T H E JOURNAL OF B I O L O G I C A L CHEMISTRS Vol. 2S8, No. 13, Issue oIJuly 10, pp. 8338-8345, 1983 I ’ r inkd In I ! S . A

Selective Overproduction of Adenosine Deaminase in Cultured Mouse Cells*

(Received for publication, November 1, 1982)

Cho-Yau Yeung$$, Diane E. IngoliaS, Chefin Bobonis$, Bonnie S. DunbarP, Mary E. RiserlT, Michael J. Siciliano11 , and Rodney E. Kellems$** From the $ Verna and Marrs McLean Department of Biochemistry and the IDepartment of Cell Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030 and the 11 Department of Genetics, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030

The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regu- lation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene ampli- fication mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed llAAU, which selected si- multaneously for adenosine deaminase and adenosine kinase. The 1 lAAU selection medium consists of alan- osine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosyn- thesis caused by adenosine at the concentration em- ployed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 1 lAAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in aden- osine deaminase activity. Adenosine deaminase ac- counted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguisha- ble from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased aden- osine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures in- dicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine de-

* This work was supported by National Institutes of Health Grants GM 30204 and ES01287, National Science Foundation Grant PCM 8104533), Robert A. Welch Foundation Grand 6-893, and by a gift from the Kleberg Foundation. Alanosine and xylofuranosyl-adenine were kindly provided by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. Deoxyco- formycin was generously provided by the National Products Branch, Developmental Therapeutics Program, Division of Cancer Treat- ment, National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Recipient of Postdoctoral Fellowship Q-893 from the Robert A. Welch Foundation.

CA00828. ** Recipient of Research Career Development Award 1 KO4

aminase levels. Based on the stepwise selection proto- col employed, the instability of the phenotype, and the presence of double-minutes, we believe that the in- creased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.

Adenosine deaminase is an enzyme of purine nucleoside metabolism that is present in virtually all mammalian tissues (1-5). The highest adenosine deaminase levels are observed in gastrointestinal and thymus tissues (1-10) and, within these tissues, adenosine deaminase levels are subject to de- velopmental regulation (3,9,10). A number of permanent cell lines retain these tissue-specific and developmental features of adenosine deaminase expression and should be useful as model systems for studying adenosine deaminase gene regu- lation (8,11,12). Human genetic disorders affecting adenosine deaminase gene expression have played a major role in eluci- dating the function of adenosine deaminase in mammalian development. Individuals lacking adenosine deaminase ex- pression suffer from an autosomal recessive genetic disorder called severe combined immunodeficiency disease which is characterized by a lack of functional B- and T-lymphocytes (13-16). On the other hand, a 45- to 70-fold increase in erythrocyte adenosine deaminase is associated with a domi- nantly inherited hemolytic anemia (17, 18).

To address a number of questions regarding adenosine deaminase gene structure, the regulation of adenosine dea- minase gene expression, and the molecular basis of adenosine deaminase associated genetic disorders, it is necessary to have protein and nucleic acid probes specific for adenosine dea- minase. As a first step toward achieving this objective, we attempted to isolate mammalian cells with amplified adeno- sine deaminase genes. Since adenosine deaminase gene expres- sion is not required for growth of cells in culture, the first step involved the development of a selection system requiring adenosine deaminase expression for growth of cultured cells. This was accomplished by modifying the adenosine kinase selection medium known as AAU (adenosine, alanosine, uri- dine) (19) by raising the adenosine concentration 11-fold to 1.1 mM, a level that is cytotoxic. The new selection system, termed 11AAU, selected not only for the expression of aden- osine kinase but also for that of adenosine deaminase, which served to detoxify excess adenosine by converting it to inosine. We used 1lAAU in conjunction with a stepwise selection for resistance to increasing concentrations of deoxycoformycin, an adenosine deaminase inhibitor (20-22), to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Based on a number of criteria (see “Dis-

8338

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Selective Overproduction of Adenosine Deaminase 8339

cussion”), we believe that the increased adenosine deami- nase is the result of amplification of adenosine deaminase genes.

EXPERIMENTAL PROCEDURES

Materials

Alanosine was obtained from the Drug Synthesis and Chemistry Branch, and 2’-deoxycoformycin was obtained from the Natural Products Branch, Division of Cancer Treatment, National Cancer Institute.

Cell Lines and Culture Conditions

Cells were maintained in DMEM’ supplemented with 15% horse serum (Grand Island Biological Company) and incubated at 37 “C in a humidified atmosphere of 95% air and 5% COz. Horse serum was used because i t has very low levels of adenosine deaminase as com- pared with fetal calf serum (23, 24). C1-1D is a thymidine kinase- deficient mouse fibroblast cell line (25). All cell lines were tested periodically and found to be free of mycoplasma contamination.

