relevance of glutamine metabolism to tumor cell growth

Molecular and Cellular Biochemistry 113: 1-15, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Relevance of glutamine metabolism to tumor cell growth

Miguel Angel Medina, Francisca Sfinchez-Jimdnez, Javier Mfirquez, Ana Rodriguez Quesada and Ignacio Ntifiez de Castro Laboratorio de Bioqufmica y Biologia Molecular, Facultad de Ciencias, Universidad de Mdlaga, E-29071 Mdlaga, Spain (t current address:Antibi6ticos Farina S.A., Antonio Ldpez 109, Madrid, Spain)

Received 5 June 1991; accepted 29 January 1992

Key words." glutamine, tumor cells, glutaminase, glutamine transport, glutamine analogues

Introduction

Tumor cells are characterized as rapidly dividing cells, and consequently they need a constant supply of both energy and nitrogen substrates. To resolve their energy requirements, they are able to use virtually any substrate: glucose [see 1 for a review; 2-4], lipids [5-7], ketone bodies [3], even amino acids [2-4, 8-10]. Never- theless, the glucose and amino acid consumption by malignant tumor cells overcomes their own needs for their metabolic requirements; thus, tumor cells appar- ently waste glucose and amino acids without any profit [1, ll]. In this context, tumor has been described as a trap for glucose and nitrogen [12-13].

Tumors compete with the host for glucose [13-14]; this competence results in a progressive hypoglycemia [15] and host hepatic glycogen depletion [13]. In the same way, tumors compete for nitrogen compounds; this process produces in the host a negative nitrogen balance and a characteristic weight loss, and in the tumor a reciprocal nitrogen increase. The biochemical mechanisms underlying these phenomena still remain unclear. There is consensus that tumors increase pro-

tein degradation and reduce protein synthesis in the host tissues [16].

Alanine and glutamine are two efficient vehicles for the transport of nitrogen and carbon-skeletons between the different tissues in the living organism [17-18]. When a tumor develops, there is a net flux of amino acids from host tissues to the tumor [19]. Since ammonium ions are very toxic for most of the cells, glutamine is the physiological non-toxic ammonium vehicle between different mammalian tissues; therefore, glutamine is the main source of nitrogen for tumor cells [2, 20-21]. Thus, the presence of a tumor must produce great changes in the metabolism of glutamine in host tissues in such a way that host nitrogen metabolism is accomo- dated to tumor enhanced requirements of glutamine. To be used, glutamine must be transported into tumor mitochondria, where it is metabolized [21]. This implies two transport processes: the transport of glutamine across the plasma membrane and across the inner mitochondrial membrane. Once glutamine has been incorporated into tumor cells, this amino acid is quickly metabolized [12, 16, 19].

Address for offprints." M.A. Medina, Laboratorio de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Mfilaga, E-29071 Mfilaga, Spain

PYRIMIDINES PURINES PROTEIN

UReA CYCLe< CAR.AMOYLP< I , TA INEI

NAD 3D PHENYLACETYLGLUTAMINE |

ASPARAGINE J NH + KETOACIDS ~ @ ~ 4

AMIN~ .AKII~IG L~UU T A!A M A T?~_~

SYNTHESIS

GLUTAMATE ~ ~-AMINOBUTYRATE

N-ACETYLGLUTAMATE

GLUTATHIONE

~ KETOACIDS i

AMINO AC,DS

~: -KETOGLUTARATE

Fig. 1. The main metabolic pathways for glutamine in animal cells, h Glutamine synthetase. 2, Glutaminase. 3, Glutamate dehydrogenase. 4, Glutamate transaminases, 5. Glutamine transaminases. 6. Amidases, 7. Carbamoyl-phosphate synthetase I and II. 8. Amidotransferases.

