Determination of thymidine metabolism in HeLa cell cultures by a combined electrophoretic and paper chromatographic method

Thymidine

645

metabolism of HeLa cells

At any rate, we are of the opinion that the human interphase chromosome is a single continuous DNA fiber. This work was supported in part by United States Energy Commission contract number AT(ll-l)-34. REFERENCES 1. CAIRNS, J., J. Mol. Biol. 6 208 (1963)., A., Proc. Natl Acad. Sci. 53, 356 (1965). 3. HUBER~~AN, J. A. and RIGGS, A. I>., Proc. Natf Acad. Sci. 55, 599 (1966). 4. KLEISSCHMIDT, A. and Z.~HN, R. K., %. Naturforsch. 14, 770 (1959). 5. PL.IUT, IV. and NASH, D., in 11. LOCKE (ed), The Role of Chromosomes p. 113. Academic Press, Sew York, 1964. 6. SCHMII~, W., Cvtogenefics 2, 175 (1963). 7. SOLARI, A. .J., Proc. Satf Acad. Sci. 53, 503 (1965). 2. HOTTA, Y. and BASSEL,

DETERMINATION

OF THYMIDINE

CELL CULTURES AND

BY A COMBINED

PAPER

W. LANGI, Institut

fiir

Medisinische

METABOLISM

D. MULLER

METHOD

and W. MAURERI

der Universitiit Wiirzburg Marburg, Deutschland

Received

IN HELA

ELECTROPHORETIC

CHROMATOGRAPHIC

Strahlenkunde Universitiit

in Dovelopnrr~!I

und Hygiene- Institut

der

June 6, 1966

W

HEN using 3H-TdR as a DNA precursor in long-term experiments with mammalian cells in culture, two main factors must be taken into account. Firstly, cultured mammalian cells are able to degrade TdR to T, DHT, and/or BUIB, which cannot-or only to a very low degree-serve as DNA precursors [6,16]. Secondly, as could be demonstrated for mouse fibroblasts in culture, several types of enzyme activity, necessary for the phosphorylation of TdR, decline before the cells pass from the exponential growth phase into the stationary phase [la, 13,211. Degradation of TdR could be demonstrated for human leucocytes [3,14,15], mouse thymocytes [ 31, and Ehrlich ascites cells [ll, 221. This paper deals with the TdR degradation in HeLa cell cultures over a period of 48 hr. HeLa cells were grown in Roux bottles (inoculated with 5 x 104 cells/ml) in 100 ml Eagle’s medium supplemented with 10 per cent calf serum and antibiotics (104 units penicillin/ml and 100 pg/ml streptomycin). 42 hr after inoculation the medium was replaced by 100 ml Eagle’s medium containing 0.25 ,uC/ml SH-TdR (spec. act. 120 mC/m&f). 3H-TdR with a spec. act. of 6700 mC/mlM (New England

Present address: Institut ftir Medizinische Isotopenforschung der Universitlt K61n. Abbreuiations: BAIB, P-amino-iso-butyric acid; BUIB, p-ureido-iso-butyric acid; DHT, dihydro-thymine; DNA, deoxyribonucleic acid; T, thymine; TMP, TDP, TTP, thymidine mono-, di-, and tri-phosphate; TdR, thymidine. Experimental

Cell Research 44

W. Lang, Il. Miiller

646

and IV. Maurer

Nuclear Corp., Boston/Mass., U.S.A.) was diluted with inactive TdR (Sigma, Chem. Comp., St. Louis/MO., U.S.A.) to a spec. act. of 120 mC/mll/l. The radiochemical purity determined by the method described below was: TdR =97.7 per cent, T =2.3 per cent. At different times after the initiation of the experiment aliquots of the medium were collected and radioactive degradation products of 3H-TdR were measured. TTP TDP BLIIB TMP start TdR.T,DHT,BAlB --all / / I I

Electrophoresis

-

I r ;:\\

_ ; -.; e \

;. .. --\, Chromatography

/ &JIB

/ front

T/pi&

BPID ’

+

AR

I / T DHT

/ ,rcnt -

I 0

10

20

30

40

50

60

70

SO

90

100

cm

Fig. l.-Separation of thymidine degradation products by a combined electrophoretic and paper chromatographic method. The electrophoretic paper was cut along two vertical dotted lines and descendingly chromatographed without elution. Elecfrophoresis: Buffer = pyridinelacetic acid/water (4 : 1 : 47, v/v); pH, 5.1; field strength = 40 V/cm; t = - 8”C, 150 min; paper: Schleicher & Schtill 2043 b MgL. Paper chromatography: Acetic acid-ethyl-ether/formic acid/ water (60 : 5 : 35, v/v, upper phase).

