Myocardial colchicine-binding proteins: Possible relation to DNA synthesis initiation

Journal of Molecularand CellularCardiology(1979) U, 1137-1150

Myocardial Colchicine-binding Proteins: Possible Relation to DNA Synthesis Initiation C O N S T A N T I N O S J. L I M A S

Department of Medicine, CardiovascularDivision, Universityof Minnesota School of Medicine Minneapolis, Minnesota 55455, U.S.A. (Received29 aVovember1978, acceptedin revisedform 8 January 1979)

C.J.I.aM~.Myocardial Colchicine-binding Proteins: Possible Relation to DNA Synthesis

Initiation. ffournalof MolecularandCellularCardiology(1979) 11, 1137-1150. Administration oftri-iodothyronine ('I"3)to 2-week-old rats resulted in enhanced [SH] thymidineincorporation into myocardial ceils (126 4- 1.7 dpm/~g DNA vs 69 zk 1.2 dpm/tzg DNA in untreated controls, P < 0.01), which reached a maximum at 48 h. This enhancement was prevented by the concomitant adminis~atlon of colchicine but not lumicolchicine. The inhibitory effect was progressivelyattenuated when colchicine was administered at increasinglylonger intervals after T3 injection. This suggested that mlcrotubular proteins may be involved in the initiation of DNA synthesis. This possibility was further evaluated by studying the extent of [3H] colchicine binding to myocardial extracts as an index of tubulin content and polymerization. [SH]colchicine-binding decreased progressively with age (49 q- 1.9 pmol/mg[h at 5 days vs 21 4- 0.7 pmol/mg/h at 180 days) with no change in the decay rate of the reaction. The extent of tubulin polymerization also decreased from 39 4- 1.2% at age 5 days to 19 4- 1.4% at age 180 days. In contrast, Ta administration to neonatal rat~ resulted in enhanced [ZH]colchicine-binding (36 4- 1.7 pmol/mg vs 27 4- 1.5 pmol/mg in controls, P < 0,01) and enhanced tubulin polymerization (38 4- 1.6% vs 29 =k: 1,8% in controls). These effects were maximal 12 to 24 h after Ta prior to the peak in thymidine incorporation. These results indicate that microtubular assembly may be involved in the early steps of myocardial DNA synthesis activation. KEY Wonvs: Tri-iodothyronine; Colchicine; Tubulin; Polymerization; Thymidine incorporation.

1. Introduction M y o c a r d i a l cells r e t a i n t h e i r p r o l i f e r a t i v e c a p a c i t y for o n l y a short t i m e following b i r t h [4, 7, 13, 18, 26]. Proliferative a c t i v i t y as reflected in [ 3 H ] t h y m i d i n e incorp o r a t i o n a n d the activities o f D N A synthetic enzymes declines r a p i d l y in the n e o n a t e [4, 7, 10] a n d , in t h e r a t , reaches t h e low a d u l t levels b y t h e t h i r d postn a t a l w e e k [4]. B e y o n d t h a t age, stimuli to c a r d i a c e n l a r g e m e n t result in m y o c a r d i a l h y p e r t r o p h y (increased cell size) w i t h o u t h y p e r p l a s i a (increased cell n u m b e r s ) . I n contrast, interstitial n o n m u s c l e cells r e t a i n t h e i r m i t o t i c p o t e n t i a l to a d u l t h o o d [6, 14]. T h e m e c h a n i s m s involved in the restriction o f m y o c a r d i a l 0022-2828/79/111137+ 14 $02.00]0

9 1979 Academic Press Inc. (London) Limited

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c.j. LIMAS

proliferative capacity with age are not well understood and are currently under intensive investigation. We recently observed that myocardial thymidine kinase and DNA synthesis could be stimulated in neonatal hypophysectomized rats by the administration of growth hormone [16]. This stimulation could be prevented by the concurrent administration of colchicine or vinblastine, substances thought to act through their interactions with microtubules [35]. Little is known about the function of cytoplasmic microtubules in the myocardium; previous studies [22] have implicated them in control of contraction rate of myocytes in culture. Our results suggest that myocardial microtubules might be involved in the early steps of DNA synthesis activation. The present report extends these observations and offers further evidence that initiation of myocardial DNA synthesis may require a microtubuledependent step.

