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Amino acid analog-resistant mammalian cells
Life Sciences, `Vol . 22, pp . 1469-1480 Printed in the U .S .A .
Pergamon Press
MINIREVIEW AMINO ACID ANALOCrRESISTANT MA1~IIiALLAN CELLS John J . Wasmuth, Ph .D . and C . Thomas Caekey, M .D . Department of Medicine, Baylor College of Medicine 1200 Mouraund Avenue, Houston, Texas 77030
Amino acid analogs have been employed very successfully as biochemical and genetic probes is Eschericia coli and Salmonella typhimurium toward an understanding of the regulation of amino acid metabolism (1) . Much of this information was derived from characterizing mutants resistant to amino acid analogs . Resistance to various analogs resulted from alterations in the transport and assimilation of amino acids, as well as from alterations in the regulation of the appropriate biosynthetic pathways (1,2,3) . Advances in somatic cell genetics in the last decade now make it possible to study similar problems in cultured animal cells with approaches analogous to those used in bacterial studies . For ezample, animal cell mutants that are auaotrophic for various amino acids have been isolated, and in some cases the defective enzyme has been identified, (4,5,6,7,8) . Amino acid analogs represent a large spectrum of potential selective agents for obtaining mutant animal cells with alterations in both structural and regulatory genes involved in various aspects of amino acid metabolism and utilization (9,10) . In view of the limited knowledge concerning the patterns and mechanisms involved in regulating these essential functions in animal cells, such studies should be most enlightening from both a biochemical and genetic standpoint . The purpose of this article is to review the information that has been derived from studying various amino acid analog resistant mutants, to discuss their significance and to speculate what types of studies might be done in the future . Since several reviews have appeared recently oa mutants affecting purine and pyrimidine biosynthesis (11,12), we have avoided discussing amino acid analogs (such ae azaserine) whose primary effect is in the biosynthesis of these compounds . An amino acid analog, by acting ae an antagonist of a natural amino acid, might inhibit cell growth by inhibiting nay essential function in which the natural amino acid ie required . Aay effect of an analog that results in a reduction of the natural amino acid's incorporation into protein would result in growth inhibition . This might result from a reduction in the cellular concentration of the amino acid by the analog's ability to inhibit the transport of an essential amino acid or the biosynthesis of a nonessential amino acid . Many analogs are known to inhibit protein synthesis directly by inhibiting the cognate aminoacyl tRNA synthetase . In some cases, the analog is incorporated into protein in place of the natural amino acid . This ie usually quite toxic to the cell since many of the resultant proteins are nonfunctional . Besides their role in protein synthesis, various amino acids serve as precursors for other essential metabolites such as S-adenosylmethionine, polyamines and phosphatidal serine . Inhibition of the formation of any such essential metabolite could also inhibit cell growth . Amino acid analogs have also been shown to inhibit various other cellular functions in animal cells (13,14,15) . Alterations in the cell that might lead to resistance to an analog are ae varied se the ways in which analogs inhibit cell growth . An enzyme essential for growth (such as an aminoacyl-tRNA synthetase) that is inhibited by as analog could be altered by mutation such that it ie lees sensitive to inhibition by 0300-9653/78/0501-1469$02 .00/0 Copyright © 1978 Pergamon Press
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the antimetabolite . Alternatively, increased resistance to an analog might be achieved by as increase in the intracellular concentration of the natural amino acid relative to the analog . This could result from a variety of alterations, including : 1) increased biosynthesis (in the case of non-essential amino acids), 2) decreased uptake or retention of the analog, 3) decreased degradation of the natural amino acid, 4) increased degradation or conversion of the analog to a nontoxic compound . It is important to point out that the mechanism by which a cell achieves resistance to an analog does not necessarily esy anything about the mechanism by which the analog inhibits cell growth . In the following sections, some of these types of inhibition and resistance will be described, while some remain theoretical possibilities . Methionine Analog Resistant Mutante Although methioniae is classified as an essential amino acid in mammals, various cultured cells can synthesize methioniae from L-homocysteiae and N5-methyl-tetrahydrofolate (NS~methyl THF) (16,17,18) (Fig . 1) . The reaction is catalyzed by N 5-methyl-tetrahydrofolate : homocysteiae methyltraneferase (HCMT), which requires S-adenosylmethionine (SAM) and a vitamin B12 coenzyme . Another enzyme which utilizes betaine as the methyl donor in an analogous reaction has not been detected in cultured cells . The former pathway is functional is cultured baby hamster kidney (BHR) cells as shown by the observation that these cells grow in methioniae-free medismt supplemented with homocysteine, B12 and a high concentration of folic acid (HC medium) (17,18) . The activity of HCMT is cultured cells is affected by both B12 and methioniae . The former apparently converts apo HCMT to holo HCMT while the latter represses the synthesis of enzyme (17,19) . In addition to its role in protein synthesis, methioniae also plays a key role in methyl group transfer via its conversion to S-adenosylmethionine (SAM) (Fig . 1) . The S-adenosylhomocysteiae (SAH) produced in methylation reactions can be cleaved to homocysteine, which in turn can be reconverted to methioniae by HCMT . In view of the role of methioniae in polypeptide chain initiation and SAM in methyl group transfers, one might expect this cyclic pathway to be highly regulated (20,21) . In an effort to learn more about this regulation, two laboratories have isolated methioniae analog-resistant BHR cells .
THF Methylene THF
r
Methionine
S-adenosylmethionlne ~Acceptor
a
Methylated S-adenosylhomocystetne
Acceptor
Methyl rHF Homocysteine
FIG . 1 The enzymes involved in the interaonversioa of methioniae and homocysteine are : 1) methioniae adenosyltrsasferase . 3) Adenosylhomocysteinase, 4) N5-methyl-tetrahydrofolate :homocysteiae methyltransferase, 5)methylene
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tetrahydrofolate r.eductase (menadione reductaee) . The conversion of S-adenosylmethionine to S-adenosylhomocyateine (Step 2) is catalyzed by a large number of enzymes that transfer the methyl group of S-adenosylmethionine to various acceptors . Kamely and Littlefield (22) selected mutants resistant to norleucine using methionine-free, HC medium . Only when selection was done at a norleucine concentration of 10 mg/ml was there an effect of mutagene on the frequency of reaiatant cells . In addition, only about 1/10 of the clones isolated remained substantially reaiatant to norleucine upon subculturing . The uptake of norleucine was examined in 20 reaiatant clones . The steady state intracellular level of norleucine was significantly lower in five of these and the initial rate of methionine uptake was altered in four of these five . However, without more detailed kinetic studies, it is not possible to determine the reason for decreased norleucine pools in the resistant clones . In certain clones, HCMT was elevated two-fold above wild type levels, when cells were grown in medium with methionine . However, since the reaiatant clones were selected in methionine-free medium, more striking differences between reaiatant and wild type cells might have been observed if the enzyme had been measured in cells grown without methionine ; especially since it is known to repress the synthesis of HCMT . Thus, although the "repressed" level of HCMT (in cells grown with methionine) was not markedly different in wild type and reaiatant clones, the "depressed" levels of HCMT (in cells grown without methionine) may be significantly different . Perhaps the moat well characterized amino acid analog reaiatant cells reported are the ethionine resistant BHR mutants isolated by Carboche (23) . These mutants were also selected in methionine-free HC medium, which contained 20ug/ml of ethionine . One mutant, ETH304, which maintains the reaiatant phenotype after prolonged growth is nonselective medium, was studied in detail . In methionine-free HC medium, ETH304 has markedly higher intracellular methionine pools than wild type cells, and excretes ten times more methionine than wild type cells . These results suggested that ETH304 synthesized methionine from homocysteine at an increased rate . ETH304 also had a significantly higher SAM pool than wild type in HC medium, but not in medium with methionine . Oae puzzling observation was that both ETH304 and wild type cells had greatly increased SAM pools when grown in HC medium with ethionine . Based on the growth response of wild type cells to aminopterin and folic acid in methionine-free medium, Caboche concluded that the rate limiting step in methionine biosynthesis is the formation of N5-methyl THF . In ETH304, the enzyme catalyzing the formation of N5-methyl THF from methylene THF (methylene THF reductaee ; or meaadione reductaee) was elevated under all growth conditions . In wild type BHR cells, menadione reductaee is inhibited by SAM . However, the enzyme from ETH304 was much leas sensitive to inhibition by SAM under all conditions tested . In addition, the menadione reductaee from ETH304 was more thermoaeneitive than the wild type enzyme, another good indication that this enzyme is altered is this mutant . In cell hybrids between wild type and ETH304, ethionine resistance behaved as a codominant trait is growth experiments . An examination of menadione reductaee from such a hybrid provided some evidence far a "hybrid" enzyme . The increased resistance of ETH304 to ethionine might thus be explained ae follows . In wild type and ETH304, ethionine causes a marked elevation in the SAM pool . In the wild type cells, but not in ETH304, this would result in inhibition of meaadione reductaee, thereby reducing the rate of methionine synthesis, for lack of N 5 -methyl THF . Since the menadione reductaee in
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BTH304 is much lean sensitive to inhibition by SAM, these cells continue to produce NS~methyl THF and methioniae . The increase is intracellular and extracellular methionine could thus antagonize the inhibition by ethionine . Proline Analog Resistant Mutants In animal cells, proline can be synthesized from either glutamic acid or ornithine through the intermediate glutamic-y-eemialdehyde (GSA) (24) (Figure 2) . Cultured human fibroblaste contain low levels of argiaaee and thus, synthesize very little ornithine from arginine (25) . We have been unable to detect any conversion of arginine to proline in Chinese hamster cells (unpublished results) . Thus, in cultured human and Chinese hamster lung (CHL) fibroblasts, the mayor, if not only, pathway for proline biosynthesis is the one from glutamic acid (26, unpublished results) . Ia addition, the CHO cell line established by Yuck at al (4) is auxotrophic for proline se a result of its inability to convert glutamate to GSA . In these cells the arginine to proline pathway moat not be functional or the cells would not require eaogeaous proline .
