Intracellular degradation of bleomycin hydrolase in two Chinese hamster cell lines in relation to their peplomycin susceptibility

Biochimica et Biophysica Acta, 1012 (1989) 29-35 Elsevier

29

B B A 12459

Intracellular degradation of bleomycin hydrolase in two Chinese hamster cell lines in relation to their peplornycin susceptibility C h i a k i N i s h i m u r a , H i d e o Suzuki, N o b u o T a n a k a a n d H i d e y o Y a m a g u c h i Institute of Applied Microbiology, University of Tokyo, Tokyo (Japan) (Received 31 October 1988)

Key words: Bleomycin hydrolase; Intrinsic resistance; Intracellular degradation; Enzyme distribution; Monoclonal antibody; (Chinese hamster cell line)

The Chinese hamster lung 0/79) cell was intrinsically 10-times more resistant to peplomycin, a bleomycin-related antitumor antibiotic, than the Chinese hamster ovary (CHO) ceil This may be associated with the 3-times higher levels of recovery of bleomycin hydrolase activity of the V79 cell The degradation of bleomycin hydrolase molecules in both V79 and CHO cells was examined using a monoclonaJ antibody specific for the enzyme. Labeling experiments showed that the bleomycin hydrolase in CHO cells was less stable than the comparable enzyme in V79 ceils, and that 48 kDa subunits comprising bieomycin hydrolase (a homohexameric enzyme) molecules were degraded into 31 kDa forms in both cell lines. The 105000 × g pellet (microsomes) fraction obtained after subceHular fractionation of CHO cells contained both 48 kDa subunit and 31 kDa forms of bleomycin hydrolase, while the 105000 x g supernatant cytoso| fraction yielded only 48 kDa subunit forms of the enzyme. Moreover, bleomycin hydrolase activity of both V79 and CHO cells was almost entirely recovered from the cytosol fraction. These results suggest that degradation of the 48 kDa subunit form of bleomycin hydrolase in these two lines of cultured cells into the 31 kDa form occurs on the plasma membrane or the endoplasmic reticulum, with which the resulting large number of bleomycin hydrolase molecules or degradated forms of the enzyme that have lost enzymatic activity are associated.

Introduction Bleomycin shows remarkable therapeutic efficacy for squamous cell carcinoma, testicular carcinoma, and malignant lymphoma. The antibiotic is known to be more or less inactivated by bleomycin hydrolase which hydrolyzes the carboxamide bond in pyrimidoblamic acid moiety of the bleomycin molecule [1]. Bleomycin hydrolase is also capable of hydrolyzing peplomycin, a bleomycin derivative, which shows higher cytotoxicity against cultured HeLa $3 cells, as well as higher antitumor effect on Ehrlich solid carcinoma in mice and AH66 ascites hepatoma in rats than that of bleomycin [2]. The enzyme is widely distributed in a variety of mammalian cells and tissues [3]. Some bleomycin-re-

Abbreviations: SDS-PAGE, sodi::m dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; lg, immunoglobulin; EDTA, ethylenediam;aetetraacetic acid; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay Correspondence: C. Nishimura, Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.

sistant mutant tumor cells displayed higher levels of bleomycin hydrolase activity than the parental cells [4,5]. The level of bleomycin hydrolase activity in such cells seems to be related to their bleomycin susceptibi'ity. Umezawa et al. [6] attempted to purify bleomycin hydrolase from the murine liver by affinity chrom~,ography with Sepharose 4B-lysinamide and obtained a 25-fold-purified enzyme. However, they failed in further purification because of the lability of the partially purified enzyme. Recently, we established a hybridoma clone which produced anti-bleomycin hydrolase monoclonal antibody by immunizing BALB/c mice with the partially purified enzyme [7]. Using immunoaffinity with this monoclonal antibody and DEAE-Toyopearl chromatography, we successfully purified bleomycin hydrolase from the rabbit liver; its molecular weight, determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 48 kDa and that by gel filtration was 300 kDa [7]. Sebti et al. [8] also reported purification of the same enzyme from the rabbit lung. In this publication, results of our further studies regarding degr,idation and localization of bleomycin hydrolase in both V79 and CHO cells are reported.

