extranucleolar and nucleolar areas, the density of silver grains in old-type nuclei was one-third or one-fourth that in young-type nuclei for the same exposition time of the autoradiograms (1.5f0.6 grains/urn* instead of 6.7k3.1 for extranucleolar areas and 4.2k2.1 grains instead of 14.2k3.6 grains/urn2 of nucleolar areas). When high resolution autoradiography was carried out after different periods of chase, at both low and high PDL, the label was localized and migrated in the usual way. Exp Cell Res 151 (1984)

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Effects of Hydrocortisone As shown before with other human fibroblast populations [6,7, 361, hydrocortisone stimulated the proliferation of the cells used in the present study. Cell counts performed each day after subcultivation in cultures grown with or without hormone (fig. 5 a) showed that the latter significantly increased the maximum cell density. This effect seems to be due in part to an increased fraction of cells entering DNA synthesis per unit of time, as revealed by a higher percentage of labelled cells after autoradiography (fig. 5 b). One series of cultures was carried from the 19 PD continuously in the presence of the hormone. The cells carried with hydrocortisone lasted seven doublings more than the controls. The effect of the hormone on this cell population was more marked when it was measured in terms of total number of cells produced. This was calculated in each group, adding the differences at each doubling between the number of cells at resting stage and the number of cells inoculated. In the group without the hormone, the figure was 2.87~ lo7 cells and in the group carried in the presence of hydrocortisone, 5.10~ lo7 cells. The effect of the hormone at the ultrastructural level was analysed. As shown in table 1, the hormone delayed the appearance of phase IV, since the pronounced increase in the percentage of nuclei with old-type chromatin which characterizes this phase [14, 151 appeared seven doublings later, in comparison with the controls. The last PD resembled that of the terminal cultures grown in a hydrocortisone-free medium (table 1); however, a few particularities were observed. The nucleolar tibrillar masses revealed by the loosening procedure were not altered (fig. 6 a) as in untreated cultures (fig. 6 b); they consisted of numerous thin and entangled RNP fibrils similar to those of ‘young’ type nuclei. Miller’s technique (fig. 7) showed that nucleosomes were better preserved than in control cells (fig. lJ), since many beaded DNP fibres were present. In addition, nucleolar (fig. 7a) and non-nucleolar (fig. 7b) transcription units were more easily found and the former displayed a frequency of RNP tibrils higher than in controls (about 25 fibrils per urn of DNA axis instead of 10 fibrils). Addition of hydrocortisone during one doubling prior to conventional fixation, loosening, or spreading procedure did not revert the chromatin lesions significantly. However, after mild loosening of nucleolar structures, the filamentous masses sometimes resembled those of young cells. To see if the hormone would reduce the thermolability of chromatin, phase IV cells carried without and with the hormone were labelled during 6 h with [14C]TdR, lysed under heating conditions and centrifuged in alkaline sucrose gradients. The radioactivity from the non-treated cells formed several peaks, the highest having MW of 49x lo6 D (fig. 8a). The radioactivity collected from

Fig. 6. Phase IV fibroblasts (a) treated; (b) not treated with hydrocortisone prepared by the loosening procedure. Exp

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Fig. 7. Phase IV fibroblasts treated with hydrocorti Isone from the 19th PDL on, prepared with Miller’s technique. (a) Nucleolar; (b) non-nucleolar transcl ription units.

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Fig. 8. Radioactivity found in the different fractions collected from an alkaline-sucrose gradient prepared (a) phase IV cells; (b) phase IV ceils which had been submitted to long-term hydrocortisone treatment.

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cells was found in one main peak with a 136x IO6 D (fig.

DISCUSSION Previous works have suggested the presence of strand breaks in chromosomal DNA of ageing fibroblasts, but it was not clear if they were present in the cell or were due to alkali-labile sites [35, 371. Ultrastructural studies also revealed chromatin alterations but could not distinguish between real breaks or fragile sites due to the alkaline pH and the shearing forces during manipulation [141. The data presented here show that the chromatin of terminal cells has fragile sites which are thermolabile. Independently of this thermolability, small MW DNA separate from bulk DNA seems to be present in phase IV cells, as revealed by different techniques. Small MW DNA was also observed by other methods in old fibroblasts [381. We found it in alkaline sucrose gradients, irrespective of the detergent used (anionic or non-ionic) and at the ultrast.ructural level, regardless of pH value and of the use of a detergent. Liberation of nucleases during manipulation is not responsible for the fractionation of chromatin fibres, since mixing cells with young- and old-type chromatin did not produce chromatin alterations in the former. A fragility of chromatin from phase IV cells is also apparent from the nuclear swelling and the nucleolar disruption induced by the detergent. The data also showed that DNA loss did not take place during preparation of cells for ultrastructural analysis and that chromatin alterations are not accompanied by changes in mean DNA content. Exp Cell

