Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

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Experimental Cell Research88 (1974) 295-302

ISOLATION

OF TEMPERATURE-SENSITIVE MURINE

LEUKEMIC

MUTANTS

FROM

CELLS (L5178Y)

K. SAT0 and T. SHIOMI Department

of Microbial

Genetics, Research Institute for Microbial Yamada-kami, Suita 565, Japan

Diseases, Osaka University,

SUMMARY Two temperature-sensitive mutants (tsl and ts3) have been isolated from murine leukemic cells, LS178Y, after mutagenesis and cytosine arabinoside selection. Both tsl and ts3 grew normally at the permissive temperature (33°C) but not at the non-permissive temperature (39°C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39°C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.

Temperature-sensitive (ts) mutants are useful in studying the detailed mechanisms of various cell functions, such as division, macromolecular synthesis, differentiation, and their regulatory processes. A number of investigators have reported on the isolation of ts mutants from fibroblastic cells [l-7], but few, if any, ts mutants are available in lymphoblastoid cell lines. Lymphoblastoid cells have been shown to retain their original functions; i.e., production of immunoglobulins and a wide variety of effector molecules of cell-mediated immunity [8]. Thus it is expected that ts mutants of lymphoblastoid cells, if isolated, may shed light on the biosynthetic processes of these molecules. Since fibroblastic cells attach to the substratum and detach from it when they become nonviable, simple washing procedure can eliminate the dead cells. In lymphoblastoid cells which grow in suspension there is some difficulty in separating viable and nonviable 20-741819

cells after the selection procedure. In addition, there is a limitation to the number of cells that can be plated per dish without reducing the plating efficiency [9]. The difficulty has been overcome by killing the cells to a moderate degree and repeating the selection procedure. We report here on the isolation and preliminary characterization of two ts mutants of the murine leukemic cells. MATERIALS

AND METHODS

Murine leukemic cells (L5178Y) were kindly provided by Dr Y. Doida (Kyoto University) who had adapted the cells to grow in Fischer’s medium 1101containine 10 % calf serum. The cells grow in this medium wi& doubling times of 13-15 h and 10-12 h at 33°C (oer.. missive- temperature) and 39°C (non-permissive temperature), respectively. Ham’s F12 medium [l l] was also used in a certain experiment. Ethylmethane sulfonate (EMS) was obtained from Nakarai Chemicals, Ltd., Kyoto, and cytosine arabinoside hydrochloride (ara-C) from Sigma Chemical Co., St Louis, MO. Microtiter plates (Cooke Engineering Co., Alexandria, Va) were used for comparing growth at different temperatures. Exptl Cell Res 88 (1974)

296

Sato and Shiomi

Table 1. Survival after ara-C treatment at 33 and 39°C” Surviving fraction after treatment with Cell line Wild-type ts1 ts3

Treatment temp. CC)

ara-C

None

Ratio ( %) ara-C/None

39 33 39 33 39 33 33b

3.4 i: 10-S 7.8 x 1O-3 2.4 x 10-l 1.4 x 10-Z 1.5 x 10-l 1.6 x 10-l 2.6 x 1OF

9.6 x 10-r 4.8 x 10-l 4.2 x 10-l 5.9 x 10-l 3.3 x 10-I 7.8 x 10-l 6.9 x 10-r

0.35 1.6 57 2.4 44 21 3.8

a 5 x lo4 cells/ml were incubated for 16 h at 39 or 33°C when the cells were treated with or without 0.2 pg/ml of ara-C for 24 h at the respective temperatures. Then the cells were washed and plated in agar at 33°C in the presence of 10 pg/ml of deoxycytidine. All the figures are average of duplicate plates. b The same as above except that the cells were treated for 48 h with ara-C. Soft agar cloning technique Cells mixed in 0.3 % agar (Bacto-agar, Difco Laboratories, Detroit, Mich.) in medium plus serum were poured onto 0.5 % base agar in Petri dishes [9].

