Znt. J. Cancer: 20, 296-302 (1977)

A MOUSE LEUKEMIA CELL MUTANT RESISTANT TO BLASTICIDIN S Michihiko KUWANO, Katsuko MATSUI,Kenji TAKENAKA, Shin-ichi AKIYAMA and Hideya ENDO Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan

After nitrosoguanidine mutagenesis we isolated from mouse leukemia L5178Y cells a mutant cell (Bla-R) resistant to blasticidin S, an inhibitor of protein synthesis. Neither growth nor leucine incorporation into hot-acid insoluble fraction of Bia-R cell was inhibited by 5 to 20 pg/ml blasticidin S, which almost completely blocked protein synthesis as well as growth of the parental L5178Y cells. However, other inhibitors such as fusidic acid, cycloheximide, r i c h D or L-asparaginase blocked protein synthesis in Bla-R cells to the same extent as in L5178Y cells, Protein synthesis in vitro using S-30 extracts from the parental cell line L5178Y was almost completely blocked in the presence of the antibiotic, while no inhibition by blasticidin S occurred when S-30 extracts from Bla-R mutant cells were used. Protein synthesis assays were made by using the SlOO fraction from rat liver together with ribosomes from either L5178Y cells or Bla-R cells. Blastkidin S inhibited protein Synthesis when ribosomes were derived from L5178Y cells, but not from Bla-R mutant.

Remarkable progress has been made in the isolation of eucaryotic mutant cells by various selective procedures such as nutritional deficiency, drug resistance or conditional lethal mutation (Basilico and Meiss, 1974; Kao and Puck, 1974; Siminovitch, 1976). For an understanding of the physiological role of the steps involved in transcription or translation processes in mammalian systems, the isolation of genetic mutants is a powerful investigative approach. For example, temperature-sensitive mutants have been useful in the analysis of ribosomal RNA maturation (Toniolo et al., 1973), function of the 60s ribosome (Haralson and Roufa, 1975) or aminoacylation of transfer RNA (Thompson et al., 1973; Sato, 1975). Chinese hamster ovary (CHO) cells resistant to a-amanitin (Chan et al., 1972) or to cycloheximide (Poche et al., 1975) have been isolated and the mutations demonstrated in the RNA polymerase and ribosomes respectively. Thus, one may expect mutations at either the translational or the transcriptional level among drug-resistant mutants if the drug used for selection has a specific action on either process. In this paper, we report a mutant of mouse leukemia cells resistant to high doses of blasticidin S (BlaS), an inhibitor of protein synthesis (Yamaguchi et al., 1965; Yamaguchi and Tanaka, 1966; Battaner and Vazquez, 1971).

MATERIAL AND METHODS

Cefl fines

Isolation of mutants was made from the parental wild-type cell, mouse lymphatic leukemia cells (L5178Y), which require L-asparagine for growth, as described previously (Cooney and Handschumacher, 1970; Nakashima et af., 1976). The procedure for isolation of blasticidin-S-resistant cells (Bla-R) is described in " Results ". The cells were cultured in MEM medium (Nissui Seiyaku Co., Tokyo) containing 10% fetal calf serum (Difco Lab., Detroit) and the required amino acids. The number of viable cells grown in the presence of various doses of chemical agents or antibiotics was determined by staining with trypan blue. Chemicals

Blastkidin S (BlaS) was kindly given to us by Kaken Chemical Co., Tokyo. Highly purified ricin D was kindly donated by Dr. G. Funatsu of the Faculty of Agriculture, Kyushu University. Cycloheximide (Wako Pure Chemical Co., Tokyo), fusidic acid (Sankyo Pharmaceutical Co., Tokyo), Lasparaginase (Kyowa Hakko Co., Tokyo) and actinomycin D (Merk Scharp, Dohme, Rahway, N. J.) were used in this study. SH-leucine (60Ci/ mmol), 14C-leucine (314 mCi/mmol), 14C-uridine (472 mCi/mmol) and SH-uridine (25.9 Ci/mmol) were purchased from New England Nuclear, Boston, Mass. Protein synthesis in intact cells

