Mechanisms of Ageing and Development, 8 (1978) 383-395

383

©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

C O M P A R I S O N O F R N A S Y N T H E S I S BY L I V E R N U C L E I F R O M R A T S OF VARIOUS AGES

THOMAS CASTLE, ALAN KATZ and ARLAN RICHARDSON Department of Chemistry and Department of Biological Sciences, Illinois State University, Normal, Hlinois 61 761 (U.S.A.)

(Received March 13, 1978)

SUMMARY RNA synthesis was studied in nuclei isolated from male Fischer F344 rats of various ages. Both the initial rate and the extent of RNA synthesis were determined in two distinct media that result in the preferential synthesis of either mRNA or rRNA by hepatocytes. Synthesis of mRNA and rRNA, as determined by [aH]-UMP incorporation into acid-insoluble material, was shown to increase 1.6- to 3-fold from 3- to 6-months. The increase in RNA synthesis during maturation was followed by a 1.6 to 2.7-fotd decrease from 6- to 31-months of age depending upon the assay medium and time of incubation. The age-related changes in RNA synthesis by rat liver nuclei were shown not to be due to changes in RNase activity, UTP uptake, or UTP degradation. RNA synthesized by liver nuclei from rats of various ages was characterized by SDS-polyacrylamide gel electrophoresis. Slight age-related differences in the size distribution of RNA isolated from liver nuclei were observed when nuclei from rats of various ages were incubated with [all] -UTP in either assay medium.

INTRODUCTION An age-related decrease has been observed in protein synthesis by liver [1--6], as well as other tissues. Because of the role of RNA in protein synthesis, the age-related decrease in protein synthesis might occur, in part, from changes in RNA metabolism. Agerelated changes in RNA metabolism might result in decreased RNA synthesis and/or an alteration in the RNA species synthesized. Unfortunately, only a limited number of studies on the effect of aging on RNA synthesis have been performed. Studies using cultured fibroblasts from mice [7] and humans [8-10] have shown decreased RNA synthesis from middle to late passage cells. However, these results might not be representative of in vivo processes because the complex interactions of the cell with the rest of the organism are absent in the culture system. Using isolated nuclei, several investigators have compared RNA synthesis with respect to age. Using isolated

384 nuclei from rat liver [11] and mouse prostrate or liver [12], early studies showed a decrease in RNA synthesis by 30-month animals compared to 5-month-old animals. Using u-amanitin sensitivity and varying ionic conditions of the assay media to differentiate between mRNA and rRNA synthesis [13-16], Britton et al. [17] reported that the rates of total, mRNA, and rRNA synthesis were all lower for nuclei isolated from liver and muscle tissue of 28-month-old mice compared to that of 8-month-old mice liver and muscle. However, Gibas and Harman [18] reported that there was no significant difference in the rates of either mRNA or rRNA synthesis by liver or brain nuclei from 5- and 22-month-old rats. Owing to the discrepancies between the fndings of Britton et al. [17] and Gibas and Harman [18], the effect of age on RNA synthesis by nuclei is uncertain at the present time. In an attempt to study age-related changes in RNA synthesis in vivo, Kanunga et al. [19] monitored the incorporation of [14C]-uracil into acid-precipitable material by several tissues from 3-, 10-, and 22-month-old rats. In liver, a decrease in RNA synthesis after maturation was observed. However, because the specific activity of the UTP pool in these animals was not determined, the actual effect of aging on RNA synthesis in vivo is uncertain. The basic purpose of this study was to determine what effect aging has on RNA synthesis by rat liver nuclei and to compare the types of RNA synthesized by nuclei from rats of different ages. The discrepancies in the findings of previous studies have been suggested to be due to differences in the procedures to isolate nuclei and assay for RNA synthesis [17]. Using improved isolation techniques and assay conditions that have been developed since the previous studies were performed, RNA synthesis by liver nuclei isolated from 3- to 31-month-old rats has been determined. The results of this study demonstrate that with increasing age there is a decrease in the synthesis of both mRNA and rRNA as well as an age-related change in the species synthesized.