To examine volatile radioactivity an aliquot of the collected medium was freezedistilled to dryness and the vapour recondensed in an solid carbon dioxide-aceton freezing mixture. The volatile activity was measured by liquid scintillation counting (TriCarb EX 314). The rest of the medium was deproteinized with equal parts of 5 per cent ZnSO, and K,Fe(CN),. The filtrate was concentrated by vacuum destillation and partly fractionated by high voltage paper electrophoresis (for conditions see Fig. 1). By this method a separation into 3 groups can be achieved (Fig. 1). The electrophoretic paper was cut into 3 parts, with the middle one containing only TMP, that can be measured directly. The right and left parts were further fractionated without elution by descending paper chromatography in the following mediums: (a) acetic acid-ethyl-ether/formic acid/water (60: 5: 35, v/v) [4], (6) isopropyl-ether/nbutanol/formic acid (30: 30: 20, v/v) [ 71. The products were identified by comparison to authentic substances and made visible according to the method given by Fink et al. [5] The compounds for comparison were obtained from Sigma (Chem. Comp., St. Louis/MO., U.S.A.); BUIB was prepared from DHT according to the method of Fink et al. [5] The radioactivity was measured by liquid scintillation counting [S]. The DNA was isolated according to the Schmidt-Thannhauser method [19] and determined by indol reaction [I]. The curve showing SH-TdR and its active degradation products as a function of time is shown in Fig. 2. The incubation period began 42 hr after inoculation of the HeLa cells and lasted over 48 hr. All the values are given as a percentage of the initial activity in the medium. During the first hours, there is a sharp decrease of Experimental

Cell Research 44

Thymidine

metabolism of HeLa cells

647

TdR in the medium and a similar sharp increase of T. Later on T is further degraded to BUIB. Only traces (1-2 per cent) of the intermediate product, DHT, could be detected. Neither thymidylic acid nor other phosphorylated products, BAIB, nor tritiated water could be found. In this series of experiments the DNA activity of the HeLa cells collected after an incubation period of 48 hr was 13.1 & 2.3 per cent (5 experiments; mean + s.D.)

Fig. 2.-Percentage of 3H-TdR and its labelled degradation products in the culture medium of HeLa cells after various time intervals of incubation with *H-TdR. Inoculation with 5 x lo4 cells/ml; 42 hr later aH-TdR was added (0.25 PC/ml, 120 mC/mM); the points represent results from three different experiments.

of the initial aH-TdR activity in the medium. In another series of 15 experiments 15.2 & 1.4 per cent of the activity was found in the DNA of the cells. These amounts of activity correspond satisfactorily to the decrease of ca 15 per cent in total activity in the medium after 48 hr of incubation (Fig. 2). Cooper and Milton [3] examined TdR degradation by human leucocytes in vitro between 0 and 90 min. In the culture medium they found TdR, T, and DHT. The faster degradation of thymidine in their experiments may be explained by the use of another cell type and a larger number of cells/ml. Marsh and Perry [14, 151 tested homogenates of normal and leucemic human leucocytes and found a degradation of TdR to T, DHT, and BUIB. They also did not findphosphorylated TdR derivatives in the medium. Jacquez [II] and Zajicek ef al. [22] showedthat under inuitro conditions Ehrlich ascites cells degrade TdR to T. These results further support existing evidence that-similar to the animal organism [2, 4, 5, 10, 17, 18, 20]-mammalian cells in culture also degrade most of the applied TdR and incorporate only a small part into their DNA [9]. REFERENCES G., J. BioZ. Chem. 198, 297 (1952). L. 0. and LOONEY, W. B., Ccmcer Res. 25, 1817 (1965). 3. COOPER, E. H. and MILTON, J. D., Brit. J. Cancer 18, 701 (1964). 1. CERIOTTI,

2. GANG,

Experimental

Cell Research 44

4. 5. 6. i. 8. 9. 10. 11. 12.

13. 14. 15.

16. 17. 18. 19. 20. 21.

22.

FINK, Ii., CLINE, R. E., HFS.DERSOS, R. U. and FINK, R. M., J. Biol. Chem. 221,425 (195ti). FINK, R. M., MCGAUGHEY, C., CLINE, R. 1-:. and FISK, K., .I. Bid. Chem. 218, 1 (1956). FRIEDKIX, M., TILSOS, D. and ROBERTS, I)., J. 1jiol. Chem. 220, 627 (1956). GERLACII, E., DHEISHACII, R. H. and DEUTICI
THE

MATHEMATICAL

OF MALIGNANT

TREATMENT

MAMMALIAN

CELLS

OF GROWTH CULTURED

CURVES IN

V’ITRO

G. L. TRITSCH Clinical

Biochemistry Section, Department of Medicine C, Roswell Park Memorial New York State Department of Health, Bu$alo, N.Y. 14203, U.S.A.

Institute,

Received June 22, 1966

A description of the growth of cells in culture should indicate the length of the lag period, the maximum rate of growth, the maximum cell density attained, and the maintenance of the viability of the culture. Although these parameters are obviously interrelated, no single expression has as yet been devised for cell culture systems which incorporates all of them. The length of the lag period and the maintenance of viability are not readily amenable to quantitation. The maximum cell density attained has been used as a measure of the growth response [7, 91. Since the maximum cell density also depends on the concentration of the limiting nutrient or accumulated toxic metabolites in the medium, cultures of different growth rates but of essentially equal viability have been observed to attain equal maximum cell densities, albeit after different periods of growth (unpublished data). Therefore, it was felt that growth rate would be the most meaningful single indicator of a growth response. During the course of work on the evaluation of the growth rate of mammalian cells in suspension culture in the presence of increasing concentrations of calf serum and peptides derived from calf serum [2, 81 it became apparent that the dose-response Experimental

Cell Research 44