Exponentially growing cells (5 x 104/ml) were incubated with 1 pCi/ml 3H-leucine in the absence or presence of antibiotics. Aliquots (1 ml) were chilled by adding 9 vol of cold phosphate-buffered saline (containing 0.2 g KCI, 8 g NaCI, 0.2 g KH2POI, 1.15g Na,HP0,.12H20 per liter of water) and centrifuged at 2,OOOg for 5 min. The sediments suspended in 2 ml of 10% trichloroacetic acid were boiled for IOmin and filtered through glass-fiber paper (Whatman GF/c), then washed twice with Received: March 11, 1977 and in revised form May 9, 1977.

297

BLASTICIDIN-S-RESISTANT MUTANT CELLS TABLE I1

10 ml of 5 % trichloroacetic acid, 0.1 M hydrochloride solution, and finally once with ethanol. The radioactivity on the filter was counted. I n the experiment described in Table I showing the effect of various antibiotics on protein synthesis of intact cells, 2 x lo6 cells/ml were exposed to various chemicals or antibiotics for 24 h and the incorporation of 1 pCi/ml 3H-Ieucine during incubation for 3 h was measured.

Rat liver L5178Y

Preparation of extracts of the cultured cells

Rat liver

Suspension cultures of L5178Y and Bla-R cells were grown in 300 ml culture medium, and the cells were harvested by centrifugation then washed twice with phosphate-buffered saline. About 2-3 x lo8 cells were suspended in 2 ml of hypotonic buffer (10 mM Tris.HCI PH 7.6, 10 mM KCI, 1 mM magnesium acetate), and the suspension was transferred to a cold Dounce homogenizer. After swelling for 20 rnin in an ice-water bath, the cells were lysed using 40 strokes of a tight-fitting pestle, and the concentration of magnesium acetate and dithiothreitol in the homogenate was adjusted to 10 mM and ImM respectively. The extracts were centrifuged at 30,000 g for 20 rnin at 4" C and the S-30 supernatant fluid was stored. SlOO and ribosomal fractions were obtained from the S-30 extracts after centrifugation at 105,000g for 120 min and the pellets corresponding to the ribosomal fraction were suspended in 10 mM Tris-HCI PH 7.6, 10 mM magnesium acetate, 1 mM dithiothreitol to a final concentration of 80 to 100 A260 units/ml. SlOO fractions from rat liver were obtained according to Baliga et al. (1968). Cell-free protein synthesis directed by endogenous mRNA

The reaction mixture contained per loop1 per assay: 50 mM Tris-HCI PH 7.6; 5 mM magnesium

TABLE 1 THE EFFECT OF VARIOUS INHIBITORS O N LEUCINE INCORPORATION IN LS178Y A N D Bla-R CELLS ~

Radioactivity incorporated (cmp) Agents added

Dose

perm'

(%)

Bla-R

(%)

1215 509 297 153 122 120 50 176 47

(100) (42) (24) (12) (10) (9) (4) (14) (3)

1152 737 403 121 153 109 56 1173 1020

(100) (63) (34) (10) (13) (9) (4) (101) (88)

Radioactivity incorporated (cpm) ' t:::!e

Ribosomes

~~

Bla-R

No treatment

Elasticidin S (10 &ml)

536 762 770 906

202 171 1013 1014

Ricin D (10 &ml)

187

217

-

~~

From each value obtained after 60 min incubation at 37" C, radioactivities at time 0 (302cpm when LS178Y ribosome was used and 282cpm when Bla-R ribosome was used) were subtracted respectively.

acetate; 50mM KCl; 5 mM ATP; 1 mM GTP; 5 mM phosphoenolpyruvate; 1 pg pyruvate kinase; 25 mM 2x M each of the glutathione; 2 pCi 14C~leucine; 19 unlabelled amino acids except leucine and 54 pg of L5178Y S-30 or 52 pg of the Bla-R S-30 fraction. When Sl00 and ribosomal fractions were used in the assay, 180pg of SlOO from rat liver was combined with 36 pg L5178Y ribosomes or with 39 pg Bla-R ribosomes in each assay (see Table 11). After incubation at 37" C, reactions were terminated by adding 2 ml of 10% trichloroacetic acid, and then the radioactivity of the boiled sample was determined as described above. Analysis of polysome profiles

