Somatic Cell Genetics, Vol. 4, No. 2, 1978, pp. 143-156

The Selection of Wild-Type Revertants from Methotrexate Permeability Mutants Wayne Flintoff and Linda Saya Cytobiology Group, Department of Bacteriology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1 R e c e i v e d 1 Au gust 1977--Final 6 October 1977

Abstract In a previous report, we described the selection and partial characterization of three distinct classes of methotrexate (Mtx)-resistant Chinese hamster ovally cells (CHO) (1). Class I cells contained a structural alteration in dihydrofolate reductase. Class H cells showed an alteration affecting the permeability of the drug. Class III cells, selected from class I cells, had an increased activity of the altered enzyme. In the work described here, the sensitivity of these lines to the diaminopyrimidines has been investigated. Class I cells are as sensitive, class H cells are 5- to lO-fold more sensitive, and class III cells are 10- to 30-fold more resistant than wild-type cells. The increased sensitivity of the class H cells provided an opportunity to selectfi)r revertants of these mutants and such phentotypic wild-type revertant cells have been selected using one diaminopyrimidine, pyrimethamine. Such cells have drug sensitivities and permeability characteristics similar to wild-type cells. A second class has been identified which has wild-type drug sensitivities to the diaminopyrimidines but Mtx class H resistance to Mtx, and drug permeabilities characteristic of Mtx-resistant class H cells.

INTRODUCTION In earlier work (1), we have shown that three classes of mutants are found among Chinese hamster ovary (CHO) cells selected for resistance to the folic acid analogue, methotrexate (Mtx). Resistance in class I cells is due to a structural alteration in dihydrofolate reductase (1, 2), whereas the resistance in class II cells involves a defect in the permeability to Mtx. Class III cells, which were derived from class I cells by a second-step selection in increased concentration of the drug showed increased levels of 143 0098-0366/78/0300-0143505.00/09 1978PlenumPublishingCorporation

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the enzyme found in class I cells. Subsequently, we have also shown that class I and class III resistances are inherited as codominant traits in somatic cell hybrids, whereas class II resistance behaves as a recessive marker (3). Similar resistant phenotypes have been described in other systems (4-9). We have been examining the sensitivity of the Mtx-resistant mutants to various structural analogues of folic acid with the rationale of obtaining more complete descriptions of the different resistant phenotypes. Since there exists a wide spectrum of such analogues, it seemed reasonable to expect that Mtx-resistant lines might be altered in their sensitivity to some compounds. Previously, we have shown that class I and class II Mtxresistant cells are cross-resistant to aminopterin (1). In this report, we describe the effects of diaminopyrimidines on Mtx-resistant cells. These compounds are low molecular weight, lypophilic inhibitors of dihydrofolate reductase (10). Class I Mtx-resistant cells are as sensitive as wild-type cells, class II cells have increased sensitivity, and class III cells are more resistant to the cytotoxicity of the diaminopyrimidines. By taking advantage of the increased sensitivity of class II cells as compared to wildtype cells, and using pyrimethamine as the selective agent, phenotypic wild-type revertants of the class II Mtx-resistant cells have been isolated and partially characterized. In addition to the revertants, cells with close to wild-type sensitivity to the diaminopyrimidines but retaining their resistance to Mtx have also been isolated. MATERIALS AND M E T H O D S

Materials. Folic acid and NADPH were obtained from Sigma Chemical Co. Mtx and aminopterin (Amino) were purchased from Nutritional Biochemicals. Pyrimethamine (Pyr) (2,4-diamino-5-p-chlorophenyl6-ethyl pyrimidine) was a gift from Dr. J. Goldie and Dr. C. A. Nichol. Metoprine (DDMP) (2,4-diamino-5-3',4'dichlorophenyl)-6-methyl pyrimidine) and DDHP (2,4-diamino-5-3',4'dichlorophenyl) pyrimidine were also gifts from Dr. C. A. Nichol, Burroughs Wellcome Co. Trimethoprim (Tri) (2,4=diamino-5-(3',4',5'-trimethoxybenzyl pyrimidine) was obtained as a gift from Miss C. Taylor, Burroughs Wellcome Ltd. and was also purchased from Sigma Chemical Co. Ethyl methanesulfonate (EMS) was purchased from Eastman Chemical Company. (G-3H) Folic acid (specific activity, 5 Ci/mmol) and (Y,5',9(n)-3H) Mtx (specific activity, 13.4 Ci/mmol) were obtained from Amersham/Searle Corporation. Both labeled compounds were at least 90% pure as determined by paper chromatography.