The AAU selection medium which selects for adenosine kinase expression has been described previously (19). The llAAU selection medium selects simultaneously for adenosine deaminase and adeno- sine kinase and consists of 1.1 mM adenosine, 50 p M alanosine, and 1 mM uridine in DMEM supplemented with 15% horse serum. The rationale of the 11 AAU selection protocol is discussed in detail under “Results” and “Discussion.”

Preparation of Cell Homogenates Cells to be analyzed were always removed from drug selection for

1 week and fed with fresh DMEM plus 15% horse serum 24 h before harvest. Cells were rinsed three times with Hank’s balanced salt solution, harvested by scraping with a rubber policeman, and pelleted by centrifugation. Cell pellets were resuspended in 1-2 volumes of homogenizing medium (0.010 M Tris-C1, pH 7.5, 1 mM P-mercapto- ethanol, and 1 mM EDTA) and disrupted by sonication (3 X 15 s ) . Cell sonicates were centrifuged for 90 min at 80,000 X gmaX to obtain high speed supernatant fractions (5-10 mg of protein/ml) which were analyzed for enzyme activities or by gel electrophoresis.

Enzyme Assays Adenosine deaminase activities were determined by the spectro-

photometric procedure of Agarwal and Parks (26) that relies on the decrease in absorbance a t 265 nm that occurs when adenosine is converted to inosine. The change in absorbance was completely inhibited by the presence of 10 nM deoxycoformycin indicating that the absorbance change was due to adenosine deaminase activity.

In some cases, adenosine deaminase activities were determined by the radiometric procedure described in the accompanying paper (12).

Adenosine kinase activities were determined by the radiometric procedure of Chan (19) with some modifications (12).

Protein concentrations were determined by the method of Lowry et al. (27).

Electrophoretic Analysis The high speed supernatant fractions of various cells were analyzed

by the following electrophoretic procedures. One-dimensional Polyacrylamide Gel Electrophoresis-Protein was

fractionated by electrophoresis through sodium dodecyl sulfate-poly- acrylamide gels according to the method of Laemmli (28). Stacking gel was 2.5% acrylamide, 0.06% bisacrylamide and the running gel was 12.5% acrylamide, 0.3% bisacrylamide. Electrophoretic separa- tion was carried out at room temperature for 6 h a t a constant current of 35 mA. Protein (- 100 pg/lane) was visualized by Coomassie blue stain after fixation with 20% trichloroacetic acid.

Two-dimensional Gel Electrophoresis-The methods used for elec- trophoresis were those described by O’Farrell (29) as modified by Anderson and Anderson (30, 31). The exact conditions used were those which have been outlined by Anderson et al. (32). Cells (ap- proximately 5 X lo6) were pelleted and washed with serum free medium. They were then suspended in a solubilization buffer con- taining 9 M urea (Bio-Rad), 4% Nonidet P-40 (Accurate Chemical

The abbreviations used is: DMEM, Dulhecco’s modified Eagle’s medium.

Co.), 2% ampholines (LKB, pH 3.5-lo), 2% (3-mercaptoethanol (Bio- Rad) for 2 h at 25 “C. Samples were centrifuged at 200,000 X g for 2 h in a Beckman Ultracentrifuge (Ti-60 rotor) and 2O-pl samples were focused for 12,000 volt hours using the ISO-apparatus (Electronu- cleonics, Inc.). Second dimension electrophoresis was carried out in 10 to 20% polyacrylamide slab gels cast using the DALT casting apparatus (Electro-Nucleonics, Inc.) as described by Anderson and Anderson (32).

Molecular weight markers included in the second dimension were obtained from Pharmacia Chemical Co. Proteins were visualized using the Gelcode silver stain (Upjohn Co.) as described by Sammons et al. (33).

Isoelectric Focusing-Isoelectric focusing was performed according to published procedures (34). Ampholines were chosen to cover the pH range of 4-6. Protein was visualized by Coomassie blue stain. Adenosine deaminase was localized by a specific histochemical stain (35, 36).

Starch Gels-Electrophoresis was conducted a t either 200 V for 16 h or 400 V for 5 h, as described (35). Following electrophoresis, the starch gel was sliced in replica sheets of 1 mm thickness. Replicas were stained for protein with bromphenol blue and for adenosine deaminase activity by a specific histochemical stain (35, 36).

Chromosome Preparation and Staining Cultures of exponentially growing cells were treated with Colcemid

(Grand Island Biological Company, final concentration 0.1 pg/ml) for 1.5 h a t 37 “C. Cells were harvested by trypsinization, collected by centrifugation, gently resuspended in 65 mM KC1 and incubated under gentle agitation for 15 min at 37 “C. Cells were removed from the hypotonic solution by centrifugation, gently resuspended, and fixed in absolute methanobacetic acid (3:l) for 20-30 min. Fixed cells were collected by centrifugation, fixed two additional times, dropped onto slides, and air-dried. Chromosomes and double-minutes were visualized by ethidium bromide staining (100 pg/ml) followed by fluorescent microscopy, and by Giemsa staining (Fisher, 40 pg/ml, 30 min) followed by light microscopy.