016%4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

30 They suggest that intracellular degradation of bleomycin hydrolase probably occurring on the endoplasmic reticulum or other membranous structures at a relatively low rate in V79 cells can be considered one of the mechanisms for their natural resistance to bleomycin.

Materials and Methods

Materials Peplomycin was kindly provided by Nippon Kayaku Co., Tokyo Japan. Anti-bleomycin hydrolase monoclonal antibody was produced by a hybridoma clone which we established [7]. V79 and CHO cell lines were generously given by Professor S. Okada, Faculty of Medicine, University of Tokyo. McCoy's 5A medium, calf serum, and fetal calf serum were supplied by GIBCO laboratories, Chagrin falls, OH. Phosphatase substrate and bovine serum albumin (BSA) were purchased from Sigma Chemical Co., St. Louis, MO. Staphylococcus aureus, goat anti-mouse immunoglobulin (Ig) M, and the IgM conjugated to alkaline phosphatase or fluorescein were supplied by Zymed Laboratories, San Francisco, CA. [14C]Leucine (specific activity, 344 mCi/mmoi) and EN3HANCE were the products of New England Nuclear, Boston, MA. RPMI-1640 medium free of glutamine and leucine was obtained from Flow Laboratories, North Ryde, N.S.W., Australia. An electrophoresis calibration kit, X-Omat AR film, and the protein assay dye reagent were provided by Pharmacia Fine Chemicals, Uppsala, Sweden, Eastman Kodak Co., Rochester, NY, and Bio-Rad Laboratories, Richmond, CA, respectively. The Aquasil SS-352N column was supplied by Senshu Scientific Co., Tokyo, Japan.

Cell culture V79 and CHO cells were maintained at 37 °C under 5~ CO 2 in McCoy's 5A medium supplemented with 10~ calf serum and 5~ fetal calf serum (cell culture medium). When necessary, cultured cells were trypsinized with 0.05~ trypsin/0.02~ ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS).

Drug sensitivity The sensitivity of cultured cells to peplomycin was measured on the basis of the number of colonies formed. 200 cells were dispensed in each well of 6-well plastic plates containing 2 ml of cell culture medium, which subsequently received various doses of peplomycin in a volume of 30 ~al (PBS). After incubation at 37 °C for 7 days, all wells were stained with 0.1% crystal violet and number of colonies was visually counted.

Assay of bleomycin hydrolase activity The bleomycin hydrolase activity was determined using high-performance liquid chromatography (HPLC)

[5,9]. The reaction mixture, in a total volume of 60/~1, containing enzyme sample and peplomycin (1.6 mg/ml) as a substrate in 10 mM phosphate buffer (pH 7.4) was incubated for 2 h at 37°C, and the incubation was terminated by the addition of 180 #1 of ice-cold methanol and 0.6 mg of cupric carbonate. The mixture was centrifuged for 5 min at 10000 x g. The resultant supernatant was injected into an Aquasil SS-352N HPLC column (4.6 x 250 mm), which was equilibrated and eluted with a mobile phase consisting of methanol/ acetonitrile/20~ ammonium acetate/acetic acid (560: 440: 100: 0.5, v/v) at room temperature. Two distinct peaks were obtained in the eluate, and were identified as peplomycin and desamido-peplomycin whose retention times were 7 and 10 min (flow rate 1.5 ml/min), respectively. The amounts of peplomycin and desamido-peplomycin were determined by the intensity of ultraviolet absorption at 290 nm. The enzyme activity extracted from the cells was not significantly lost during the incubation for 2 h at 37 o C. The activity of bleomycin hydrolase which can hydrolyze 1 /~g of peplomycin/min at 37 °C was defined as 1 unit of the enzyme.