MATERIALS AND METHODS Fischer F344 male rats, which have a mean life-expectancy of 25-27 months, were purchased from the National Institute of Aging colony maintained by Charles River Breeding Laboratories. Rats were fed ad libitum and maintained under pathogen-free conditions in our laboratory. All animals were decapitated between 10.00 a.m. and noon to minimize diurnal variation. Isolation o f nuclei

Rat liver nuclei were prepared by a modification of the procedure of Blobel and Potter [20]. Dithiothreitol (1.5 raM) was substituted for 2-mercaptoethanol, and spermidine was added to the buffer system (0.2 raM) to preserve structural and functional integrity of the nuclei and chromatin as suggested by Chevaillier and Philippe [21]. Liver tissue was minced and then homogenized in 5 volumes of buffer l which contained 50 mM Tris-HCl, pH 7.5, 3 mM MgCI2, 2.5 mM CaCI2, 25 mMKC1, 1.5 mM dithiothreitol,

385 0.2 mM spermidine, and 0.25 M sucrose. After filtering the homogenate through cheesecloth, nuclei were obtained by centrifuging the homogenate at 100,000 × g for 90 min through a discontinuous sucrose gradient composed of 6 and 13 ml layers of buffer I containing 1.67 M and 2.3 M sucrose, respectively. The pelletted nuclei were resuspended in 20% (v/v) glycerol-water. Aliquots of nuclei were removed to determine: RNA synthesis, the nuclei count using a hemocytometer after staining with 0.2% trypan blue, and the DNA concentration by the method of Cerotti [22].

RNA synthesis by isolated nuclei RNA synthesis was determined by incubating isolated nuclei at 37 °(3 with [5,63H]-UTP (34 Ci/mmol) obtained from Amersham/Searle and monitoring the incorporation of labelled UMP into acid-precipitable material. RNA synthesis was assayed using two separate media. Medium I was similar to that described by McNamara et al. [23] and has been shown to result in the preferential synthesis of rRNA by rat liver nuclei. Assay medium II was similar to that described by Gibas and Harman [18] and Britton et al. [17] and has been shown to result in the preferential synthesis of mRNA [16]. Medium I contained 100 mM Tris-HC1, pH 7.5, 3.5 mM MgC12, 0.5 mM CaC12, 5 mM NaC1, 10 mM KC1, 1 mM dithiothreitol, 3 mM spermidine, 0.5 mM L-cysteine, 3.5 mM creatine phosphate, 15/ag creatine phosphokinase, 5/aCi [3H] -UTP, 0.8 mM ATP, 0.8 mM CTP, 0.8 mM CTP, and 3 × 106 nuclei in a final volume of 0.2 ml. Medium II contained 100 mM Tris-HC1, pH 8.0, 200 mM (NH4)2SO4, 3.0 mM MnC12, 0.3 mM NaF, 0.5 mM Locysteine, 3 mM spermidine, 1 mM dithiothreitol, 5 /ICi [3H]-UTP, 0.8 mM ATP, 0.8 mM CTP, 0.8 mM CTP, and 3 X 10 6 nuclei in a final volume of 0.2 ml. In these systems, RNA synthesis was found to be linear with respect to nuclei concentrations from 1 X 10 6 to 3 × 107 nuclei/0.2 ml. Nuclei in the two media were incubated at 37 °C for incubation times up to 50 rain after a 5 min preincubation period. The reactions were stopped by the addition of 2 ml of 10% trichloroacetic acid, and acid-insoluble material was collected by centrifugation at 3000 X g for 10 min. The pellets were washed three times with 10% trichloroacetic acid, solubilized with tissue solubilizer, and the radioactivity in the sample was determined by liquid scintillation counting. Characterization o f RNA synthesized RNA synthesized by isolated rat liver nuclei in the two media was extracted by the phenol-chloroform method of Aviv and Leder [24]. The RNA was then separated on the basis of size using SDS-polyacrylamide gel electrophoresis as described by Edmonds and Carmela [25]. RNA (I00/ag) was applied to 10 cm, 2.7% gels and subjected to electrophoresis at 8 mA/tube for 4 h at room temperature. The gels were sliced into 2 mm fractions, and the radioactivity in each slice was determined by scintillation counting with Beckman Ready-Solv GP as the solvent. RESULTS Figures 1 and 2 show typical time courses for RNA synthesis using medium I and II by liver nuclei from 12- and 30-month-old rats. RNA sTnthesis by nuclei in these two

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Fig. 1. Time course of RNA synthesis per mg DNA by rat liver nuclei. RNA synthesis in medium I (A) and medium II (B) was carried out as described in Materials and Methods for nuclei isolated from a 12month (O) and a 30-month-old (Ix) rat. Fig. 2. Time course of RNA synthesis per 106 nuclei by rat liver nuclei. The same data presented in Fig. 1 are re-expressed per 106 nuclei.