CItruIline
Arginlnosucclnlc Acid
Arginine
lo
Ornlthine -" Putresclne
!m
Glutamic ----~Glutamic- i- ~-- ~e`-Pyrroline-5- -iProline Acld © Semialdehyde Carboxylic Acid
FIG . 2 Metabolic interrelationship of arginine, proline and ornithine . The enzymes involved in these metabolic pathways are : 1) Argiainoauccinic Acid Synthetase, 2) Argininosuccinic Acid Lyaee, 3) Arginase, 4) Oraithine Traneaminase, 6) A'-Pyrroline-S-Carboxylic Acid Reductase, 7) Ornithiae Decarborylaee . The enzyme or enzymes that convert glutamic acid to glutamicy-semialdehyde (Step 5) have not been demonstrated in animal cells . The uncertainty concerning this part of the proline biosynthetic pathway ie indicated by the dashed line . The iaterconversion of glutamic-y-semialdehyde and A'-pyrroline-5-carborylic acid ie apparently a spontaneous, non-enzyme catalyzed reaction .
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Very little is known about the enzymology or regulation of the glutamate to proline pathway is animal cells . Only t°he last enzyme in the pathway, D-1pyrroline-5-carbozylic acid reductase (PCA reductase) has been demonstrated in vitro (27) and no intermediates between glutamate and GSA have been identified . Since this pathway is relatively unbranched, and the end product is not further metabolized, (cultured fibroblasta appear to lack proline oxidase activity), it seemed amenable to biochemical and genetic studies using analogs . We isolated CBL mutants resistant to the proline analog, L-azetidine 2-carboxylic acid (AZCA) (28) . Clones isolated as resistant to 0 .6mM AZCA were often only marginally resistant to that the concentration of the analog in subsequent plating efficiency tests at low cell densities . However, in a more sensitive teat to determine the cytotozicity of high concentrations of AZCA, a clear cut difference was demonstrated between wild-type and AZCA resistant (AZCAr) clones . The AZCA resistant phenotype ie maintained after many months of continuous culture in nonselective medium . In wild type cells, AZCA markedly decreased the intracellular proliae pool . In two AZCAr clones much higher intracellular levels of AZCA had little effect on the soluble proline pool . These same AZCAr clones excreted much more proline into the medium than wild type cells . Using e sensitive in vivo assay, we demonstrated that the AZCAr cells convert l4C-glutamic acid to 14 C-proline at a greater rate than wild type cells . Furthermore, while AZCA greatly reduced the conversion of glutamate to proline in wild-type cells, it had no demonstrable effect on this conversion in AZCAr cells . In more recent experiments, we have demonstrated that AZCA inhibits the comreraion of glutamate to proline at some point before the formedqn of GSA. This conclusion is based upon atudiea measuring the conversion of 14 C-glutamate to 14 C-ornithine [via the conversion of GSA to ornithiae by ornithine transaminase (.OTA), see Fig. 2] . The results of experiments comparing the rate of formation of 14C-ornithiae from 14C-glutamate in wild type and AZCAr cells, paralleled those measuring the conversion of glutg~pato to proline . Thus, AZCA inhibited the conversion of l4C-glutamate to 14 C-ornithine in wild type cells but not in the AZCAr mutants (without affecting the activity of OTA) . In addition, the AZCAr cells excreted 14 C-ornithine at about twice the rate of wild-type cells . As can be seen from Figure 2, since AZCA inhibits the coaveraion of glutamate to both proline and ornithine (and does not inhibit OTA) its site of action moat praceed the formation of GSA . One would predict, therefore, that ornithine should reverse the growth inhibition caused by AZCA in wild type cells, since it could be readily com~erted to GSA. Ornithine reverses AZCA inhibition, but only at a very high concentration (5mM) . This could likely be due to poor uptake of ornithine by CHL cells in medium containing lysine and arginine, which share a common transport system with ornithine in some animal tissues . Indeed, we and others (29) have observed poor uptake of ornithine by CHL cells. It is also of acme interest to point out that CHI. cells can synthesize the polyamines (apermine, spermidine and putrescine) from glutamic acid, following its conversion to ornithine . Thus, CBI. cells labeled with 14C_glutamate contained 14 C-spermine, apermidine and putrescine . In these cells, therefore the glutamate to proliae biosynthetic pathway may also be quite important in the biosynthesis of these compouade . Since our initial report, we have found that AZCA resistance behaves as a dominant trait in cell hybride . In growth atudiea, AZCAs z AZCAr hybrida are as resistant to AZCA as the diploid AZCAr parent . In such a hybrid, the conversion of glutamate to proline (and ornithine) is completely refractory to inhibition by AZCA, again like the AZCAr parent .
