Characterization of a Nutritive Synchrony in L-cells

Pathologisches Institut (Ludwig-Aschoff-Haus) der Universitat Freiburg (Direktor: Prof. Dr. W. SANDRITTER)

Characterization of a Nutritive Synchrony in L-cells Charakteristik einer nutritiven Teilungssynchronisation DYANN FERGUS*, PETER KADEN,

and

CHRISTIAN MITTERMAYER**

With 2 Figures' Received February 26, 1973 . Accepted March 26, 1973

Summary A synchronization method in L-cells producing nutritive stagnation of the cells in G 1 is presented. After reinnoculation, cells proceed through the cycle in the normal 24 hours. The method has the advantage that no chemical blockers are added to the system and a large number of cells is obtained. In a comparison with other synchronizing systems, this system rates in the foreground. Desynchronization due to individual cell physiologies makes investigations of G 2 difficult with this system. The method is uncomplicated and probably applicable to similar cell types.

There are few cell systems which exhibit a natural synchrony of division available for biological study. Therefore, in order to answer particular questions, several methods of artificial synchronization have been developed. These include various physical methods and various chemical blocks applied to the system. In this paper, we are presenting a synchronization method which is uncomplicated, produces a large number of cells and uses no chemical blocks. It uses the simple principle of nutritive starvation of a cell culture.

Materials and Methods L-cells (line 929) grown in monolayers were used in the experiments. They were suspended in Eagle Basal Medium containing 5% calf's serum and cultured in Roux flasks and Leighton tubes. Fifty ml of medium, containing approximately 4 X 106 cells, were

* Fulbright Foundation Fellow (1972-1973)' On leave from the Universiy of Wisconsin, U. S. A. ** Partially supported by Sonderforschungsbereich 46 Molgrudent.

242 . D. FERGUS, P. KADEN, and CH. MITTERMAYER

placed in each Roux flask. After 6 days, the cell density was 4 X 10 7 in each flask. The pH of the medium had fallen from 7.3 to 6.8. The cells were loosened from the glass surface with 5 ml of 0.25% trypsin and resuspended in 1000 ml of fresh medium. This new suspension was divided between Roux flasks (100 mljflask) and Leighton tubes (10 mljtube), and the cultures were allowed to grow undisturbed, except for labeling and eventual sampling. For sampling purposes, the cells were scraped from the glass surface with a rubber policeman. An electronic cell counter was used to determine the cell count and individual cell volume for each sample. The cells were stained according to BOHM et al. (1966) for the quantitative Feulgen cytophotometric measurement with the microdensitometer (DEELEY, 1955). The cells had previously been labeled (1 0' 1 [J.Cfml 3H_ TdR) for an autoradiographic analysis. The preparations were covered with a liquid photographic emulsion (Ilford KS) and exposed for 7 days. In order to determine the exact amount of newly DNA 'synthesized, 25 [J.gjmlof 5-Brdeoxyuridine was added to the medium. The DNA was isolated using MARMUR'S method (1961). The basic methods of esCi centrifugation as described by FLAMM et al. (1966) were used in our experiments. The fractionation method was that of MADREITER et al. (1971). The protein mass after reinoculation and throughout the cycle was determined colorimetrically (LOWRY et al., 1951).

Results The following reported results and curves represent an average of three attempts. The variation between attempts did not exceed 8%. The original cell culture shows a growth pattern of an initially logarithmically growing culture which reaches a stationary phase. During the logarithmic stage, the pH of the medium is 7.3, while, at the end (approximately 144 hours), it has fallen to 6.8. Individual cell volumes and cultural protein content show G 1 values once the culture has reached its stationary phase. If fresh medium is added, the culture begins to grow again, indicating that a depletion of the medium rather than contact inhibition is the major stagnation cause. If the cell concentration is greatly diluted in used medium, a slight growth results; however, after one cycle, stagnation occurs again. This indicates that the depletion of the medium depends to a certain extent on the cell concentration, i.e., the limiting nutrients are not completely depleted, but instead present in such small amounts per cell that further growth does not occur. A simple change of the pH from 6.8 to 7.3 in used medium does not initiate further growth. The morphology of the cells in logarithmic and stagnated cultures is quite different. Inhomogenous, asynchronous cells are found in the logarithmic stage, whereas, a homogenous population and a singularity of cell size are found in a nutritive stagnated culture.

Nutritive Synchrony' 243 2c

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Fig. 1. a) Feulgen histograms of synchronized L-cells as measured with an integrating microdensitometer. Hours (right) after reinoculation. Main band density ~ 1.70.

A Feulgen cytophotometric analysis was performed to determine the DNA content of individual cells throughout the cell cycle after reinoculation. The results are presented in Figure I a. The 2 C line indicates the single, G 1 DNA content, while the 4C line indicates the double, G 2 DNA content. It should be noted that 95 %of the cells have the G 1 DNA content until the ninth hour after reinoculation, followed by a period of increasing DNA content until 2 I hours after reinoculation. The cytophotometric results are supported by the CsCl gradient DNA synthetic results (Fig. I b). Twenty-five [lgJml 5-Br-deoxyuridine was added

244 . D.

FERGUS,

P.

KADEN,

and CH.