Cells (2-3 x 106/ml) in 10 ml medium containing L-asparagine, preincubated for 15 h with 14C-uridine (1 pCi/ml), were pulse-labelled for 20 min with 3Huridine (10 pCi/ml) in the presence of actinomycin D, 0.02 pg/ml. Cytoplasmic extracts were prepared in buffer (50 mM Tris-HCI PH 7.4, 100 mM KCI, 5 mM magnesium acetate) containing 0.2 % Triton X-100 as described previously (Kuwano et al., 1973). The supernatant fraction of cell lysates was placed onto a linear density gradient of 10 to 40 percent sucrose (w/v) and centrifuged as reported previously (Nakashima et al., 1976). RESULTS

None Fusidic acid

50pg 1OOpg Cycloheximide 2pg

Rick D 0.5pg L-asparaginase 5 units 10 units Blasticidins S 5pg 20 Pi3 1

L5178Y

THE EFFECT OF BLASTICIDIN S A N D RICIN D ON PROTEIN SYNTHESIS IN VITRO

Radioactivity at time 0 (LOO cpm) was subtracted from each value.

Isolation of a blasticidin-S-resistant mutant

In order to isolate mutants of mammalian cells altered at the translational level of protein synthesis, BlaS, (Yamaguchi et al., 1965; Yamaguchi and Tanaka, 1966; Battaner and Vazquez, 1971), was used for the selection. Cell survival of mouse leukemia L5178Y cells to BlaS is shown in Figure 1 . The fraction of viable leukemia cells was logarithmically decreased after treatment for 18 h or 30 h with

298

KUWANO ET AL.

The effect of BlaS on viability of L5178Y and Bla-R cells was compared. As shown in Figure 2, the number of surviving parental L5178Y cells decreased to below 3 % of the control value with 5 ,ug/ml BlaS, while BlaS had no effect on the Bla-R mutant at this concentration. However, survival of Bla-R fell to about 50% when the cells were exposed t o 50 pglml BlaS for 3 days (Fig. 2). Thus, Bla-R cells were highly resistant to BlaS and the mutation was stable. To study in greater detail the effect of BlaS on both cell lines, kinetics of cell growth was observed in the presence of varying doses of BlaS (Fig.3). The Bla-R cells grew at a slightly reduced rate even in the presence of 50 pg/ml of BlaS (Fig. 3 ~ )However, . the growth of the parental cell was fully blocked even with 5,ug/ml of BlaS (Fig. 3 ~ ) Since . temperature sensitivity is sometimes found among drug-resistant

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The effect of various doses of blasticidin S on cell survival of L5178Y cells. Cells (3 x 105/ml)were incubated without or with blasticidin S and survival was calculated and 30 h (0-0) respectively. at 18 h (e-0)

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various concentrations of BlaS. The viable cell number was below 0.3 % of the control after 30 h incubation in 30pg/ml BlaS. No viable colonies remained after incubation for 2 days (see also Fig. 3). Thus, the BlaS concentration at which mutants could possibly be selected was in the range of 10 to 20 ,ug/ ml. Exponentially growing L5178Y cells (about lo7 cells) cultured in MEM medium containing 10%fetal calf serum (Nakashima et a[., 1976) were treated with nitrosoguanidine (3 pgfml) for 20 h. The medium was changed and the cells cultivated to maximal density (lo6 cells/ml) for 2 days. These cells were plated in agar (Macpherson and Montagnier, 1964) containing lOpg/ml BlaS. Several colonies appearing in the agar were cultured independently for 4 months in MEM containing 10 ,ug/ml BlaS. Finally, resistant cells were cloned in agar and the isolated strain was called Bla-R.

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3

10

n

E

3

= lo2 1020 50 100 Bla S Concentration (pg/mi) FIGURE 2

Comparison of the effect of various doses of blasticidin S cell survival of L5178Y and Bla-R cells. Cells ( 5 x lo4 cells/ml) were incubated for 3 days in blasticidin S and a number of viable cells were measured.