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The rapidity with which AZCA inhibits the synthesis of GSA from glutamate suggests it is inhibiting an enzyme in some manner, ae opposed to repressing enzyme synthesis . It thus appears analogous to a "false" feedback inhibition. The inhibition by AZCA may not be that simple, however, since exogenous proline has a much lean dramatic effect on the conversion of glutamate to proline (in short time periods) . In addition, another proline analog, 3,4-D, L-dehydroproline, which is at least as toxic to CHI. cells as AZCA, has no effect on the synthesis of proline from glutamic acid . Whatever the mechanism of AZCA inhibition, the data suggests that an enzyme involved in converting glutamate to GSA may be altered in the AZCAr cells . The overproduction of proline by these mutants also suggests that they lack some type of control mechanism that operates in wild type cells . These cell lines should, therefore, be valuable tools for ezamiaing the enzymology and regulatory mechanisms im~olved in the conversion of glutamate to GSA and proline . Arginine Analog Resistant Mutaata While cultured animal cells cannot synthesize arginine de novo from ornithine and carbamyl phosphate, some cell lines can convertcitrulline to arginine (Fig . 2) and thus grow in arginine-free medium supplemented with citrulline (30,31,32,33) . The two enzymes involved in the conversion of citrulline to arginine, argininosuccinic acid synthetase (ASAS) and arginiaoauccinic acid lyase (ASAL) have been studied in several types of cultured cells, including human lymphoblast cell linen (30,31,32) . A brief report has appeared on the isolation of human lymphoblast cell lines that are resistant to the arginine analog, canavanine (34) . These mutants were aehected in arginine-free medium containing 0 .6mM citrulline and .012mM canavanine . The transport of arginine did not appear to be altered in the canavanine resistant mutants but they had higher levels of ASAS than wild type cells under all growth conditions . The level of ASAS in these mutants was the same in medium containing citrulline, arginine or canavanine . In contrast, the level of ASAS in wild type cells is greatly reduced in medium containing arginine rather than citrulline . These initial results, therefore, suggest that the canavanine resistant mutants may have a defect is some regulatory element controlling the synthesis of ASAS . Previous studies with various cultured mammalian cell lines have shown that ASAS and ASAL increase in a coordinate manner when arginine is made growth limiting (32,35) . Interestingly, A3AL was not markedly increased in the canavanine reaietant mutants, indicating that the activity and/or synthesis of ASAL and ASAS can be regulated independently . Transport Mutants Engelsberg and coworkers have used two approaches to isolating mutant A9 mouse cells altered in amino acid transport . The first method employed 5-fluorotryptophan (5-FT) as a selective agent while the second employed tonic concentrations of the natural amino acid, phenylalanine (36,37) . Mutants resistant to 5-FT had a reduced ability to concentrate the analog as well as tryptophaa . Detailed kinetic analysis of tryptophan uptake in wild type cells indicated there are two transport systems for this amino acid . Determination of kinetic constants (Rm and Vim) for the two proposed transport systems in the analog resistant mutants indicated that one or both systems are altered in all the mutants examined . Similar alterations in phenylalanine uptake were observed in mutants reaietant to high concentrations of this amino acid . As in the case with tryptophan, they obtained kinetic evidence for two phenylalanine transport systems, both of which were altered in the mutants studied . The observation that both transport systems can be altered in the same mutant (that ie, both tryptophan systems or both phenylalanine systems) suggested that the two systems share a common component .
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Alternatively, there may be just one system which eahibita negative cooperativity, or has one high and one low affinity binding site . Although the frequency of cells resistant to pheaylalaniae was high in unmutagenized cultures, fluctuation tests indicated that the appearance of pheaylalaniae resistant clones was a random event . In addition, the frequency of pheaylalaniae resistant clones was increased by treatment of cello with mutagens . Protein Synthesis Mutants As mentioned in the introduction, analogs that are incorporated into protein are usually quite toxic since many of the resultant proteins are nonfunctional . We were therefore interested to determine if the analogs we found to be cytotoxic to CHL cells (see next section)eaerted their toxic effects by virtue of being incorporated into protein . One simple test to examine this possibility is to reversibly inhibit protein synthesis during the time cells are exposed to the analog in question . Following removal of the analog and the protein synthesis inhibitor, cell viability ie determined by the number of single colonies that appear 7-10 days later . We tented several cytotoxic analogs in this manner, using 2uM cycloheaimide to reversibly inhibit protein synthesis . We found that indeed the cytoxicity of many analogs could be prevented if protein synthesis was inhibited while cells were exposed to the analogs . Typical results are shown is Figure 3 for toxic analogs of lysine (S-2-aminoethyl-L-cyeteiae), tryptophan (5-fluorotryptophan), and pheaylalaniae (p-fluorophenylalanine) . The viability of cells exposed to one of these analogs for 48 hours was decreased by several orders of magnitude in the absence, but not in the presence of 2pli cycloheaimide (Pig . 3a) . Furthermore, the toxicity of S-2-amiaoethyl-L-cyateine and 5-fluorotryptophan is markedly increased by omitting the corresponding natural amino acid from the medium while cells are exposed to the analog (Fig . 3b) . Toxicity under these conditions is also prevented by cycloheximide . The moat likely interpretation of these results is that these analogs must be incorporated into protein to be cytotoxic . Another possibility that cannot be completely ruled out is that the inhibition of protein synthesis by cycloheximide causes an increase in the intracellular amino acid pools, which allows a more effective competition with the analogs . The observation that the cytotoxicity of certain analogs could be prevented by inhibiting protein synthesis suggested to us that these antimetabolites might be useful to select cell lines with conditionally lethal, tempera ture sensitive (te) mutations affecting protein biosynthesis . We have used the most toxic analog examined, S-2-aminoethyl-L-cyateine (thialysiae) in this manner and have obtained several is CHL protein synthetic mutants (38) . Five of sin to mutants isolated in such a selection (none of which are gibe) have defects in asparagyl-tRNA aynthetase (AsnRS), and fall into a single complementation group in cell hybridization experiments . In three of these mutants, increased thermolability of AsnRS is demonstrable _in vitro . Oae mutant, which is a separate complementation group, remains uncharacterized . It is intereatiag that AsnRS mutants appear to be common type of mutant in CHO as well ae CHL cells, while other aminoacyl-tRNA aynthetase mutants are much more difficult to obtain (with the eaceptioa of leucyl-tRNA aynthetase mutants in CHO cells) (39) . The reason for the preponderance of this particular type of protein biosynthetic mutant in two different Chinese hamster cell lines ie unknown . While we initially considered the possibility that this locus might be X-linked is Chinese hamsters (38), recent experiments is this laboratory have excluded that possibility . We have now obtained hybrids between one to CHL AanRS mutant and normal human leucocytea (selected under conditions that require them to retain the human gene for AenRS) in an attempt to assign this gene to a particular human chromosome .
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FIG . 3 Cytotoxicity of amino acid analogs . A . Wild type Chinese hamster lung cells were distributed to 60mm culture dishes at densities of 102 to 106 cells/ dish in Eagle's minimal essential medium (MEM) . Twenty-four hours later the medium was removed and replaced with similar medium containing 1 .5x10 - 3M 5-2aminoethyl-L-cyeteine (thialysine) (0) ; thialysine plus 2uM cycloheaimide ( " ) ; 2x10 -3M D, L 5-fluorotryptophan (0) ; 5 fluorotryptophan plus 2uM cycloheaimide (~) ; 2x10 - 3M D, L p-fluorophenylalaaine (Q ) ; or p-fluorophenylalanina plus 2uM cycloheaimide (/ ) . At the indicated times thereafter the analogcontaining medium was replaced with nontoxic medium (DIEM) . Tea days later, the number of cells that survived the various treatments (n) was determined by the number of colonies in the dishes . The surviving fraction (N/No) was then determined by dividing N by the number of colonies (No) on control dishes that were not exposed to an analog . B . Experimental conditions were as is A, with the following exceptions : 1 . The concentration of both thialysine and 5fluorotrypiophan was reduced to 10- 3M . 2 . Lysine was omitted from the medium containing thialysine and tryptophan was omitted from the medium containing 5-fluorotryptophan . Symbols are se in A . While the toaicity of thialysine and other analogs may very from one cell line to another, the basic selective procedure should be applicable to almost In addition, the selective technique can be modi any type of cultured cell . fied in many ways to select for more specific types of protein synthetic mutants . The isolation of mutants with defects in the various soluble factors involved in protein synthesis, as well as in ribosomal proteins, will enhance our ability to construct a human genetic map of protein synthetic factors . It should, therefore, be possible to determine if any of these functionally related genes are clustered . In this regard, it should be noted that the gene for tryptophanyl-tRNA synthetase has been assigned to human chromosome 14 (40,41) .