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Cell Lines and Cell Culture. The proline-requiring CHO auxotroph was originally obtained from Dr. W. C. Dewey. The wild-type, Pro-3 and Pro-4, and the Mtx-resistant C H O cell lines and their nomenclature have been described previously (1). Pro-3 Mtx RI 3-3 is a cloned line of class I resistant cells obtained in a single-step selection from wild-type cells. The Pro-3 Mtx RuI 1-2 cell line refers to a class III mutant obtained in a secondstep selection from the clone Pro-3 Mtx RI 3-3. Pro-3 Mtx an 5-3, Pro-3 Mtx Rn 7-5, and Pro 4 Mtx RII 2-1 are 3 independent clones of class II resistant cells which were obtained in a single-step selection from 2 subclones of EMS mutagenized wild-type cells. A phenotypic wild-type revertant of Mtx RII is designated as, for example, rev. Pro-3 Mtx RII 1. Clone " X , " derived in nonselective medium as a subclone of the rev. Pro-3 Mtx mI 1 line, is described as rev. Pro-3 Mtx RII 1-X. Similarily, an isolate selected from class II Mtxresistant cells in which there is no alteration in resistance to Mtx is, for example, referred to as Pro 3 Mtx RI~ Pyr R 1. Cells were grown in suspension culture at 34 ~ or 37 ~ in complete medium (11) supplemented with 10% fetal bovine serum (FBS) (Microbiological Associates) according to the methods previously described (1). For growth on plastic surfaces, the cells were placed in the same medium in a humidified atmosphere containing 5% CO2. Cloning of cells was performed in 96-well Microtest 11 trays (Falcon Plastics). Selections were carried out in ~ special medium (12) supplemented with 40 ~g/ml proline and 10% dialized FBS. This medium is referred to as selection medium. Mutagenesis. Exponentially growing cells were pretreated with 150 /xg/ml EMS for 18 h, centrifuged, and resuspended in fresh medium. Survival was usually about 60%. The cells were then allowed to grow for 4 days to allow expression of putative mutants before selections were carried out. Plating Efficiencies. Relative plating efficiencies were determined either by complete d o s e - r e s p o n s e curves or by plating 50 and 500 cells at various drug concentrations in 24-well multidishes as previously described (1). The resistance or sensitivity of the various cell lines to the drugs is expressed bY the D10 value, the drug concentration that reduces cell survival to 10%. Relative resistance is expressed as the ratio of the D10 value for the cell line of interest to the D10 value for the wild-type line. Extract Preparation and Enzyme Assay. The preparation of cell extracts for the determination of folate reductase was as previously described (1). Folate reductase activity was measured with [aH] folic acid as substrate as previously described except that the incubation temperature was 34 ~ (2). One unit of folate reductase activity is defined as the

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amount of enzyme necessary to reduce 1 nmol of folate in 15 min under standard assay conditions. Protein was measured by the method of Lowry et al. (13) using bovine serum albumin as a standard. Determination of Drug Uptake. The uptake of labeled Mtx or folic acid into monolayers of the various cell lines was determined as previously described except that the incubation temperature was 34~ (1). Briefly, monolayers were washed with phosphate buffered saline (PBS) and uptake buffer (PBS + 0.01 M Hepes, pH 7.4) was added. At various times, the uptake buffer was removed and replaced with uptake buffer containing either 2 x 10-~M pH] Mtx, specific activity 3.3 Ci/mmol, or 5 x 10-TM [3H] folic acid, specific activity 5 Ci/mmol. Uptake was terminated by washing the monolayers with large volumes of cold PBS and 1 ml of 0.1 N NaOH was added. The monolayers were solubilized at 4~ for at least 2 h. Aliquots of 0.3 ml were counted in toluene-Omnifluor (New England Nuclear) scintillation fluid containing 10% Protosol (New England Nuclear) using a Beckman LS-350 liquid scintillation counter. Protein was determined on each sample using the method described above. The amount of labeled drug taken up is expressed as cpm/mg protein. Karyotype Analysis. Cells growing exponentially in suspension culture were incubated with 0.25/xg/ml colcemid (Grand Island Biological Co.) for 1,5-2 h at 34 ~ or 37~ The cells were washed with hypotonic and fixing solutions. Slides were prepared, stained with Giemsa, and 20 representative chromosome spreads were counted.