299

BLASTICIDIN-S-RESISTANT MUTANT CELLS

In vitro cell-free protein synthesis with extracts from L5178 Y and Bla- R cells

24 48 72

24 48 72

T i m e (hr) FIGURE 3

Growth of L5178Y (A) and Bla-R (B) cells in the absence (0-0) or the presence of 5pg/ml (0-o), 20pg/ml (A-A) or 50pg/ml blasticidin S (A-A).

Before determining whether the mutation in the Bla-R strain affected the protein synthesizing apparatus, we analysed the polysome sedimentation pattern of cytoplasmic extracts of exponentially growing L5178Y and Bla-R cells by sucrose density gradient centrifugation. Newly synthesized messenger RNA has been shown to be incorporated into polysomes after 20 min labelling with 3H-uridine of growing L5178Y cells (Nakashima et a/., 1976). The rate of incorporation of pulse-labelled messenger RNA into polysomes of Bla-R cells was similar to that of L5178Y cells (Fig. 5 ~ B)., However, in six independent experiments we observed a remarkable accumulation of monosomes as shown in Figure 5B, while no such accumulation could be found in the parent strain (Fig. 5 ~ ) Figure . 5c shows that the accumulation of monosomes in the cytoplasm of Bla-R cells was almost completely reversed when a low dose of cycloheximide was added to the cells during incubation. In order to detect a possible deficiency in protein synthesis due to the mutation, we prepared cell extracts (S-30 fraction) from wild-type and Bla-R cells. Endogenous messenger RNA-dependent incorporation of leucine into peptides was found to be strongly inhibited by 10 pg/ml BlaS when S-30 of the parental L5178Y cells was used, while the antibiotic at the same concentration failed to inhibit Bla-R extracts (Fig. 6 ~ B)., On the other hand, r i c h D,

I

I

I

I

I

growth rate of the two cell lines, and Bla-R cells were not temperature-sensitive at 41 C (data not shown).

-'E

Handschumache;, 1970; Nakashima et al., i976). Protein synthesis of the mutant cell was inhibited by these agents to the same extent as that of the parental strain.

Protein synthesis in L5178Y and Bla-R cells in the Presence of blasticidin S . L5178Y (A) and Bla-R (B) cells (5x104/ml) were incubated without (0-0) or with or 20 pg/ml pg/ml (A-A), 10pg/ml (A-Al (0-0) of blasticidin S and 1 pCi/ml SH-leucine.

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300

KUWANO ET AL. I

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10

20 30 Fraction Number

30

20

30

FIGURE 5

Sedimentation profiles of polysomes of exponentially growing L5178Y cells (A), Bla-R cells (B) and Bla-R cells cultured in the presence of 0.1 ,ug/ml cycloheximide (c). 4 x 10" cells were pre-exposed to 14C-uridine(0- - - 0 ) for 15 h and then labelled for 20 min with SH-uridine(0-0) in the presence of 0.02 ,ug/ml actinomycin D. Cytopolasmic extracts were analysed by sucrose density gradient centrifugation; fraction number is shown from bottom to top (lighter) region and arrow indicates monosomal region.

which is a strong inhibitor of mammalian protein synthesis in vitro (Refsnes et al., 1974), inhibited peptide formation in both L5178Y and Bla-R extracts. An obvious question was what fraction of the protein synthesizing system (S100 or ribosomes etc.) is altered in the Bla-R strain. We examined the effect of BlaS in a cell-free protein synthesis system catalysed by the SlOO fraction from rat liver and ribosomes prepared either from wild-type or from Bla-R mutant cells (Table 11). Significant inhibition by BlaS appeared only when ribosomes were prepared from the wild-type strain, while rich D inhibited both systems to the same extent. The effect of BlaS and r i c h D on both cell lines was therefore similar in intact cells and cell extracts (see Table I). A slightly higher incorporation of leucine into peptides was observed in BlaS-treated than in control cells (Table 11) when ribosomes of Bla-R were used. This might be due to the enhanced binding of amionacyl transfer RNA to ribosomes caused by BlaS as reported previously (Yukioka and Morisawa, 1969). We confirmed that BlaS blocked protein synthesis in cell-free extracts using rat liver S-30 and also that data similar to these, as in Table 11, were obtained when SlOO from L5178Y cells was used (data not shown). These data may suggest that the mutation of the Bla-R strain cells is in the ribosome.

