Rate of DNA synthesis in Ehrlich ascites tumor cells

246

BIOCHIMICA ET BIOPHYSICA ACTA

SHORT COMMUNICATIONS BBA 93546

Rate of D N A synthesis in Ehrlich oscites t u m o r cells The rate of DNA synthesis in Ehrlich ascites cells, the increments of synthesis needed to complete the genome of S period cells, and synthesis needed to complete replicons once initated, provide standards by which synthesis by isolated nuclei can be assessed. Cells were grown as described 1. Mice were injected with [3H]thymidine (Schwarz Bio Research; specific activity II.O mC/mole). Smears were made at various times, fixed ], Feulgen stained and autoradiographed ]. Cells were scored as labeled or not. Fig. I shows the percentage of labeled interphase cells at different times after injection of label. The slope of the regression represents the rate of increase in the frequency of labeling with time and slightly underestimates the rate of DNA synthesis. The rate t l l l l l l l ? l l ]

5O

c

_a I0

I

I

t

I

i

I

i

I

I

3

2

I

I

4

I

I

5

6

Time (hours) I

I

1

I

[

I

I

I

I

I

I

I

--

~ I00

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3

75

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5o

25 1.0

0.5

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I 4

I

I 6

I

I 8

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I I0

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12

G1

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G2

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Time ( h o u r s )

Figs. I and 3

Fig. 2

Fig. I. F r e q u e n c y of labeling of interphase cells at different intervals after pulse labeling. Q , calculated mean and intercept; O , experimental points. Fig. 2. Schematic representation of the a m o u n t s of D N A in cells at different stages during the cell cycle. The abscissa shows the frequencies of ceils in a population t h a t have not yet reached the designated stage since the last division. The areas u n d e r the curve are proportional to the total D N A contained in the cells within t h a t stage. Fig. 3- F r e q u e n c y of labeling of division figures at different intervals after pulse labeling. The conditions of the experiment are similar to those used in the e x p e r i m e n t described in Fig. i. The limits m a r k i n g the different stages of the cell cycle were a p p r o x i m a t e d b y estimating the times at which levels h a l f w a y between the m a x i m a and m i n i m a were attained, b o t h in the u p s w i n g s a n d down swings.

Biochim. Biophys. Acta, 224 (197 o) 246-248

SHORT COMMUNICATIONS

247

was 2.4 %/h. An independent estimate obtained using short term conditions i n vitro was 1. 7 %/h. Another value for ascites cells grown i n vivo is 5 %/h (ref. 2), and values ranging from 2 to 6 %/h can be derived from published data 3-5. The shaded portion of Fig. 2 shows the increment expected if nuclei in the S period were to complete DNA synthesis, but no non-S period nuclei were to initiate synthesis. This increment, in terms of percentage increase, was calculated as follows: Increment =

0.5 Fs × ioo F~I + 1.5Fs + 2FG2 + 2FM

(i)

where FG1, Fs, FG2, and FM are the frequencies of cells in the G1, S, G 2 and mitotic periods 6. Nongrowing cells are lumped with the G1 cells. The 0.5 in the numerator stems from the assumption that the average S period nucleus contains 1.5 times the diploid amount of DNA and should synthesize an amount equal to 1/3 of this value. In the denominator, the frequencies of the stages are multiplied b y their DNA contents using the diploid amount as the unit. The frequencies of cells in G~ and G~ were obtained b y establishing the durations of the G~, G~ and S periods and the total cycle time, and relating the frequencies to time: Fs F~ = T~×~ Fs

FGI = T~x × - Ts

(2) (3)

T~x = total cycle time--Ts--Tc2--Tra The frequency of S period cells, 0.28, is given b y the intercept of the regression line in Fig. I. Other values for ascites cells are 0.31 (see ref. 2) and 0.33 (see ref. 5). TG2, T s and Te were obtained b y pulse labeling and following the wave of label through division in the manner of HOWARD AND PELC6, and others4, 7 (Fig. 3). An independent assessment of duration was also made b y A. ZWEIDLER (unpublished observations). 6 days after tumor innoculation, 4 mice were given 3 injections of 5-fluorodeoxyuridine (total amount 6. IO-s moles) equally spaced over 6 h and then a single dose of cold thymidine (4.5" lO-6 moles). Small samples were taken at different times and the percentage of cells in division determined. The time between the onset of the first and second waves of division following release of the block was 18 h. The following estimates were made from the data of Fig. 3. The time for the entire cycle was taken from the synchronization experiment. Ts = 5h TG2 = 4 h Teyele ~