RESULTS

Simultaneous Selection for Adenosine Deaminase and Aden- osine Kinase: The 1lAAU Selection System-In the accom- panying manuscript (12), we reported the use of xylofurano- syl-adenine to select human choriocarcinoma cells with in- creased adenosine deaminase levels. However, that selection protocol suffered from the drawback that adenosine kinase- deficient cells could also survive. To circumvent potential problems caused by the presence of adenosine kinase-deficient mutants, we developed a new protocol that selected simulta- neously for adenosine deaminase and adenosine kinase. The new protocol is a modification of a previously described selec- tion system (termed AAU) (19) that was designed to select for adenosine kinase. The modification involved an 11-fold increase in adenosine concentration to 1.1 mM (hence the name 11AAU). The important components of 1lAAU medium are alanosine (0.05 mM) which serves to block de novo AMP synthesis, adenosine (1.1 mM) which provides a salvage route for AMP synthesis via the adenosine kinase reaction, and uridine (1.0 mM) which alleviates the block in UMP biosyn- thesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM (37, 38, 45), adenosine deaminase is required to detoxify excess adenosine by converting it to inosine. (See Fig. 1 for relevant metabolic pathways.) Thus, cells that can survive llAAU selection must not only be able to utilize the exogenous adenosine via aden- osine kinase but must also be able to detoxify the excess adenosine via the unidirectional adenosine deaminase path- way.

Mouse Cl-1D cells selected for resistance to llAAU ex- pressed adenosine deaminase a t a level approximately 1.4- fold that of parental CI-1D cells as determined by spectropho- tometric assay (Table I). This level of adenosine dea- minase expression was presumably essential for these cells to

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8340 Selective Overproduction of Adenosine Deaminase CAMP

t de novo synthesis ATP "Nucleic Acids

11 t ADP -dADP"dATP

, IYP"ASA"AMP t I

adenosine ' I S-adenosyl- homocysteine

\ " HYP hom&ysteine

FIG. 1. Metabolic pathways associated with adenosine (Ado). Note that alanosine (see "Results") blocks the conversion of IMP to ASA adenylosuccinic acid (ASA) and thus blocks the de nouo synthesis of AMP.

TABLE I Selection for increased adenosine deaminase using 1 I A A U and

deoxycoformycin

Electrophoretic Analysis of Proteins-High speed superna- tant extracts of C1-1D cells and cells resistant to llAAU plus increasing concentrations of deoxycoformycin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 4). The results indicate that the increased adenosine deaminase activity was accompanied by the increased abun- dance of a protein having an apparent molecular weight of 41,000. This protein was not readily detectable in the parental cells but was the most abundant protein in the high speed supernatant fraction of 1 lAAU/6.4 PM deoxycoformycin-re- sistant cells. When the high speed supernatant fraction was boiled under nonreducing conditions for 15 min prior to analysis, most of the 41,000 molecular weight protein was converted to a form running at approximately 36,000 (Fig. 4, B-1/6.4*). Densitometric analysis of Coomassie blue-stained gels indicated that the decrease in the abundance of the

Selection

None llAAU llAAU + 0.08 p~ dCF" llAAU + 0.16 pM dCF llAAU + 0.4 p~ dCF llAAU + 0.8 p~ dCF llAAU + 1.6 pM dCF 1 lAAU + 3.2 p~ dCF llAAU + 6.4 PM dCF llAAU + 12.8 UM dCF

Cell designation

C1-1D B-1 B-1/0.08 B-1/0.16 B-1/0.4 B-1/0.8 B-1/1.6 B-1/3.2 B-1/6.4 B-1112.8

Adenosine de- Adenosine

tivity ity

nmolfrninfmg X

aminase ac- kinase activ-

0.08 1.56 0.11 0.52 1.1 0.61 2.7 0.57

15 6.1 0.63

0.75 26 0.71 55 NTb

130 N T 220 N T

a dCF, deoxycoformycin. * NT, not tested.

survive in llAAU since suppression of cellular adenosine deaminase activity by 10 nM deoxycoformycin killed these cells in llAAU selection medium. In the absence of 1lAAU selection medium, adenosine deaminase expression was not required judging from the fact that cells were not killed by up to 50 p M deoxycoformycin.

The adenosine kinase expression of the 11AAU-resistant cells was retained at a level approximately 40% that of paren- tal Cl-1D cells (Table I). Thus, resistance to llAAU selection was associated with an increase in adenosine deaminase ac- tivity and a decrease in adenosine kinase activity.

