Isolation and Characterization of Alkaline PhosphataseConstitutive Variants from Chinese Hamster Ovary Cells (CHO-K1) KIYOSHI NOSE AND HAJIM KATSUTA Department of Cancer Cell Research, Institute of Medical Science, University of T o k y o , P.O. Takanawu, Tokyo-108, J a p a n

ABSTRACT Pure clones with high alkaline phosphatase (ALP) activity were isolated from Chinese hamster ovary (CHO-K1) cells which showed no detectable ALP activity. ALP-positive cells appeared at the frequency of 1 0 - 4 in the N-methyl-N'-nitro-N-nitrosoguanidine-treated cell population. With respect for cellular morphology, plating efficiency and several other enzymatic activities, no distinct difference was found between the original CHO-K1 cells and its ALP-positive variants, although some alterations in karyotype were found. Levels of ALP activity in these clones was stably maintained during serial cultivation. Some of the enzymological properties of ALP in the isolated clones were similar to those in bone or kidney.

Activity of alkaline phosphatase (ALP) is distributed among various organs of mammals (Fernley, '71) and is regarded as one of the differentiated functions of cells. Enzymological properties of ALP found in various tissues are different from each other, as confirmed by several authors (Lin and Fishman, '72; Ghosh, '69). In cultured cells, ALP of constitutive or prednisolone-induced HeLa cells is known to have similar enzymological properties to human placental isozyme (Ghosh et al., '72; Singer and Fishman, '74). We have previously shown that cultured mammalian cells possess at least two kinds of ALP activities, ALP-I and ALP-11, different from each other in their enzymological properties (Nose et al., '73). Various kinds of cell strains exhibited a wide range of variation in the activity of ALP-I, from almost none to high level. ALP is one of the inducible enzymes in cultured cells, and the activity was induced by the treatment of cells with prednisolone (Cox and MacLeod, '61, '62), phenylphosphate (Cox and Pontecorvo, '6 l ) , 5-bromodeoxyuridine (Koyama and Ono, '71), or adenosine 3':5'monophosphate (Koyama et al., '72; Nose and Katsuta, '74). On addition of 3':5'cyclic AMP to rat liver cells, ALP-I activity was induced whereas ALP-I1 activity did not increase (Nose and Katsuta, '74). J. CELL. P H Y S I O L . , 86: 253-260.

A clonal heterogeneity in ALP activity was reported in a cell population of fibroblasts (Maio and De Carli, '62; Papayannopoulou and Martin, '66). Isolation of such variants that stably maintain high level of ALP-activity from the population of ALP-negative cells would be useful for the study of the mechanisms of phenotypic expression of cells. The present paper describes the method of isolation of ALPconstitutive clones from ALP-negative Chinese hamster ovary (CHO-K1) cells, and some properties of the isolated clones. MATERIALS AND METHODS

Cells and m e d i a The original clone CHO-Kl, Chinese hamster ovary cells (Kao and Puck, '68), was kindly supplied by Dr. R. Nozawa of The Juntendo University School of Medicine, Tokyo. A subclone was isolated in our laboratory and was used in the present study. The culture medium consisted of Eagle's MEM (Eagle, '59, a commercial product of Nissui Seiyaku, Tokyo) supplemented with 5% fetal calf serum (GIBCO, Grand Island, N.Y.) and 0.1 mM each of nonessential amino acids. Cells were grown at 37°C in stationary monolayer culture Received Dec. 23, '74. Accepted Feb. 5, '75.

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KIYOSHI N O S E A N D HAJIM K A T S U T A

and the medium was renewed tmce a week. TD-40 type of flasks (Katsuta et al., '57) were used for the maintenance of cells, and plastic tissue culture dishes (Falcon Co., Calif., 60 mm) for experiments. The dishes were incubated in a n atmosphere of 95% air-5% COz. Cells were dislodged by trypsin-digestion (200 unitslml, Mochida Seiyaku, Tokyo).

tively. Frequency of ALP-positive cells were calculated as described above.