2. Materials and Methods Experiments were carried out on male Sprague-Dawley rats. In order to study the effects oftri-iodothyronine (T3) administration on [sH]thymidine incorporation, 2week-old animals were injected with 10 ~g T3 i.p. and controls received 0.5 ml of saline alone. In some experiments, rats aged 5 days to 6 months were used, as described under Results.

Isolation of myocardial cells Myocardial cells were isolated from rat hearts by a modification of the method described by Glick et al. [11]. Cardiac ventricles from either young or adult rats were diced into 3 mm slices and washed twice with calcium-free phosphate buffer (pH 7.4). The tissue was then placed into 50 ml Erlenmeyer flasks containing 5 ml of phosphate buffer and enzymes (1 mg collagenase and 1 mg hyaluronidase per ml). The flasks were shaken at 100 strokes/rain at 37~ Cells were decanted from the tissue pieces at 20-min intervals (harvests I to 4) and the tissue was resuspended in fresh buffer. Harvests 1 and 2 were mostly erythrocytes and fragmented cells and were discarded. Cells from harvests 3 and 4 were washed twice with calciumfree phosphate buffer, pooled, and centrifuged through a 3% Fieoll solution containing 0.1% albumin at 60 • g for 5 rain. The myocytes thus isolated were washed twice with buffer and were immediately used for the enzyme isolation. Adequacy of myocyte separation was checked by light microscopy of formalinfixed cell preparations. Approximately 93 to 95 % of the cells were myocytes and 65 % of the latter excluded Trypan blue.

MICROTUBULES A N D D N A SYNTHESIS

1139

[3H] thymidine incorporation Control or growth hormone-treated rats were given 2 ~Ci/g s.c. [methyl-3H] thymidine (Amersham/Searle, Arlington Heights, Illinois, specific activity 60 mCi/ mmol) and were sacrificed 1 h later. The ventricles were washed and weighed, cardiac myocytes were isolated as described above and homogenized an ice-cold 0.15 ~ NaC1. To the homogenate was added an equal volume of 10% perchloric acid and the acid-insoluble material, collected by centrifugation at 2000 • g for 10 rain, was washed twice with ice-cold 5% perchloric acid and twice with ethanol : ether (3 : 1, v/v). It was then resuspended in 5% perchloric acid and heated to 70~ for 30 rain. The hydrolyzed DNA in the acid-soluble fraction was measured by Button's modification of the diphenylamine reaction [3]. Aliquots of the acid-soluble fraction were also dissolved in NCS solubilizer and radioactivity was counted in 10 ml of a toluene-based scintillation fluid.

Uptake and catabolism of [SH]thymidine Rats were injected with 2 ~Ci/g [SH]thymidine. Cardiac myoeyte homogenates were prepared and 10% cold perchloric acid was added as described above. Following eentrifugation, aliquots of the acid-soluble material were dissolved in NCS solubilizer and counted. Thymidine and pyrimidine derivatives in aliquots of the acid-soluble fraction were separated from nonaromatic degradation products by charcoal adsorption [2]. A 2.0-ml aliquot of the perchloric acid-soluble fractions was added to 50 mg of activated charcoal; mixing was achieved by frequent (every 5 min) vortexing for 30 min. Following centrifugation at 1000 • g for 10 min, an aliquot of the supernatant was dissolved in NCS and counted to measure unadsorbed tritlated derivatives. Radioactivity adsorbed on charcoal represents labeled thymidine and aromatic pyrimidine derivatives and gives an estimate of the [3H]thymidine taken up and not catabolized into nonaromatlc compounds.

