Isoleucine-Mediated Regulation of Genome Replication in Various Mammalian Cell Lines1

[CANCER RESEARCH 31, 46â€”51, January 1971] Isoleucine-mediated Regulation of Genome Replication in Various Mammalian Cell Lines1

R. A. Tobey and K. D. Ley2

Biomedical ResearchGroup, Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87544

SUMMARY medium (1 5). In the absence of sufficient quantities of isoleucine and glutamine, the cells initially in S, G@, and M When suspension cultures of Chinese hamster cells (line continued traverse of the cell cycle and accumulated in @, CHO) are grown in isoleucine-deficient F-l0 medium but cells in G1 were unable to initiate DNA synthesis and supplemented with dialyzed sera, the entire population of cells resume cell-cycle traverse in synchrony. Deliberate omission of @ accumulates in a state of arrest within 24 to 36 hr. Merely other amino acids from the F-l0 medium resulted in a random by the adding back of isoleucine, the entire population distribution of cells throughout the cell cycle ( 15). It was initiates DNA synthesis and subsequently divides in suggested, therefore, that isoleucine and glutamine played a synchrony. This phenomenon is not readily observed in cells role in regulation of CHO cell genome replication. It was also infected with pleuropneumonia-like organisms . Cultures of shown that resuspension of exponentially growing cells in mouse L and Syrian hamster BHK2 1 cells also accumulate in medium deficient in both isoleucine and glutamine provided a @ G1 under conditions in which isoleucine is specifically rapid and convenient means of producing cells in arrest depleted from the culture medium, and, upon addition of without the attendant risk of partial exhaustion of other @ isoleucine, synthesis of DNA and resumption of cell-cycle nutrients that occurs during arrest in high-density cultures. traverse commence in synchrony. These results suggest that In this report, we present evidence indicating that a isoleucine-mediated regulation of genome replication may be a reversible state of G1 arrest in cultures of Chinese hamster generalized phenomenon in mammalian cells. Speculations are cells may be rapidly produced by merely removing isoleucine presented regarding possible roles for isoleucine (or its from the medium. Further evidence is provided to derivatives) in initiation of genome replication. demonstrate that this isoleucine-mediated regulation of genome replication can be demonstrated in cell lines of Syrian INTRODUCTION hamster and mouse origin as well, provided that isoleucine is specifically depleted from the culture medium and the cells are For facilitation of the study of mechanisms regulating not infected with PPLO.3 mammalian genome replication, it is essential to provide large quantities of cells under conditions in which the entire population can be induced to initiate DNA synthesis in MATERIALS AND METHODS synchrony. We had previously described a system of this type in which Chinese hamster cells (line CHO) accumulated in G1 A hypodiploid (modal chromosome number of 21) line of in suspension cultures that were grown to high cell densities in Chinese hamster cells (line CHO) was grown in F-10 medium F-b medium (21). Evidence that the cells were in G1 was supplemented with 10% calf and 5% fetal calf sera, penicillin, provided by (a) autoradiographic studies, which indicated that and streptomycin. The original F-b formulation (9) has been these stationary phase cells did not incorporate labeled modified in this laboratory by omitting FeSO4 , CuSO4, thymidine into DNA; (b) microfluorometric measurements, ZnSO4 , CaC12, and sodium pyruvate. Syrian hamster @ which indicated that the arrested cells contained the BHK21/Cl3 cells were obtained from Dr. Alexander Kisch and complement of DNA; and (c) data which signified that, in were grown in F-b medium. Mouse L-929 cells were grown in cultures resuspended in fresh medium, DNA synthesis always minimum essential medium (Grand Island Biological Co., preceded cell division (21). Reversal of G1 arrest was Grand Island, N. Y.) supplemented with 10% fetal calf serum, accomplished by resuspending the cells in fresh medium, penicillin, and streptomycin. In certain experiments, cells were which stimulated the cells to initiate DNA synthesis and to grown in an isoleucine-deficient medium. The media used were divide in synchrony. identical in composition to those described above except that @ Further studies indicated that accumulation in resulted they contained no isoleucine and were supplemented with from a depletion of isoleucine and glutamine from the culture dialyzed sera. The sera (10% calf and 5% fetal calf) were dialyzed against Earle's balanced salt solution, pH 7.2, at a 1 This work was performed under the auspices of the U. S. Atomic 1:10 volume for 6 days at 3Â°,with changes of salt solution on Energy Commission. alternate days. All of the above cells were examined routinely 2 Recipient of NIH Postdoctoral Fellowship i-F02-CA43809-01 for contamination with PPLO by the method of Chanock et al. from the National Cancer Institute. Received July 10, 1970; accepted September 18, 1970. 3The abbreviation used is: PPLO, pleuropneumonia-like organism.