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The results presented here show that DNA synthesis is preserved in human embryonic fibroblasts aged in vitro, despite profound changes in both chromatin and nucleolar organization. High resolution autoradiographical studies did not reveal large variations in the percentage of cells synthesizing DNA in old and young cultures, but indicated a reduced activity in old nuclei, since silver grains were more sparsely spaced than in young nuclei for the same exposition time of the autoradiograms. In pulse-chase experiments, the increased percentage of labelled cells in young and old cultures could be due to the division of labelled cells, or to the labelling of new cells through the release of [3H]TdR. Although old-type chromatin is always dispersed, as revealed by conventional fixation [15, 27, 391, the two distributions of labelling, peripheral and internal labelling, were still observed even after chase experiments. They could be related to the timing of DNA synthesis during the S period in relation to the degree of chromatin condensation [40]. In addition, the experimental dispersion of chromatin in young and old cells did not induce dispersion of the labelling. These data are in agreement with the presence of complexes of replication attached to the internal nuclear matrix and suggest preservation of their relative steric position during loosening. The persistence of the two types of labelling sites even after 10 h of chase is rather difftcult to explain if DNA is reeled through fixed sites of replication [41, 421. However, the incorporation of radioactive precursor incompletely eliminated during the washing step at the end of the 10 min pulse, or a reincorporation of [3H]TdR released by the cells during the chase step cannot be excluded. The sites of transcription were localized by the use of short pulse (5 min) labelling with tritiated uridine, whereas pulse-chase experiments allowed the study of RNA processing in young- and old-type nuclei. A net decreased density (one-third or one-quarter) of the number of silver grains was observed over both nucleolar and non-nucleolar areas of old-type nuclei, as compared with youngtype nuclei after a short pulse of tritiated uridine. Since all nuclei are increased in size, the reduced number of grains could be due to dilution. In another system, using mouse embryonic fibroblasts [20], long-term incubations (2 h) with the radioactive precursor revealed a net decrease in both nucleolar and non-nucleolar labelling in late-passage fibroblasts, whereas cytoplasmic labelling was unmodified. A decreased RNA synthesis was also found after analysis of autoradiographs by optical microscopy [12, 18, 191. Taken together, these results suggest that the increased RNA content [43] during ageing must be due either to changes in turnover [43-45] or to an accumulation of RNA in the cytoplasm in the absence of mitosis. Cortisone [5, 461 and hydrocortisone [6, 7, 9, 361 are known to stimulate the growth of human fibroblasts and to delay the ageing process, although cell populations differ in the response to the hormone. In the cell population used here, the hormone increased the rate of entrance into division, prolonged the population lifespan by seven doublings, and almost doubled the total number of Exp Cd

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cells produced due to an increase in maximal density. The total number of cells produced has been shown to be a more sensitive indicator of changes exerted on cellular ageing [473. The protractive effect of hydrocortisone on the fibroblast growth potential is accompanied by delayed increases in cell volume [9] and cell permeability [48] and a sustained effect on rRNA synthesis [7, 91, parameters which are known to evolve with cellular senescence. The data reported herein show that a short treatment of old cells with hydrocortisone could partially reverse the nucleolar lesions-but never the chromatin ones. Long-term treatment with the hormone delayed the appearance of the chromatin alterations characteristic of the terminal phase and rendered chromatin less thermolabile. Ultimately, however, the decondensed nuclei from hydrocortisone-treated cells presented the same chromatin changes as did the controls. The nucleolar masses, however, maintained the same structure as that observed in young cells; this was associated with a higher level of nucleolar RNA synthesis as revealed in Miller’s spreads by the easier detection of transcription units displaying contiguous RNP fibrils. This is direct evidence of an increased gene expression in the presence of hydrocortisone and could explain the sustained effect of hydrocortisone on rRNA synthesis previously reported [7, 91. We have shown that transcription units of high PDL cultures have an unusually low density of lateral RNP fibrils (about ten fibrils per pm of DNA fibre) [15]. rRNA synthesis seems to be one of the factors involved in the arrest of division during cell crowding and it seems reasonable to assume that the high saturation densities reached by hydrocortisone-treated cells is due to the action of the hormone at that level. On the other hand, the protective effect on chromatin could be due to the action on histone synthesis and acetylation [49], which regulate the binding of histones to DNA. The hormone has, however, other effects on cell metabolism which may also contribute to improving the growth potential of human embryonic lung fibroblasts. The authors are indebted to Professor M. Van der Ploeg for all help with the cytophotometry measurements and to Mrs K. Boumoutian and Mrs J. Pedron for their efficient technical assistance. This work was partly supported by grants from the Centre National de la Recherche Scientifique (ATP no. 6082751), the Institut National de la Sante et de la Recherche MCdicale (CRL no. 802030). Euratom (B10352-F), and by the Fondation pour la Recherche MCdicale Francaise.

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Received July 15, 1983 Revised version received October 27, 1983

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