Labeling of V79 and CHO cells with [! 4C]leucin e Both V79 and CHO cells were labeled with []4C]leucine in leucine-free labeling medium. 2 ml of cell culture medium containing 3.105 cells was inoculated into each well of 6-well plates and cultured at 37 o C. The following day, the cells grown in the wells were washed three times with leucine-free RPMI-1640 medium (labeling medium). Then they were labeled for 1 h at 37 °C in 2 ml of labeling medium containing 2 /~Ci/ml [14C]leucine, for 8 h at 37 °C in the above-mentioned medium supplemented with 10~o calf serum and 5~ fetal calf serum, or for 24 h at 37°C in the abovementioned medium supplemented with 10~ calf serum, 5~ fetal calf serum and 4/~g/ml unlabeled leucine. The labeled cells were washed three times with serum-free cell culture medium and fed with cell culture medium at 37 ° C. At selected intervals, the cells were washed with PBS and lysed with 1 ml of 50 mM Tris-HCl buffer (pH 7.4) supplemented with 0.05~ SDS, 0.2~o Triton X-100, and 120 mM NaCI (buffer A). The lysates were centrifuged at 10000 × g for 10 min, and the resulting supernatants containing labeled proteins were used for immunoprecipitation analyses.

Immunoprecipitation of labeled bleomycin hydrolase Anti-bleomycin hydrolase monoclonal antibody was first adsorbed on S. aureus cells coated with anti-mouse IgM as follows. A 10~ suspension of S. aureus cells in a volume of 250 #1 was incubated with 50 /~1 of goat anti-mouse IgM serum and 300/LI of a 10 mM phosphate buffer (pH 7.4) containing 100 mM NaCI !~or 1 h at 4°C, and then centrifuged f~t 5 rain at 10~00 × g.

31 The sedimented S. aureus cells were resuspended in the hybridoma culture fluid (1 ml) containing the antibleomycin hydrolase monoclonal antibody. The mixture was incubated for 1 h at 4 ° C, centrifuged for 5 rain at 10000 ×g, and the resultant pellet was washed twice with PBS. The proteins extracted from V79 and CHO cells (approx. 1 • 106 cells), prelabeled with [14C]leucine as described above, were incubated with anti-bleomycin hydrolase monoclonal antibody adsorbed on S. aureus cells for 1.5 h at 4°C. After centrifugation, the pellets were washed with PBS containing 0.05~ Tween 20 and again with PBS alone and then mixed with 150 #1 of 120 mM Tris-HCl buffer (pH 6.8) supplemented with 2~ SDS and 25~ glycerol (buffer B) at 4°C. After sedimentation, the supernatants were subjected to SDS-PAGE.

cubated for I h at 4 °C with goat anti-mouse IgM serum (10 #1) in 10 mM phosphate buffer (100 #1) (pH 7.4) containing 100 mM NaCI, sedimented and reacted with anti-bleomycin hydrolase monoclonal antibody (0-15 /tg). The mixture was incubated for 1 h at 4°C, centrifuged for 5 rain at 10000 × g, and the resulting precipitate was washed twice with PBS by centrifugation. The 10000 × g supernatants of V79 and CHO cells homogenated in 10 mM phosphate buffer (pH 7.4) or the 105 000 x g supernatants of V79 and CHO cells homogenated in the buffer containing 0.25 M sucrose were incubated with the anti-bleomycin hydrolase monoclonal antibody adsorbed on S. aureus cells for 1.5 h at 4 ° C. After sedimentation, bleomycin hydrolase activity remaining in the supernatant was assayed as described above.

SDS.PAGE SDS (12.5~) -PAGE was carried out as described by Laemmli [10], followed by fluorography. The gels were exposed to preflashed X-Omat AR film at - 8 0 ° C . Molecular weight markers were used as the standard.

lmmunofluorescence V79 and CHO cells were grown on glass coverslips placed in the bottom of 60-mm cula~re dishes. The specimens were fixed with 3.7~ formaldehyde h~ PBS for 20 min at 37 °C and washed three times with PBS. Then they were treated with 0.2~ Triton X-100 in PBS for 5 min at 20°C and washed three times with PBS alone. Anti-bleomycin hydrolase monoclonal antibody was overlaid and kept for 1 h at 37°C. After being washed three times with PBS, the cells were stained with goat anti-mouse IgM conjugated to fluorescein for 1 h at 37 o C. The specimens were rinsed with PBS, mounted with 80% glycerol and observed under a fluorescent microscope (125 × ).