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(1), with the modified technique of House and Waddell (12) for preparation of yeast extract. No PPLO were detected in any of these lines during the course of these experiments. A culture of CHO cells was deliberately infected with the PPLO Mycoplasma hyorhinis (kindly provided by Dr. Elliot Levine). This culture (designated PPLO-CHO) became persistently infected and, whenever plated on the agar z described above, gave rise to PPLO colonies. 0 Cell concentrations were determined in monodisperse C) populations of CHO and L-cells to a precision of 1% or better 4 with an electronic particle counter, as described previously 0 w (19). The fraction of cells incorporating thymidine-methyl-3 H 0 > (6 Ci/mmole,SchwarzBioResearch,Inc.,Orangeburg,N.Y.) 0 was measured autoradiographically and scored as described previously (21). 0 0 â€˜U -I RESULTS

For conclusive demonstration of the primary role of isoleucine in reversibly inducing a state of G1 arrest, it is necessary to provide evidence for the pheomenon in cultures maintained in medium singly deficient in isoleucine. Consequently, CHO cells from an exponentially growing culture were washed and resuspended in isoleucine-deficient F-lO medium containing twice the normal concentration of glutamine to prevent glutamine from becoming depleted, HOURS supplemented with dialyzed serum, in a spinner flask. As was Chart 1. Reversal of G1 arrest in cultures of CHO cells maintained shown elsewhere (1 5), there was an immediate reduction in in isoleucine-deficient F-i0 medium. Cultures were grown for 48 hr rate of division, with the cell number increasing by 33% (the in isoleucine-deficient medium containing 2 times the normal approximate number of cells initially in 5, G2 , and M) during glutamine concentration and dialyzed serum, then, at t 0, cells were the first 18 hr after transfer to isoleucine-deficient medium; resuspended in fresh complete medium (A), or to the cultures in thereafter, the cell concentration remained constant, and the deficient medium were added back either 2 times the normal fraction of cells incorporating thymidine-3 H into DNA concentration of isoleucine and glutamine (B) or 2 times the normal dropped essentially to 0. Upon resuspension of these cells in isoleucine concentration only (C). Thymidine-methyl-3 H was added to all cultures at t = 0 to a final concentration of 0.05 zCi/ml, and fresh complete medium (Chart 1A) or after addition to the samples were removed at intervals for autoradiographic determination isoleucine-deficient cultures of either isoleucine and glutamine of the labeled fraction. In this and subsequent charts, labeled or (Chart lB) or isoleucine only (Chart lC), the entire population divided fraction represents N/N0 â€”1. â€¢, fraction labeled with of cells first initiated DNA synthesis (measured by thymidine-3 H; 0, the divided fraction. autoradiographic determination of the fraction of cells labeled with thymidine-3 H, the label being added at t 0) and then contamination on isoleucine-specific synchrony induction was divided in synchrony. In this and later figures, â€œlabeledor determined by growing infected cells to high density in divided fractionâ€• represents N/N0 â€”1. The rate at which the complete medium or by resuspending PPLO-CHO cells in cells entered S and subsequently divided is essentially the same isoleucine-deficient medium supplemented with dialyzed serum in all 3 cultures in Chart 1. The time course and rates of entry (Chart 2A). Also shown in Chart 2A is a noninfected control into S and mitosis in Chart 1 are comparable to values culture grown to high density in complete medium. The obtained in suspension cultures of CHO cells prepared by growth pattern of infected cells in isoleucine-deficient medium mitotic selection, in which it is known that the initial was similar to that observed in noninfected cultures in population occupied less than 1% of the total cell cycle (5, 6, complete medium (1 5), but the infected cells grown in 18, 20, 22) or an initial Engelberg (4) synchrony index of complete medium exhibited 2 grossly different properties 0.97. These results clearly indicate that, in suspension cultures from their noninfected counterparts. The maximum cell of CHO cells, it is possible to arrest cells in G1 and then to concentration in the infected culture was only slightly more induce initiation of genome replication merely by the sole than half that observed in the uninfected culture, and the addition of isoleucine in the medium. PPLO-CHO cells survived much shorter periods in stationary In view of the high incidence of contamination of common phase. Coincident with the fall in cell count was a cell lines with PPLO and documented evidence for proportionate increase in number of cells permeable to trypan competition between cells and PPLO for nutrients [for a blue, which indicates cell death. review of effects of PPLO on cultured cells, see the work of When the high-density infected and control cultures and the MacPherson (16)] , cultures of CHO cells were deliberately culture maintained in isoleucine-deficient medium were infected with the PPLO M. hyorhinis. The effect of PPLO resuspended in fresh complete medium, the resultant division