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Survey of Inhibit or~ Analogs In vies of our limited knowledge concerning the genetic and biochemical mechanisms that regulate the transport, biosynthesis, catabolism and assimilation of amino acids in animal cells, further ezploitation of amino acid analogs as selective agents seems well worthwhile . In Table I ie a listing of amino acid analogs that we found to be cytoetatic or cytotoxic to the clonal growth of CHL cells in Eagle's minimal essential medium . Cytoetaticity ie defined as a reduction in the plating efficiency to at least 10- 2 when cells are grown in the continued presence of the analog . Cytotoxicity is defined as a reduction in the plating efficiency to at least 10- 2 when cells are exposed to the analog for a period of 1-3 days, then eubeequently grown for at least seven days in normal medium . The purpose of these studies was to screen a large number of analogs to determine which would be useful as selective agents . They were therefore usually done over a limited range of cell densities and at three or four concentrations of the analog, ragging from .Olmg/ml up to 2mg/ml . The effectiveness of many of the analogs of essential amino acid may be increased in medium containing lower concentrations of the natural amino acid . Likewise, many analogs that are not inhibitory in normal medium might be effective inhibitors in medium with lower concentrations of a given amino acid . As mentioned in the preceding sections on methionine and arginine resistant mutants, it is sometimes possible to remove essential amino acids from the medium completely, and supply the cells with a precursor that can be comrerted to the missing amino acid . Similar possibilities exist for other essential amino acids, depending upon the ability of various types of cultured cells to utilize precursors of different amigo acids (33) . Thin allows more flexibility in the types of mutants one might attempt to isolate . One is left with the question, of what use are the various types of amino acid analog resistant mutants? Transport mutants will undoubtedly aid our understanding of the very complex systems involved in the uptake of amino acids in animal cells, defects in some of which are the basis for certain inherited human disorders (42,43) . The biosynthetic regulatory mutants can be used to learn more about the physiology of the appropriate pathways and what types of mechanisms regulate them . For example, the ability of various types of malignant and transformed cell lines to utilize homocysteine is place of methionine appear to be impaired, even though all the biosynthetic enzymes are present in normal amounts (44,45) . Thus, a better understanding of the factors regulating the uptake of homocyeteine and its conversion to methionine could prove valuable in understanding this unusual property of malignant cells . The regulation of arginine and prolixe biosynthesis is of interest from several standpoints . In some cells, arginine serves ae the major precursor of prolixe while in others glutamate fulfills this role . What mechanisms regulate the relative contribution of the two pathways? Do metabolites of one pathway regulate carbon flow over the other? Can the regulation of either of these pathways affect the cellular level of polyamines, compounds which have wide ranging metabolic effects? Ae the technology for transferring limited amounts of genetic material from one cell line to another advances (46,47,48,49,50,51), it should be possible to examine the nature of some regulatory mutations ix greater detail . Systems can be developed in which one can ask whether structural and regulatory genes are closely linked or unlinked by determining if the two can be cotraxsferred with small amounts of DNA . Such approaches will be necessary in order to define regulatory elements in animal cells more precisely at the molecular level .
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TABLE I Cytostatic and Cytotoaic Amino Acid Analoge Amino Acid
Analog
Selection of Mutante ESSENTIAL AMINO ACIDS (34)
Arginine
canavanine *,+
Cyateine
aelenocysteine* cyeteine hydroxamate
Isoleucine
4-thiaisoleucine*
Leucine
leucine amide, leucine hydroxamate
Lyaine
S-2-aminoethyl-L-cyeteine*,+
(38)
Methionine
ethionine+ Norleucine+
(23) (22)
Phenylalanine
p-fluorophenylalanine*, ß-thienylalanine
Threonine
a-amino-ß-hydroxyvaleric acid* threonine hydroxamate
Tryptophan
5-fluorotryptophan *,+, 6-fluorotryptophan* tryptamine, 6~ethyltryptophan
Tyrosine
3-aminotyroaine*, 3-nitrotyroaine*, tyramine
Valine
heaafluorovaline*
(36)
NONESSENTIAL AMINO ACIDS Aspartic Acid
S-methylcyateine
Proline
azetidine-2-carboxylic acid*,+, line*, 4-thioproline
Serine
a-methylaerine, serine hydroxamate
3,4-dehydro-(28)
The analogs indicated by an asterisk were found to be cytotoxic, ae well as cytostatic, in experiments analogous to those shown in Fig. 3A . Analogs that have been successfully used to select mutants are indicated by a +, followed by the appropriate reference in the right hand column .
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ACKNOWLEDGMENTS Research in the authors' laboratory, the Howard Hughes Laboratory for the Study of Genetic Disorders, was supported by the Howard Hughes Medical Institute and by a grant from the National Science Foundation . We thank Dr . Ellis Englesberg for sending us manuscripts prior to their publication . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 . 36 . 37 . 38 .
H .E . UMBARGER . Adv . in Genetics 16 119-140 (1971) . J .E . Brenchley and L .S . WILLIAMS . Ann . _Rev . Microbiol . _29 251-274 (1975) . Y .S . HALPERN . _Ann . _Rev . Genet . _8 103-133 (1974) . F .T . RAO and T .T . PUCK . Genetics _55 513-524 (1967) . F .T . KAO, L .A . CHASIN and T .T . PUCK . Proc . _Nat . Aced . Sçi . _USA _69 12841291 (1969) . C . JONES and T .T . PUCK . _J . Cell Physiol . 81 299-304 (1973) . C . JONES aad E .E . MOORE . Somatic Cell Genet . 2 235-243 (1976) . 0 . HANKINSON . Somatic Cell Genet . 2 497-507 (1976) . L . FOWDEN, D . LEWIS and H . TRISTRAM . _Adv . _in Eazymol . _29 89-163 (1967) . W . SHIVE and C .G . SKINNER . Amino Acid Analogs in Metabolic Inhibitors , Vol . 1, pp . 1-73 . Academic Press, New York (1963) . .H K . ÂSTRIN sad C .T . CASREY . Arch . Biochem . Biophye . _176 397-410 (1976) . G .B . CLEMENT . Advan . Cancer Res . 21 273-390 (1975) . J .E . SISKEN and T . IWASAKI . Eux . Cell Res . 55 161-167 (1969) . S .S . KERWAR and A .M . FELL%. _J . Biol . Chew . _251 503-509 (1976) . S .S . KERGTAR, R .J . MARCEL and R .A . SALVADOR . Biochem . Biophye . _Kea . Comm . _66 1275-1280 (1975) . J .H. MANGUM aad J .A . NORTH . Biochem . Biophye . _Ras . Comm . _32 105-110 (1968) . D . KAMELY, J .W . LITTLEFIELD and R .W . ERBE . Proc . _Nat . Aced . Sçi . USA _70 2585-2589 (1973) . J .H . MANGUM, B .R . MURRAY and J .A . NORTH . Biochemistry _8 3496-3499 (1969) . S .S . RERWAR, C . SPEARS, B . McAUSLAN and H . WEISSBACH . Arch . Biochem . Biophye . _142 231-237 (1971) . J .D . FINRELSTEIN, W .E . KYLE and B .J . HARRIS . Arch . Biochem . Biophye . _146 84-92 (1971) . C . KUTZBACH and L .R . STORSTAD . Biochem . Biophye . Acts 139 217-220 (1967) . D . KAMELY and J .W . LITTLEFIELD . Espt . Cel l Res . _89 154-160 (1974) . CARBOCHE, M . J . Cell . Phyaiol . _87 321-336 (1976) A . MEISTER . Biochemistry _of _the Amino Acids , _Vol . _2 Academic Press, New York (1965) . _ V .V . MICHELS and A .L . BEAUDET . Pediat . Rea . 11, 461 (1977) . T .-F . SHEN and H .J . STRECKER . Biochemical_J . _150 453-461 (1975) . J . PEISACH and H .J . STRECIÜrR . _J . Biochem . 237 2255-2260 (1962) . J .J . WASMUTH aad C .T . CASREY . Cell 8 71-77 (1976) . D .H . RUSSELL and P .J . STAMBROOK . Proc . Nat . Acad . Sci . USA 72 1482-1486 (1975) . T .A . TEDESCO and W .J . MELLMAN . Proc . Nat . Aced . Sci . _USA _57 829-834 (1967) . E .B . SPECTOR, 0 . LOCRRIDGE and A .D . BLOOM . Biochem. Genet . 1 3 471-485 (1975) R . T . SCHIMKE . _J . Biol . Chem . _239 136-145 (1964) . S . L . NAYLOR, L .L . BUSBY and R .J . KLEBE . Somatic Cell Genet . 2 93-111 (1976) . L .B . JACOBY . J . Cell Biol . 70 134a (1976) . R .T . SCHI?IICE . Biochem . Biophye . Acts _62 599601 (1962) . B . TAUB and E . ENGLESBERG . Somatic Cell Genet . _2, 441-452 (1976) . E . ENGLESBERG, R . BASS and W . HEISER . Somatic Cell Genet . 2, 411-428 (1976) . J .J .WASMUTH and C .T .CASKEY . Cell 9 655-662 (1976) .