Selection for Increased Adenosine Deaminase Production Using Increasing Concentrations of Deoxycoformycin in Con- junction with 11AAU-The level of adenosine deaminase required for growth in llAAU was approximately 40% greater than that normally present in Cl-1D cells. It was possible to isolate from 11AAU-resistant cells those which were resistant to 1lAAU plus increasing concentrations of deoxycoformycin, starting at a deoxycoformycin concentration of 10 nM. Selec- tion pressure was increased a t 4- to 6-week intervals by doubling the deoxycoformycin concentration used in conjunc- tion with 11AAU medium. Continued selection in llAAU for increasing deoxycoformycin resistance resulted in the isola- tion of highly drug-resistant cells as judged by relative plating efficiencies in the presence of 1 lAAU and various concentra- tions of deoxycoformycin (Fig. 2). The increased adenosine deaminase activity in the drug-resistant cells was linearly correlated with the level of deoxycoformycin resistance (Fig. 3, Table I) with a slope of exactly 1. This indicated that increased deoxycoformycin resistance was fully accounted for by a corresponding increase in cellular adenosine deaminase levels. Throughout the series, adenosine kinase expression remained relatively stable at a level approximately 40% that of parental Cl-ID cells (Table I).

%"I Y 100

u- u 75 U

W

w 2 25 \ \ - I W 5 Il"L.+,L,I E 10-10 10-9 10-8 10.7 10-6 10-5 10-4

DEOXYCOFORMYCIN (MI

FIG. 2. The relative plating efficiencies of 11 AAU-resistant (B-I) and 11 AAU plus 6.4 PM deoxycoformycin-resistant (B- Z/6.4) cells in l lAAU medium, plus various concentrations of deoxycoformycin. Relative plating efficiencies were determined by innoculating approximately 2,000 cells/60-mm (diameter) culture dish containing 3 ml of l lAAU selection medium (see "Experimental Procedures") plus increasing concentrations of deoxycoformycin. Fol- lowing growth for two weeks, colonies were fixed with methano1:acetic acid (3:l) and visualized by staining with Giemsa. Each value repre- sents the average of a t least three determinations and is expressed relative to control values for cells plated in 11AAU medium without deoxycoformycin.

I

t ._

t

101 In-l

DEOXYCOFORMYCIN (pM1

FIG. 3. The relationship betwety ADA activity and deoxy- coformycin resistance in 11 AAU-resistant cells. Cells were grown for at least two months at each level of llAAU/deoxycofor- mycin selection prior to analysis. Cells to be assayed were passaged one time (1:8 cell dilution) in drug-free medium and grown for approximately one week prior to assay for ADA activity. The drug- free medium was replaced several times during the week in an attempt to remove deoxycoformycin which would interfere with the ADA assay. The values shown represent the mean and standard deviation of at least three determinations. ADA, adenosine deaminase.

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Selective Overproduction of Adenosine Deaminase 8341

41,000-Da protein was exactly accounted for by the increase in abundance of the 36,000-Da protein. This led us to believe that the 36,000-Da protein is derived from the 41,000-Da protein. The molecular basis for this electrophoretic shift is as yet undetermined. Together, these bands accounted for approximately 15% of the soluble protein in B-1/6.4 cells. In more highly resistant cells these bands accounted for approx- imately half the soluble protein (data not shown). Another change associated with selection for llAAU/deoxycoformycin resistance was an increase in the abundance of a minor band having an apparent molecular weight of 61,000 (Fig. 4).

A more detailed comparison of the proteins present in Cl- 1D cells and an 11AAU-deoxycoformycin-resistant line (B-l/ 3.2) was achieved by two-dimensional gel electrophoretic anal- ysis involving isoelectric focusing in the first dimension and electrophoresis in a gradient polyacrylamide gel containing sodium dodecyl sulfate in the second (Fig. 5). The silver stain method of Sammons et al. (33) was used as the method of protein detection. This method stains different proteins dif- ferent colors. Although the molecular basis of this stain has yet to be determined, the color differentiation as well as the sensitivity of the stain allow for increased resolution in two- dimensional electrophoresis. Using this method, a 41,000- 43,000-Da peptide was readily apparent in the drug-resistant cells and ran with an apparent PI of 4.5. Although other peptide differences were apparent, this spot accounted for the most prominent difference between the parental and drug- resistant cells.

Histochemical Staining for Adenosine Deaminase Activity- To determine if the 41,000-Da protein having a PI of 4.5, co-

-61

FIG. 4. Sodium dodecyl sulfate-polyacrylamide gel electro- phoretic analysis of the soluble protein from CI-1D cells and cells selected for resistance to 1 lAAU (designated B-I) and increasing concentrations of deoxycoformycin. The number following B-1 refers to the p~ level of deoxycoformycin resistance. The protein in the lane designated B-1/6.4* was frozen and thawed several times and incubated for 1 h at 37 "C under oxidative condi- tions (see "Results") prior to analysis. Molecular weight standards are (top to bottom): phosphorylase b, 92,500; bovine serum albumin, 66,200; ovalbumin, 45,000; and carbonic anhydrase, 31,000. The ar- rows serve to draw attention to proteins having apparent molecular weights of 61,000, 41,000 (believed to be ADA), and 36,000 (believed to be derived from ADA).

