Adaptation of cultured human lymphoblasts to growth in citrulline

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Experimental Cell Research 84 (1974) 167-174

ADAPTATION

OF CULTURED HUMAN LYMPHOBLASTS IN CITRULLINE LEE BRECKENRIDGE

TO GROWTH

JACOBY

Genetics Unit, Massachusetts General Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Mass. 02114, USA

SUMMARY Growth and enzymic studies of the urea cycle have been carried out in long-term human lymphoblast cultures. Lymphoblasts could utilize argininosuccinate in place of arginine; however, only 2 of 8 lines could immediately utilize citrulline in place of arginine, the other 6 lines exhibiting lag periods of a few days to a few weeks before growing in arginine-deficient medium supplemented with citrulline. Adaptation of the entire population rather than the outgrowth of exceptional variants during the lag period was suggested by the ability of cells to form clones in citrulline as efficiently as in argininesupplemented media and by the phenotype of clones selected in complete medium, most of which exhibited delays before growth in citrulline similar to those of the parental lines. This ability to grow in citrulline was not lost after growth for 64X-80generations in arginine. Citrulline itself in the presence of arginine did not act as an inducer. Argininosuccinate synthetase activity appeared to be significantly enhanced in the citrulline-adapted derivatives, while argininosuccinase activity remained constant in the parental lines and variants. Selective adaptation of urea cycle enzymes has not been observed in other human diploid lines in culture, and it may reflect a flexibility in gene expression characteristic of these immunoglobulin-producing cells.

Cultured human lymphoblasts are particularly useful in the study of cellular control mechanisms becausethey grow in suspension,produce immunoglobulins and other cell-specific immune factors, do not senescein culture as do human fibroblasts, and retain a diploid or near-diploid chromosome complement, at least for many months [9, 141.Relatively little, however, is known yet about their intermediary metabolism in comparison with human fibroblasts. The present studies concern the urea cycle enzymes in these cells. Studies on the samepathway have also begun elsewhere [l]. In liver, the complete urea cycle generates urea from carbon dioxide and ammonia and also produces arginine for protein and polyamine synthesis. For cells in culture, however, arginine is an essential amino acid because

their complement of urea cycle enzymesis incomplete [5, 161.Human fibroblasts in culture cannot utilize ornithine in place of arginine becausethey lack ornithine transcarbamylase, but they can utilize citrulline or argininosuccinate becausethey contain argininosuccinate synthetase [ 151 and argininosuccinase [ 131. The present work shows that lymphoblast lines vary in their ability to grow in citrulline and that some demonstrate a novel and specific adaptation involving argininosuccinate synthetase. MATERIALS AND METHODS Cells and culture conditions Lymphoblast lines MGL3 and MGL4 (normal males), MGLS (female, infectious mononucleosis patient) and MGL6 (male, translocation and ring chromosome) were initiated in this laboratory using lysates of Exptl Cell Res 84 (1974)