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39 . L .G . THOMPSON, C .P . STANNERS and L . SIMINOVITCH . Somatic Cell Genet . _1 187-208 (1975) . 40 . R .M . DENNEX and I- . W. CRAIG . Biochem . Genet . _14 99-117 (1976) . 41 . N . SHIMIZU, R,S . KUCHERLAPATI and F .H . RUDDLE Somatic Cell Genet . _2 345-357 (1976) . 42 . C .R . SCRIVER and L .E . ROSENBERG . Amino Acid Metabolism _and Its Diaorders . W . B . Saunders, Philadelphia (1973) . 43 . J .B, STANBURY, J .B . WYNGAARDEN and D,S . FREDRICKSON . The Metabolic Baeie _of Inherited Disease, 3rd Edition, McGraw-Hill, New York(1972) . 44 . D, RAMELY, H . WEISSBACH and S .S . KERWAR . Arch . Biochem . Biopcs- 179 43-45 (1977) . 45 . R .M . HOFIMAN and R,W . ERBE, Proc . Nat . Acad . Sci . USA 73 1523-1527 (1976) . 46 . W .O . McBRIDE and H .L . OZER . Proc . _Nat . Acad, Sci . USA _70 1258-1262 (1973) . 47 . R . WILLECKI, R, LANGE, A . KRÜGER and T . REBER . Proc . Nat . Acad . Sci . US A _73 1274-1278 (1976) . 48 . R .E . FOURNIER and F . H . RUDDLE . Proc . Nat . Acad . Sci . _USA _74 319-323 (1977) . 49 . G .J . WULLEMS, J . van der HORST and D . BOOTSMA . Somatic Cell Genet . _2, 155164 (1976) . 50 . S .J . GOSS and H . HARRIS . Nature 255 680-684 (1975) . 51 . V,A . McKUSICK and F,H . RUDDLE . Science 196 390-405 (1977) .
Pergamon Press
MINIREVIEW AMINO ACID ANALOCrRESISTANT MA1~IIiALLAN CELLS John J . Wasmuth, Ph .D . and C . Thomas Caekey, M .D . Department of Medicine, Baylor College of Medicine 1200 Mouraund Avenue, Houston, Texas 77030
Amino acid analogs have been employed very successfully as biochemical and genetic probes is Eschericia coli and Salmonella typhimurium toward an understanding of the regulation of amino acid metabolism (1) . Much of this information was derived from characterizing mutants resistant to amino acid analogs . Resistance to various analogs resulted from alterations in the transport and assimilation of amino acids, as well as from alterations in the regulation of the appropriate biosynthetic pathways (1,2,3) . Advances in somatic cell genetics in the last decade now make it possible to study similar problems in cultured animal cells with approaches analogous to those used in bacterial studies . For ezample, animal cell mutants that are auaotrophic for various amino acids have been isolated, and in some cases the defective enzyme has been identified, (4,5,6,7,8) . Amino acid analogs represent a large spectrum of potential selective agents for obtaining mutant animal cells with alterations in both structural and regulatory genes involved in various aspects of amino acid metabolism and utilization (9,10) . In view of the limited knowledge concerning the patterns and mechanisms involved in regulating these essential functions in animal cells, such studies should be most enlightening from both a biochemical and genetic standpoint . The purpose of this article is to review the information that has been derived from studying various amino acid analog resistant mutants, to discuss their significance and to speculate what types of studies might be done in the future . Since several reviews have appeared recently oa mutants affecting purine and pyrimidine biosynthesis (11,12), we have avoided discussing amino acid analogs (such ae azaserine) whose primary effect is in the biosynthesis of these compounds . An amino acid analog, by acting ae an antagonist of a natural amino acid, might inhibit cell growth by inhibiting nay essential function in which the natural amino acid ie required . Aay effect of an analog that results in a reduction of the natural amino acid's incorporation into protein would result in growth inhibition . This might result from a reduction in the cellular concentration of the amino acid by the analog's ability to inhibit the transport of an essential amino acid or the biosynthesis of a nonessential amino acid . Many analogs are known to inhibit protein synthesis directly by inhibiting the cognate aminoacyl tRNA synthetase . In some cases, the analog is incorporated into protein in place of the natural amino acid . This ie usually quite toxic to the cell since many of the resultant proteins are nonfunctional . Besides their role in protein synthesis, various amino acids serve as precursors for other essential metabolites such as S-adenosylmethionine, polyamines and phosphatidal serine . Inhibition of the formation of any such essential metabolite could also inhibit cell growth . Amino acid analogs have also been shown to inhibit various other cellular functions in animal cells (13,14,15) . Alterations in the cell that might lead to resistance to an analog are ae varied se the ways in which analogs inhibit cell growth . An enzyme essential for growth (such as an aminoacyl-tRNA synthetase) that is inhibited by as analog could be altered by mutation such that it ie lees sensitive to inhibition by 0300-9653/78/0501-1469$02 .00/0 Copyright © 1978 Pergamon Press
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the antimetabolite . Alternatively, increased resistance to an analog might be achieved by as increase in the intracellular concentration of the natural amino acid relative to the analog . This could result from a variety of alterations, including : 1) increased biosynthesis (in the case of non-essential amino acids), 2) decreased uptake or retention of the analog, 3) decreased degradation of the natural amino acid, 4) increased degradation or conversion of the analog to a nontoxic compound . It is important to point out that the mechanism by which a cell achieves resistance to an analog does not necessarily esy anything about the mechanism by which the analog inhibits cell growth . In the following sections, some of these types of inhibition and resistance will be described, while some remain theoretical possibilities . Methionine Analog Resistant Mutante Although methioniae is classified as an essential amino acid in mammals, various cultured cells can synthesize methioniae from L-homocysteiae and N5-methyl-tetrahydrofolate (NS~methyl THF) (16,17,18) (Fig . 1) . The reaction is catalyzed by N 5-methyl-tetrahydrofolate : homocysteiae methyltraneferase (HCMT), which requires S-adenosylmethionine (SAM) and a vitamin B12 coenzyme . Another enzyme which utilizes betaine as the methyl donor in an analogous reaction has not been detected in cultured cells . The former pathway is functional is cultured baby hamster kidney (BHR) cells as shown by the observation that these cells grow in methioniae-free medismt supplemented with homocysteine, B12 and a high concentration of folic acid (HC medium) (17,18) . The activity of HCMT is cultured cells is affected by both B12 and methioniae . The former apparently converts apo HCMT to holo HCMT while the latter represses the synthesis of enzyme (17,19) . In addition to its role in protein synthesis, methioniae also plays a key role in methyl group transfer via its conversion to S-adenosylmethionine (SAM) (Fig . 1) . The S-adenosylhomocysteiae (SAH) produced in methylation reactions can be cleaved to homocysteine, which in turn can be reconverted to methioniae by HCMT . In view of the role of methioniae in polypeptide chain initiation and SAM in methyl group transfers, one might expect this cyclic pathway to be highly regulated (20,21) . In an effort to learn more about this regulation, two laboratories have isolated methioniae analog-resistant BHR cells .