Several previous reviews have considered the metabolism of the tumor cell itself and the metabolic changes which take place in the host [13, 16, 21-27]. The aim of the present review is to summarize what is currently known on the role of glutamine in the interactions between tumor cells and host tissues. All the three major points mentioned above - namely, changes in host tissues glutamine metabolism, transport of glutamine into tumor mitochondria, and glutamine metabolism in tumor cells - will be reviewed. Finally, the potential therapeutic interest of glutamine related chemotherapy will be also reviewed.

Changes in glutamine metabolism in host tissues

As mentioned above, the presence of a tumor induces deep alterations in the metabolism of glutamine in host tissues. In this section, glutamine metabolism in the main normal tissues and organs in the absence and in the presence of a tumor will be summarized.

Glutamine metabolism in the main host tissues and organs

Glutamine is considered to be the most versatile and

useful amino acid [28-29]. The main metabolic pathways for glutamine in animal cells are mentioned in Fig. 1. The amide nitrogen of glutamine is utilized in the synthesis of purine and pyrimidine bases and aminosu- gars [29]. The importance of glutamine as a respiratory substrate and as a source of anabolic metabolites has been stressed [30--31]).

Glutamine synthetase (EC 6.3.1.2) and phosphate- activated glutaminase (EC 3.5.1.2) are the two main enzymes in the metabolism of glutamine in animal cells [32-33]. Glutamine synthetase is a cytosolic enzyme that catalyzes the formation of glutamine from glutamate and ammonium ions coupled to an energy consumption as a breakdown of ATP to produce ADP and inorganic phosphate [28]. Phosphate-activated glutaminase is the first and main enzyme in glutaminolysis [33-34]. This enzyme is localized in the inner mitochondrial membrane [35-36] and catalyzes the hydrolysis of glutamine, with the production of glutamate and ammonium ions. The rate of glutamine utilization by skeletal muscle is high [37]; but there is evidence that glutamine is synthesized and released by skeletal muscle [38]. A substrate cycle operates in the direction of net glutamine synthesis and release by the muscle. Recent- ly, glutamine has been proposed to participate in the regulation of protein turnover in skeletal muscle [39]. Glutamine and alanine represent more than 50% of total amino acids exported by skeletal muscle [17, 40].

Both amino acids are produced in muscle from the metabolism of other amino acids, mainly branched- chain ones and aspartate. More than 80% of total glutamine produced by the muscle goes through the glutamine synthetase [40].

Liver has both high levels of glutamine synthetase and glutaminase activities. The net balance, that is, glutamine synthesis or consumption, depends on metabolic conditions [41-43]. Thus, liver plays a main role as a regulator in glutamine homeostasis [44]. Liver glutamine synthetase is a cytosolic enzyme exclusively localized in a little subpopulation of pericentral parenchy- real cells [45]. On the other hand, liver glutaminase is an inner mitochondrial membrane enzyme mainly localized in the periportal hepatocytes [46-49]. The intercellular compartmentation of the two main enzymes in glutamine metabolism makes possible the existence of an intercellular substrate cycle [44, 50-51], with the regulatory advantages that this type of cycle presents [52]. According to Newsholme and Start [53], substrate cycles enhance the sensitivity of a metabolic pathway to the action of regulators. The intercellular liver substrate cycle glutamine synthetase/glutaminase has a main role in the regulation of the levels of circulating glutamine [51] because it makes possible the production or consumption of glutamine according to the metabolic requirements [54].

The role of kidney glutamine metabolism in normal metabolic conditions is still not well defined [55]. Stud- ies on arterious-venous differences of amino acid concentrations show that man, dog and monkey kidney consumes glutamine [56-58], whereas Guinea pig kidney produces glutamine, and rat and mouse kidney consumes or produces very little glutamine in normal conditions [59]. These differences reflect the relative activities of glutamine synthetase and glutaminase. In fact, glutamine synthetase is absent in man, dog, cat, and pig kidney [57, 60-61]. In metabolic acidosis, there is glutamine consumption by the kidney, due to a decrease in glutamine synthetase activity when this enzyme is present, and an increase in glutaminase activity [36, 62].