[sH]colchicine binding assay Myocardial cell suspensions were homogenized in 2 volumes of MES buffer (0.1 ~ morpholinoethane sulfonic acid, pH 6.5, 0.5 mM MgCI~, 1 rnM EGTA, 1 rnM GTP) at 4~ The homogenates were then spun at 48 000 • g for 1 h at 4~ and the assay was performed in the supernatant. Samples were incubated at 30~ (unless otherwise indicated) with 10-4M [3H]methoxyl-colchicine (4 Ci] ~mol, Amersham/Searle, Arlington Heights, Illinois). Blanks contained labeled colchicine but no protein. At different time intervals, 0.2 ml aliquots were transferred to 1 ml of packed cold DEAE-Sephadex equilibrated with PMC buffer

1140

c.j. L~S

(0.1 g phosphate buffer, pH 6.5, 5 rnM MgG1,, 10 mM colchicine), stirred, and added after 10 rain with 5 ml of cold PMC buffer. After 5 min occasional stirring, the resin was sedimented by centrifugafion, then freed of unbound colchieine by five successive washes and spun with cold PMG buffer. Bound colchicine was directly eluted from DEAE-Sephadex by addition of 10 ml of Bray's solution and counted. Microtubule assembly was estimated by the method of Patzelt et al. [24]. Myocardial cell suspensions were homogenized at 4~ in 4 volumes of MES buffer in the presence (A) or absence (B) of 4 M glycerol. The homogenates were spun at 48 000 • for 1 h and [aH]colchicine binding reaction was performed in the supernatants, as described above. Since, in the presence of glycerol, microtubules are stabilized, the [3H]colchicine binding assay in A homogenates measures free tubulin. In the absence of glycerol, microtubules are depolymerized and the assay in B homogenates measures total amount of tubulin. The difference (B--A) gives an estimate of the extent oftubulin polymerization. Microtubular protein was prepared by the method of Weisenberg et al. [32, 33]. Myocardial cells were homogenized in 2 volumes of P M G buffer (0.01 M sodium phosphate, pH 6.5, 0.01 M MgCI~, and 0.1 mM GTP) and the homogenate was spun at 15 000 g for 30 rain. To the supernatant ammonium sulphate (18 g/100 ml) was slowly added and was allowed to stand for 10 rain before centrifuging at 10 000 g for 20 rain. Ammonium sulphate (25 g/100 ml) was again added to the supernatant, the mixture was allowed to stand for 10 rain and then was centrifuged at 10 000 g for 20 man. The pellet was suspended in P M G buffer, stirred intermittently for 30 rain and spun at 2000 g. The ensuing pellet was washed twice in 0.4 re KC1 in P M G for 10 rain followed by 0.8 M KC1 in PMG. The last two supernatants were pooled and (NH,)uSO, (25 g/100 ml) was added with constant stirring, resuspended in PMG and dialyzed overnight in excess P M G ; 0.5 M MgC12 was then added slowly to a final concentration of 0.05 re. The mixture was allowed to stand in the cold for I0 rain. The pellet was washed once in 0.05 M MgCI2, resuspended, and dialyzed against 0.01 M sodium phosphate, pH 7.4, 10-4 re GTP.

3. R e s u l t s

(A) Thymidine incorporation into myocardial DNA Thymidine incorporation into myocardial DNA was significantly enhanced by tri-iodothyronine (Table 1), indicating stimulation of DNA symthesis. This stimulation became statistically significant 24 h following T~ and plateaued at 48 h. Differences in uptake and catabolism of injected [aH]thymidine did not account for the results: recovery of radioactivity in the acid-soluble fraction was similar in control (1401 -t-92 drop/rag net weight) and Ta-treated (1459 -4- 83 dmp/

MICROTUBULES AND DNA SYNTHESIS

1141

TABLE I. [SH]thymldine incorporation into myocardial DNA of control and tri-iodothyronine treated rats Time following tri-iodothyronine

[3H] thymidine incorporation (dpm]~g DNA)

(h) 0 12 24 48 72

694-1.2 734-1.3 1014-1.8" 1264-1.7" 108 + 1.4"

Animals received 2 FGi/g of [methyl-aH]thymidine i.p. and sacrificed 1 h later; treated rats were given 10 [tg Ta and sacrificed at varying intervals thereafter. Results are given as mean 4- S.E.M.for seven experiments in each group. *P < 0.01.