a

0

I

20

I

40 0

b

I

I

20

40

'

T i m e (min) FIGURE 6 Effect of blasticidin S and rich D on protein synthesis

in cell-free extracts. Endogenous mRNA-dependent incorporation of 14C-leucineinto peptides in L5178Y (A) and Bla-R (B) extracts was measured at 37°C in the absence (0-0) or the presence of 10,ug/ml blasor lO,ug/ml rich D (A-A). ticidin S (0-0)

BLASTICIDIN-S-RESISTANT

DISCUSSION

The detection of thermosensitive mutants among drug-resistant somatic cell mutants may lead to a rapid advance in the study of cell proliferation in vitvo. In fact, Harris and Whitmore (1974) have isolated a stable line in CHO cells called Aza-R ts, which is temperature-sensitive for 8-azaguanidine resistance. A recent study by Siminovitch and his colleagues showed that a CHO mutant among many a-amanitin-resistant mutants carried a mutated RNA polymerase 11, which was more thermolabile than the wild-type enzyme (Lobban et al., 1976). Although our isolated Bla-R mutant is not temperature-sensitive, further studies on many Bla-R mutants might favor the isolation of a Bla-R ts mutation. On the other hand, Poche et al. (1975) isolated a cycloheximideresistant mutant from CHO cell lines, and suggested that the mutation might affect the ribosomes. However, we failed to isolate stable cycloheximideresistant mutants from mouse leukemia L5178Y cells (Matsui, unpublished data). We are tempted to ask why so many mutants have been isolated from CHO cell lines (Puck et al., 1958). The CHO cell genome might tend to express mutated genes more frequently than other cell lines. The increased frequency of stable mutations in CHO cell lines might be due to their aneuploidity or hemizygosity, as discussed recently (Siminovitch, 1976).

MUTANT CELLS

301

BlaS, like gougerotin or puromycin, is known to belong to the class of aminoacyl aminonucleoside antibiotics and inhibits protein synthesis through interactions with 50s and 60s ribosomal subunits from procaryotic and eucaryotic cells (Yamaguchi and Tanaka, 1966; Battaner and Vazquez, 1971). Although cycloheximide is known to overcome disaggregation of polysomes in mammalian cells cultivated under conditions of amino acid or serum starvation (Stanners, 1966; Kuwano et al., 1973, 1976), BlaS seemed not to alter the polysome disaggregation (Takenaka and Kuwano, unpublished data). Disappearance of polysomal disaggregation in the Bla-R cell following addition of a low dose of cycloheximide (Fig. 5c) may suggest decreased binding of altered ribosomes to messenger RNA as discussed previously (Kuwano et a/., 1976). In addition to data shown in Figure 5 ~ the , level of alkaline phosphatase activity in Bla-R mutant cells was found to be 1/3 of that in L5178Y cells (unpublished data). Thus, an altered translation apparatus may be responsible for the accumulation of monosomes and low enzyme activity in the mutant strain. ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture, Japan.

CELLULES MUTANTES DE LEUCEMIE MURINE RESISTANT A LA BLASTICIDINE S Apres mutagenbse par la nitrosoguanidine, nous avons is016 L partir de cellules L5178Y de leuc6mie murine un mutant (Bla-R) resistant B la Blasticidine S, qui inhibe la synthbse des prot6ines. La croissance cellulaire et I’incorporation de leucine dans une fraction de cellules Bla-R insoluble dans l’acide n’ont pas 6t6 inhibks par 5 A 20 cg/ml de Blasticidine S, qui bloque presque totalement la synthkse des prottines et la croissance cellulaire dans les tests avec la lignee L5178Y. Par contre, d’autres inhibiteurs tels que l’acide fusidique, la cycloheximide, la ricine D ou la L-asparaginase ont bloqu6 egalement la synthese des protCines dans les cellules Bla-R et dans les L5178Y. Avec des extraits S-30 de la lign6e parentale L5178Y, la synthbse des proteines in vitro a BtC presque completement bloqde en presence de l’antibiotique, alors qu’aucune inhibition n’a Ct6. observee lorsque I’on a utilisk les extraits S-30 du mutant Bla-R. Des tests de synthbse des prottines ont 6t6 effectuts avec la fraction SlOO de foie de rat et des ribosomes de cellules L5178Y ou Bla-R. La Blasticidine S n’a inhibt cette synthese que lorsque les ribosomes provenaient des cellules parentales.

