Effect of aging on acetate incorporation in nuclei of lymphocytes stimulated with phytohemagglutinin

Effect of aging on acetate incorporation in nuclei of lymphocytes stimulated with phytohemagglutinin

Life Sciences Vol . 11, Part II, pp. 877-884, 1972. Printed in Great Britain Pergamon Press EFFECT OF AGING ON ACETATE INCORPORATION IN NUCLEI OF LY...

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Life Sciences Vol . 11, Part II, pp. 877-884, 1972. Printed in Great Britain

Pergamon Press

EFFECT OF AGING ON ACETATE INCORPORATION IN NUCLEI OF LYMPHOCYTES STIMULATED WITH PHYTOHSMAGGLUTININ Yang H . Oh and Robert A. Conard Medical Department Brookhaven National Laboratory Upton, New York 11973

(Received 18 April 1972; in final form 7 June 1972) suMMARY Aqe-correlated decrease in lymphocyte transformation in blood cultures treated with phytohemagglutinin (PHA) had been noted previously in a Marshallese population . In this same population, acetylation of nuclei in PHAtreated lymphocytes was measured at various times as a function of lymphocyte transformation and aging . Decreased acetylation of nuclei was associated with increasing age and roughly paralleled the decrease in lymphocyte transformation . Total acetate incorporation in nuclei during the first hour of culture was correlated with aoetylation of histones, but by 20 hours acetylation of other nuclear material had also occurred .

Imrestigations of nuclear histone acetylation during the wurse of gene activation in mammalian cells suggest that such modifications may be involved in the regulation of chraaatin structure which may influenoe nuclear biosynthetic activities, particularly with respect to RNA synthesis (1-5) .

Pogo et al .

(3,4) and Cooper and Rubin (6) reported that human or equine lymphocytes increase their rates of RNA synthesis during the first hour after exposure to phytohemagglutinin (PHA) .

Histone

acetylation is greatly increased in human sad equine lymphocytes responding to PHA though equine granulocytes show a decreased activity (3,4) . We previously reported reduced transformation of cultured lymphocytes in response to PHA stimulation with increasing age in 877

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a Marshallese population (7) .

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in this paper we also report a

correlation between the rate of acetylation in lymphocyte nuclei and PHA-induced transformation in this same population as a function of age . Materials and Methods Lymphocyte cultures and isotopic labeling : During routine hematological examinations a 30-m1 aliquot of heparinized blood was collected from each of 44 Marshallese people (not previously exposed to fallout radiation) in three age groupas 18 people aged 15 to 29 ; 17 aged 30 to 49 ; and 9 aged 50 to 79 .

The blood was allowed to settle for 1 hour and centrifuged

at 1000 Xq for 10 minutes . smears from the buffy coat .

Differential counts were done on The buffy coat was then suspended in

Eagle's minimum essential medium supplemented with 1! glutemine, 158 fetal calf serum, penicillin (100 units/ml) and streptomycin (0 .1 mg/ml) .

Five separate 5-ml cultures were seeded with 10 6

leukocytes/ml, then 0 .1 ml each of PHA-M Difco and soditms (methyl3H) acetate (50 ~nCi, specific activity 100 mCi/mmole) were added, and the cultures were incubated at 37 0 .

At 30 minutes, 1 hour,

and 20 hours, 0 .25 ml of non-radioactive 2 M sodium acetate (41 mq, 1000-fold excess isotope) were added to each culture, and cells were harvested.

A separate 20-m1 culture (2 .5 X 10 6 leukocytes

per ml) fran several donors was similarly treated with PHA and 3H-acetate and incubated for extraction of nuclear histories .

In

addition, the percentage of transformed lymphocytes (blast-like cells) was determined in 72-hour cultures by electronic sizing according to the method described previously (8) .

Autoradiagrams

were made of cells from smears from 1-hour and 20-hour cultures . The slides were dipped in Rodak nuclear emulsion, type NTH2 , developed at 5 days for 20-hour cultures and 17 days for 1-hour

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cultures, and stained with Giemsa . Isolation of lymphoc~e nuclei and extraction of histones : Following incubation of 5-ml cultures at 30 minutes, 1 hour and 20 hours, the nuclei were isolated by a modification of the citric acid procedure of Pogo et al .

(3) .

The histories were ex-

tracted fray the isolated nuclei of 50 X 106 leukocyte cultures by the procedure of Pogo et al . (3) . The nuclei from the 5-ml cultures, isolated as described above, were dissolved in 3 to 5 ml of Protosol tissue solubilizer (New England Nuclear, Cat . No . NEF-935) 3iI-acetate .

to determine incorporation of

Each sample (0 .2 to 0 .5 ml) was dissolved in 10 ml

of a toluene-based liquid scintillation solvent (9) to which a few drops of glacial acetic acid had been added.

The samples were

left overnight at 5° to allow for decay of chemihaainescence . (Chemiluminescence with Protosol is about the same as with other alkaline solubilizers .)