RESULTS

Resistance o,f Mtx-Resistant Lines to Other Folate Analogues. As indicated earlier, both class I and class II Mtx-resistant cell lines are crossresistant to aminopterin, a compound that is structurally similar to Mtx (1, 14). Class I resistant cells, which are about 20-fold resistant to Mtx, are about 50-fold resistant to aminopterin, and class II cells, about 50-fold resistant to Mtx, are about 200-fold resistant to aminopterin (Table 1). This differential sensitivity of these cell lines to the 2 drugs has provided a rapid means of distinguishing between the phenotypes (3). It was of further interest to determine whether the Mtx-resistant cells had altered resistance patterns to other folate analogues with sinqilar sites of action (14). Thus, the cytotoxicity of the diaminopyrimidines, pyrimethamine, trimethoprim, DDHP, and DDMP, on representative clones carrying the different Mtx-resistant markers, was determined by the plating efficiencies in increasing drug concentrations. Comparisons were quanti-

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Table 1. Relative Resistance of MTX-Resistant Cells to Folic Acid Anologues Relative Resistance ~ to Cell line

Mtx

Amino

Pyr

Tri

DDHP

DDMP

Pro ~ Pro -3 Mtx Rr 3-3 Pro -3 Mtx Rnl 1-2 Pro 3 MtxRH 5-3 Pro -3 Mtx Rn 7-5 Pro -4 Mtx Rn 2-1

1.0 17.5 375 55 58 38

1.0 10 350 205 235 290

1.0 0.8 10 0.13 0.19 0.19

1.0 1.0 16 0.24 0.20 0.18

1.0 1.3 30 0.15 0.10 0.08

1.0 1.1 33 0.18 0.13 0.13

Relative resistances are expressed as the ratio of the Di0 value (drug concentration reducing cell survival to 10%) for the Mtx-resistant cell lines to the Dlo value for the wild-type line. The D10 values for the wild-type line were 1.2 x 10 8M for Mtx, 1.7 • 10-9M for amino, 2.6 x 10-7M for pyr, 1 x 10-3M for tri, 2 x 10-SM for DDHP, and 4 x 10-9M for DDMP.

tated using a D10 value, that is, the concentration of drug which reduced relative cell survival to 10%. The resistance level of the Mtx-resistant phenotypes to these compounds is summarized in Table 1. The class I resistant cell is as sensitive to killing by the diaminopyrimidines as the wild-type cells. The class III resistant cell, on the other hand, is about 10- to 30-fold more resistant. Of greater interest, however, is the observation that the 3 independently selected class II resistant cells have an increased sensitivity to the diaminopyrimidines. Depending on the clone and drug, the class II cells are 5- to 10-fold more sensitive than the wild-type cells to the cytotoxicity of these drugs. Selection fi'om Class II Mtx-Res&tant Cells of Cells Having WildType Pyrimethamine Resistance. The increased sensitivity of the class II Mtx-resistant cells to the cytotoxic action of the diaminopyrimidines suggested that it might be possible to use these compounds to select phenotypic revertants of the class II Mtx-resistant cells. Pyrimethamine was used for this purpose. Class II resistant cells, at various cell numbers, were plated in various concentrations of pyrimethamine at 34~ or 37~ The frequency of survival in 2 x 10-TM pyrimethamine varied between 10-4 and 10-5 depending upon which Mtx an clone was used. Colonies surviving at this particular drug concentration were picked, grown in the absence of pyrimethamine and retested. Over 90% of these colonies showed wildtype sensitivity to the drug, rather than the increased sensitivity of the Mtx R" cells. Prior treatment of the class II resistant cells with EMS increased the frequency of surviving colonies 2- to 5-fold. Under these selection conditions, the number of colonies which retained their resistant phenotype (i.e., wild-type sensitivity) on retesting was again over 90%.