Subcellular fractionation V79 and C¢tO cells were homogenized in 10 volumes of 10 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose with a Potter-Elvehjem homogenizer. Subcellular fractions were separated by sequential centrifugation [11]: 10 rain at 800 x g for nuclei and unbroken cells, 20 rain at 10000 × g for large granules and 60 rain at 105000 x g for the microsomal fraction. The final supernatant was designated as the cytosol fraction. Enzyme.linked immunosorbent assay (ELISA) for the fixed antigen Polystyrene plates (96 well) were coated with 50 #1 of each subceUular fraction (2.5-10-40 #g protein/ml diluted with PBS) and kept for 2 h at 37°C. After ,,,ashing twice with PBS, nonspecific sites were blocked by treatment with 0.5~ BSA for 1 h at 37°C. The coated wells were incubated with hybridoma culture fluid containing anti-bleomycin hydrolase monoclonal antibody for 1 h at 37 o C. After rising, goat anti-mouse IgM conjugated to alkaline phosphatase was added to the wells and incubated for 1 h at 37 o C. The wells were washed three times with 0.05~ Tween 20 dissolved in PBS and subsequently three times with PBS alone. Then alkaline phosphatase substrate was added and the enzyme activity was measured spectrophotometrically at 405 nm. Immunoprecipitation of bleomycin hydrolase from V79 and ClIO cells Immunoprecipitation of bleomycin hydrolase was performed as described above with some modifications. A 10~ suspension (50 #1) of S. aureus cells was in-

Protein content determination The content of protein was determined by the methoc! of Lowry et al. [12] using BSA as a standard. Results

Sensitivity to peplomycin and bleomycin hydrolase activity of two Chinese hamster lines The peplomycin sensitivity and bleomycin hydrolase activity of growing V70 cells were compared with those of CHO cells (Table I). The ICs0 value represents the concentration of peplomycin which was required to reduce plating efficiency of a cell to 507o of control. V79 cells were approx. 10-times more resistant to peplomycin than CHO cells. The 105 000 x g supernatant from V79 cells homogenated in 10 mM phosphate buffer (pH 7.4) yielded a level of bleomycin hydrolase activity which was 3-times higher than that from CHO cells. Any bleomycin hydrolase activity was not detected in the ambient medium even after a substantially large number of V79 cells or CHO cells had been grown (data not shown), suggesting that bleomycin hydrolase is not secreted outside from these cells.

32 TABLE l

tabet

lh

24h

V 7 9 CHO

V 7 9 CHO t

67K- ~

Sensitivity for peplomycin and bleomycin hydrolose activity of V79 and ClIO cells The lCs0 value represents the concentration of peplomycin which reduced plating efficiency of a cell to 50% of control. V79 and CHO cells were homogenated in 10 mM phosphate buffer (pH 7.4). The homog~.aates were centrifuged for 1 h at 105000 x g and bleomycin hydrolase activity in the resulting supematant was measured as described in Materials and Methods.

o

~.

43K-

Cells

ICso

Bleomycin hydrolase activity (/~g/min per mg protein)

V79

1.l

3.8

CHO

0.12

1.3

3OK- ....... ZO.1KFig. 1. Fluorogram analysis of bleomycin hydrolase from V?9 and CHO cells labeled with [t4C]leucine for [ h or 24 h. V79 and CHO cells were cultured at 3.105 cells/well in 6-well plates. The following day, both cells were labeled for ] h in labeling medium containing 2 /~Ci/ml [Z4C]leucine and for 24 h in te above-mentioned medium further supplemented with 10% calf serum, 5% fetal calf serum and 4 /~g./ml unlabeled leucine. After labeling, the cells were lysed and the lysates were incubated with anti-bleomycin hydrolase monoclonal antibody adsorbed on S. aumus cells for 1.5 h, Following centrifugation, the immunopredpitates were eluted with buffer B and then subjected to SDS (12.5%) .PAGE, followed by fluorography.