IOO

+

cx 75

50

84

O O

50

I O0

150

Fg/lO0 g

FIGURE 1. Effects of lumicolchicine (A) and coichicine (O) on Ts=induced enhancement of [3I-1]thymidine incorporation into myocardial cells. Animals were injected with 10 ~g T8 alone or with the indicated doses of lumicolchicine and colchicine and [3H]thymidine incorporation was measured 48 h later. Results are expressed as percentage of control (Ts alone) values [134 4- 1.8 dpm/t~g DNA]. Values represent mean 4- s.~..m for six experiments in each group. mg) rats. Similarly, the p e r c e n t a g e o f r a d i o a c t i v i t y r e t a i n e d b y c h a r c o a l 1 h after [3H] t h y m i d i n e a d m i n i s t r a t i o n was 23-4-11% (n = 8) in controls a n d 244-1.2 % (n = 8) in T s - t r e a t e d animals. A d m i n i s t r a t i o n o f c o l c h i c i n e , b u t n o t l u m i c o l c h i c i n e w h i c h does n o t affect m i c r o t u b u l e s , in c o n j u n c t i o n w i t h t r i - i o d o t h y r o n i n e prev e n t e d s u b s e q u e n t s t i m u l a t i o n o f D N A synthesis (Figure 1) in d o s e - d e p e n d e n t m a n n e r . T h i s i n h i b i t o r y effect was m o d i f i e d b y d e l a y i n g colchicine a d m i n i s t r a t i o n ( F i g u r e 2); i n h i b i t i o n was progressively less as the t i m e since T8 injection increased. T h e s e results i n d i c a t e d t h a t a colchicine-sensifive step was involved in the i n i t i a t i o n o f m y o c a r d i a l D N A synthesis.

1142

C.J.L[MAS

'~

150

g g

Ioo

I0

4

8

12

16

20

Time (h)

FIGURE 2. Inhibition of Ts-induced enhancement of [sift] thymidine incorporation as a function of tlmlrtg colchicine administration. Animals were given 10 I~gTs and 0.5 ~g/g colchlcine at varying intervals thereafter. [SH]thymidine incorporation was measured 48 h after Ts administration.

Characteristicsof [SH]colchicine.binding reaction

(B)

T h e p r o p e r t i e s o f [SH]colchicine b y m y o e y t e extracts were f o u n d to be in agreem e n t w i t h results previously p u b l i s h e d b y o t h e r a u t h o r s [24, 25, 27]. [SH]colchicine b i n d i n g to m y o c a r d i a l cell extracts was b o t h t e m p e r a t u r e a n d timed e p e n d e n t . T h e c o l c h i c i n e - b i n d i n g activity is u n s t a b l e a n d decays in a n a p p a r e n t l y 2O --

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Preincubation time ( h )

FIGURE 3. Decay of [SH]colchicine binding activity with time. Myocardial extract from 3-weekold rats was incubated 37~ At the indicated times, aliquots were removed and incubated with 10-4 M[SH'Jcolchicineat 37~ for 2 h. Bound colchiclne was assayed as described in the text.

1143

M I C R O T U B U L E S A N D D N A SYNTHESIS

first-order m a n n e r (Figure 3). T h e half-time for decay calculated from the slope of the curve was a p p r o x i m a t e l y 4 h. T h e a m o u n t of colchicine t h a t w o u l d have b e e n b o u n d if n o decay h a d occurred (initial colchicine b i n d i n g capacity) c a n be d e t e r m i n e d b y a 2-h extrapolation a n d is i n d e p e n d e n t of the decay rate. 20 m

IO

T

I

I

I

IC

20

30

40

Temperature (~

FIGURE 4. Temperature dependence of the decay rate of colchicine-binding activity. Myocardial extract from 2-week-old rats (6 mg/ml) was pre-incubated at different temperatures; incubation in the present of 10-4 u [aH-]colchicinewas then carried out for 3 h at 37~ 5 h at 30~ 6 h at 25~ 8 h at 20~ 9 h at 13~ and 14 h at 5~ Colchicine binding was measured as described in the text. Half-times were calculated from the slope of the first-order decay curve obtained for each temperature.