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Phenotype Characterization. Spontaneous and mutagen-induced colonies with wild-type sensitivity to pyrimethamine have been selected from 3 independently cloned lines of Mtx Ru cells. These colonies have been cloned and maintained in nonselective medium. Two of the pyrimethamine-resistant clones have been kept in continuous culture in the absence of the drug for 4 months. Over this period of time, the characteristic response of the cells to pyrimethamine in terms of plating efficiency was reproducible. Dose-response curves for a wild-type line, Pro -3, a class II resistant line, Pro -3 Mtx aII 5-3, and 2 class Mtx R~ cell lines independently selected for resistance to pyrimethamine, rev. Pro -* Mtx a~ 1-4 and Pro-3 Mtx RHPyr R 7-2, are shown in Figure 1. It is clear that

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Table 2. Relative R e s i s t a n c e to Folic A n a l o g u e s of P y r i m e t h a m i n e - R e s i s t a n t Cells Selected from Class II M T X - R e s i s t a n t Cells Relati ve re s i s t a nc e~ to

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rev. Pro a Mtxmt 1-4 rev. Pro a Mtx~H 2-4 rev. Pro a MtxR,I 4-1 rev. Pro -4 Mtx R'1%2 rev. Pro -4 Mtx R'' 11-3 Pro -a Mtx m' Pyr R 7-2 Pro -4 Mtx R;' Pyr R 5-3 Pro 4 MtxR[i PryR 4-4 Pro -4 Mtx ea' Pry R 2-3

+ + + + + + + -

1.2 0.8 0.75 0.8 1.0 25 42 33 42

1.3 1.0 0.8 0.9 1.2 295 265 235 206

0.9 0.8 1.1 0.8 0.8 1.1 0.7 0.7 0.8

0.8 0.8 1.0 1.0 0.9 0.8 0.8 1.2 0.7

0.8 0.9 0.8 0.85 0.9 0.5 0.7 0.6 0.6

1.3 0.8 1.0 0.8 0.9 0.6 0.5 0.6 0.6

a Relative r e s i s t a n c e s are e x p r e s s e d as the ratio of the D,0 value for the p y r i m e t h a m i n e resistant cell lines to the Dlo value for the wild-type line. The Dlo va l ue s for the wild-type line are indicated in the legend to Table 1.

in contrast to the sensitivity of the Mtx RH cells, the selected lines were more resistant to the drug and b e h a v e d similarly to wild-type cells in this respect. The D10 values for Pro-3, Pro-3 Mtx R'' 5-3, rev. Pro-3 Mtx R'I 1-4, and Pro-3 Mtx alI Pyr R 7-2 were determined f r o m the curves in Figure 1 to be 2.6 • 10-r M, 3.3 x 10-8 M, 2.5 x 10-7 M, and 3 x 10-r M, respectively. Table 2 summarizes the relative resistances of several cell lines that were selected for resistance to pyrimethamine. Since the sensitivity to p y r i m e t h a m i n e had been altered in the Mtx class II resistant cells selected for resistance to this drug, it was of interest to determine whether the resistance to Mtx had also been altered. Thus, the sensitivity to Mtx was determined by plating various n u m b e r s of cells in varying amounts of Mtx. The survival as a function of the Mtx concentration for the cell lines indicated in Figure 1 is shown in Figure 2. It is apparent that the cell line rev. Pro-3 Mtx Rn 1-4 has a sensitivity to M t x that is similar to the wild-type cells. The cell line Pro-3 Mtx RII Pyr R 72, however, has retained its resistance to Mtx. The D10 values for Pro-3, Pro-3 Mtx m~ 5-3, rev. Pro-3 Mtx m' 1-4, and Pro-3 Mtx RH Pyr R 7-2 were determined from the curves in Figure 2 to be 1.2 x 10-s M, 6.6 x 10-7 M, 1.5 x 10-s M, and 3 • 10-r M Mtx, respectively. As the data in Table 2 indicate two p h e n o t y p e s can be obtained f r o m Mtx RI' cells by selection for resistance to pyrimethamine. One p h e n o t y p e , revertant Mtx m', has wildtype sensitivity to both p y r i m e t h a m i n e and Mtx; the other, Mtx RII Pyr R, has wild-type sensitivity to p y r i m e t h a m i n e but retains the class II resistance to Mtx. It was next of interest to c o m p a r e the sensitivities of the pyrimethamine-resistant cells selected f r o m the class Mtx aII cells to the other folate structural analogues. The data are shown in Table 2. As can be seen,