JANUARY 1971 47

patterns in Chart 2B were strikingly different. (The 00 high-density PPLO-infected CHO culture was resuspended in fresh medium at a time equivalent to t 100 hr in Chart 2A

before cells began dying, and the other infected culture was @0.80 resuspended in fresh medium after 48 hr in suspension I- culture.) Cells in the control culture began dividing 12 hr after resuspension in fresh medium, and the time required for cell U. 0 0.60 number to double was approximately 10 hr. In contrast, both LU of the PPLO-infected cultures began dividing early after 0 > transfer to fresh medium, and the doubling times for the 0 isoleucine-deficient and hIgh-density infected cultures were @0.40 approximately 17 and 18 hi, or approximately that of the 0 Ui nonsynchronized exponentially growing cells from which these -J Ui cultures were set up. It appears, therefore, that it is difficult or impossible to demonstrate the isoleucine-mediated effect upon @0.20 genome replication in cells infected with PPLO, even in cultures maintained in isoleucine-deficient medium. Attempts were then made to determine whether or not

isoleucine played a role in regulating genome replication in HOURS other cell lines. In preliminary experiments, it was determined that arrest of cells in G1 did not occur in cultures allowed to Chart 3. Reversal of G1 arrest in cultures of L-cellsmaintained in isoleucine-deficient minimum essential medium. Cultures of L-cells were grown for 90 hr in isoleucine-deficientminimum essentialmedium containing 2 times the usual glutamine concentration and dialyzed serum. At t 0, cells were resuspended in fresh complete medium (A), or to the isoleucine-defIcient medium were added back either isoleucine and glutamine (B) or isoleucine only (C). Labeling conditions with thymidine-3 H were those described in Chart 1. ., fraction labeled with thymidine-3H; o, divided fraction calculated as described in Chart 1.

grow to high density in standard tissue culture media. These results were not totally unexpected, since the isoleucine content of most tissue culture media is 10 to 200 times that of z the F-lO medium used in our studies with CHO cells. As has been shown previously (1 5), when the concentration of isoleucine in F-b medium was increased 3-fold over normal, CHO cells in suspension culture grew to higher concentrations but, in contrast to cultures grown in normal F-b, did not survive as well, could not be induced to resume traverse of the cycle by addition of isoleucine, and resumed traverse of the cycle in an essentially asynchronous manner. These results @ suggest that, merely by an increase in the isoleucine concentration of 3-fold over normal, cells reach stationary phase due to a depletion of other essential medium components under conditions in which cells stop at various portions throughout the entire cell cycle. Thus, in examination 80 120 @8 16 24 of the isoleucine-mediated phenomenon in other cell lines, it HOURS HOURS was necessary to reduce the concentration of isoleucine to the Chart 2. Effect of PPLO infection on isoleucine-mediated point where depletion of isoleucine only was assured. This was synchrony induction in the CHO cell. A culture of CHO cells was accomplished most conveniently by resuspending cells in deliberately infected with M. hyorhinis. In A , infected cells were medium deficient in isoleucine, supplemented with dialyzed placed in either fresh complete F-10 medium (.) or serum. isoleucine-deficient F-b medium containing 2 times the normal A PPLO-free strain of mouse L cells was maintained for 90 glutamine concentration and dialyzed serum (.), and cell hr in isoleucine-deficient minimum essential medium concentrations were determined with an electronic counter; o, uninfected cells in fresh complete F-10 medium. At a time equivalent supplemented with dialyzed serum. Then the cells were either to t 100 hr in A , the high-density cultures were resuspended in resuspended in fresh complete medium (Chart 3A), or to the fresh complete medium, and the infected culture was placed in isoleucine-deficient culture were added either isoleucine and complete medium after 48 hr in deficient medium. B, resultant patterns glutamine (Chart 3B) or isoleucine only (Chart 3C). All 3 of cell division for the 3 cultures. The divided fraction was calculated as cultures initiated DNA synthesis and cell-cycle traverse in in Chart 1. Symbols are as shown in A. synchrony. As in Chart 1, the fraction of cells incorporating