168 Lee Breckenridge Jacoby MGL33C19 and/or stimulation with PHA [3]. MGL33C19 is a thrice-cloned derivative of PGLC33H (or MGL33) (female, infectious mononucleosis patient) obtained from Glade [2]; UM-10 (or MGL35) (male, Lesch-Nyhan syndrome) was obtained from Bloom [4]; MGL38Al is a once-cloned derivative of P3HR-l(Buj. a 5-bromodeoxvuridine-resistant mutant of PjHk-1 (male, Burkitt’slymphoma), obtained from Hamoar 171:and CCRF-CEM (or MGL39) (female, acute lympiddlastic leukemia) was obtained from Lazarus [6]. (According to the nomenclature system in use in this laboratory, once-cloned lymphoblast lines are assigned the letter “A”, e.g. MGLSAl, and twice-cloned lines the letter “B”, e.g. MGLSBl, etc.) Lvmohoblasts were routinely maintained in log phase in RPM1 1640 medium (GIBCo) supplemented with 15 % fetal calf serum (Gray, MBA). Most experiments were performed in arginine-free Eagle’s minimal essential medium (GIBCo) supplemented with nonessential amino acids, 15 % fetal calf serum, and 0.6 mM L-ornithineHC1. 0.6 mM L-citrulline. 0.6 mM L-argininosuccinate (prepared from the bar&n salt by precipitation with potassium sulfate immediately before use), or 0.6 mM L-arginine-HCl. The serumsupplemented Eagle’s medium without arginine (MEMA) is termed ‘arginine-deficient’ rather than ‘arginine-free’ since the 15 % fetal calf serum supplement contributes a trace amount of a&nine, about 6 pM (Shih, unpublished). Citrulline-adapted lines were routinely maintained in MEMA plus citrulline. For nutritibnal tests, exponentially -growing cells were harvested bv centrifuaation. concentrated lo-fold in MEMA, and-inoculateh intd T30 flasks (Falcon) with 1.0 to 1.5 x lo5 cells/ml, usually in duplicate. Cultures were counted daiiy and were refed at least once weekly by centrifugation and resuspension in an equal volume of fresh medium. Lymphoblasts were cloned in 0.22 % agarose as previously described [ll] with a feeder layer of human fibroblasts MGF326, deficient in hypoxanthine-guanine phosphoribosyltransferase, or MGF407, deficient in argininosuccinate synthetase, in experiments with MEMA. All lines were routinely monitored for contamination with mycoplasma in the laboratory of L. Dienes [8] and tested negative.

Enzyme assays Argininosuccinate synthetase was kindly assayed by Dr N. G. Kennaway (Portland, Ore.) in lysates of late log phase cells according to the radiochemical procedure of Schimke [12], with minor modifications found to give optimal results with human fibroblasts. The conversion of L-[ureido-14C]citrulline to [14C]urea was measured in a coupled assay system containing excess argininosuccinase and arginase. L-(Ureido-*4Cjcitrulline was nurchased from Amersham Searle and freed from contaminating urea by chromatography on Dowex 50-X12. Argininosuccinase was prepared from human liver by themethod of Ratner [lo] & far as the heat denaturation step, followed by repeated ammonium sulfate precipitations at 30% saturation. Arginase was purchased from Worthington. Blanks containing boiled cell extract gave the same values as

Exptl Cell Res 84 (1974)

blanks containing no extract. The backgrounds were usually about 300 dpm/assay and, in the case of fibroblasts, the assay was linear to over 2 000 dpm. Argininosuccinase was measured spectrophotometrically in sonicates of late log phase cells as the rate of urea formation from argininosuccinate in the presence of excess arginase, as previously described [131.

RESULTS Nutritional tests in arginine-deficient medium (MEMA) supplemented with ornithine, citrulline, argininosuccinate or arginine were initially carried out with MGL33C19 (fig. 1). This line grew with a doubling time of about 24 h in MEMA plus arginine or argininosuccinate, as it did in RPM1 1640 medium (data not shown), but it did not grow in MEMA plus citrulline or ornithine during the first 7 days of incubation. Growth was observed, however, in MEMA plus citrulline after 28 days of incubation. Therefore the growth of several lymphoblast lines in citrulline and arginine was examined (fig. 2). Two lines, CCRF-CEM and UM-10, showed no lag before growth in citrulline, while four lines, MGL3, MGL4, MGL5 and MGL6, showed a lag of 3 to 6 days. In addition, MGL33C19 again showed a long lag, and one line, MGL38A1, failed to grow at all after 35 days of incubation in MEMA plus citrulline. In all cases, the control cultures in arginine grew at rates comparable to those observed in citrulline but with no appreciable lag period. To determine whether growth in MEMA plus citrulline resulted from adaptation of the mass culture or from selection of pre-existing citrulline prototophs from a mixed population of cells, we examined two representative lines, MGLS, a recently established diploid line that adapted quickly, and MGL33C19, a pseudo-diploid line in culture for several years that adapted slowly in citrulline. The frequency of citrulline prototrophs pre-existing in the populations of MGL33C19 and MGL5 was estimated by cloning directly in