MITTERMAYER

Hours

Heovy

Gradient

Light

Fig. 1. b) DNA synthesis in synchronized L-cells measured by 5-Br-deoxyuridine incorporation and seperated in a esCl gradient.

to the medium and any newly synthesized DNA was specifically heavier than the existing DNA and could, therefore, be separated in a CsCl gradient. Here, hybrid DNA first appears 8 hours after reinoculation and almost all "light" DNA disappears after 24 hours. Reinoculated and diluted cultures double in number with an average generation time of 24 hours (Fig. 2 a). The acutal mitotic wave takes 6 hours, indicating that the asynchrony at this point is about 25 %. DNA synthetic activity was measured on the basis of 3H-TdR incoporadon throughout the cell cycle. After autoradiographic preparation, the per cent labeled cells were determined for each experimental point. A tenfold increase is found between 9 and 15 hours (Fig. 2 b). The integral of the CsCl gradient optical density absorption provides a means of comparison be-

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Fig. 2. a) The growth pattern of G 1 arrested cells after reinoculation. b) Synchronized L-cells: ----- DNA synthesis caH-TdR, percent labeled cells), - - DNA mass increase per 4 hours (5-BrdU incorporation), .... Mitotic Index.

tween the 5-BrdU incorporation and the 3H-TdR incorporation. Both show a maximum at 15 hours preceeded by a steep increase and followed by a steep decrease. The mitotic index was also determined and has its maximum at 25 hours after reinoculation. 17 Beitr. Path. Bd. 149

246 . D.

FERGUS,

P.

KADEN,

and CR.

MITTERMAYER

Both the individual cell volume and protein content increase linearly over the cell cycle, and double within 25 hours. This indicates that, first, there is no lag period following reinnoculation, and secondly, the cells were definitely stagnated in early G 1, directly after telophase. Our results indicate that stagnation in G 1 through nutritive depletion does not irreversibly injure the cells, since immediately after reinnoculation, they resume the cell cycle. The cycle's duration is 24 hours under our culture conditions. The asynchrony reaches 25% by the first mitosis, and therefore, results in the G 2 phase should be viewed with this in mind. Further, the stagnation in G 1 is due to nutritive depletion in the medium and not to contact inhibition or pH decrease.

Discussion The characteristics of synchronized cell system produced by nutritive stagnation in G 1 have been presented in this paper. We have shown that the system provides a large number of cells synchronized from the beginning of G 1 with increasing asynchrony, through mitosis. The system is easliy controllable through nutritive depletion. It has certainly proved to be a very usable method. Other methods of synchronizing cell cultures use different controlling mechanisms. In mitotic selection (TERASIMA, et aI., 1963; MITTERMAYER et aI., 1968) and sedimentation (SCHINDLER et aI., 1972), cells are specifically selected at one stage of the cycle, separated and cultured further. Here, of course, the number of obtainable cells is limited. DNA synthetic inhibitors, FUDR (PRIEST et aI., 1967), double thymidine (GALAvAZI et aI., 1966), hydroxyurea (ADAMS et aI., 1967), N0 2 (RAo, 1968), amethopterin (MUELLER et aI., 1966), stop the cell population at the beginning of the S-phase. However, two major disadvantages appear. First, foreign chemical substances are introduced into the metabolism and secondly, recent work (STUDZINSKI et aI., 1969) has shown that unbalanced growth occurs while the cells are under the influences of certain DNA-inhibiting substances. Metaphase inhibitors, colchicine (BRENT et aI., 1966) and vinblastine (KIM et aI., 1966) both have the disadvantage of introducing foreign substances. In order to determine how our starvation method compared with other synchrony producing systems, we attempted to compare the systems on the basis of the cell growth curve for the cycle immediately following synchronization. It must be noted that this is in no way a rigorous comparison due to the differences on cell material and culture conditions. In each case investigated, the DNA synthetic activity (when reported) showed a ten-fold increase during the S-phase.

Nutritive Synchrony' 247

A comparison was made between two time intervals. First, the average time from synchronization to the first mitosis was determined and this compared to the time elapsed in which 75 % of the cells go through division. In a perfectly synchronized system, this ratio would be zero. Surprisingly, results obtained from published data showed the majority to have a ratio of 25 % to 33%. This is a measure of the relative asynchrony after one cycle. Our method rated among the best with 25%. This result suggests that the de synchronization is mainly due to an inherent difference in individual cell physiologies and generation times. In the present literature no method has been reported which eliminates this individual variation. On the basis of this comparison, our nutritive synchrony stands in the foreground. It has the advantage that no external chemical substances enter the metabolic pathways, but at the same time provides a large number of cells for biochemical analysis. Asynchrony in this system, as in others, increases with time and therefore, is not designed for experiments involving the G 2 phase or mitosis. This method, however, is simple and uncomplicated and probably applicable to similar cell types. The question which remains unanswered is, why do the cells stop growing? The possibility of contact inhibition, alone, has been eliminated since the addition of fresh medium to a stagnated culture reinitiates growth. The obvious answer lies in the depletion of the medium. It is much easier to deplete Eagle Basal Medium than most others, since it has the lowest amino acid content. It was beyond the realm of this experiment to investigate which nutrient or nutrients are limiting.

Zusammenfassung Diese Arbdt zeigt einen Weg zur Gewinnung von synchron-wachsenden L-Zellen, die durch Mediumverbrauch in G 1 arretiert werden. Nach Umsetzen mit frisch em Medium wachsen die Zellen synchron, mit dner Generationszeit von 24 Stunden. Diese Methode hat den Vorteil, groBe Mengen von Zellen unter Umgehung von Synchronisationsblockern zu gewinnen. ]edoch entsteht durch individuelle Generationszeit eine Asynchronie von etwa 25% innerhalb eines Zyklus. Vergleicht man dieses System mit anderen Synchronisationsmethoden, so gehort es in die Reihe der brauchbarsten.

Acknowledgement: Thanks are due to Dr. H. Madreiter for his many useful suggestions.

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