42

7 30 9 X L

5 69

30 ?., i.. c * . e, r B

- "

FIG. 5. Two-dimensional gel electrophoretic analysis of the proteins in CI-1D cells (A) as compared with those of the 1 lAAU/deoxycoformycin resistant line, B-1/3.2 (B). Patterns represent approximately one-third of the proteins resolved by isoe- lectric focusing in the first dimension followed by separation in 10- 20% gradient polyacrylamide slab gels in the second dimension. Spots numbered 1-7 are included as reference markers since they are common to both cell types. The arrow indicates the 41,000-43,000- Da peptide which is readily apparent in the B-1/3.2 cell line but not in the CI-1D line.

migrated with adenosine deaminase activity, the following experiment was performed. High speed supernatant proteins were separated by isoelectric focusing in the 4 to 6 pH range. Visualization of protein by Coomassie blue staining revealed the presence of a protein band at PI 4.5 that was present at high abundance in llAAU/3.2 PM deoxycoformycin-resistant cell extracts but not readily detectable in extracts of Cl-1D cells (Fig. 6). Histochemical analysis indicated that adenosine deaminase activity co-migrated with this protein. When sim- ilar amounts of enzyme activity were analyzed, no apparent differences in the electrophoretic properties of adenosine de- aminase from parental and highly drug-resistant lines were observed (Fig. 6). However, analysis of undiluted or partially diluted samples from the drug-resistant cells revealed the presence of minor bands a t isoelectric points higher than 4.5. Since insufficient adenosine deaminase activity from CI-1D cell extracts had been loaded to allow detection of the minor bands, we cannot say at this time whether the minor forms were present in CI-1D cells. However, the main ADA activity in the drug-resistant cells did co-migrate with the parental adenosine deaminase activity and the PI 4.5 protein.

The adenosine deaminase activity of Cl-1D cells was also compared with that of llAAU/12.8 PM deoxycoformycin-

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8342 Selective Overproduction of Adenosine Deaminase

$$$ tion up to 0.3 p ~ . Over the linear portion of the curve, the

$0 p @+ \ac mycin inhibited adenosine deaminase from parental and drug- o+ 0s- $' slopes were indistinguishable. This indicated that deoxycofor-

resistant cells to the same extent. Beyond 0.3 p ~ , the decrease

deoxycoformycin concentration. However, even in this region of the curve, the effect of deoxycoformycin on adenosine deaminase activities from both the Cl-1D and B-1/1.6 cells was indistinguishable. These results suggest that the molec- ular basis for deoxycoformycin resistance was not due to a structural alteration in adenosine deaminase which resulted in a reduced sensitivity to deoxycoformycin inhibition.

Cytogenetic Analysis of Cells with Increased Adenosine De- aminase-llAAU/deoxycoformycin-resistant cells were ex- amined for the presence of double-minutes, cytogenetic struc- tures often associated with the presence of unstable amplified DNA in animal cells (39). Examination of metaphase cells from B-1/0.8 cells by fluorescent microscopy following ethid- ium bromide staining, or by light microscopy following

3% ?). % % % ? ) % ?). ?).

G S G S S S ' S ' ,. ,\' ,Q ,\' A' \' \' in adenosine deaminase was not linearly related to increased

- "-

-pH 4.5

PROTEIN ADA ACTIVITY FIG. 6. Isoelectric focusing of soluble protein from Cl-1D

cells and cells selected for resistance to l lAAU + 3.2 p~ deoxycoformycin (B-1/3.2). Protein was visualized by Coomassie blue stain; ADA was localized by a specific stain for ADA activity (see "Experimental Procedures"). Because of high ADA levels, ex- tracts from the drug-resistant cells were analyzed a t multiple dilu- tions. ADA, adenosine deaminase.

resistant cells (B-1/12.8) by electrophoretic analysis on starch gels. The results (Fig. 7) indicate that a major protein which was present at increased abundance in extracts of the B-l/ 12.8 cells co-migrated with adenosine deaminase activity (Fig. 7). The extracts of B-1/12.8 cells displayed a pronounced increase in adenosine deaminase activity over the Cl-1D cell extracts, as judged from the size and intensity of the histo- chemical stain. One minor electrophoretic variant (indicated by the arrow in Fig. 7) was observed. The minor variant did not co-migrate with adenosine deaminase activity and its exact nature was undetermined. The identical electrophoretic mobility of the adenosine deaminase derived from Cl-1D cells and the llAAU/deoxycoformycin-resistant cells suggests that the increased ADA activity in the highly drug-resistant cell line was not associated with a structural alteration in adeno- sine deaminase that resulted in a change in electrophoretic mobility in starch gels.