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the revertant Mtx RtI cells have wild-type sensitivity to aminopterin, whereas, the Mtx RII Pyr R cells retain the class II resistance to this compound. In addition, the revertant Mtx mI cells show wild-type sensitivity to trimethoprim, D D H P , and DDMP. Although the Mtx alI Pyr R shows an altered r e s p o n s e to these other c o m p o u n d s , the sensitivity does not return to wild-type for all drugs tested. Rather, in most cases for D D M P and D D H P , it is intermediate between wild-type and Mtx m~. Uptake of [all] Mtx by Cell Lines. Previously, we showed that the class II Mtx-resistant cells were altered in their permeability to Mtx (1). To determine whether the ceils of the revertant Mtx RH and Mtx RH Pyr R p h e n o t y p e s were now p e r m e a b l e to the drug, the uptake of [aH] Mtx into

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monolayers of the various cell lines was examined. The incorporation of [3HI Mtx by the wild-type line Pro-3, two class II Mtx-resistant cells, Pro-3 Mtx R~ 5-3, Pro-4 Mtx Rn 2-1, and four independently selected pyrimethamine-resistant cells, rev. Pro-3 Mtx Rn 1-4, rev. Pro-4 Mtx Rn 113, Pro-3 Mtx Ru Pyr R 7-2 and Pro-4 Mtx R~I Pyr R 5-3 is shown in Figure 3. The revertant Mtx mI cells, which have wild-type drug sensitivities, accumulate the drug in a similar manner as wild-type cells. In contrast, accumulation of the drug in the Mtx RH Pyr R cells was greatly depressed and was similar to that in class II Mtx-resistant cells. Uptake of [3HI Folie Acid by Cell Lines. Various studies in other mammalian systems suggest that Mtx and folic acid share in part the same carrier mechanism (15). However, other work suggests that there may be different transport systems for folic acid and Mtx (16-18). To determine whether the differences in uptake of Mtx exhibited by the various cell phenotypes (Figure 3) were also found with folic acid, the uptake of labeled folic acid was investigated. The incorporation of PHI folic acid by the wild-type cell line, Pro-3, and two independently selected class II Mtx-resistant cells, Pro-3 Mtx R~ 5-3 and Pro-4 Mtx aII 2-1, is shown in Figure 4. Both the wild-type cells and the class II Mtx-resistant cells are permeable to folic acid. However, the accumulation of the compound at saturation in the class II Mtx-resistant cells was 30-50% of that in the wild-type cells. It is apparent that the alteration in the cell affecting the 24

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permeability of Mtx also affects the permeability offolic acid. Cells of the revertant Mtx R" phenotype, rev. Pro-3 Mtx ~II 1-4 and rev. Pro-4 Mtx R" 11-3, are readily permeable to folic acid. Cells of the second phenotype, Mtx RxIPyrR , Pro-3 Mtx R" Pyr R 7-2, and Pro-4 Mtx R" Pyr R 5-3, accumulate the compound in a similar manner as the class II Mtx-resistant cells. Folate Reductase of Cell Lines. From the results described so far, it appears that selection of cells resistant to pyrimethamine from class II Mtx-resistant cells gives rise to two different classes of cells. One phenotype, revertant Mtx Rn, is a phenotypic wild-type revertant of Mtx RII since it has the drug sensitivity and drug permeability properties of wildtype cells. The other phenotype, Mtx m~ Pyr R, has wild-type sensitivity to some of the diaminopyrimidines, but its resistance to Mtx and aminopterin and permeability properties are those of the Mtx aII cells. The intracellular site of action of the diaminopyrimidine analogues of folic acid is similar to that of Mtx, i.e., the folate reductase e n z y m e (10, 14, 19, 20). The possibility of an alteration in this e n z y m e was investigated in two independent isolates of the Mtx R~' Pyr R phenotype. As is apparent from Table 3, there was no significant difference in the amount of reductase in this phenotype compared to wild-type or revertant Mtx Rn cells. To examine whether the affinity of the reductase for pyrimethamine or Mtx was altered in the Mtx RH Pyr R cells, the enzyme in crude extracts

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Table 3. Folate Reductase of Pyrimethamine-Resistant Cells Selected from Class II MTX-