Content of labeled bleomycin hydrolase in V79 and ClIO cells Cellular extracts were prepared from V79 and CHO cells labeled for 1 h in labeling medium containing 2 /~Ci/ml [n4C]leucine and for 24 h in the above-mentioned medium further supplemented with 10~ calf serum, 5~ fetal calf serum and 4 /tg/ml unlabeled

leucine, and then the bleomycin hydrolase existing therein was immunoprecipitated and developed on SDS-PAGE (Fig. 1). When labeled for 1 h, only the 48 kDa subunit form of the enzyme was detected in cellular extracts from both V79 cells and CHO cells. It was also found that the amount of bleomycin hydrolase recovered from CHO cells was almost equal to that from V79 cells. However, after 24 h of labeling, the radioactivity of the 48 kDa subunit form from CHO cells was significantly lower than that from V79 cells.

lntracellular degradation of bleomycin hydrolase into lower molecular weight form Immunochemical analyses of bleomycin hydrolase in cellular extracts from V79 and CHO cells that were pulse-labeled for 1 h with [n4C]leucine and chased for

V79 pu[se chase

lh

lh

CHO lh

2h 15h ,.8h

lh lh

lh lh lh 2h 15h 28h

67K....

45K-

qm=R=P

5OK20.IKFig. 2. Kinetics of degradative [n4c]leucine-labeled bleomycin hydrolase in V79 and CHO cells. V79 and CHO cells were cultured at 3.105 ~ l l s / w d l in 6-well plates. The following day, the cells were labeled for 1 h with 2 p C i / m l [14C]leucine and fed with cell culture medium. At ~elected intervals after labeling, the cells were lysed and the lysates were incubated with anti-bleomycin hydrolase monoclonai antibody adsorbed on $. aureus cells for 1.5 h. After centrifugation, the immunoprecipitates were eluted with buffer B and then subjected to SDS (12.5%) -PAGE, followed by fluorography.

33 various times revealed two major bands of proteins with molecular weights of 48 k D a and 31 kDa during the chase period (Fig. 2). After 2 h of chase, the radioactivity of the 48 kDa subunit form was detected from both V79 and C H O cells. After 15 h of chase, the label of the 48 k D a subunit form was clearly seen in the extracts from V79 cells, but was no longer visible in the extracts from C H O cells. The faint band of the 48 k D a subunit form was still visible in V79 cells even after 28 h of chase. The 31 kDa form was seen after 2 h of chase in V79 cells, reaching the maximal level after 15 h of chase. In C H O cells, the amount of 31 k D a form detected after 2 h of chase was larger than that detected after 1 h of chase and almost equal to that detected after 15 h of chase.

Intracellular localization of bleomycin hydrolase in V79 and ClIO cells To examine the intracellulax localization of bleomycin hydrolase, several subcellular fractions prepared from both V79 and C H O cells were examined for their immunoreactivity with anti-bleomycin hydrolase monoclonal antibody and for bleomycin hydrolase activity. ELISA performed with varying amounts of the fixed bleomycin hydrolase antigen showed a linear relationship between absorbancy at 405 nm and antigen content in the range between 2.5 and 40 p g / m l . As seen in Table If, the results of the ELISA study demonstrated that the 105000 x g supernatant fraction prepared from V79 and C H O cells contained 52% and 29%, respectively, of the total amount of those proteins which were reactive with bleomycin hydrolase-specific monoclonal antibody; the value for the 105000 x g pellet

TABLE I1 Subcellular distribution of bleomycin hydrolasedetectedby ELISA and enzymatic assay After subceilular fractionation, the amount of the bleomycin hydrolase antigen in each fraction was measured by ELISA and bleomycin hydrolase activity enzymatically as described in Materials and Methods. Fractions

Distribution (£) ELISA V79

Nuclear or unbroken cells (800 x g pellet) Large granules (10000 x g pellet)

Microsomes (105000 x g pellet) Cytosol (105000 x g sup.)