Deoxycoformycin Titration of Adenosine Deaminase Activ- ity-Adenosine deaminase activity from Cl-1D and 11AAU/ 1.6 PM deoxycoformycin-resistant cells (B-1/1.6) was com- pared with regard to sensitivity to deoxycoformycin inhibi- tion. Similar amounts of ADA activity from high speed su- pernatant fractions of parental and drug-resistant cells were incubated for 10 min a t room temperature with increasing concentrations of deoxycoformycin, then assayed for adeno- sine deaminase activity. The results (Fig. 8) show that the inhibition of adenosine deaminase activity from each source was linearly related to increasing deoxycoformycin concentra-

&$ .$ oo+ oo+

0 &O Q0 Q ,@'* \2. \o \\@ c..' %.' o/ %/\\ c?'

49

-E c 0

t

Protem ADA Activity

FIG. 7. Starch gel electrophoretic analysis of soluble pro- tein from C1-1D cells and cells selected for resistance to l lAAU + 12.8 pM deoxycoformycin (B-1/12.8). Protein was visualized by bromophenol blue stain. ADA was localized by a specific stain for ADA activity. Because of high ADA levels, extracts from drug-resistant cells were analyzed a t multiple dilutions. A protein (see arrow) which was not associated with ADA activity was also present a t increased abundance in the B-1/12.8 cell extracts. ADA, adenosine deaminase.

C. CI-1D M 8-110.8 -

- -

1 I I I I 0.1 0.2 0.3 0.4 0.5 DEOXYCOFORMYCIN (nM)

FIG. 8. Deoxycoformycin titration of ADA activity from C1- 1D cells and cells selected for resistance to l lAAU + 1.6 pM deoxycoformycin (B-l/l.6). Similar amounts of enzyme activity from parental and drug-resistant cells were incubated with increasing amounts of deoxycoformycin (10 min, 20 "C), then assayed for ADA activity. ADA, adenosine deaminase.

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Selective Overproduction

Giemsa staining revealed the presence of large numbers of double-minutes (Fig. 9, C and D). The number of double- minutes/cell varied substantially, and the size of individual double-minutes within a single cell also varied over a wide range. This suggested heterogeneity in the quantity of protein and DNA/double-minute. Double-minutes were not observed in Cl-1D cells (Fig. 9, A and B ) .

Analysis of Drug-resistant Cells Following Growth in the Absence of Selection Pressure-Cells that were selected for resistance to llAAU and 0.8 p~ deoxycoformycin had a 190- fold increase in adenosine deaminase levels (Table I, B-l / 0.8). To determine if this phenotype was stable, adenosine deaminase activities were measured at various intervals fol- lowing growth in the absence of selection pressure. The results (Fig. 10) indicate that by four passages (1:8 cell dilution/ passage) in the absence of selection, adenosine deaminase

FIG. 9. Cytogenetic analysis of parental and 1 lAAU/deox- ycoformycin-resistant cell lines. Metaphase chromosomes from CI-1D cells and B-1/0.8 cells were analyzed by light microscopy following Giemsa staining ( A and C ) or by fluorescent microscopy following ethidium bromide staining ( B and D) as described under "Experimental Procedures." Numerous small double-minutes were readily apparent in the B-1/0.8 cells (C and D) and no double-minutes were apparent in the CI-1D cells ( A and B) .

I C ' I I I

2 4 6

GROWTH IN THE ABSENCE OF SELECTION Ips-)

FIG. 10. Analysis of ADA activity following growth of llAAU/deoxycoformycin-resistant cells in the absence of se- lection pressure. Cells selected for resistance to llAAU + 0.8 p~ deoxycoformycin (designated B-2/0.8) were grown in the absence of selection for the indicated number of passages and analyzed for ADA activity. At each passage, cell cells were diluted 1:8; thus, each passage corresponds to at least three generations. ADA, adenosine deaminase.

of Admosine Deaminase a343

A B

, . FIG. 11. Cytogenetic analysis of 1 lAAU/deoxycoformycin-

resistant cells following removal of selection pressure. A, a representative metaphase of B-1/0.8 cells grown under continuous selection pressure in llAAU plus 0.8 pM deoxycoformycin. B, a representative metaphase of B-1/0.8 cells grown for two months in the absence of 1 lAAU/deoxycoformycin selection pressure. Meta- phase chromosomes were visualized as described under "Experimental Procedures."

activity dropped to a level approximately 10% that originally present in the B-1/0.8 cells. The decrease in adenosine de- aminase levels that accompanied the growth of B-1/0.8 cells in the absence of selection was associated with the loss of double-minutes in metaphase cells (Fig. 11).