In the nervous system, glutamine is the precursor for glutamate and GABA, two important neurotransmitters [63-64]. In addition, glutamine synthesis is the only way to eliminate ammonium ions in the brain [65-68]; this is an important fact because brain is very sensitive to ammonium ion toxicity [32]. Glutamine synthetase is located mainly in astrocytes, whereas glutaminase is present in neuronal terminals; thus, an intercellular

glutamine synthetase/glutaminase cycle could also exist in the brain [69].

Small intestine plays a main role in the metabolism of circulating glutamine [70]. It has been shown that more than 30% of the nitrogen used in the synthesis of urea is derived from the products of glutamine metabolism in intestine [71]. On the other hand, glutamine is the main energy and respiratory substrate for small intestine cells [42], Glutamine oxidation contributes to the energy required in the active transport of nutrients and electro- lytes from the lumen. Phosphate-activated glutaminase has been found in both villus and crypt cells; it is similar in many respects to kidney glutaminase, but unlike the kidney enzyme it is not induced during acidosis [72].

Alterations induced by the tumor in the host glutamine metabolism

As Felig [17] indicates, plasma-free amino acid concentrations under normal conditions show relatively little intra- or interindividual variations; they are kept at constant levels by a net balance between amino acid uptake and release by the tissues. This balance can be disturbed in the presence of the tumor by a number of means: (a) by variations in the ingested protein [73]; (b) by changes in the intestinal absorption [74]; (c) by alterations of the nonessential amino acid biosynthesis in liver [74]; (d) by changes in tissue oxidative breakdown of amino acids [75]; (e) by differences between protein synthesis and tissue proteolytic activities; and (f) by tumor demand for the essential and nonessential amino acids needed for tumor proliferation [16].

Actually, the plasma-free amino acid pool must be the main direct source of nitrogen compounds for tumor cells [14]. In a recent study on plasma and ascitic fluid concentrations of amino acids during Ehrlich ascites carcinoma development, it is shown that plasma glutamine concentrations are higher than ascitic fluid glutamine concentration for the entire life span of tumor- bearing mice [19]. The low concentrations of glutamine in ascitic fluid attest to the avidity of tumor cells for this amino acid [2-3, 8].

Tumor elicits a specific response in the host nitrogen metabolism so that the whole host organism is mobili- zed to augment circulating glutamine, namely, the prime source of nitrogen for tumor cells [2, 20-21, 29, 54, 76]. A great secretion of glutamine by host tissues has been described in rabbits bearing the Brown-Pierce carcinoma [13], and in mice bearing the Lewis lung

carcinoma [77] or the Ehrlich ascites carcinoma [19]. In fact, only 24 h after Ehrlich ascites tumor transplantation, there is a significant increase in plasma glutamine concentration [19], but in the last days of host life the free glutamine concentrations decreased in plasma, liver, and kidney [76]. In rats bearing Walker-256 carcinosarcoma, tumor burden results in a 23% increase of the arterial concentration of glutamine [78]. The plasma glutamine increase observed in the first day after tumor transplantation could reflect the simultaneous modula- tion of glutamine synthetase and glutaminase activities in liver and kidney, conducive to a net production of glutamine by the host tissues observed in mice bearing Ehrlich ascites carcinoma [76]. No changes in the levels of hepatic glutamine synthetase are observed in the first days after tumor inoculation; however, glutaminase sharply decreases in the first 24h after tumor transplantation, an indication that the glutamine production capability of the whole liver has increased [76]. In other tumors, as methylcholantrene induced sarcoma in rat, a decrease in plasma glutamine concentration is observed [79]. A significant increase in the hepatic protein synthesis has been reported in mice bearing Ehrlich ascites carcinoma [80-81], and in Morris hepatoma [82]; however, liver glutamine concentrations remain unchanged or they decrease as tumor progresses [83-84]. After tumor implantation, other mitochondrial enzyme activities, as well as acid proteinase pattern, change [85-86]; in the same way, key enzyme activities in hepatic metabolism are changed. Hexokinase and phosphofructo- kinase show two-fold increased activities in liver of Walker-256 carcinosarcoma-bearing rats [84]; total proteinase activity is also increased in Ehrlich tumor-bearing mice [86]; on the contrary, liver citrate synthase and cytochrome c oxidase decrease during tumor progres- sion [86]; urea cycle and polyamine metabolism are also altered, as reflected by the great increases in arginase and ornithine decarboxylase activities during tumor growth [87].