3a

CHO

bleomycin hydrolase activity V79

CHO

2

0

0

8

10

1

0

37

59

14

15

52

29

85

85

a Number represents percentage in the cell homogenate.

67K:i!J!jl

43K-

'

3OK-

20JK-

1

2

3

4

Fig. 3. Subcellular distribution of [14C]leucine-labeled bleomycin hydrolase in CHO cells. The CHO cells were labeled for 8 h with 2 ~Ci/ml [t4C]leucine. After labeling, the cells were digested with 0.05~ trypsin/0.02~ EDTA and homogenized in 10 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was separated by sequential centrifugation: 10 min at 800× g, 20 min at 10000 x g and 60 rain at 105000 x g. Then each fraction was digested for 30 rain at 4°C with buffer A. The digested fractions were centrifuged at 10000x g for 10 min and the resulting supernatants containing labeled proteins were incubated with anti-bleomycin hydrolase monoclonal antibody adsorbed on S. aureus cells for 1.5 h. After centrifugation, the immunoprecipitates were eluted with buffer B and then subjected to SDS (12.59[) .PAGE, followed by fluorogra° phy. 1,800 × g pellet fraction (nuclear or unbroken cells); 2, 10000 × g pellet fraction (large granules); 3, 105000x g pellet fraction (microsomes); 4, 105000x g supernatant fraction (cytosoi).

fraction from V79 cells was 37% and that from C H O cells was 59%. 10% or lower amounts of the bleomycin hydrolase-specific antibody-reactive proteins were recovered from the 800 × g and 10000 × g pellet fractions in both cell lines. However, approx. 85% of the total bleomycin hydrolase activity was recovered from the 1 0 5 0 0 0 × g supernatant fractions and only 15% from the 105 000 x g pellet fractions of both species of cells (Table II). The enzymatic activity was scarcely detected in the 800 x g and 10000 × g pellet fractions from both of the cell lines. The subcellular distribution of bleomycin hydrolase in labeled C H O cells was studied by S D S - P A G E followed by fluorography. As shown in Fig. 3, no band was detected in the 800 x g and 10000 x g pellet fractions. The 105 000 x g supernatant fraction yielded only the 48 k D a band, whereas the 105000 x g pellet fraction gave both the 31 k D a and the 48 kDa band. The 31 kDa band was not detected in any other fraction.

Immunoprecipitation of bleomycin hydrolase from V79 and CHO cells Immunoprecipitation was performed using 10 000 × g supernatants of cell homogenates prepared in 10 m M

34 cell-derived enzyme was not precipitated unless a 15 pg or greater amount of the monoclonal antibody was added. Moreover, immunoprecipitation was performed using 105 000 x g supernatants of cell homogenates prepared in 10 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose. There was no difference in the reactivity with the monoclonal antibody between the V79 cell-derived bleomycin hydrolase and that derived from CHO cells as above (data not shown).

6

>,c"

~_o

i>_-x 4

.t,0 "$-

o'E

oe. 0 ® ~" 0

5

10

1"5

'¢Antibody (/~g) Fig, 4. lmmunoprecipitation of blcomycin hydrolase extracted from

V79 and CHO cells. Bleomycin hydrolase in the 10000x g supernatants of the two cell typeshomogenatedin 10 mM phosphate buffer (pH 7.4) was immunoprecipitated with increasing amounts of antibleomycin hydrolase monocional antibody as described in Materials and Methods. The remaining bleomycin hydrolase activity in the supernatant fromV79(®) and CHO (o) cellswas assayed. phosphate buffer (pH 7.4). Increased amounts of bleomycin hydrolase were precipitated from the homogenates from both V79 and CHO cells with application of increasing amounts of the anti-bleomycin hydrolase monoclonai antibody (Fig. 4). When 5.10 -2 units of bleomycin hydrolase was reacted with 5 pg of the monoclonal antibody, the V79 cell-derived enzyme was almost completely precipitated. By contrast, the CHO

Fig. 5. Immunofluorescenceof bleomycin hydrolase in V79 and CHO cells. V'/9 (A) and CHO (B) cells were subjected to immunofluorescence staining as described in Materials and Methods. Specimens were observed under a fluorescent microscope (125 x ).