Measurement of macromolecular synthesis All the radioactive materials were obtained from Daiichi Pure Chemicals Co., Ltd., Tokyo. Cells were labeled for 1 h with 1 ,&i/ml of either 3H-thymidine (spec. act. 14.5 Ci/mmole), 3H-deoxycytidine (26.5 Ci/ mmole), 3H-uridine (15.0 Ci/mmole), or 3H-leucine (32.0 Ci/mmole). When 3H-uridine was used, unlabeled thymidine and deoxycytidine were added together at 10 pg/ml each to reduce the incorporation of uridine into DNA. Media containing one-tenth the normal concentration of leucine were used to label protein with 3H-1eucine. Cells were then chilled and received lOO-fold excess unlabeled precursors. They were applied to the presoaked glass fiber filters (Whatman, type GF/C) and treated successively with 60 ml of phosphate-buffered saline, 40 ml of ice-cold 5 % trichloroacetic acid and 40 ml of methanol [12]. To the dried filters in vials was added 1 ml of scintilamine-OH (Wako Pure Chemical Ind., Ltd., Osaka) and the vials were incubated at 45°C overnight. After adding 15 ml of toluene scintillator, the radioactivity was determined in a Beckman LS-200B liquid scintillation system. The results were expressed as cpm/cell relative to time zero.

RESULTS Isolation of temperature-sensitive mutants Murine L5178Y cells were treated with EMS at 400 ,ug/ml for 16 h, which reduced the Exptl Cell Res 88 (1974)

surviving fraction to 0.1. After the treatment the cells were washed, resuspended in fresh medium and incubated at 33°C for 2 days. They were then transferred to 39°C and kept there for 16 h, followed by addition of ara-C to a final concentration of 0.2 pg/ml. After 24 h of incubation, the cells were washed and grown in fresh medium. The ara-C treatment was repeated. Finally the cells were plated in soft agar in the presence of 10 ,ug/ml of deoxycytidine which was added to reverse the residual ara-C effect [13] and the plates were incubated at 33°C. When the colonies developed, they were transferred to two sets of microtiter plates; one was incubated at 33°C and the other at 39°C. Cells were recovered from the wells which showed growth at 33°C but not at 39°C. Two ts mutants, tsl and ts3, were isolated among more than a thousand colonies tested. They were used after recloning in soft agar. Survival after ara-C treatment Since the mutants have been isolated after selection with ara-C, they should be less sensitive to the drug at the nonpermissive temperature. If this is the case, isolation of

ts mutants from L5178Y many ts mutants would become feasible. Therefore sensitivity of the mutant cells to the drug was studied at low and high temperatures. Cells were pre-incubated at either 33°C or 39°C for 16 h, when ara-C added to a final concentration of 0.2 pg/ml and incubation was continued for another 24 h at the respective temperatures. Cells were washed and plated in soft agar at 33°C in the presence of deoxycytidine (10 ,ug/ml). The results are presented in table 1. The parental wild type cells were killed by the drug to a survival of less than 2 % at either temperature, whereas about half the mutant cells survived the toxic effect of ara-C when incubated at 39°C. At 33°C most of the tsl cells were killed by the drug within 24 h and the ts3 cells killed after 48 h. The reason for the difference in killing time is not clear but the finding was confirmed by repeating the experiment. These results show that mutant cells are apparently less sensitive to ara-C I

I

0

1

2

3

/

4

I

5

Fig. 1. Abscissa: time (days); ordinate:

cell number. Growth curves of wild-type (A-A, A-A), tsl (O-Z, H--¤),andts3(0-0, l -•)cellsat33”C (open symbols) and 39°C (solid symbols). 2 x lo4 cells were inoculated on day zero and cell number was scored daily with hemocytometer.