48 CANCER RESEARCH VOL. 31

thymidine-3 H measured autoradiographically was taken as a suspension with trypsin and/or DNase also failed to yield measure of the fraction of cells entering S. Direct reproducible estimates of cell concentrations. Therefore, determination of cell number with an electronic particle demonstration of isoleucine effects in suspension cultures of counter was possible, since the cultures of L cells were grown Syrian hamster cells was based solely on autoradiographic as monodisperse populations. DNA synthesis preceded cell data. When suspension cultures of Syrian hamster BHK cells division, again indicating that, in isoleucine-deficient medium, were maintained in isoleucine-deficient F-l0 medium L cells had been accumulated in G1 , and the entire population supplemented with dialyzed serum for 48 hr, then resuspended was stimulated to resume traverse of the cycle in synchrony. in fresh complete medium at t 0, the results presented in There was very little variation in pattern of DNA synthesis and Chart 4 were obtained (0, a control culture grown in complete division among the 3 cultures. The time required for cell F-b medium). Under these growth conditions, the time number to double in these cultures (by extrapolation, required for the entire exponentially growing population to disregarding â€œtailingâ€•)wasapproximately 15 hr, compared to become labeled would be approximately 30 hr (i.e., the a doubling time of24 hr for the exponential stock culture. culture doubling time). In contrast, upon transfer from Isoleucine-mediated effects upon cell-cycle traverse were isoleucine-deficient medium to fresh medium, the fraction of also examined in suspension cultures of Syrian hamster cells synthesizing DNA initially was essentially 0, and the time BHK2 1/C 13 cells. Because the BHK cells grew as polydisperse for labeling the entire population would be approximately 18 populations in which the size distribution of clumps of cells hr. Thus, as was observed in CHO and L cells, maintenance of was extremely heterogeneous, it was impossible to obtain BHK cells in isoleucine-deficient medium and subsequent objectively an accurate determination of cell concentration return to fresh medium results in a synchronous initiation of with either an electronic particle counter or with the aid of an DNA synthesis. optical counting chamber. Treatment of aliquots of cell DISCUSSION

The isoleucine-mediated phenomenon originally observed in a Chinese hamster cell line has now been demonstrated in Syrian hamster and mouse cell lines as well. Although a great deal of work has been expended in determination of the @ optimum conditions for induction of reversible arrest in the CHO cell, relatively little work has been carried out with other types of cells examined in the investigation, and we strongly suspect that the conditions described for induction of G1 arrest in Syrian hamster and mouse lines are far from ideal. Manipulation of the experimental parameters (isoleucine concentration, time of incubation in isoleucine-deficient media, etc.) should result in an improved technique for U. regulating cell cycle traverse. Our data clearly indicate that 0 LU isoleucine-mediated regulation of genome replication is not -I LU peculiar to the CHO cell line and may represent a general 4 â€”I regulatory mechanism in vitro. Our data also suggest 2 reasons why this phenomenon has not been demonstrated previously. In view of the high concentrations of isoleucine in standard cell culture media, other media components are far more likely to become depleted. Depletion of other amino acid components of F-l0 medium results in a halting ofCHO cells at stages throughout the cycle (1 5). Furthermore, many commonly used cell lines are persistently infected with PPLO. Our results indicate the difficulty in demonstrating isoleucine effects upon cell-cycle traverse in cultures of cells infected with PPLO. In view of the 8 preceding considerations, it is perhaps not unexpected that a HOURS similar regulatory mechanism has not been detected previously in other in vitro systems. Chart 4. Reversal of G1 arrest in BHK21/C13 cells. A culture was Obviously, a great amount of additional research must be grown for 48 hr in isoleucine-deficient F-10 medium containing 2 carried out before the general biochemical significance of times the usual glutamine concentration and dialyzed serum. At t 0, the cells were resuspended in fresh complete medium, and the 5 ol e u c me-mediated regulation of mammalian genome fraction labeled with thymidine-3 H (.) was determined as described in replication may be appreciated. Amino acid-regulatory Chart 1. An exponentially growing stock culture of BHK cells in mechanisms are of special interest at this time in view of the complete F-10 medium was also labeled with thymidine-3H as an demonstrated growth-inhibitory effects of L-asparaginase on asynchronous control culture (o). certain neoplastic cells [see review by Cooney and