Immunocytochemical localization of bleomycin hydrolase in V79 and ClIO cells In order to obtain information on the intraceilular localization of bleomycin hydrolase, V79 and CHO cells were cytologically examined by the indirect immunofluorescence method with anti-bleomycin hydrolase monoclonal antibody (Fig. 5). In both cell types, specific fluorescence was observed in the cytoplasmic area surrounding the nucleus. Fluorescence that looked vaguely granular in appearance was densely localized on the endoplasmic reticulum. In some CHO cells, fluorescence appeared to be associated with the plasma membrane. In general, CHO cells were more strongly stained than V79 cells. Discussion

The intracellular level of bleomycin hydrolase activity is considered to be related to the bleomycin susceptibility of the cell. We examined how metabolic degradation of bleomycin hydrolase occurs in two Chinese hamster cell lines with different bleomycin hydrolase activity using a bleomycin hydrolase-specific monoclonal antibody. When labeled with [~4C]leucine for 1 h, the radioactivity of the band of the 48 kDa subunit form recovered from CHO cells was almost equal to that recovered from V79 cells, suggesting that the rate of de hove synthesis of bleomycin hydrolase in the two cell lines is similar. However, after 24 h of labeling, the radioactivity of the 48 kDa subunit form recovered from CHO cells was significantly lower than that from V79 cells. These results lead us to postulate that the 48 kDa subunit form occurring in CHO cells is less stable than that in V79 cells. This postulation is favorably supported by the results of pulse-chase labeling experiments showing that the label of the 48 kDa subunit form was clearly seen in the extracts from V79 cells but not seen in the extracts from CHO cells after 15 h or more of chase. Although the possible involvement of gene amplification or regulation of mRNA related to bleomycin hydrolase has not been explored, greater stability of bleomycin hydrolase in V79 cells than in CHO cells seems to be associated with the existence of higher levels of bleomycin hydrolase in the former. The stability of bleomycin hydrolase in a given cell line may be related to its intrinsic resistance to bleomycin.

35 Pulse-chase labeling experiments demonstrated the retardation of the intracellular appearance of the 31 kDa form compared with that of the 48 kDa subunit form. Moreover, after subcellular fractionation, the 31 kDa band was detected only in the 105000 × g pellet fraction and not in any other fraction. These results suggest that the 31 kDa form is a cleavage product of the 48 kDa subunit form, and that the degradation of bleomycin hydrolase into the 31 kDa cleavage form takes place on some membranous structures, most probably the plasma membrane or the endoplasmic reticulum where some proteinases may play a role. The resulting 31 kDa form appears to be rapidly subjected to further degradation, because the present study also detected radioactivity on the 48 kDa subunit form but not on the 31 kDa form of the enzyme in V79 and CHO cells prelabeled with [14C]leucine for 24 h. Using 105 000 x g supernatants of cell homogenates prepared in 10 mM phosphate buffer containing 0.25 M sucrose, there was no difference of the reactivity with te monoclonal antibody between the V79 cell-derived bleomycin hydrolase and the ClIO cell-derived bleomycin hydrolase. This result suggests that V79 cellderived bleomycin hydrolase and C l i o cell-derived bleomycin hydrolase are recognized by the monoclonal antibody with the same efficiency. However, since greater portions of bleomycin hydrolase activity were removed from the 10000 × g supernatant fraction prepared from V79 cells than that from CHO cells with the same amount of bleomycin hydrolase-specific monoclonal antibody, it is hypothesized that the fraction prepared from CHO cells contains a larger quantity of the enzymatically inactive form of bleomycin hydrolase molecules or degradative forms of the enzyme which is reactive to the bleomycin hydrolase-specific monoclonal antibody. This hypothesis is supported by the results of experiments showing a significant discrepancy between the immunoreactivity with bleomycin hydrolase-specific monoclonal antibody and bleomycin hydrolase activity of subcellular fractions, especially in the 105000 x g pellet fraction (microsomes) prepared from ClIO cells. The finding from the cytochemical study using the indirect immunofluorescence method that CHO cells, especially on their plasma membrane and endoplasmic reticulum, were more strongly stained by fluorescence than were V79 cells, may be explained by the existence of increased amounts of the above-mentioned bleomycin hydrolase-derived inactive substances in the cells. The subcellular distribution of bleomycin hydrolase and related inactive substances in labeled C l i o cells was more precisely demonstrated by SDS-PAGE followed by fluorography; the 105 000 x g pellet fraction gave 31 kDa and 48 kDa bands, although it is unclear in