Biochemical assays Cells were washed three times with phosphate buffered saline, suspended in 10 mM Tris, pH 7.4 containing 5 mM MgC12, and sonically disrupted. The lysate was centrifuged at 800 rpm for five minutes and the supernatant fraction was used as an Detection of ALP-positive cells enzyme source. and colonies Activities of ALP-I, ALP-11 and acid phos(1) The cells of the original CHO-K1 phatase were assayed as described previshowed no detectable ALP-I actvity. For ously (Nose et al., '74). Activity of ALP-I the analysis of populational heterogeneity was optimal at pH 10.0 and inhibited by of ALP activity, monolayers of cloned CHO- cyanide, whereas that of ALP-I1 was the K1 were stained histochemically with a highest at pH 8.6 inhibited by p-chlorosolution containing 1.O mg of a-naphthyl- merculibenzoate. Lactate dehydrogenase phosphatase AS-MX (Sigma Chemicals, St. (LDH) was measured following the proceLouis, Mo.) and 4.0 mg of Fast Red Vio- dure of Kornberg ('55) and protein was let LB salt (Sigma) in 10 ml of 0.1 M Tris, determined by the method of Lowry et al. pH 9.6 (Burnstone, '58). ALP-positive cells ('51). were stained red, and the number of Preparation of extract from various organs of Chinese hamster stained cells in each dish was counted under the microscope and the frequency Adult Chinese hamster (male) was anesof ALP-positive cells was calculated by thetized with ether. Liver, kidney, intesdividing the number of positive cells by the tine and femur were taken out to be minced total number of cells per dish. with scissors and homogenized in distilled (2) For the isolation of ALP-positive water with a Polytron homogenizer (Kineclones, cells were inoculated into plastic matica GMBH, Luzern-Schweiz). Cold n-budishes (Falcon, 100 mm) a t the cell den- tanol (0.4 vol) was added to the extracts, sity of 500 cells/dish. After ten days, colo- and the mixture was stirred for ten minnies were overlayered with Hanks solution utes in ice and then for ten minutes at (Hanks and Wallace, '49) supplemented 37°C. After centrifugation at 2,000 rpm with 0.1 M Tris (pH 9.4), 2 mum1 of p- for 10 minutes, the aqueous layer was renitrophenylphosphate and 1.5% bactoagar moved and lyophilized. Dried material was (Difco Lab., Detroit, Mich.) which had dissolved in 10 mM Tris, pH 7.4 containing been prewarmed at 45°C (Maio and De 5 mM MgCIP. Carli, '62). They were allowed to stand at Chromosomal analysis room temperature for ten minutes, and the ALP-positive colonies stained yellow Cells were treated with colchicine (5 x were picked up. Three ALP-positive clones 10-6 M) at 37°C for four hours. Specimens (AL-151, AL-323, and AL-431) wereisolated for chromosome analysis were prepared by by this procedure. Pure clones were iso- the dropping and flaming method (Yosida lated from these ALP-positive clones by the et al., '65). The chromosomes were aruse of Micro Test Plate (Falcon) following ranged by the method of Kao and Puck the method by Suzuki and Horikawa ('73). ('69). RESULTS Fluctuation tests Frequency of ALP-positive cells in the Studies were made on the spontaneous population of C HO-Kl mutation rate from ALP-negative to ALPCells of CHO-Kl were seeded into 60 mm positive cells according to Luria and Delbruck ('43). Seven independent clones were Falcon plastic dishes at a density of about isolated from CHO-Kl, and were grown 5 x 105 cells/dish. After one day, cells to total population of 1.2 X 106 cells/DISH were treated with N-methyl-N'-nitro-Nin four Falcon dishes (60 mm), respec- nitrosoguanadine (MNNG; Daiichi Pure

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ALKALINE PHOSPHATASE-POSITIVE CELLS IN CULTURE TABLE 1

Frequency of ALP-positive cells i n t h e population of C H O - K l Number of cellsldish Treatment with MNNG (pg/ml)

Survival (’lr 1 2

ALP-positive

Total ( X 10-6)

0

100 59 45 22 9.4

5 72 60 45 82

3.6 3.5 3.4 2.8 2.2

0.2 0.5 1.0 2.0

Frequency of

ALP-positivecells 1.4 X 10-6 20.9 17.5 16.0 30.4

Cells were treated with N-methyl-”nitro-N-nitrosoguanidine (MNNG) for two hours. Cells were trypsinized four days after MNNG-treatment and inoculated into dishes to form colonies. Number of colonies were counted ten days after plating. Number of cells was counted four days after MNNG-treatment. 1

Chemicals, Tokyo) at various concentrations foy two hours, and the medium was renewed. Cells were stained for ALP four days after the MNNG-treatment. Number of total cells and ALP-positive cells were counted. The result is shown in table 1. CHO-K1 used in this experiment was a clonal line. However, the untreated population contained a few ALP-positive cells per 106 cells. The frequency of ALP-positive cells increased when the cells were treated with MNNG. The result of fluctuation test (table 2) shows that the appearance of ALP-positive cells was a random event.

Isolation and characteristics of ALP-positive clones The result in table 1 suggested the possibility of isolating ALP-positive clones from CHO-Kl which is biochemically ALPnegative. CHO-K1 cells were treated with MNNG (0.5 pg/ml) for two hours a t 37°C and seeded into Falcon plastic dishes to form colonies. Among 33,200 colonies examined, five colonies were found to be ALP-positive as revealed by p-nitrophenylphosphate-agar method. Three ALP-positive colonical clones, AL-151, AL-323, AL-431, were isolated and characterized. No marked difference was noted in cellular morphology and plating efficiency among the clones. Modal numbers of chro-

mosomes of CHO-K1, AL-151, AL-323, and AL-431 were 20, 21, 20, and 20, respectively. Karyotypes of these clones are shown. in figure 1 . Consistent alterations in chromosomes were observed in all the ALPpositive clones: disappearance of two small metacentric chromosomes and increase in the number of submetacentric chromosomes. Enzymological properties of each clone are summarized in table 3. The level of ALP-I activity increased several hundred fold, whereas ALP-I1 activity increased only three to four fold in isolated clones as compared with those in the parent CHO-Kl cells.