Shortly after tumor transplantation, glutamine synthetase is increased and glutaminase is decreased in the kidney of tumor-bearing mice [76]. Nevertheless, in the last days of life, glutamine synthetase decreases and glutaminase increases to control values in kidney. Squires and Brosnan [62] report that renal synthesis of glutamine decreases in rats treated with NH4CI solution in drinking water. In similar conditions, Tong et al. [88] demonstrated that kidney glutaminase levels rose due to an increase in the rate of its synthesis. Thus, the ammonemia detected in Ehrlich ascites tumor-bearing

mice [8] could explain the raise in glutaminase levels found in the kidney in the last days of life. Changes in mitochondrial marker enzymes and proteinase activity are similar to those found in liver of tumor-bearing mice [861.

There is also an increase in skeletal muscle glutamine synthetase in the first days after tumor inoculation [76], when the specific growth rate of tumor reaches a peak [89]. Skeletal muscle protein synthesis is significantly decreased in Ehrlich tumor-bearing mice very early after tumor implantation, before tumor metabolism could have a direct impact on the substrate supply to host tissues [81].

In healthy organisms, there is a glucose-glutamine cycle between glyconeogenic organs and glutaminogen- ic tissues and organs. This cycle is altered by the presence of the tumor (see Fig. 2).

After tumor implantation, nitrogen metabolism is also changed in other host organs, such as intestine, spleen, or brain. Unfortunately, current data are very scarce [54, 81].

Transport of glutamine to the tumor cell

A century ago, Mtiller [90] described a negative balance of nitrogen in patients with cancer. Mider [11] showed that tumors assimilate not only the nitrogen from the diet, but also the nitrogen proceeding from host proteins. In this way, tumors can be considered as 'nitrogen traps'. Shrisvastava and Quastet [91] incubated brain tissue with Ehrlich ascites tumor cells and observed that there was a net glutamine flux from brain cortex cells to tumor cells. These experiments were considered as an 'in vitro' model for the role of tumors as nitrogen sinks.

As stated above, tumors induce a general mobili- zation of the host tissues to produce glutamine [29]. Thus, glutamine can be considered as the main non toxic vehicle of nitrogen between host tissues and tumor. Before its metabolism by tumor cells, glutamine must be transported through both the plasma membrane and the inner mitochondri",l membrane.

Glutamine transport through the plasma membrane

It is obvious that amino acid transport plays an important role in cell growth. This importance is enhanced in

KIDNEY

N H ~ ~ ~ - - ~ - - - ~ ' . Glutamine

Gluco#e ~ _ . . . . - w "- ~ Serine

~t- j ...... / ~ ' ~ i u t a m i n e Alanine " ~ . _ . "

4 . . . . . . . . . . . . . . . /- . . . . . . . . . . . .

MUSC L E~--.. /

"~"- Glutamine

/

Alanine

" , /

INTESTINE

Urea.

¢ LIVER

Fig. 2. Glucose-glutamine cycle in the healthy organism,

tumor cells by the fact that these cells synthesize less amino acids from carbohydrate-carbon skeletons than normal tissues do [92-93]. Consequently, tumors depend more than normal cells on an adequate amino acid supply from extra-cellular fluids. For this reason, tumor cells are more vulnerable to the action of substances that interfere with the transport of amino acids. This fact is the basis for some therapies in which amino acid analogs have been used [54]. All the statements pre- sented in this section clearly show the main role of amino acid transport processes in tumor survival. Par- ticularly relevant is the role of glutamine, a nonessential amino acid for normal cells that becomes essential for tumor cells. This statement will be discussed below.