~= 20

Io CO

_o ~

o

9

/ I

I

I

I

4

8

12

16

Time ( h )

FIGURE 5. Colchicine binding by myocardial extracts of 2-week-oldrats. Reactions were carried out at 13~ for the indicated time. Specific binding refers to initial binding capacity calculated from the decay curve.

1144

c . j . LIRAS

T h e rate of decay (as reflected in half-time) is strongly temperature-dependent (Figure 4) as has been described for other systems [24, 25]. At lower temperatures, colchicine binding was a slow process with equilibrium reached only after 16 h (Fig. 5). T h e effects of protein concentrations on half-time and specific activity of [SH]colchicine binding are shown in Figure 6; specific activity was independent of protein concentration while decay rates increased with protein concentrations up to 200 ~g/ml and plateaued thereafter. 20 ~ l ~ u l l

l~l

F

all.

30O c

-,o

200

_~

T

~

lO0 -

l

l

l

50

I00

150

l 200

l

l

2.50

300

Protein concentration (p.g/ml)

FIGURE 6. Effect of protein concentration on half-time ( 0 ) and initial colchicine-binding capacity (1) of myocardial extracts from 3-week-old rats. Reactions were carried out at 30~ as described in the text. T h e effects oflumicolchicine, podophyllotozin, and vinblastine on the colchicinebinding reaction are shown in T a b l e 2. Lumicolchicine, a compound structurally similar to colchicine but unable to bind to tubulin, had no effect on colchicine binding, in contrast to podophyUotoxin which competitively inhibits colchicine binding to tubuIin. Vinblastine had a biphasic effect with low concentrations stabilizing and high concentrations inhibiting colchicine binding. TABLE 2. Effects of lumicolchicine, podophyllotoxin, and vinblastine on initial colchicinebinding capacity of myocardial extracts from 3-week-old rats Additions None Lumicolchicine (0.5 mM) Podophyllotoxin (4 g~) Vinblastine (4 g~) Vinblastine (4 mM)

[SH]colchiclne binding (pmol/mg prot.) 23 4-0.9 21-4-0.9 7.6 4-0.2 28 4-0.8 12.7 ~z0.9

Reactions were carried out at 30~ as described in the text. Results represent mean 4- s.E.~, for six experiments in each group.

1145

M I C R O T U B U L E S A N D D N A SYNTHESIS

(C)

Effects of post-natal growth and 7"3 administration

T h e eolchieine-binding reaction of myocardial homogenates was studied at different stages during post-natal development. T h e requirements for the reaction were not different a m o n g the varying age-groups. As shown in T a b l e 3, however, there was considerable progressive decline in [SH]colchieine binding capacity with ages, whereas decay rates were unaltered. T h e degree of tubulin polymerization was also affected with a progressive decrease from 39% at age 5 days to 19% in adulthood. T h e opposite changes were seen when tri-iodothyronine was administered to 2-week-old rats: increased [3H]eolchicine binding and tubulin polymerization was found (Table 4). Moreover, T8 effects were maximal 12 to 24 h following administration, prior to the peak in [3H] thymidine incorporation shown in Table 1. T o examine whether T3 effects depended on altered tubulin properties, comparison was made of colchicine binding by purified rat myocardial tubulin from control and T3-treated animals (Figure 7). T h e dose-response curves for the binding of eolehicine to these preparations were almost identical with half-maximal binding occurring at 6.5 X 10 -5 M and saturation at 10 -4 M. TABLE 3. Effects of age on myocardial colchicine-binding Age (days) 5 14 21 180

[sI-I]colchicine binding Half-time (pmol/mg prot.) (min) 49-4-1.9 284-1.8 264-0.9 21 4-0.7