169

Adaptation of lymphoblasts to citrulline

I 0

I 2

I

I 4

I

1 6 t 0

t 0

I

,t,

t 10

,t,

20

t,

30

Fias 1. 2. 3. 4. 5. 6. Abscissa: incubation time (days); or&t&:‘ceilsjmi x KP. Fip. 1. Growth of MGL33C19 in A, MEMA+ornithme:; 4, citrulline; A, argininosuccinate; or 0, argmme. Fig. 2. Growth of lines x , CCRF-CEM; 0, UM-10; 0. MGM: W. MGLS: A. MGW: A. MGL4: 0, MGL33C19; or 0, MG’L3gAl in MEMA +citruhine: Cells were inoculated into MEMA +citrulline and MEMA +arginine (not shown) and refed weekly (indicated by arrows). Fig. 3. Growth of MGL33C19 clones in MEMA+ citrulline. Cells were inoculated into MEMA+ citrulline and MEMA + arginine (not: shown) rand refed weekly (arrows). MGL33C19 (o--0)lwas included as control.

, t , t, ,t , t, 1 10

20

30

MEMA plus citrulline. For each line a citrulline-adapted derivative served as the control. Under conditions in which citrullineadapted MGL33D3 formed clones after 3 weeks incubation, less than 0.1% of the MGL33C19 population was able to grow in citrulline after 8 weeks incubation (table 1). No adaptation to growth in citrulline was observed in MGL33C19 during this period, although it might have been expected by

extrapolation from:the mass culture data if the adaptation itself required 3 or 4 weeks, and the clones required an additional 3 weeks for growth. MGLS, on the other hand, cloned poorly in all media, including RPM1 1640, as generally observed for recently established lines, but it cloned as well in citrulline as in arginine, just like a citrulline-adapted subline, MGL5Al (table 1). The MGL5 clones in citrulline, however, were visible only after Exptl Cell Res 84 (1974)

170 Lee Breckenridge Jacoby Table 1. Cloning ability in supplemented MEMA

media

Supplement Ornithine

Citrulline

Arginine

Cell line

Clones

C.E.C %

Clones

C.E. %

Clones

C.E. %

MGL33C19 MGL33D3 MGLS MGL5Al

8::

< 0.07 < 0.08 co.04 co.02

Ob 229a IlOb 253’

c 0.04 25 2.3 3.1

523a 161= 64’ 107=

40 18 2.7 2.4

Lines were cloned in MEMA + ornithine, citrulline or arginine as described in Materials and Methods. 150 cells of MGL33C19 and MGL33D3 and 300-600 cells of MGLS and MGLSAl were seeded per 60 mm Petri dish. a Clones appearing after 3 weeks incubation. b Clones appearing after 8 weeks incubation. ’ Cloning efficiency.

about 4 weeks, in contrast to the MGLS clones in arginine and the MGL5Al clones in citrulline which were fully grown after 3 weeks. The delayed appearance of all the clones in citrulline suggested that the whole population was able to adapt to growth in citrulline. Moreover, the fact that none of the four lines was able to form clones in MEMA plus ornithine indicated that growth in MEMA plus citrulline was due to utilization of citrulline and not due to utilization of trace amounts of arginine contributed by the medium or the feeder layer of fibroblasts. The phenotype of clones of MGL33Cl9 and MGLS, independently isolated in RPM1 1640 medium containing arginine, was then examined. At least 9 of 11 MGL33C19 clones and 7 of 7 MGLS clones exhibited delays before growth in citrulline similar to those of the parental lines (figs 3, 4). Control cultures in arginine grew at rates comparable to those in citrulline but with no appreciable lag, except for 3 slow-growing MGL5 clones, which exhibited lags in all media and therefore were not included in fig. 4 for clarity. Failure of 2 MGL33Cl9 clones to grow in citrulline could have resulted from low viability after several weeks in a small volume of starvation medium, and it was considered more Enptl Cell Res 84 (1974)