Solid tumors are poorly vascularized, and they should be endowed with very efficient transport systems in order to compete with normal host tissues for nutrients. Different specific amino acid transport systems have been described [94-95]. As a neutral amino acid, glutamine can be transported by both Na+-dependent sys-

terns A and ASC, and Na+-independent system L [96]; the Na*-dependent component is quantitatively the most important one. In some tissues, there is another Na+-dependent system specific for glutamine, asparagine~ and histidine, namely, the N system in hepatocytes [97], and the N m system in muscle [98]. However, there are few works made on glutamine transport by tumor cells. In Ehrich cells, it was possible to show the existance of two transport systems for glutamine in intact cells; the apparent K m for glutamine were 0.31 mM and 0.74 raM. The concentration of glutamine in ascitic fluid was much less than these apparent U m

values, suggesting a first order kinetics for glutamine transport 'in vivo' [8].

To avoid problems with the high rates of glutamine metabolism, glutamine transport can be easier studied with isolated plasma membrane vesicles. Recently, glutamine transport through the plasma membrane of Ehr- lich cells has been studied by using both native and reconstituted vesicles [99-100]. There were two Na +-

6

dependent transport systems implicated in glutamine transport, most probably A, and ASC systems, previously described in Ehrlich cells in studies with the non- metabolizable amino acid AIB [101-102]. In tumor cells the main way of glutamine uptake seems to be the derepressed A system [96]. There is experimental evidence for an essential sulfhydryl group at the substrate binding site of this transporter, since sulfhydryl reagents inhibit the net uptake of glutamine into native vesicles [100, 103]. In normal cells with no N or N m systems, glutamine is mainly transported via ASC system. In tumor cells, plasma membrane glutamine transport is inhibited by glutamine analogs; 2.5 mM acivicin and 2.5 mM azaserine inhibit 67 and 70%, respectively [100]. Huber et al. [104] report that acivicin transport is inhibited by glutamine. Sastrasinh and Sastrasinh [105] show the inhibition of glutamine transport by acivicin in rat renal brush border membrane vesicles. However, acivicin is a relatively poor inhibitor of glutamine uptake into liver membrane vesicles [106]. These differences in responses to acivicin by tumor and normal cell membrane vesicles appear to be due to the fact that acivicin is mainly an inhibitor of system A activity, which is derepressed in tumor cells [96]. Azaserine be- haves as an inhibitor of both system A and system L, and consequently is a good inhibitor of glutamine transport in liver plasma membrane [106]. Recently, McCor- mick and Johnstone [107] claim for a purification of system A protein from Ehrlich ascites cell plasma membrane; the major component present in the purified fraction has a molecular mass of 120-130 kDa.

Glutamine transport through the inner mitochondrial

membrane

Once incorporated into the cell, glutamine must be transported through the inner mitochondrial membrane to be metabolized in the glutaminolytic pathway [33]. The study of transport processes through the inner mitochondrial membrane presents additional difficul- ties to those that occur in the study of transport through the plasma membrane: this mitochondrial transport is usually 1-2 orders of magnitude faster than the transport in the whole cell [108]. A number of different mitochondrial carriers, usually antiporters, have been described and characterized by using different experimental approaches [108-110]; the glutamine transporter is still not well characterized. The special problem of

glutamine transport through the inner mitochondrial membrane has been previously reviewed [32,111-112].

There is no doubt that glutamine transport in mitochondria is a carrier-mediated process. Glutamine seems to be transported into the mitochondria through a neutral uniport mechanism. The existance of such a transporter was originally proposed by Kovacevid et al.