The loss of adenosine deaminase activity in the absence of selection pressure was also associated with a decrease in the abundance of the prominent 41,000 molecular weight protein (data not shown). This observation is consistent with the suggestion that this polypeptide is adenosine deaminase. A protein with an apparent molecular weight of 61,000 also decreased in abundance when selection was removed.

DISCUSSION

The specific objective of the work reported here was to develop a selection protocol to isolate cell lines with amplified adenosine deaminase genes. Such cell lines should be useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Previous successful attempts at isolating cell lines with amplified genes have relied on the use of specific inhib- itors of essential enzymes (40,41) or on the ability of a specific enzyme or protein to detoxify or sequester a cytotoxic com- pound (42). To obtain cells with increased adenosine deami- nase, we relied on a combination of these approaches. Hoffee et al. (43) have recently described a substantially different selection protocol that enabled them to select adenosine ki- nase-deficient rat hepatoma cells with a 20-fold increase in adenosine deaminase.

Since adenosine deaminase is not an essential enzyme for growth of cells in culture, it was necessary to develop a selection protocol requiring adenosine deaminase expression. I t should then be possible to use such a selection protocol in conjunction with deoxycoformycin, a potent adenosine de- aminase inhibitor (44), to select for cells with amplified aden- osine deaminase genes. Our first efforts a t developing an adenosine deaminase selection protocol relied on the ability of adenosine deaminase to detoxify the cytotoxic adenosine analogs, xylofuranosyl-adenine or arabinofuranosyl-adenine, by converting them to their respective inosine analogs (12). Subsequent metabolism of the inosine analogs, via the purine nucleoside phosphorylase reaction, yielded hypoxanthine and the sugars xylose or arabinose, all harmless metabolites (see Fig. 1 for relevant metabolic pathways). As shown in the accompanying manuscript (12), resistance to these cytotoxic adenosine analogs may result from either increased adenosine deaminase (which serves to detoxify the drug) or loss of

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8344 Selective Overproduction of Adenosine Deaminase

adenosine kinase (thus preventing phosphorylation of the drug to its toxic metabolites).

Since we were interested only in isolating mutant cells with increased adenosine deaminase expression, we devised a new adenosine deaminase selection protocol that would also select against any spontaneously appearing adenosine kinase-defi- cient mutants. The new selection protocol involved the use of adenosine as the cytotoxic adenosine deaminase selecting agent in conjunction with AAU selection for adenosine kinase expression (19). The AAU selection protocol utilizes alanosine to block de nouo AMP biosynthesis (19, 46, 47), adenosine to relieve the AMP block via the adenosine kinase pathway, and uridine to alleviate the block in UMP biosynthesis caused by adenosine. By increasing the adenosine concentration 11-fold (to 1.1 mM), we constructed a new selection protocol termed llAAU that selected not only for adenosine kinase which provided a salvage route for AMP synthesis but also for adenosine deaminase which served to detoxify excess adeno- sine. The adenosine cytotoxicity at this concentration is be- lieved to be due to secondary effects on several metabolic pathways including 1) inhibition of pyrimidine nucleotide biosynthesis, 2) inhibition of cell growth resulting from in- creased synthesis of cyclic AMP, 3) inhibition of ribonucleo- tide reductase resulting from dATP accumulation, and 4) inhibition of methylation reactions resulting from the accu- mulation of S-adenosylhomocystine (reviewed in Refs. 37, 38, and 45). Cl-1D cells surviving selection in llAAU had a 40% increase in adenosine deaminase and a 60% reduction in adenosine kinase. The increased adenosine deaminase and the reduced adenosine kinase both presumably serve to min- imize the cytotoxic effects of the high adenosine concentration in the selection medium.

In the presence of llAAU mouse GI-1D cells were killed by the addition of 0.01 FM deoxycoformycin indicating that, in the presence of 11AAU medium, adenosine deaminase is an essential enzyme. Selection for resistance to 1lAAU plus increasing concentrations of deoxycoformycin resulted in the isolation of highly resistant cells with a 6000-fold increase in adenosine deaminase. Thus, the successful approach to ob- taining increased adenosine deaminase relied on 1) the devel- opment of an effective adenosine deaminase selection protocol and 2) the use of a potent and highly specific adenosine deaminase inhibitor.