TABLE I DNA CONTENT OF LIVER NUCLEI FROM RATS OF VARIOUS AGES Age (months]

na

•g DNA /10 6 nuclei b

3 4 6 13 22 31

2 5 7 5 3 5

14.6 26.7 29.8 28.4 36.2 34.0

+ 0.8 ± 1.0 ± 1.7 ± 2.1 ± 2.6 + 1.3

aTriplicate assays were performed for the number of animals listed for each group. bDNA content of the nuclei determined by the method of Cerotti [22] represents the mean + SEM.

systems was linear f r o m 10 to 30 m i n d e p e n d i n g o n the assay m e d i u m and t h e age o f the a n i m a l used. R N A s y n t h e s i s was linear for longer periods o f i n c u b a t i o n for nuclei f r o m 12-month-old

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rats. In all cases, R N A

synthesis p l a t e a u e d after 50 m i n . R e c e n t l y , van Bezooijen et al. [26] r e p o r t e d t h a t t h e p l o i d y o f rat liver nuclei changes w i t h age. Table I s h o w s t h e changes in D N A c o n t e n t o f nuclei for t h e age g r o u p s studied in this investigation. T h e D N A c o n t e n t per 106 nuclei was f o u n d to b e a p p r o x i m a t e l y 2-fold greater in 4 - m o n t h - o l d rats t h a n in 3 - m o n t h - o l d rats. The D N A c o n t e n t f r o m 4- to 1 3 - m o n t h s r e m a i n e d c o n s t a n t . A n increase in D N A

387 content was observed in nuclei from 13- to 22-months and then the DNA content remained constant from 22- to 31-months o f age. Because the DNA content o f rat liver nuclei increased with age, it was insufficient to express RNA synthesis by nuclei in terms of DNA content per assay only, as previous investigators have done [11, 17, 18]. Therefore, RNA synthesis was expressed both in terms of DNA content and per 104 nuclei because there is no information on the transcriptional activity o f the additional DNA in polyploid nuclei. In Fig. 1, RNA synthesis is expressed per mg DNA, while in Fig. 2 RNA synthesis is expressed per 104 nuclei. Figures 1 and 2 show that both the rate and the extent o f RNA synthesis by nuclei from 30-month-old rats were lower than nuclei from 12-month-old rats. In both medium I and II, the rate of RNA synthesis by nuclei from 12-month-old rats was 1.3- to 1.5-fold higher than nuclei from 30-month-old rats. The extent o f RNA synthesis by nuclei from 12-month-old rats was 1.8-to 2.2-fold and 2.1- to 2.5-fold higher than nuclei from 30-month-old rats in medium I and II, respectively. Therefore, a decrease in the synthesis of b o t h m R N A and rRNA was observed in nuclei isolated from senescent rats compared to adult rats. To study changes in RNA synthesis with age, nuclei were isolated from male Fischer F344 rats ranging in age from 3- to 31-months-old, and 10 and 50 minute samples were taken to determine the initial rate and the extent of RNA synthesis. Figures 3 and 4 show an increase in the extent of RNA synthesis between 3- and 6-months for rRNA (3fold) and m R N A (1.6- to 1.8-fold). This increase in RNA synthesis during maturation was followed by a decrease in the extent of rRNA synthesis (2.5- to 2.7-fold) from 6- to 31months and in the extent of m R N A synthesis (2.1- to 2.2-fold) from 13- to 31-months o f 1

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Fig. 3. Comparison of the rates and extents of RNA synthesis per mg DNA by nuclei from rats of various ages. Nuclei from rats of various ages were incubated in medium I (A) and medium II (B) for 10 ( - - ) and 50 ( - - ) min to determine the rate and extent of RNA synthesis, respectively. The number of animals in each age group are given in Table I. Each point represents the mean with the SEM, denoted by the vertical bars. Fig. 4. Comparison of the rates and extents of RNA synthesis per 106 nuclei by nuclei from rats of various ages. The same assays as presented in Fig. 3 are re-expressed per 106 nuclei.

388 age. The same trends were observed for the initial rates o f R N A synthesis. A 2-fold increase was observed for initial rates o f R N A synthesis from 3- to 6-months of age in b o t h media. This increase is followed by a 1.6- to 2.3-fold decrease in R N A synthesis from m a t u r i t y to 31-months o f age depending on the assay m e d i u m and expression o f results. Table II shows the results o f the statistical t r e a t m e n t o f the data presented in Figs. 3 and 4 using analysis o f variance. It can be seen that the age-related changes in b o t h the e x t e n t and rates o f R N A synthesis in b o t h media represent statistically significant changes b e t w e e n the age groups at the P < 0.001 level. The e x c e p t i o n to this observation was the rate o f m R N A synthesis when expressed per mg DNA. This set o f data yielded only borderline levels o f significance for changes with age at 0.05 < P