which the enzymatically inactive bleomycin hydrolase exists - the 31 kDa form, 48 kDa form, or both. Since the 31 kDa form seems to be a cleavage product of the 48 kDa form, the 31 kDa form can be considered as the most probable candidate for the inactive form. However, there is another possibility that bleomycin hydrolase may interact with some endogenous bleomycin hydrolase inhibitor existing in the cells, eventually being converted to the 48 kDa masked form, as is the case for calpain which was demonstrated to be inhibited by an endogenous inhibitor, calpastatin [13,14]. It also cannot be ruled out that the 48 kDa subunit form resulting from dissociation of the intact hexameric form of bleomycin hydrolase may be inactive, because it is unclear whether it is from the dissociated 48 kDa subunit form or the intact hexameric form of the enzyme that the 48 kDa band seen on SDS-PAGE is derived. The molecular size of inactive bleomycin hydrolase remains to be determined. Acknowledgements This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan. The authors express their deep thanks to the late Dr. H. Umezawa for his kind advice and cooperation in the present study. References 1 Sugiura, Y., Muraoka, Y., Fujii, A., Takita, T. and Umezawa, H. (1979) J. Antibiot. 32, 756-758. 2 Ebihara, K., Ekimoto, H., ltchoda, Y., Abe, F., Inoue, H., Aoyagi, S., Yamashita, T., Koyu, A., Takahashi, K., Yoshioka, O. and Matsuda, A. (1978) Jpn. J. Antibiot. 31,872-885. 3 Umezawa, H., Takeuchi, T., Hod, S., Sawa, T., lshizuka, M., Ichikawa, T. and Komai, T. (1972) J. Antibiot. 25, 409-420. 4 Miyaki, M., Ono, T., Hod, S. and Umezawa, H. (1975) Cancer Res. 35, 2015-2019. 5 Akiyama, S., Ikezaki, K., Kuramochi, H., Takahashi, K. and Kuwano, M. (1981) Biochem. Biophys. Res. Commun. 101, 55-60. 6 Umezawa, H., Hori, S., Sawa, T., Yoshioka, T. and Takeuchi, T. (1974) J. Antibiot. 27, 419-424. 7 Nishimura, C., Tanaka, N., Suzuki, H. and Tanaka, N. (1987) Biochemistry 26, 1574-1578. 8 Sebti, S.M., DeLeon, J.C. and Lazo, J.S. (1987) Biochemistry 26, 4213-4219. 9 Lazo, J.S., Boland, C.J. and Schwartz, P.E. (1982) Cancer Res. 42, 4026-4031. 10 Laemmli, U.K. (1970) Nature (London) 227, 680-685. 11 De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Applemans, F. (1955) Biochem. J. 60, 604-617. 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 13 Waxman, L. and Krebs, E.G. (1978) J. Biol. Chem. 253, 5888-5891. 14 Kominami, E., Wakamatsu, N. and Katunuma, N. (1982) .L Biol. Chem. 257, 14648-14652.