291

Table 2. Plating efficiencies of wild type and mutant cells at 33 and 39°C Plating efficiency ( %) at Ratio (O/b) Cell line

39°C

33°C

39”/33”

Wild-type ts1 ts3

48

87 61 88

55

0.05 0.08

0.08 0.09

at 39°C than at 33°C and also less sensitive than wild type cells at 39°C. They are not resistant to ara-C, since they were killed by the drug at 33°C. The apparent insensitivity to ara-C at the restrictive temperature may be related to the absence of DNA synthesis at high temperature in the mutants. The above experiment shows that the ara-C treatment at the nonpermissive temperature facilitates selection of ts mutants. Cell growth at the permissive and nonpermissive temperatures Cell growth was measured at 33°C and 39°C with mutants, tsl and ts3, and parental strain L5178Y. As shown in fig. 1, the parental cells multiplied well at both temperatures with doubling times of 12 h and 15 h at 39 and 33°C respectively. Mutant cells grew at 33°C with a doubling time of 18-20 h, whereas at 39°C they did not grow and even a progressive decline in cell number was observed as the incubation continued. The initial cell density was 2 x lo* cells/ml in fig. 1, but essentially identical growth patterns were obtained with a starting density of 1 x IO5 cells/ml as will be shown in the temperature shift experiments. These results demonstrate that the cell growth of both mutants was susceptible to high temperature. Effect of temperature on cell growth was also examined by determining the plating Expti Cell Res 88 (1974)

298

Sato and Shiomi

Fig.

2.

Abscissa: time (days);

cell number. Growth curves of tsl (a) and ts3 (b) cells after temperature shift from39”C(W-W; l -•)to33”C 1 x lo5 cells were (O-O, O-O). incubated at 39°C. At daily intervals they were shifted down to 33°C and cell growth was followed. The ordinate:

arrows indicate when shifts from

0

2

4

efficiencies of the mutants at the permissive and non-permissive temperatures. Appropriate number of cells were plated in soft agar and incubated at 33 and 39°C. After 3 weeks the number of colonies formed was scored. The results are given in table 2. At 33°C there was no appreciable difference in plating efficiencies among wild-type, tsl and ts3 cells. At 39°C wild-type cells plated with approximately half the plating efficiency seen at 33°C. By contrast only 5 and 8 min colonies were barely visible at 39°C when as many as lo4 cells were plated in tsl and ts3 cells, respectively. As far as plating efficiencies are concerned, more than a thousandfold difference is evident between wild-type and mutant cells as well as between the permissive and non-permissive temperatures in mutant cells. The minute colonies formed at high temperature could not be identified Table 3. Survival of mutant cells after exposure to the non-permissive temperature for varying periods ts3

Days at 39°C before shift to 33°C

ts1 Plating efficiency (%)

% survival

Plating efficiency ( %)

% survival

1 2 3 4

39.0 19.4 7.9 5.4 3.7

100 50 20 14 10

38.5 40.5 7.3 6.3 4.7

100 105 19 16 12

Exptl Cell Res 88 (1974)

6

a

IO

39°C to 33°C were made.

as revertants, since they never increased their size on further incubation and could not be picked. Alternatively they might have developed from cell aggregates present at the time of plating and if so, mutant cells help each other grow to a limited extent even at the restrictive temperature when cells are close in contact. Temperature shift experiments Mutant cells (1 x lo5 cells/ml) kept at 39°C were transferred to 33°C at various times and the growth response was examined. Fig. 2 shows that upon down-shift, cells rapidly resume growth achieving a doubling time comparable to that of control, although the cell viability, growth rate, and maximal cell densities attained gradually decrease with time of incubation at 39°C. Nevertheless, a considerable fraction of cells were viable and grew again when shifted to 33°C after maintenance at 39°C for as long as 6 days. The results indicate that mutant cells retained their potential for division during exposure to high temperature. The temperature shift experiment was also carried out with respect to colony forming ability of the cells kept at 39°C for varying periods. As shown in table 3, plating efficiencies in agar decreased rather quickly with time and on the 4th day only 10% of the cells plated formed colonies. This is not in accord with the shift experiments in liquid