significant that none of the clones from either line grew in citrulline without a lag period. These data suggestedthat the acquisition of ability to grow in citrulline was occurring rapidly (MGLS) or slowly (MGL33C19) in most if not all cells of the culture, thus indicating that adaptation of the mass culture rather than selection of exceptional cells was the basis of the lag phenomenon. The slightly longer lag times of the 9 clones derived from MGL33C19 compared with their parent and of the 7 clones derived from MGL5 compared with their parent suggested that cloning itself might select for cells able to grow less well in MEMA plus citrulline. Indeed, reexamination of the data in fig. 2 revealed that the two lines with long lags in citrulline, MGL33C19 and MGL38A1, were the only two lines that had been recloned. Accordingly, the behavior of PGLC33H [2], the progenitor of MGL33C19, was examined. It had a lag of only 4 to 6 days before growth in MEMA plus citrulline, in support of this conclusion. To determine if the stimulus for adaptation was arginine starvation or the presence of citrulline, MGL33C19 and MGL5 were grown in MEMA plus both citrulline and arginine for 10 to 14 generations and then

Adaptation of lymphoblasts to citrulline

171

5

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t0

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t

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IO

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subcultured in MEMA plus citrulline. The times required for adaptation in these cultures were identical to those in control MGL33C19 and MGL5 cultures grown in MEMA plus arginine alone, thus suggesting that the presence of citrulline was not sufficient for adaptation to take place. Reversal of this adaptation in two citrulline-adapted lines, MGL33D3 and MGLSAl,

t 0

I

I 2

I

I 4

I

I 6

Fig. 4. Growth of MGLS clones in MEMA + citrulline Cells were inoculated into MEMA + citrulline and MEMA +arginine (not shown) and refed weekly (arrows). MGL5 (O--O) was included as control. Fig. 5. Growth of MGL33D3 in MEMA + citrufline after l , 0, n , 2, A, 10; A, 40, or 0, 80 generations in MEMA + arginine. Fig. 6. Growth of MGL5 and MGL5Al in MEMA + citrulline or MEMA + arginine after 60 generations in MEMA +arginine. MGLS in O-O, MEMA + MGLSAl in MEMA +arginine; arginine; o-o, 0 -- 13, MGLSAl in MEMA + citrulline; and U--W, MGLS in MEMA + citrulline.

was then studied. MGL33D3 did not completely lose its ability to grow in citrulline after 80 generations in arginine (fig. 5); however, it did have a slightly altered phenotype, with a lag of about 25 h (range 14-29 h) before growth in citrulline, in contrast to the fully adapted line with no lag or the parent with a lag of about 3 weeks. MGLSAI similarly did not completely deadapt after 60 generaExptl Cell Res 84 (1974)

172 Lee Breckenridge Jacoby

Table 2. Argininosuccinate argininosuccinase activities

synthetase and

Cell line

Argininosuccinate synthetase

Argininosuccinase

MGL33C19’ MGL33D3b MGLS= MGL5Al b

< 0.001 0.021 < 0.001 0.024

0.051 0.035 0.056 0.058

Assays were performed on extracts of late log phase cells grown in an RPM1 1640 or ‘MEMA + citrulline. Activities are expressed as pmoles product/mg protein/h.

values of the specific activities presented in table 2 must be considered preliminary, however, because although enzyme activity was proportional to protein concentration at low concentrations, at higher concentrations enzyme activity diminished disproportionately. Further studies are in progress to determine whether an inhibitor of the synthetase activity is present. Argininosuccinase activities, in contrast, were essentially the same in the variants as in the parental lines (table 2). DISCUSSION