Percent polymerization

1094-6 1154-8 1214-9 1184-7

394-1.2 304-1.6 224-1.1 194-1.4

[SH]colchicine binding represents initial colchicine binding capacity determined from time-decay assays 30~ as described in the text. Results represent mean 4- s.E.m for six experiments in each group. TABLE 4. Effects of 10 ~g Ts on myocardial colchicine binding, decay rate, and extent of tubulin polymerization Time after T; (h)

[SH]colchicine binding (pmol/mg)

0

274-1.5

8

30 + 1.4"

12 24 48

344-1.2++ 36 4-1.7++ 32 4-1.8"

Half-time (min)

Tubulin polymerization (%)

1164-7 112 -4-7 109 4-6 109 4-6 1144-5

29• 32 4-1.7 t 364-1.6++ 38 4-1.6++ 33 4-1.8

Results represent mean 4- S.E.M.for six experiments in each group. *P < 0.05. 1"Not statistically significant. ~P < 0.01.

1146

c . j . LIMAS

20

--

-

4

A / A

-

///t IO

-2

5

--I

10-6

I

I

I

10-5

10-4

i0-~

Colchicine ( M)

FIGURE 7. Dependence of colchiclne-bindingactivity of myocardial extracts (A) and purified tubulin from control (O) and Ts-treated (0) 2-week-old rats, on colchicine concentration. Reactions were carried out at 30~ as described in the text. The curve for myocardial cell extracts was similar to that for purified tubulin except that a sharp rise occurred at colchicine concentrations higher than 10-s M (data not shown). This activity at high colchicine concentrations was not precipitated by vinblastine and is unlikely to be related to the microtubular system. The data in Figure 7 yield a specific binding of about 0.36 mol colchicine per mol tubulin, assuming a molecular weight of about 110 000 for dimeric tubulin.

4. D i s c u s s i o n Several factors, alone or in combination, may contribute to the age-dependent decline in proliferative capacity of cardiac cells: (a) loss of enzymes necessary for DNA replication; (b) restriction of nuclear template activity by virtue of changes in chromafin structure and composition; (c) loss of putative signals to cell proliferation. O f these, the first possibility has received the most attention and it is generally recognized that a significant decrease in the activities of DNA polymerase and thymidine kinase occurs during early postnatal development [4, 7, 10]. This decrease, however, is more likely to reflect the progressively falling number of cells engaged in mitosis and may not be an adequate explanation for the inability of adult ceils to undergo mitosis. Support for this conclusion comes from two observations: (a) 3- to 4-week-old rats are able to synthesize DNA and undergo mitosis