Resistant Cells

Ceil line

Relative Enzyme activity~

Pro -a Pro -3 MtxR~3-3 Pro -3 MtxEII5-3 rev. Pro-~ MtxE~I1-4 rev. Pro -3 MtxEII4-1 Pro -3 Mtxm~PyrE 7-2 Pro -~ Mtx EEEPyrE 5-3

1.0 0.9 0.85 0.7 1.3 1.2 1,5

Affinities of folate reductase for Pyr I~g (M) 2.0 • 2.5 • 2.5 x 2.0 x 1,5 x 2.0 x 2.0 x

10 6 10-8 10-6 10-6 10 6 10-8 10 6

Mtx I50~ (M) 4.0 x 3.0 x 3.5 x 4.0 • 3.5 x 4.0 • 3.5 x

10-9 10-8 10-9 10-~ 10-9 10-9 10-~

Crude cell extracts were prepared and assayed for folate reductase as described in Materials and Methods. Relative activity is expressed as the ratio of activities in the various cell line extracts to wild-type extract. The wild-type line had an average activity of 4 units/mg protein, as measured in several independent experiments. Drug concentration inhibiting the enzyme 50%. was a s s a y e d as a f u n c t i o n of either the p y r i m e t h a m i n e or Mtx c o n c e n t r a tion. A s i n d i c a t e d in T a b l e 3, the p y r i m e t h a m i n e c o n c e n t r a t i o n that r e s u l t e d in 50% i n h i b i t i o n of the r e d u c t a s e (I50 value) was similar for e n z y m e p r e p a r a t i o n s f r o m all the cell lines, S i m i l a r l y , t h e r e was n o diff e r e n c e in the I~0 v a l u e for M i x of t h e s e e n z y m e s . T h e e n z y m e f r o m the class I M t x - r e s i s t a n t ceil, P r o - 3 Mtx ~I 3-3, as p r e v i o u s l y s h o w n (1, 2), is a b o u t s e v e n times less s e n s i t i v e t h a n w i l d - t y p e e n z y m e to i n h i b i t i o n b y Mtx. Chromosome Number of Cell Lines. K a r y o t y p e a n a l y s i s s h o w e d that all the r e p r e s e n t a t i v e s tested of the two p h e n o t y p e s , r e v e r t a n t M t x R~ a n d M t x RII Pyr R, c o n t a i n e d 21 c h r o m o s o m e s as did the w i l d - t y p e a n d p a r e n t a l class II M t x - r e s i s t a n t lines, DISCUSSION W e h a v e s h o w n that C H O cells s e l e c t e d for r e s i s t a n c e to Mtx show different r e s p o n s e s to the c y t o t o x i c i t y o f the d i a m i n o p y r i m i d i n e s , A r e p r e s e n t a t i v e class I r e s i s t a n t cell is as s e n s i t i v e as the w i l d - t y p e cells, T h e r e p r e s e n t a t i v e class I I I r e s i s t a n t cell is a b o u t 10 to 30 t i m e s m o r e r e s i s t a n t , d e p e n d i n g o n the drug. This i n c r e a s e d r e s i s t a n c e p r o b a b l y r e s u l t s f r o m the i n c r e a s e d a m o u n t of the i n t r a c e l l u l a r target, the folate r e d u c t a s e e n z y m e (1, 2). T h r e e i n d e p e n d e n t l y s e l e c t e d class II r e s i s t a n t cells, in c o n t r a s t , a r e 5 to I0 times m o r e s e n s i t i v e t h a n the w i l d - t y p e cells to the c y t o t o x i c a c t i o n of these c o m p o u n d s , B e c a u s e of the c o m p l e x nature of the M t x t r a n s p o r t s y s t e m (see below) it s h o u l d b e p o i n t e d out that all M t x - r e s i s t a n t cells d i s p l a y i n g the class II p h e n o t y p e m a y n o t b e sensitive to the d i a m i n o p y r i m i d i n e s .