ts mutants from L5178Y

0

L--I 0

4

8

/ 24

O/

100

100

50

50

0

4

8

24

299

0

4

8

24

0

4

8

24

Fig. 3. Abscissa: time (hours); ordinate: radioactivity/cell

relative to time zero. Relative uptake of 3H-thymidine (a), 3H-deoxycytidine (b), 3H-uridine (c), and W-leucine (d) in wild-type (,1--n, A-A), tsl (z-3, M-B), and ts3 (O-O, O-O) cells at 33”C(opensymbols) and 39”C(solid symbols). 5 x lo4 cells were labeled with 1 @X/ml of radioisotopes for 1 h after incubation for various lengths of time at 33 or 39°C and the radioactivity incorporated into acid-insoluble material was determined.

medium (fig. 2) where more than 60 % of the cells appeared to be viable on the 4th day and began to grow soon after the down-shift. The inconsistency between growth in liquid medium and in agar may indicate that a higher integrity of cells is required for growth in agar. Macromolecular synthesis at the non-permissive temperature Effect of temperature on the synthesis of macromolecules was studied. Mutants tsl and ts3 and wild type cells were incubated at either 33°C or 39°C and at various times

radioactive precursors were added to the cultures. In fig. 3a, incorporation of 3Hthymidine into acid-precipitable material was compared among mutants and wild-type cells at the permissive and non-permissive temperatures. At 39°C incorporation activity of thymidine per cell fell steadily with incubation time in both tsl and ts3 cells. Incorporation activity was also reduced in wild type cells during the early time of incubation but it was resumed by 24 h following up-shift. The result was confirmed by repeating the experiment. In order to exclude the possibility that thymidine metabolism is specifiExptl Cell Res 88 (1974)

300

Sato and Shiomi I

I

I

I

b

lo5

I OS

104,,

1 o4

time (days); ordinate: cell number. Growth curves of tsl (a) and ts3 (b) cells in Ham’s F12 medium (n--a, A-A) as compared with O-O, e-0) Fischer’s medium (G-I:, m-m; at 33°C (open symbols) and 39°C (solid symbols).

Fig. 4. Abscissa:

tally affected, incorporation of 3H-deoxycytidine was studied. As shown in fig. 3b, deoxycytidine incorporation was also inhibited in the mutant cells at high temperature. Uptake of 3H-uridine and 3H-leucine did not much differ between 33°C and 39°C and among mutants and wild-type cells during 24 h (fig. 3c, d). Although leucine uptake was depressed slightly in the mutants after incubation at 39°C for 24 h (fig. 3d), incorporation of thymidine and deoxycytidine was inhibited much earlier and to a greater extent (fig. 3a, b). These results suggest that synthesis of DNA, but not of RNA or protein, is affected at the restrictive temperature in the mutants.

medium was used in place of Fischer’s (fig. 4b). The growth rate in F12 medium at 39°C was nearly comparable to that at 33°C until day 2 and slowed down thereafter. The results indicate that tsl and ts3 are independent mutations and that some components included in F12 but not in Fischer’s are responsible for growth of ts3 cells at 39°C. In order to identify the responsible components, growth of ts3 cells at 39°C was measured in the reconstituted media which contained all the F12 components except one. The results demonstrated that alanine, hypoxanthine, and pyruvate in combination restored the growth of ts3 cells at 39°C (table 4). The mechanism of the multiple requirement is not clear. DISCUSSION From a lymphoblastoid cell line L5178Y, two ts mutants have been isolated after EMS mutagenesis and ara-C selection. EMS has been shown to be effective for increasing a mutation frequency in lymphoblastoid cells [9]. The ara-C selection was originally Table 4. Growth of ts3 cells in media containing various supplementsa Temp. (“C) 39”