The samples were then counted in n

Beckman liquid scintillation counter . were dissolved

The isolated histone samples

in 1 ml of 0 .1 N HC1 and added to 10 ml of a

toluene-based solvent and counted . Results The total radioactivity incorporated into the nuclei was adjusted for the number of lymphocytes in the culture (obtained from differential counts) and was expressed as dpm per lymphocyte : Fiq . 1 (a,b,c) shows the acattergrams of the percent transformation of lymphocytes at 3 days versus the acetate incorporation of lymphocyte nuclei . and 20-hour cultures

Correlation was seen only in the 1-hour (coefficient of correlation L r~ 0 .86 and 0 .85

respectively) . Fiq . 2 shows a decreasing response with increasing age of the people in both acetate incorporation (canbined data) and percent

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Phytohemagglutinin and Acetate Incorporation

FIG . 1 Scattergrams of the acetate incorporation of nuclei per lymphocyte (y) versus the percent transformation of lymphocytes at 3 days (x), (a) in 30-min . cultures, (b) in 1-hr cultures, (c) in 20-hr cultures .

transformation of lymphocytes .

It was unfortunate that more

samples were not available from older people .

Also, we were

unable to obtain a larger quantity of blood from every donor for precise acetylation studies at the histone level .

Radioactivity

of extracted nuclear histones, however, fran several large cultures was found to be similar to total activity of the whole nuclei of 1-hour cultures .

Considerable difference in values was

noted with 20-hour cultures . Tha label seen in autoradiograms (Fig . 3) appeared to be largely localised in the nuclei of the lymphocytes .

The red blood

cells were not labeled, and only slight labeling was seen in the neutrophils .

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s"Y

QF t; ni 0 0

z.s

I trouNOi Is-za usl

I ~woo~.et so-+s un

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FIG . 2 Aqe-related change in lymphocyte transformation and in total acetate incorporation .

FIG . 3 Autoradiogram of leukocytes frown a 1-hour culture. represent 3g- acetate incorporation of the cells .

The grains

ßS1

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Discussion In past studies age-related changes in immunohematological criteria were reported in this Marshall island population .

With

increasing age, a reduction in blood lymphocytes and an increase in gamma globuline (reflected also in increases in IgG and IgA immunoglobuline with increase of R light chains) were noted (7) . We believe these changes to be associated with age-accumulated effects of lymphocyte attrition, repeated exposure to infectious agents, and possibly an increase in autoimmune reactions .

Of

particular interest was the additional finding of reduced blastogenesis of cultured lymphocytes fn response to PHA stimulation with increasing age .

This suggested the possibility that acti-

vation of nuclear histones might similarly be affected by aging . The results of the present studies, carried out during the last medical examination of the Marshalleae at Rongelap Atoll, support this contention . The presence of contaminating granulocytes in the cultures is not believe to seriously affect the results since (1) autoradiographic studies showed only very slight acetylation occurring in the granulocytea either before or after PHA addition whereas nuclear labeling of lymphocytes was predominant, and (2) the results were based on absolute lymphocyte counts .

The 3H-acetate

labeling technique used in this experiment represents acetylation of histonea as well as other nuclear materials .

However, during

the first hour the incorporation appears to be largely in the histones while by 20 hours the incorporation represents other nuclear camponente as well as the histones . The reason for reduced PHA transformation of lymphocytes, (including acetylation of nuclear histones) associated with the

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aging process, may be related to impairment of immune mechanisms that is known to occur with aging .

It is known also that such

reduced PHA effects occur in certain diseases characterised by impairment of cellular immunity such as chronic lymphocytic leukemia, Hodgkin's disease, sarooidosis, etc.

Perhaps similar

lymphocytic defects occur in these conditions . The increased histone acetylation that occurs following PHA stimulation is no doubt a part of the chain of events in the lymphocyte leading to blast formation and cell division . precise nature of these events remains obscure .

The

Increased acetyl-

ation of non-histone nuclear components is probably also repreaentative of the increased metabolism associated with the process of transformation . Acknowledgement-We wish to thank Messrs . W . Scott and M . Maker for their excellent technical assistance .

This research was

supported by the United States Atomic Energy Cammiasion and NIH Grant Np . HD 00199 .

References 1.

V . G. ALLFREY, R . FAULRNER and A. E . MIRSRY, Proc . Nat . Acad . Sci . U . S . 51, 786 (1964) .

2.

B . G. T. POGO, A . O. POGO, V . G. ALLFREY and A. E. MIRSKY, Proc . Nat. Acsd . Sci . U. S . 59, 1337 (1968) .

3.

B . G . T. POGO, V . G. ALLFREY and A. E . MIRSRY, Proc . Nat. Acad . Sci . U . S . 55, 805 (1966) .

4.

B . G . T. POGO, V . G. ALLFREY and A. E . MIRSRY, J. Cell . Biol . 35, 477 (1967) .

5.

V. G . ALLFREY, Çancer Res . 26, 2026 (1966) .

6.

H . L . COOPER and A. D . RUBIN, Blood 25, 1014 (1965) .

7.

R. A . CONARD, C. F . DEMOIS, W . A. SCOTT and M . MAKER, J. Gerontol . 26, 28 (1971) .

88~

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8.

Y . H . OH and R. A. CONARD, Arch . Bioohem. Biophya . 146, 525 (1971) .

9.

Y . H . OH, W . E . LEITCH, S . AXELROD, S . M . SMALL, R. J . WIN8LER and B. E . SANDERS, Biochem . Pharmacol . _16, 849 (1967) .