THF Methylene THF
r
Methionine
S-adenosylmethionlne ~Acceptor
a
Methylated S-adenosylhomocystetne
Acceptor
Methyl rHF Homocysteine
FIG . 1 The enzymes involved in the interaonversioa of methioniae and homocysteine are : 1) methioniae adenosyltrsasferase . 3) Adenosylhomocysteinase, 4) N5-methyl-tetrahydrofolate :homocysteiae methyltransferase, 5)methylene
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tetrahydrofolate r.eductase (menadione reductaee) . The conversion of S-adenosylmethionine to S-adenosylhomocyateine (Step 2) is catalyzed by a large number of enzymes that transfer the methyl group of S-adenosylmethionine to various acceptors . Kamely and Littlefield (22) selected mutants resistant to norleucine using methionine-free, HC medium . Only when selection was done at a norleucine concentration of 10 mg/ml was there an effect of mutagene on the frequency of reaiatant cells . In addition, only about 1/10 of the clones isolated remained substantially reaiatant to norleucine upon subculturing . The uptake of norleucine was examined in 20 reaiatant clones . The steady state intracellular level of norleucine was significantly lower in five of these and the initial rate of methionine uptake was altered in four of these five . However, without more detailed kinetic studies, it is not possible to determine the reason for decreased norleucine pools in the resistant clones . In certain clones, HCMT was elevated two-fold above wild type levels, when cells were grown in medium with methionine . However, since the reaiatant clones were selected in methionine-free medium, more striking differences between reaiatant and wild type cells might have been observed if the enzyme had been measured in cells grown without methionine ; especially since it is known to repress the synthesis of HCMT . Thus, although the "repressed" level of HCMT (in cells grown with methionine) was not markedly different in wild type and reaiatant clones, the "depressed" levels of HCMT (in cells grown without methionine) may be significantly different . Perhaps the moat well characterized amino acid analog reaiatant cells reported are the ethionine resistant BHR mutants isolated by Carboche (23) . These mutants were also selected in methionine-free HC medium, which contained 20ug/ml of ethionine . One mutant, ETH304, which maintains the reaiatant phenotype after prolonged growth is nonselective medium, was studied in detail . In methionine-free HC medium, ETH304 has markedly higher intracellular methionine pools than wild type cells, and excretes ten times more methionine than wild type cells . These results suggested that ETH304 synthesized methionine from homocysteine at an increased rate . ETH304 also had a significantly higher SAM pool than wild type in HC medium, but not in medium with methionine . Oae puzzling observation was that both ETH304 and wild type cells had greatly increased SAM pools when grown in HC medium with ethionine . Based on the growth response of wild type cells to aminopterin and folic acid in methionine-free medium, Caboche concluded that the rate limiting step in methionine biosynthesis is the formation of N5-methyl THF . In ETH304, the enzyme catalyzing the formation of N5-methyl THF from methylene THF (methylene THF reductaee ; or meaadione reductaee) was elevated under all growth conditions . In wild type BHR cells, menadione reductaee is inhibited by SAM . However, the enzyme from ETH304 was much leas sensitive to inhibition by SAM under all conditions tested . In addition, the menadione reductaee from ETH304 was more thermoaeneitive than the wild type enzyme, another good indication that this enzyme is altered is this mutant . In cell hybrids between wild type and ETH304, ethionine resistance behaved as a codominant trait is growth experiments . An examination of menadione reductaee from such a hybrid provided some evidence far a "hybrid" enzyme . The increased resistance of ETH304 to ethionine might thus be explained ae follows . In wild type and ETH304, ethionine causes a marked elevation in the SAM pool . In the wild type cells, but not in ETH304, this would result in inhibition of meaadione reductaee, thereby reducing the rate of methionine synthesis, for lack of N 5 -methyl THF . Since the menadione reductaee in
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BTH304 is much lean sensitive to inhibition by SAM, these cells continue to produce NS~methyl THF and methioniae . The increase is intracellular and extracellular methionine could thus antagonize the inhibition by ethionine . Proline Analog Resistant Mutants In animal cells, proline can be synthesized from either glutamic acid or ornithine through the intermediate glutamic-y-eemialdehyde (GSA) (24) (Figure 2) . Cultured human fibroblaste contain low levels of argiaaee and thus, synthesize very little ornithine from arginine (25) . We have been unable to detect any conversion of arginine to proline in Chinese hamster cells (unpublished results) . Thus, in cultured human and Chinese hamster lung (CHL) fibroblasts, the mayor, if not only, pathway for proline biosynthesis is the one from glutamic acid (26, unpublished results) . Ia addition, the CHO cell line established by Yuck at al (4) is auxotrophic for proline se a result of its inability to convert glutamate to GSA . In these cells the arginine to proline pathway moat not be functional or the cells would not require eaogeaous proline .
CItruIline
Arginlnosucclnlc Acid
Arginine
lo
Ornlthine -" Putresclne
!m
Glutamic ----~Glutamic- i- ~-- ~e`-Pyrroline-5- -iProline Acld © Semialdehyde Carboxylic Acid
FIG . 2 Metabolic interrelationship of arginine, proline and ornithine . The enzymes involved in these metabolic pathways are : 1) Argiainoauccinic Acid Synthetase, 2) Argininosuccinic Acid Lyaee, 3) Arginase, 4) Oraithine Traneaminase, 6) A'-Pyrroline-S-Carboxylic Acid Reductase, 7) Ornithiae Decarborylaee . The enzyme or enzymes that convert glutamic acid to glutamicy-semialdehyde (Step 5) have not been demonstrated in animal cells . The uncertainty concerning this part of the proline biosynthetic pathway ie indicated by the dashed line . The iaterconversion of glutamic-y-semialdehyde and A'-pyrroline-5-carborylic acid ie apparently a spontaneous, non-enzyme catalyzed reaction .