Effect of enriched medium on cell growth So far all the experiments were performed by using Fischer’s medium supplemented with 10 y0 calf serum. To examine if enriched medium might favor the growth of mutant cells at the non-permissive temperature, Ham’s F12 medium was compared with Fischer’s. As shown in fig. 4a, tsl cells did not grow at 39°C even in F12 medium, whereas ts3 cells grew fairly well at 39°C if F12 Exptl Cell Res 88 (1974)

33”

Supplement None Alanine Hypoxanthine Pyruvate Alanine + hypoxanthine Alanine t pyruvate Hypoxanthine T pyruvate Alanine + hypoxanthine + pyruvate None

Cell number/ml (x 10-4) 0.6 1.0 0.4 1.4 1.9 2.8 3.1 6.1 8.5

a Fischer’s medium containing 10 % dialysed calf serum was supplemented with L-alanine (9 Pglml), hypoxanthine (4 fig/ml), sodium pyruvate (110 ,ng/ ml), or their combinations. Mutant ts3 cells (1 x 104/ ml) were grown in these media and cell number after 3 day incubation was scored.

ts mutants from LS178Y used in combination with tritiated thymidine suicide [2]. In the present report we have shown that ara-C alone works in selecting mutants. Ara-C has been reported to inhibit DNA synthesis in mammalian cells [13] and specifically kill cells which are undergoing DNA replication [14]. Although some mutants which have been selected after ara-C treatment are not more resistant to the drug under the conditions of selection than the parent [4], mutants described in the present report are more resistant to ara-C than parental cells at 39°C but as sensitive to the drug as the parent at 33°C (table 1). These results are consistent with the finding that mutant cells do not incorporate DNA precursors at the restrictive temperature (fig. 3a, 6). There is a great difference in thymidine uptake between mutant and wild type cells at 24 h after temperature shift to 39°C but at 4 and 8 h thymidine incorporation declined in wild-type cells as well. The initial decline may be caused by transient metabolic disturbances due to heat shock and the later resumption may be accounted for by adaptation to high temperature, although nothing is known as for heat shock and adaptation. Inability to incorporate thymidine or deoxycytidine at 39°C does not necessarily indicate that the DNA replicating machinery has become temperature-sensitive in these mutants, since both RNA and protein synthesis are required for the DNA replication to continue [15] and any defect in these processes may result in cessation of DNA synthesis. Accordingly it can be assumed that an alteration of a single RNA or protein molecule which is related to thymidine and deoxycytidine incorporation causes no appreciable changes on the overall uptake of uridine or leucine (fig. 3c, d). In this connection ts3 cells are interesting, for their ts phenotype was substantially suppressed by the supplementation of three nutrients,

301

alanine, hypoxanthine, and pyruvate (table 4). Mutants which require only alanine have been isolated [16]. Requirement for many supplements for growth at the non-permissive temperature is to be studied further. There are three reports on multiple auxotrophs of Chinese hamster cells [17, 18, 191, but they have not been characterized yet. They might be caused by either multiple mutations or a single mutation which exerts a pleiotropic effect. These two possibilities may be discriminated by reversion experiments which attempt to obtain a revertant for one phenotype and examine another phenotype if it is also reverted. By this method we can hopefully pinpoint the affected functions in ts3 cells. We are grateful to Dr Y. Doida for providing us L5178Y cells and to Dr A. Matsushiro for critical review of the manuscript. This work was supported by grants-in-aid for Cancer Research and for General Research from the Ministry of Education, Japan.

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18. Chu, E H Y, Sun, N C & Chang, C C, Proc natl acad sci US 69 (1972) 3459. 19. Suzuki, F & Horikawa, M, Methods in celI biol (ed D M Prescott) vol. 6, p. 127. Academic Press, New York (1973). Received January 18, 1974 Revised version received May 14, 1974