JANUARY 1971 49

Handschumacher (3)]. In future experiments, the although obtained in bacteria and therefore subject to fundamentally important question that must be considered is criticism regarding extrapolation of data from prokaryotes to the biochemical role that isoleucine plays in genome eukaryotes with differences in chromatin organization and regulation. We do not at present possess information on the composition, suggest the existence of an isoleucine-rich isoleucine-requiring mechanism but, among a variety of protein involved in regulation of genome replication. Our possible roles for isoleucine, we consider the following results with CHO cells might suggest the presence of a similar speculations to be most likely in view of current knowledge. isoleucine-requiring factor in mammalian cells. Isoleucine may be an essential constituent of a specific nucleoprotein intimately involved in genome replication. There do not appear to be any gross peculiarities in isoleucine ACKNOWLEDGMENTS content of acidic (1 1) or basic (10, 23) nucleoproteins, although isoleucine-rich regulatory species might comprise a We thank Mrs. Evelyn Campbell, Mrs. Susan Carpenter, and Mrs. very small fraction of the total proteins in the nucleus and, Phyllis Sanders for excellent technical assistance. therefore, might not be detectable in amino acid analyses of bulk nucleoprotein material. Alternatively, isoleucine may be an important constituent REFERENCES of factors implicated in stimulation of DNA synthesis [for example, the â€œwedgeâ€•initiatorreported in the ascitic fluid of 1. Chanock, R. M., Hayflick, L., and Barile, M. F. Growth on mice bearing Ehrlich ascites cells (7, 8)] . Erhan et aL (8) have Artificial Medium of an Agent Associated with Atypical speculated that the initiator from ascitic fluid might play an Pneumoniae and Its Identification. Proc. Natl. Acad. Sci. U. 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17. Meister, A. Valine, Isoleucine, and Leucine. In: A. Meister (ed.), Cultures of Chinese Hamster Cells. J. CellullarComp. Physiol., 70: Biochemistry of the Amino Acids, Ed. 2, Vol. 2, pp. 729â€”757. 63â€”68,1967. New York: AcademicPress,Inc., 1965. 21. Tobey, R. A., and Ley, K. D. Regulation of Initiation of DNA 18. Petersen, D. F., Anderson, E. C., and Tobey, R. A. Mitotic Cells as Synthesis in Chinese Hamster Cells. I. Production of Stable, a Source of Synchronized Cultures. In: D. M. Prescott (ed.), Reversible G1-arrested Populations in Suspension Culture. J. Cell Methods in Cell Physiology, Vol. 3, pp. 347â€”370. New York: Biol.,46: 151â€”158,1970. AcademicPress,Inc., 1968. 22. Tobey, R. A., Petersen, D. F., and Anderson, E. C. Energy 19. Petersen, D. F., Tobey, R. A., and Anderson, E. C. Essential Requirements for Mitosisin Chinese Hamster Cells.In. R. Baserga Biosynthetic Activity in Synchronized Mammalian Cells. In: G. M. (ed.), Biochemistry of Cell Division, pp. 39â€”56.Springfield, Ill. Padilla, G. L. Whitson, and I. L. Cameron (eds.), The Cell Cycle: Charles C Thomas, Publisher, 1969. Gene-Enzyme Interactions, pp. 34 1â€”359.New York: Academic 23. Venderly, R. The Enzymatic Degradationof Nucleohistones.In: J. Press, Inc., 1969. Bonner and P. Ts'o (eds.), The Nucleohistones, pp. 307â€”314.San 20. Tobey, R. A., Anderson, E. C., and Petersen, D. F. Properties of Francisco: Holden-Day, Inc., 1964. Mitotic Cells Prepared by Mechanically Shaking Monolayer

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R. A. Tobey and K. D. Ley

Cancer Res 1971;31:46-51.

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