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The basis for the increased sensitivity to the diaminopyrimidines demonstrated by the class II Mtx-resistant cells shown here is not understood since, at present, the molecular basis for the alteration affecting the permeability of Mtx has not been identified. This increased sensitivity, however, may provide some important clues as to the nature of this alteration. The diaminopyrimidines are lipid-soluble inhibitors of the reductase enzyme (10). For example, the logarithms of the octanol-water partition coefficients, which are an indication of the hydrophobic nature of the compounds, are 2.69 and 2.82 for pyrimethamine and DDMP, respectively, as compared to -1.85 for Mtx (10). In addition, the diaminopyrimidines appear to be transported into the cell by a different mechanism than Mtx (21, 22). Both the transport of the diaminopyrimidines and Mtx appear in part to involve a carrier-mediated process (21-23). However, there is no competition for uptake between DDMP, pyrimetham!ne, and Mtx (22). The increased sensitivity of the class II Mtx-resistant cells to the diaminopyrimidines has provided a simple back-selection system. Using pyrimethamine, stable cell lines have been selected from the class II Mtxresistant cells that have wild-type sensitivity to not only pyrimethamine but also to the other diaminopyrimidines (trimethoprim, DDHP, and DDMP) examined. Such selections, however, produce two classes. One class, revertant Mtx RH, shows wild-type sensitivity to Mtx, aminoptelin, and the diaminopyrimidines. Furthermore, such cells are able to accumulate both Mtx and folate in a similar manner as wild-type cells. Thus, the diaminopyrimidines can be used to select phenotypic wild-type revertants of class II Mtx-resistant cells. The second pyrimethamine-resistant phenotype, Mtx RII Pyr R, shows wild-type sensitivity to pyrimethamine and trimethoprim and a sensitivity to DDHP and DDMP intermediate between wild-type and Mtx R~I, but retains the Mtx RHlevel of resistance to both Mtx and aminopterin. In addition, these cells are defective in their ability to accumulate both Mtx and folate; a feature of class II Mtxresistant cells. No qualitative or quantitative differences were found in the reductase from these cells compared to that from wild-type cells. The basis for the alteration in the Mtx R~IPyr R cells is unknown. Both phenotypes are present in spontaneous and mutagen-induced selections. At the present time, however, it is difficult to determine the relative distribution of these two phenotypes since only a small number of isolates have been picked and characterized from any one selection. Both phenotypes were obtained from three lines of class II cells. Preliminary results indicate that each line does not give rise to an equal number of each phenotype. In other systems, revertant phenotypes have been selected in which

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the reverted function is temperature sensitive (24-26). To enhance the possibility of a temperature-sensitive alteration in the back-selection system described in this report, pyrimethamine selections were carried out at 34~. To date, however, no temperature-sensitive mutants have been isolated. The mechanism of Mtx transport into mammalian cells has been the subject of several studies because of the widespread use of this drug for cancer chemotherapy. It is apparent that the transport process is complex, perhaps involving a carrier-mediated process at low drug concentrations, a passive diffusion process at high drug concentrations, and an efflux system (23, 27-29). There are conflicting reports as to whether Mtx and folic acid are transported by the same system in mammalian cells. In some systems, it is apparent that both compounds share in part the same carrier mechanism since they compete with each other for uptake (15). The observations with the class II Mtx-resistant CHO cells tend to support this since the accumulation of both Mtx and folic acid is altered. However, other work suggests that separate transport systems may be involved since mercurials block the transport of Mtx, but not folate (18). In addition, there are Mtx-resistant LI210 routine leukemia cells that have a decreased uptake of Mtx but the uptake of folic acid is similar to the wildtype cells (30). Thus, it is apparent that additional work is required to define this complex transport process. The further characterization of the Mtx-resistant CHO cells and their revertants should provide important information as to the relationships between altered membrane structure and function, and to some of the molecular details of Mtx and folic acid permeability. ACKNOWLEDGMENTS The authors thank Drs. V. Ling and L. Siminovitch for their editorial comments and suggestions. This research was supported by the Medical Research Council of Canada. One of us (W.F.) is a Scholar of the Medical Research Council of Canada. LITERATURE CITED 1. 2. 3.

Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976). Somat. Cell Genet. 2:245-261. Gupta, R. S., Flintoff, W. F., and Siminovitch, L. (1977). Can. J. Biochem. 55:445452. Flintoff, W. F., Spindler, S. M., and Siminovitch, L. (1976). In Vitro 12:749-757.

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The selection of wild-type revertants from methotrexate permeability mutants.

Somatic Cell Genetics, Vol. 4, No. 2, 1978, pp. 143-156 The Selection of Wild-Type Revertants from Methotrexate Permeability Mutants Wayne Flintoff a...
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