REFERENCES

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KUWANO,M., NAKASHIMA, T., IKEHARA, Y., and ENDO,H., Regulation of RNA metabolism in malignant cells. In: W. E. Criss, T. Ono, and J. R. Sabine (ed.), Control mechanism in cancer, p. 269, Raven Press, New York (1976). LIN, J. Y., KAO,W. Y., and TSERNG, K. Y., Effect of crystalline abrin on the biosynthesis of protein, RNA, and DNA in experimental tumors. Cancer Res., 30, 2431-2433 (1970). LOBBAN, P. E., SIMINOVITCH, L., and INGLES, C. J., The R NA polymerase I1 of an a-amanitin-resistant Chinese hamster ovary cell line. CeN, 8, 65-70 (1976). MACPHERSON, I., and MONTAGNIER, L., Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology, 23, 291-294 (1964). NAKASHIMA, T., KUWANO, M., AKIYAMA, S., and ENDO,H., Effect of L-asparaginase and asparagine deprivation on RNA metabolism in mouse leukemia L5178Y cells in suspension culture. Cancer Res., 36, 3616-3621 (1976). PBCHE,H., JUNGHAHN, I., GEISSLER, E., and BIELKA, H., Cycloheximide resistance in Chinese hamster cells. 111. Characterization of cell-free protein synthesis by polysomes. Molec. gen. Genet., 138, 173-177 (1975). PUCK, T., CIECIURA, S., and ROBINSON, A,, Genetics of somatic mammalian cell. 111. Long-term cultivation of euploid cells from human and animal subjects. J . exp. Med., 108, 945-955 (1958). REFSNES,K., OLSNES,S., and ALEXANDER, P., On the toxic proteins abrin and ricin. J . Biol. Chem., 249, 3557-3562 (1974).

SATO,K., A leukemia cell mutant with a thermolabile alanyl transfer R N A synthetase. Nature (Lond.), 257, 813-815 (1 975). SIMINOVITCH, L., On the nature of hereditable variation in cultured somatic cells. Cell, 1, 1-11 (1976). STANNERS, C. P., The effect of cycloheximide on polyribosomes from hamster cells. Biochem. Biophys. Rrs. Commun., 24, 758-764 (1966). THOMPSON, L., HARKINS, J., and STANNERS, C., A mammalian cell mutant with a temperature-sensitive leucyl-transfer RNA synthetase. Proc. nat. Arad. Sci. (Wash.), 70, 3094-3098 (1973). TONIOLO,D., MEISS,H. K., and BASILICO, C., A temperaturesensitive mutation affecting 28s ribosomal RNA producing in mammalian cells. Proc. nat. Acad. Sci. (Wash.), 70, 12731277 (1973). YAMAGUCHI, H., and TANAKA,N., Inhibition of protein synthesis by blasticidin S. 11. Studies on the site of action in E. coli polypeptide synthesizing systems. J. Biochem., 60, 632-642 (1966). YAMAGUCHI, H., YAMAMOTO, C., and TANAKA, N., Inhibition of protein synthesis by blasticidin S. 1. Studies with cell-free systems from bacterial and mammalian cells. J. Biochem., 57, 667-677 (1965). YUKIOKA, M., and MORISAWA, S., Studies on the mechanism of action of gougerotin. 111. Enhancement of enzymatic binding of phenylalanyl-tRNA t o ribosomes by gougerotin. J. Biochem., 66, 241-247 (1969).