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Very little is known about the enzymology or regulation of the glutamate to proline pathway is animal cells . Only t°he last enzyme in the pathway, D-1pyrroline-5-carbozylic acid reductase (PCA reductase) has been demonstrated in vitro (27) and no intermediates between glutamate and GSA have been identified . Since this pathway is relatively unbranched, and the end product is not further metabolized, (cultured fibroblasta appear to lack proline oxidase activity), it seemed amenable to biochemical and genetic studies using analogs . We isolated CBL mutants resistant to the proline analog, L-azetidine 2-carboxylic acid (AZCA) (28) . Clones isolated as resistant to 0 .6mM AZCA were often only marginally resistant to that the concentration of the analog in subsequent plating efficiency tests at low cell densities . However, in a more sensitive teat to determine the cytotozicity of high concentrations of AZCA, a clear cut difference was demonstrated between wild-type and AZCA resistant (AZCAr) clones . The AZCA resistant phenotype ie maintained after many months of continuous culture in nonselective medium . In wild type cells, AZCA markedly decreased the intracellular proliae pool . In two AZCAr clones much higher intracellular levels of AZCA had little effect on the soluble proline pool . These same AZCAr clones excreted much more proline into the medium than wild type cells . Using e sensitive in vivo assay, we demonstrated that the AZCAr cells convert l4C-glutamic acid to 14 C-proline at a greater rate than wild type cells . Furthermore, while AZCA greatly reduced the conversion of glutamate to proline in wild-type cells, it had no demonstrable effect on this conversion in AZCAr cells . In more recent experiments, we have demonstrated that AZCA inhibits the comreraion of glutamate to proline at some point before the formedqn of GSA. This conclusion is based upon atudiea measuring the conversion of 14 C-glutamate to 14 C-ornithine [via the conversion of GSA to ornithiae by ornithine transaminase (.OTA), see Fig. 2] . The results of experiments comparing the rate of formation of 14C-ornithiae from 14C-glutamate in wild type and AZCAr cells, paralleled those measuring the conversion of glutg~pato to proline . Thus, AZCA inhibited the conversion of l4C-glutamate to 14 C-ornithine in wild type cells but not in the AZCAr mutants (without affecting the activity of OTA) . In addition, the AZCAr cells excreted 14 C-ornithine at about twice the rate of wild-type cells . As can be seen from Figure 2, since AZCA inhibits the coaveraion of glutamate to both proline and ornithine (and does not inhibit OTA) its site of action moat praceed the formation of GSA . One would predict, therefore, that ornithine should reverse the growth inhibition caused by AZCA in wild type cells, since it could be readily com~erted to GSA. Ornithine reverses AZCA inhibition, but only at a very high concentration (5mM) . This could likely be due to poor uptake of ornithine by CHL cells in medium containing lysine and arginine, which share a common transport system with ornithine in some animal tissues . Indeed, we and others (29) have observed poor uptake of ornithine by CHL cells. It is also of acme interest to point out that CHI. cells can synthesize the polyamines (apermine, spermidine and putrescine) from glutamic acid, following its conversion to ornithine . Thus, CBI. cells labeled with 14C_glutamate contained 14 C-spermine, apermidine and putrescine . In these cells, therefore the glutamate to proliae biosynthetic pathway may also be quite important in the biosynthesis of these compouade . Since our initial report, we have found that AZCA resistance behaves as a dominant trait in cell hybride . In growth atudiea, AZCAs z AZCAr hybrida are as resistant to AZCA as the diploid AZCAr parent . In such a hybrid, the conversion of glutamate to proline (and ornithine) is completely refractory to inhibition by AZCA, again like the AZCAr parent .
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The rapidity with which AZCA inhibits the synthesis of GSA from glutamate suggests it is inhibiting an enzyme in some manner, ae opposed to repressing enzyme synthesis . It thus appears analogous to a "false" feedback inhibition. The inhibition by AZCA may not be that simple, however, since exogenous proline has a much lean dramatic effect on the conversion of glutamate to proline (in short time periods) . In addition, another proline analog, 3,4-D, L-dehydroproline, which is at least as toxic to CHI. cells as AZCA, has no effect on the synthesis of proline from glutamic acid . Whatever the mechanism of AZCA inhibition, the data suggests that an enzyme involved in converting glutamate to GSA may be altered in the AZCAr cells . The overproduction of proline by these mutants also suggests that they lack some type of control mechanism that operates in wild type cells . These cell lines should, therefore, be valuable tools for ezamiaing the enzymology and regulatory mechanisms im~olved in the conversion of glutamate to GSA and proline . Arginine Analog Resistant Mutaata While cultured animal cells cannot synthesize arginine de novo from ornithine and carbamyl phosphate, some cell lines can convertcitrulline to arginine (Fig . 2) and thus grow in arginine-free medium supplemented with citrulline (30,31,32,33) . The two enzymes involved in the conversion of citrulline to arginine, argininosuccinic acid synthetase (ASAS) and arginiaoauccinic acid lyase (ASAL) have been studied in several types of cultured cells, including human lymphoblast cell linen (30,31,32) . A brief report has appeared on the isolation of human lymphoblast cell lines that are resistant to the arginine analog, canavanine (34) . These mutants were aehected in arginine-free medium containing 0 .6mM citrulline and .012mM canavanine . The transport of arginine did not appear to be altered in the canavanine resistant mutants but they had higher levels of ASAS than wild type cells under all growth conditions . The level of ASAS in these mutants was the same in medium containing citrulline, arginine or canavanine . In contrast, the level of ASAS in wild type cells is greatly reduced in medium containing arginine rather than citrulline . These initial results, therefore, suggest that the canavanine resistant mutants may have a defect is some regulatory element controlling the synthesis of ASAS . Previous studies with various cultured mammalian cell lines have shown that ASAS and ASAL increase in a coordinate manner when arginine is made growth limiting (32,35) . Interestingly, A3AL was not markedly increased in the canavanine reaietant mutants, indicating that the activity and/or synthesis of ASAL and ASAS can be regulated independently . Transport Mutants Engelsberg and coworkers have used two approaches to isolating mutant A9 mouse cells altered in amino acid transport . The first method employed 5-fluorotryptophan (5-FT) as a selective agent while the second employed tonic concentrations of the natural amino acid, phenylalanine (36,37) . Mutants resistant to 5-FT had a reduced ability to concentrate the analog as well as tryptophaa . Detailed kinetic analysis of tryptophan uptake in wild type cells indicated there are two transport systems for this amino acid . Determination of kinetic constants (Rm and Vim) for the two proposed transport systems in the analog resistant mutants indicated that one or both systems are altered in all the mutants examined . Similar alterations in phenylalanine uptake were observed in mutants reaietant to high concentrations of this amino acid . As in the case with tryptophan, they obtained kinetic evidence for two phenylalanine transport systems, both of which were altered in the mutants studied . The observation that both transport systems can be altered in the same mutant (that ie, both tryptophan systems or both phenylalanine systems) suggested that the two systems share a common component .
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Alternatively, there may be just one system which eahibita negative cooperativity, or has one high and one low affinity binding site . Although the frequency of cells resistant to pheaylalaniae was high in unmutagenized cultures, fluctuation tests indicated that the appearance of pheaylalaniae resistant clones was a random event . In addition, the frequency of pheaylalaniae resistant clones was increased by treatment of cello with mutagens . Protein Synthesis Mutants As mentioned in the introduction, analogs that are incorporated into protein are usually quite toxic since many of the resultant proteins are nonfunctional . We were therefore interested to determine if the analogs we found to be cytotoxic to CHL cells (see next section)eaerted their toxic effects by virtue of being incorporated into protein . One simple test to examine this possibility is to reversibly inhibit protein synthesis during the time cells are exposed to the analog in question . Following removal of the analog and the protein synthesis inhibitor, cell viability ie determined by the number of single colonies that appear 7-10 days later . We tented several cytotoxic analogs in this manner, using 2uM cycloheaimide to reversibly inhibit protein synthesis . We found that indeed the cytoxicity of many analogs could be prevented if protein synthesis was inhibited while cells were exposed to the analogs . Typical results are shown is Figure 3 for toxic analogs of lysine (S-2-aminoethyl-L-cyeteiae), tryptophan (5-fluorotryptophan), and pheaylalaniae (p-fluorophenylalanine) . The viability of cells exposed to one of these analogs for 48 hours was decreased by several orders of magnitude in the absence, but not in the presence of 2pli cycloheaimide (Pig . 3a) . Furthermore, the toxicity of S-2-amiaoethyl-L-cyateine and 5-fluorotryptophan is markedly increased by omitting the corresponding natural amino acid from the medium while cells are exposed to the analog (Fig . 3b) . Toxicity under these conditions is also prevented by cycloheximide . The moat likely interpretation of these results is that these analogs must be incorporated into protein to be cytotoxic . Another possibility that cannot be completely ruled out is that the inhibition of protein synthesis by cycloheximide causes an increase in the intracellular amino acid pools, which allows a more effective competition with the analogs . The observation that the cytotoxicity of certain analogs could be prevented by inhibiting protein synthesis suggested to us that these antimetabolites might be useful to select cell lines with conditionally lethal, tempera ture sensitive (te) mutations affecting protein biosynthesis . We have used the most toxic analog examined, S-2-aminoethyl-L-cyateine (thialysiae) in this manner and have obtained several is CHL protein synthetic mutants (38) . Five of sin to mutants isolated in such a selection (none of which are gibe) have defects in asparagyl-tRNA aynthetase (AsnRS), and fall into a single complementation group in cell hybridization experiments . In three of these mutants, increased thermolability of AsnRS is demonstrable _in vitro . Oae mutant, which is a separate complementation group, remains uncharacterized . It is intereatiag that AsnRS mutants appear to be common type of mutant in CHO as well ae CHL cells, while other aminoacyl-tRNA aynthetase mutants are much more difficult to obtain (with the eaceptioa of leucyl-tRNA aynthetase mutants in CHO cells) (39) . The reason for the preponderance of this particular type of protein biosynthetic mutant in two different Chinese hamster cell lines ie unknown . While we initially considered the possibility that this locus might be X-linked is Chinese hamsters (38), recent experiments is this laboratory have excluded that possibility . We have now obtained hybrids between one to CHL AanRS mutant and normal human leucocytea (selected under conditions that require them to retain the human gene for AenRS) in an attempt to assign this gene to a particular human chromosome .