Deoxycoformycin resistance (in conjunction with llAAU selection) could potentially have resulted from at least three mutant phenotypes: 1) increased adenosine deaminase, 2) reduced deoxycoformycin transport, or 3) a structural altera- tion in adenosine deaminase resulting in reduced affinity for deoxycoformycin. The fact that the increased drug resistance at each selection step was exactly accounted for by a corre- sponding increase in adenosine deaminase activity in the resistant cells indicated that we had not isolated either deox- ycoformycin transport or adenosine deaminase structural mu- tants using this selection protocol. This probably reflects the fact that transport mutants as well as adenosine deaminase structural mutants are unlikely to survive the 11AAU/deox- ycoformycin selection protocol for the following reasons. Since deoxycoformycin is an adenosine analog, it presumably enters the cell by the same transport mechanisms as adeno- sine. The llAAU selection protocol would select against aden- osine (and deoxycoformycin) transport mutants since cells must be able to utilize exogenous adenosine to meet the adenine nucleotide needs of the cells (via the adenosine kinase reaction). Similar considerations apply to the possibility of selecting adenosine deaminase structural mutants having re- duced deoxycoformycin affinity. Such mutants would proba- bly be impaired in their ability to bind and detoxify the

cytotoxic adenosine and thus be selected against in 11AAU. A structural alteration in adenosine deaminase resulting in reduced affinity for deoxycoformycin was formally ruled out as an explanation for drug resistance by comparing the deox- ycoformycin inhibition of adenosine deaminase activity from parent and highly drug-resistant cell lines. Thus, the design of the llAAU/deoxycoformycin selection appeared to make it well suited for selecting only adenosine deaminase over- producers and not nucleoside transport mutants, adenosine deaminase structural mutants and, as discussed above, aden- osine kinase-deficient mutants.

The increased adenosine deaminase activity resulting from selection for 1 lAAU/deoxycoformycin resistance was accom- panied by the increased abundance of a soluble protein having a molecular weight of 41,000 and a PI of approximately 4.5. These studies also revealed that this protein co-migrated with adenosine deaminase activity and that no change in the electrophoretic properties of the enzyme had accompanied selection for deoxycoformycin resistance. However, we were surprised to find that when undiluted extracts from 11AAU/ deoxycoformycin-resistant cells were examined by isolectric focusing, numerous minor adenosine deaminase bands were observed. These minor adenosine deaminase species do not appear to correspond with satellite adenosine deaminases (48, 49) observed in other mammalian systems. We do not know if these minor bands were present in extracts from parent cells since we could not apply a sufficient amount of adenosine deaminase activity in extracts of parent cells to determine if the minor bands were present. The minor bands may repre- sent precursors, degradation products, or modified forms of adenosine deaminase.

The llAAU/deoxycoformycin-resistant cells share a num- ber of properties associated with a class of mutants termed gene amplification mutants (39-42). A well studied example of this type of mutant are methotrexate-resistant cells with amplified dihydrofolate reductase genes (40). Methotrexate- resistant mutants are generally characterized by the stepwise selection protocol required for their isolation (50, 51), phen- otypic instability (50, 52), and by the presence of double- minutes (53). Based on a similar set of phenotypic properties associated with the llAAU/deoxycoformycin-resistant cells described here, we believe that the 6000-fold increase in adenosine deaminase levels that accompanied selection for 1 lAAU/deoxycoformycin resistance resulted from a corre- sponding amplification of adenosine deaminase genes.'

Gene amplification mutants are very useful because they produce large quantities of specific enzymes or proteins which are produced in small quantities in most cells. Since the increased enzyme content results from increased gene num- ber, such cell lines provide obvious advantages for DNA cloning due to enriched starting material and the ability to obtain nucleic acid probes specific for the amplified genes and their transcripts (54,41,42). Gene amplification mutants also facilitate studies of gene organization, gene regulation, and mRNA metabolism since, in most cases, the amplified genes are active and regulated normally (55-58). Because amplified genes often comprise specific cytogenetic structures such as double-minutes and/or homogeneously staining chromosomal regions, they serve as important model systems for cytogenetic studies relating chromosome or chromatin structure to gene activity (59, 60). They also provide enriched starting material

We have recently cloned a mouse adenosine deaminase cDNA sequence and used this nucleic acid probe to show that adenosine deaminase gene amplification fully accounts for the increased aden- osine deaminase levels in these cells (C.-Y. Yeung et al., manuscript in preparation).

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Selective Overproduction of Adenosine Deaminase 8345

for the isolation and characterization of double-minutes. We have continued to impose additional llAAU/deoxycoformy- cin selection pressure in an effort to obtain cell lines which overproduce adenosine deaminase to the highest extent pos- sible. In some of the most highly resistant cell lines, adenosine deaminase levels were elevated approximately 6000-fold rel- ative to the parental cells and the protein accounted for approximately 50% of the soluble protein. Such cell lines will make it possible to address a number of questions concerning the structure and regulation of adenosine deaminase genes in animal cells.

Acknowledgments-We are especially grateful to Dr. Robert Ferrel for help in running the isoelectric focussing gel. We appreciate the assistance of Meredith Riddell in the preparation of this manuscript.

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KellemsC Y Yeung, D E Ingolia, C Bobonis, B S Dunbar, M E Riser, M J Siciliano and R E

Selective overproduction of adenosine deaminase in cultured mouse cells.

1983, 258:8338-8345.J. Biol. Chem. 

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