Stability of ALP-positive phenotype Several pure clones of single cell origin were isolated from ALP-positive clones as described in MATERIALS AND METHODS, and the stability of their ALP-positive phenotype was studied. Cells of pure clones were subcultured every two weeks in a dilution rate of 1:20. Specific activity of ALP-I was determined at various times. A s shown in table 4, specific activity of ALP-I in each pure clone was maintained fairly constant TABLE 3

E n z y m a t i c activities of various clones Activity (units/mg protein) Clone

TABLE 2

Variation in ALP-activity a m o n g various c l o n e s of C H O - K l

Number of clones Mean frequency of ALP-Dositive cellsll05 cells

CHO-K1 AL-151 AL-323 AL-431

7 0.598 5.32 < 0.05

~

ALP-I

ALP-I1

Acid Pasel

LDH2

x c

.Q

50

al

.-> L

Cel Is

Y

-I Bone

5

10 L-Homoarginine

20 ( mM )

Fig. 3 Effects of L-homoarginine on ALP-I activity from various sources. ALP-I activity was measured in the presence of L-homoarginine in the reaction mixture. Cell extracts were similar to those in figure 2.

We compared the enzymological properties of ALP in isolated clones and various organs of a Chinese hamster in order to examine the nature of ALP-I in ALP-positive subclones. Extracts of various organs and cells were prepared, and sensitivity of ALP to heat or to L-homoarginine was examined. As shown in figures 2 and 3 , ALP-I of liver and intestine was unstable at 50°C and was not inhibited by L-homoarginine, in contrast to that of bone and kidney. ALP in the extracts of cells of ALP-

positive clones all belonged to the type characteristic of bone or kidney. DISCUSSION

Many kinds of variants with altered heritable phenotypes have been isolated from cultured mammalian cells (Thompson and Baker, '73). It is at present difficult to determine whether or not the isolated variants are mutants as defined in prokaryotes (Harris, '71; Mezger-Freed, '72). The variants described in the present paper are

ALKALINE PHOSPHATASE-POSITIVE CELLS IN CULTURE

such that acquired a new phenotype which lacked in the original cells. Although it is premature to conclude that the variants were produced by a mutational event, the result shown in table 1, in which a mutagen increased the frequency of appearance of ALP-positive cells, seems to lend support to this contention. Since the sensitivity of CHO-K1 and ALP-positive cells to MNNG was essentially the same, the selection on the basis of resistance to the chemical seems to be improbable (data not shown). There could be several mechanisms for the appearance of ALP-positive clones: (1) correction of defective structural gene for ALP-I, (2) inactivation of “repressor gene” for ALP-I, ( 3 ) activation of “positive-regulator gene.” Prednisolone, 5-bromodeoxyuridine or dibutyryl adenosine 3 ’ :5’-mOnOphosphate failed to induce ALP-I activity when added to CHO-K1 cells (data not shown). The fact that ALP-1 in all of the isolated clones showed similar enzymological properties (figs. 2, 3 ) seems to be at variance with the possibility (1) that the variants appeared by a mutational event in the structure gene. All the hybrid cells produced by hybridization between ALPpositive and -negative cells were phenotypically ALP-positive, and therefore the former phenotype seemed to be dominant (manuscript in preparation). It seems most probable that ALP-positive cells were developed through the activation of a “positive regulator.” Cox and MacLeod (’62) reported that there was no appreciable difference in the karyotypes of HeLa strains possessing different ALP activity. On the other hand, DeCarli et al. (’63) and Bottomley et al. (’69) found that consistent chromosomal variation was associated with alterations in ALP activity. In the present study, the common alterations in the karyotype were found in all the ALP-positive clones (disappearance of two small metacentric chromosomes and increase in the number of submetacentric chromosomes). Further study has yet to be done to establish the correlation between the chromosomal change and ALP activity. Human bone ALP is known to be heat labile and inhibited by L-homoarginine (Lin and Fishman, ’72). The result shown in figures 2 and 3 indicate that ALP in Chinese hamster bone or kidney was inhibited

259

by L-homoarginine, but heat stable, whereas that in Chinese hamster liver or intestine was resistant to L-homoarginine and heat labile. We also examined ALP activity in various organs of rat, and found that ALP in liver or intestine was resistant to Lhomoarginine, whereas ALP in bone or kidney was sensitive (data not shown). These observations suggest that the enzymological properties of ALP isozymes are different among species. Considering the ALP-I activity as one of the differentiated functions of cells, the appearance of ALP-positive cells would be regarded as an example of cellular differentiation. One of the interesting properties of the isolated ALP-positive clones was that the ALP-I activity was of bone-type. It seems reasonable that the fibroblasts of CHO-K1 expressed ALP-I of bone-type, since the line is of mesenchymal origin. ACKNOWLEDGMENT

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