Human lymphoblast lines are heterogeneous with respect to their growth patterns in tions in arginine (fig. 6), although it too ac- citrulline-supplemented medium. For such quired a lag of about 26 h (range 19-36 h) lines as MGLS that adapted in 2 or 3 days to before growth in citrulline. The phenotype of growth in citrulline, uniform adaptation of MGLSAl remained distinct from that of the entire population, rather than the outMGLS, however, which had a further lag in growth of exceptional variants, was suggested citrulline of at least 21 h when examined in by (1) the failure of MGLS cells to clone immediately in citrulline; (2) the delayed apparallel with MGLSAI (fig. 6). The growth patterns of MGL33D3 and pearance of all MGLS clones in citrulline with MGL5Al in MEMA plus citrulline after a final plating efficiency equal to that in growth in MEMA plus arginine were re- arginine; (3) the growth in citrulline of indemarkably similar. To exclude inadvertant pendently isolated clones of MGLS with a cross-contamination each line was checked lag similar to that of MGLS. This adaptation for sensitivity to 1 X 1O-4M thymidine. Like wasremarkably stable since,evenafter 60geneMGL33C19 [17], MGL33D3 after long-term rations in citrulline-free medium, MGLSAl passagein MEMA plus citrulline or arginine could adapt faster than the parental line to was thymidine-sensitive, while MGLSAl in citrulline. In contrast, MGL33C19 adapted to growth MEMA plus citrulline or arginine, as with MGLS, was thymidine-resistant, making in citrulline only after 3 weeks. No cells (less than 0.1%) cloned directly in citrulline, cross-contamination unlikely. but this was not a conclusive test as prolonged Growth in citrulline requires argininosuccinate synthetase and argininosuccinase to starvation may have prevented the outgrowth convert citruhine to arginine. Therefore the of truly competent members of the populalevels of these two enzymes in MGL33C19, tion. When individual clones of MGL33C19 MGLS and their derivatives were measured were tested, at least 9 of 11 grew in citruIline (table 2). While neither of the parental lines after a long lag period similar to that of the had detectable argininosuccinate synthetase parent. Furthermore, all nine lines demonactivity, the citrulline-adapted variant of each strated similar growth kinetics. If the eventual line had significant activity (table 2). The outgrowth of mutants were the basis of the Exptl Cell Res 84 (1974)

Adaptation of lymphoblasts to citrulline prolonged lag, greater variation in lag times would have been expected, as individual clones would have contained various numbers of mutant cells. After growth in citrullinefree medium, MGL33D3 did not revert to the parental phenotype but, like MGLSAI, came to have a lag of about 1 day. This may represent the obligatory time required for adaptation in these lines, while for somelines, like MGL33C19, further time is required for one or more additional events which are prerequisites for adaptation, such as the formation of a citrulline permease. Further studies will be required to determine why some lymphoblast lines have lost the ability to adapt rapidly to citrulline. There appears to be a correlation between recloning and a longer lag before growth in citrulline-supplemented medium. Thus PGLC33H, the parent of MGL33C19, had a lag in citrulline-supplemented medium of 4 to 6 days, while individual clones of MGL33Cl9 had slightly longer lag periods than their parent. A similar type of adaptation was observed recently in a human lymphoblast line derived from a patient with citrullinemia, a metabolic disorder characterized by a deficiency in liver argininosuccinate synthetase [l]. This line, like MGL33C19, grew in arginine-deficient medium plus citrulline after a lag of about 4 weeks, raising the possibilities that the lymphoblast argininosuccinate synthetase is distinct from the liver enzyme or that there is an alternate pathway of citrulline utilization in these cells. While the genetic basis for the growth of MGL33C19 and MGLSAI in citrullinecontaining medium remains unsettled, the biochemical basis seemsto be increased argininosuccinate synthetaseactivity. Since citrulline in the presence of arginine did not promote adaptation, this increase in activity is tentatively termed a ‘depression’, although its molecular basis is as yet unknown. This de-