18 h

TM = I h (determined from Fm=o.6) TGx = Teyele--To2--Ts--TM = 8 h

These values are in accord with similar ones obtained by others: T s ----6.5 h, TG2 = 4 h, Teyele = 15 h (ref. 4); Ts ----8.5 h, T e y e l e = 18 h (ref. 8). From these the following frequencies were obtained from Formulae 2 and 3. FGx = 0.44 ~'G2 ~

0.22

Biochim. Biophys. Acta, 224 (197o) 246-248

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The increment of DNA calculated from Formulae I is 9-9 %. Thus a rate of synthesis of 2.4%/h and an increment of IO °/o serve as "goals" to be attained in a nuclear system that approximates the performance of the whole cells in DNA duplication. A more realistic prediction m a y be based on the expectation that isolated nuclei m a y complete but not initiate synthesis of new replicons. A limit can be calculated by considering the rate of synthesis along a DNA strand, the number of replicons in a cell, their size and the number of replicons synthesizing DNA at a n y instant. HUBERMAN AND RIGGS9 found a "typical" replicon in a H e L a cell to be 50 # long. Taking the weight of DNA in a mouse cell as 6. lO -12 g10,11, the calculated weight of the typical replicon is 15 • lO -17 g. The number of such replicons in a cell is about 4" I0a. HUBERMAN AND RIGGS also found the rate of synthesis along a strand to be about I/~/min. From the length of DNA in a cell, the time needed for a fork to traverse the length of the genome is calculated to be 3" Io 4 h. Since the S period is only 6 h, there must be about 5" IO3 forks in a S period nucleus. If there are 4" lO4 replicons and a pair of forks per replicating replicon 9, the proportion of replicating to nonreplicating replicons should be 2.5" IOa to 4" lO4, or I in 16. By the reasoning described in Fig. 2, the increment expected by completion of replicons is approx. 0.6 °/o of the total DNA in the sample. This value has been approached in the nuclear systems 1. This work was supported in part by grants No. DRG 905 from the Damon Runyon Fund for Cancer Research and GM 09654 from the U.S. Public Health Service. It was done under tenure of Research Career Development Award 5 K 3 GM 04253 held by one of us (D. B.).

Department o/Botany and Cell Research Institute, University o/ Texas at Austin, Austin, Texas 78712 (U.S.A.) I 2 3 4 5 6 7 8

C. L. G. V. S. A. J. J.

J. 9 J. i o R. i i E.

CHRISTINA TENG* D . P . BLOCH

TENG, D. P. BLOCH AND R. ROYCHOUDHURY', Biochim. Biophys, .4cta, 224 (197 o) 232. R. BROWN, P h . D . D i s s e r t a t i o n , The U n i v e r s i t y of Texas, A us t i n, 1969. KLEIN AND L. REVI~SZ, J. Natl. Cancer Inst., 14 (1953) 229. DI~FENDI AND L. A. MANSON, Nature, 198 (1963) 359. TOLNAI, Lab. Invest., 14 (1965) 7Ol. HOWARD AND S. R. PELC, Heredity, 6, Suppl. (1953) 261. H. TAYLOR AND R. D. IV[cMASTER, Chromosoma, 6 (1954) 489. L. EDWARDS, A. L. KOCH, P. Y o u c l s , H. L. FREESE, ~][. B. LAITE AND J. T. DONALSON, Biophys. Biochem. Cytol., 7 (t96o) 273. A. HOBERMAN AND A. D. RIOGS, J. Mol. Biol., 32 (1968) 327 . VENDRELY AND C. VENDRELY, Experientia, 5 (1949) 327 • M. DEN TONKELAAR AND P. VAN DuIJN , Histochemie, 4 (1964) I.

Received June 23rd, 197o " P r e s e n t ad dress : R o c k e f e l l e r U n i v e r s i t y , N. Y. 10012, U.S.A.

Biochim. Biophys. Acta, 224 (197 o) 246-248