MICROTUBULES AND D N A SYNTHESIS

1147

in response to anema [23], exercise [2], and aortic constriction [6], whereas, adult animals adapt to the same stimuli with hypertrophy rather than hyperplasia [14]. Since the activities of DNA polymerase-~ and thymidine kinase do not differ significantly between 1 month of age and adulthood [18], they cannot be major determinants of the difference in profiferative response; (b) cardiac overload of adult animals results in polyploidism [14] which requires DNA synthesis and is, therefore, incompatible with an irreversible loss of DNA synthetic enzymes. Restriction of DNA availabifity for transcription by DNA polymerases might also contribute to the constraint on mitotic potential. Evidence on this issue is conflicting. We have recently examined chromatin structure of myocardial cells during postnatal growth [17] by studying thermal stability, resistance to DNase digestion, circular dichroism spectra, protein composition, and template activity for RNA synthesis. The results indicated a progressive decline in chromatin DNA accessibility pari p~su with the loss of proliferative capacity. In contrast to these findings, Claycomb [5] has reported that exogenous DNA polymerase directed DNA synthesis equally well in nuclei of neonatal and adult rats and condluded template availabifity was not a major raetor in mitotic restriction. The reason for these discrepant results are, at present, unclear; the major difference between the two studies was the use by us of isolated myocytes instead of whole ventricular homogenates. This is important since it avoids the contribution of interstitial cells which retain their proliferative capacity to adulthood. The possibility that, in adult animals, cardiac overload is not transcribed into slgnal(s) for cell proliferation has not been adequately examined. In most cell types, transition from quiescence to mitosis following a proliferative stimulus typically involves a lag period of variable length. This pre-replicative period is replete with metabolic changes that accompany the commitment of the cell to proliferation and include changes in membrane permeability for ions and metabolites, induction of specific RNA and protein species, increased phospholipid turnover and changes in cyclic nucleofide content. Inability to propagate the proliferative stimulus from the plasma membrane to the cell interior would prevent initiation of DNA synthesis and subsequent mitosis. Unfortunately, our knowledge of the early events of the pre-replicafive phase is fragmentary and entirely derived from non-muscle cell types. The results of the present study provide indirect evidence supporting a role of colchicine-binding proteins in the early steps of myocardial DNA synthesis activation. The first suggestion of such involvement was provided by the inhibitory effects of colchicine on Ts-induced enhancement of [SH]dTR incorporation into myocardial DNA. This inhibition was not only dose-, but, also, time-dependent. Delaying colchicine administration for more than 10 h after T~ virtually abolished the inhibitory effect. This suggests that colchiclne acts during the period of commitment to mitosis; once this commitment has been made, the presence of the proliferative stimulus may not be required and vinca alkaloids are no longer

1148

c.j. L~S

effective. DNA synthesis is not affected once it has been initiated (Figure 2) and colchicine does not modify the rate of DNA synthesis by isolated nuclei (results not shown). An inhibitory effect of colchicine on initiation of DNA synthesis and mitosis has previously been reported for phytohemagglufinin--or concanavalin A--stimulated lymphocytes [9, 3/], cultured fibroblasts [29], and regenerating liver [17, 25, 26]. A corollary of the thesis that colchicine-binding proteins are intimately involved in DNA synthesis activation is that their amount should fluctuate with proliferative activity. Colchicine binding to tubulin monomers in equilibrium with formed microtubules results in a shift of the equilibrium and depolymerization of formed microtubules. This possibility that tubulin polymerization is also affected was examined under conditions of declining (postnatal growth) and increased (Ta administration) DNA synthesis. The results presented in this report are consistent with the proposed role of the microtubular assembly since both [aH]colchicine binding and mbulin polymerization declined with age and increased when neonatal rats were injected T8 under experimental conditions that lead to enhanced DNA synthesis. The mechanism by which microtubular proteins interact with DNA synthetic apparatus is not clear but does not appear to involve metaphase arrest or nonspecific cytotoxicity [3/]. Since cyclic AMP changes are not affected [12] and induction of ornithine decarboxylase is only slightly delayed [30], the effects must be highly selective. Colchicine and related alkaloids do not interfere with thymidine transport [21] and do not inhibit DNA synthesis directly. They do however, block the increase in amino acid transport following the proliferative stimulus to both lymphocytes [12] and liver cells [29]. Colchicine-binding sites are present in nuclear chromatin [27] and are partly associated with the nonhistone nuclear proteins [8, 15]. Not all sites are related to tubulin, however, and may, instead, be involved in other (e.g. ionic) transport processes important in DNA synthesis initiation. Since microtubule inhibitors affect the function of a number of transport proteins [35], the block of DNA synthesis may be mediated by affecting the propagation of the proliferative stimulus from the plasma membrane to the cell interior. Alternatively, the effects ofcolchicine may be secondary to inability of the cell to enter mitosis which depends on adequate spindle or tubulin proteins. The difference between neonatal and adult myocardium may be related to a decrease in the total amount of tubulin, a change in the microtubule-associated proteins [36] which are needed for microtubule growth, or absence of regulatory factors that influence polymerization. In particular, transport of calcium ions across the cell membrane which is a pre-requisite for DNA synthesis activation [1, 20, 28] may be related to microtubular assembly. Distinction between these alternatives requires further study.

MICROTUBULES AND DNA SYNTHESIS

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