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FIG . 3 Cytotoxicity of amino acid analogs . A . Wild type Chinese hamster lung cells were distributed to 60mm culture dishes at densities of 102 to 106 cells/ dish in Eagle's minimal essential medium (MEM) . Twenty-four hours later the medium was removed and replaced with similar medium containing 1 .5x10 - 3M 5-2aminoethyl-L-cyeteine (thialysine) (0) ; thialysine plus 2uM cycloheaimide ( " ) ; 2x10 -3M D, L 5-fluorotryptophan (0) ; 5 fluorotryptophan plus 2uM cycloheaimide (~) ; 2x10 - 3M D, L p-fluorophenylalaaine (Q ) ; or p-fluorophenylalanina plus 2uM cycloheaimide (/ ) . At the indicated times thereafter the analogcontaining medium was replaced with nontoxic medium (DIEM) . Tea days later, the number of cells that survived the various treatments (n) was determined by the number of colonies in the dishes . The surviving fraction (N/No) was then determined by dividing N by the number of colonies (No) on control dishes that were not exposed to an analog . B . Experimental conditions were as is A, with the following exceptions : 1 . The concentration of both thialysine and 5fluorotrypiophan was reduced to 10- 3M . 2 . Lysine was omitted from the medium containing thialysine and tryptophan was omitted from the medium containing 5-fluorotryptophan . Symbols are se in A . While the toaicity of thialysine and other analogs may very from one cell line to another, the basic selective procedure should be applicable to almost In addition, the selective technique can be modi any type of cultured cell . fied in many ways to select for more specific types of protein synthetic mutants . The isolation of mutants with defects in the various soluble factors involved in protein synthesis, as well as in ribosomal proteins, will enhance our ability to construct a human genetic map of protein synthetic factors . It should, therefore, be possible to determine if any of these functionally related genes are clustered . In this regard, it should be noted that the gene for tryptophanyl-tRNA synthetase has been assigned to human chromosome 14 (40,41) .
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Survey of Inhibit or~ Analogs In vies of our limited knowledge concerning the genetic and biochemical mechanisms that regulate the transport, biosynthesis, catabolism and assimilation of amino acids in animal cells, further ezploitation of amino acid analogs as selective agents seems well worthwhile . In Table I ie a listing of amino acid analogs that we found to be cytoetatic or cytotoxic to the clonal growth of CHL cells in Eagle's minimal essential medium . Cytoetaticity ie defined as a reduction in the plating efficiency to at least 10- 2 when cells are grown in the continued presence of the analog . Cytotoxicity is defined as a reduction in the plating efficiency to at least 10- 2 when cells are exposed to the analog for a period of 1-3 days, then eubeequently grown for at least seven days in normal medium . The purpose of these studies was to screen a large number of analogs to determine which would be useful as selective agents . They were therefore usually done over a limited range of cell densities and at three or four concentrations of the analog, ragging from .Olmg/ml up to 2mg/ml . The effectiveness of many of the analogs of essential amino acid may be increased in medium containing lower concentrations of the natural amino acid . Likewise, many analogs that are not inhibitory in normal medium might be effective inhibitors in medium with lower concentrations of a given amino acid . As mentioned in the preceding sections on methionine and arginine resistant mutants, it is sometimes possible to remove essential amino acids from the medium completely, and supply the cells with a precursor that can be comrerted to the missing amino acid . Similar possibilities exist for other essential amino acids, depending upon the ability of various types of cultured cells to utilize precursors of different amigo acids (33) . Thin allows more flexibility in the types of mutants one might attempt to isolate . One is left with the question, of what use are the various types of amino acid analog resistant mutants? Transport mutants will undoubtedly aid our understanding of the very complex systems involved in the uptake of amino acids in animal cells, defects in some of which are the basis for certain inherited human disorders (42,43) . The biosynthetic regulatory mutants can be used to learn more about the physiology of the appropriate pathways and what types of mechanisms regulate them . For example, the ability of various types of malignant and transformed cell lines to utilize homocysteine is place of methionine appear to be impaired, even though all the biosynthetic enzymes are present in normal amounts (44,45) . Thus, a better understanding of the factors regulating the uptake of homocyeteine and its conversion to methionine could prove valuable in understanding this unusual property of malignant cells . The regulation of arginine and prolixe biosynthesis is of interest from several standpoints . In some cells, arginine serves ae the major precursor of prolixe while in others glutamate fulfills this role . What mechanisms regulate the relative contribution of the two pathways? Do metabolites of one pathway regulate carbon flow over the other? Can the regulation of either of these pathways affect the cellular level of polyamines, compounds which have wide ranging metabolic effects? Ae the technology for transferring limited amounts of genetic material from one cell line to another advances (46,47,48,49,50,51), it should be possible to examine the nature of some regulatory mutations ix greater detail . Systems can be developed in which one can ask whether structural and regulatory genes are closely linked or unlinked by determining if the two can be cotraxsferred with small amounts of DNA . Such approaches will be necessary in order to define regulatory elements in animal cells more precisely at the molecular level .
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TABLE I Cytostatic and Cytotoaic Amino Acid Analoge Amino Acid
Analog
Selection of Mutante ESSENTIAL AMINO ACIDS (34)
Arginine
canavanine *,+
Cyateine
aelenocysteine* cyeteine hydroxamate
Isoleucine
4-thiaisoleucine*
Leucine
leucine amide, leucine hydroxamate
Lyaine
S-2-aminoethyl-L-cyeteine*,+
(38)
Methionine
ethionine+ Norleucine+
(23) (22)
Phenylalanine
p-fluorophenylalanine*, ß-thienylalanine
Threonine
a-amino-ß-hydroxyvaleric acid* threonine hydroxamate
Tryptophan
5-fluorotryptophan *,+, 6-fluorotryptophan* tryptamine, 6~ethyltryptophan
Tyrosine
3-aminotyroaine*, 3-nitrotyroaine*, tyramine
Valine
heaafluorovaline*
(36)
NONESSENTIAL AMINO ACIDS Aspartic Acid
S-methylcyateine
Proline
azetidine-2-carboxylic acid*,+, line*, 4-thioproline
Serine
a-methylaerine, serine hydroxamate
3,4-dehydro-(28)
The analogs indicated by an asterisk were found to be cytotoxic, ae well as cytostatic, in experiments analogous to those shown in Fig. 3A . Analogs that have been successfully used to select mutants are indicated by a +, followed by the appropriate reference in the right hand column .
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ACKNOWLEDGMENTS Research in the authors' laboratory, the Howard Hughes Laboratory for the Study of Genetic Disorders, was supported by the Howard Hughes Medical Institute and by a grant from the National Science Foundation . We thank Dr . Ellis Englesberg for sending us manuscripts prior to their publication . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 . 33 . 34 . 35 . 36 . 37 . 38 .
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