173

repression was specific for the synthetase, since argininosuccinase activity remained unchanged and ornithine transcarbamylase and arginase activities remained undetectable in the citrulline-adapted lines (table 2; Snodgrass & Jacoby, unpublished). This specific derepression contrasts with the coordinate regulation of argininosuccinate synthetase and argininosuccinase observed in several heteroploid human lines [12]. In HeLa or KB cells grown under conditions of limiting arginine, the argnininosuccinate synthetase and argininosuccinase levels increased 1.5- to 15-fold in a coordinate fashion. Moreover, the increaseswere complete within one generation and were diluted out rapidly by subsequent growth under conditions of high arginine in the medium. This type of derepression contrasts as well with that of normal human fibroblasts in culture, which showed no increasein argininosuccinate synthetaseactivity when grown in arginine-deficient medium with or without citrulline [15]. A citrullinemic fibroblast strain, believed to have a defective argininosuccinate synthetase with a higher K,,, for citrulline, was also examined and similarly showed no increase in synthetase activity in these media [15]. Indeed, thesestudies, together with attempts in this laboratory to alter the arginininosuccinase levels in normal or argininosuccinase-deficient fibroblasts (Shih & Littlefield, unpublished), suggestthat bacterial-like regulation of these two urea cycle enzymes does not exist in human diploid fibroblasts. The adaptation observed in human lymphoblasts is therefore a novel finding, and it may reflect a flexibility in gene expression also thought to underly the ability of these cells to produce immunoglobulins. I am grateful to Dr J. W. Littlefield for helpful advice, to Drs N. G. Kennaway and P. J. Snodgrass for some of the enzyme assays, and to Mrs V. Owyangland Miss S. Tsairis for expert technical assistance. This investigation was supported by ACS (Mass. Div.) grant 1406 and by USPHS grant CA14534. Exptl Cell Res 84 (1974)

174 Lee Breckenridge Jacoby REFERENCES 1. Spector, E B & Bloom, A D, Pediat res 7 (1973) 700. 2. Chessin, L N, Glade, P R, Kasel, J A, Moses, H L, Herberman, R B & Hirshaut, Y, Ann internal med 69 (1968) 333. 3. Choi, K W &Bloom, AD, Nature 227 (1970) 171. 4. - Science 170 (1970) 89. 5. Eagle, H, Science 130 (1959) 432. 6. Foley, G E, Lazarus, H, Farber, S, Uzman, B G, Boone, B A, & McCarthy, R E, Cancer 18 (1965) 522. 7. Hampar, B, Derge, J G, Martos, L M & Walker, J L, Proc natl acad sci US 68 (1971) 3185. 8. Madoff, S, Ann NY acad sci 79 (1960) 383. 9. Povey, S, Gardiner, S E, Watson, B, Mowbray, S, Harris, H, Arthur, E, Steel, C M, Blenkinsop, C & Evans, H J, Ann hum genet 36 (1973) 247.

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10. Ratner, S, Methods in enzymology (ed S P Colowick & N 0 Kaplan) vol. 2, p. 365. Academic Press, New York (1955). 11. Sato, K, Slesinski, R S & Littlefield, J W, Proc natl acad sci US 69 (1972) 1244. 12. Schimke, R T, J biol them, 239 (1964) 136. 13. Shih, V E, Littlefield, J W & Moser, H W, Biochem genet 3 (1969) 81. 14. Steel, C M, Nature 233 (1971) 555. 15. Tedesco, T A & Mellman, W J, Proc natl acad sci US 57 (1967) 829. 16. Tytell, A A & Neuman, R E, Exptl cell res 20 (1960) 84. 17. Zielke, H R & Littlefield, J W, Methods in cell physiology (ed D M Prescott) voi. 8, p. 107. Academic Press, New York (1973). Received September 17, 1973