[113], who found that mitochondria swell in an isoos- motic solution of L-glutamine in the absence of other metabolites, ions, or ionophores. This finding was con- firmed by other group [114]. Another result that strongly suggests the existance of carrier-mediated glutamine transport in mitochondria is the inhibition of the transport by mersalyl, a sulfhydryl-group-blocking reagent [115-118].

Applying automated rapid mixing and rapid filtration techniques, Goldstein and Botlan [119] found in kidney mitochondria that at pH 7.4 and 23°C the K m of the carrier was 2.7 mM and the Vmax = 150--300 nmol glutamine/rag protein. It must be remarked that in these experiments the metabolic process was interfering with the transport. To avoid this problem, Kovacevid and Bajin [120] loaded rat liver mitochondria with 14C-glutamine and measured the rate of its efflux at 0 ° C using a phosphate-free medium, so that the glutaminase activity was negligible as compared to the rate of transport. Under these conditions, the rate of glutamine efflux was 5-times faster than the rate of glutaminase activity. Very recently, Sastrasinh and Sastrasinh [121], using submitochondrial particles from rat kidney, report that the mitochondrial glutamine transport is inhibited by sulfhydryl reagents and trans-stimulated when submitochondrial particles are preloaded with L-glutamine or L-alanine.

Glutamine transport into mitochondrial inner membrane vesicles has been recently studied [122]. The process shows a saturation kinetics with K m = 3.3 mM, and a partial inhibition by the sulfhydryl reagents mersalyl and PCMBS (p-chloromercuribenzene sulfonate).

Glutamine metabol ism in tumor cells

It has been shown that tumor glutamine concentration negatively correlates with tumor growth rate [123]. In rapidly growing hepatoma the low glutamine concentration was attributed, at least in part, to very increased activities of the glutamine-utilizing enzymes of purine and pyrimidine biosynthesis and to a decrease in the

TISSUES

GLUTAMINE . ~.

P L A S M A

GLUTAMINE }

GLUTAMATE 4

\ NH~

C,T,C LUID / TUMO. CELL ' G L U T A M I N E T GL~AMINE ]

"GLUTAMATET GLTMATE ] NH; { \ NH~

"2c°2

® ® L-GLUTAMINE

A ® (!)

L-GLUTAMATE [ L-GLUTAMATE I + NH + ATP I + NH 3 I 3 I

Fig. 3. Enzymic imbalance in glutamine metabolism in tumor cells. A) Main fluxes between host tissues and tumor cells. B) Imbalance in a rapidly growing hepatoma. The width of the arrows represents the relative activity of the enzyme. l. Glutaminase. 2. Phosphoribosyl-formyl-glycinamidine synthetase and amidophosphoribosyltransferase. 3. Carbamoyl-phosphate synthctase 11. 4. GMP synthetase. 6. Glutamine synthetase. 8. CTP synthetase. Numbers 5 and 7 are the exchange reactions leading to the production of UTP and ATP from UMP and IMP, respectively.

activity of glutamine synthetase and an increase in the activity of glutaminase [124].

In fact, glutamine synthetase was considered by Knox [74] as a 'prescindible' enzyme for tumors, that is, the activity is scarce or totally absent in tumor cells. When a neoplastic transformation occurs in rat astrocytes, the glutamine synthetase-inducfion capability of these cells is lost [125]. In hepatoma cells, glutamine synthetase activity is decreased by comparison with normal liver tissues [126]. In Ehrlich cells, glutamine synthetase is practically undetectable [55].

The activities of amidophosphoribosyltransferase, GMP synthetase, carbamoyl phosphate synthetase II, and CTP synthetase were increased in various neo- plasms [123]. However, glutaminase is the main glutamine-utilizing enzyme in cancer cells. The enzymic imbalance in glutamine metabolism in tumor cells is de- picted in Fig. 3.

Tumor glutaminase

Many experimental evidences support that phosphate-

activated glutaminase activity is correlated with malignancy in tumor cells [74, 123,126-127], and with growth rate in rapidly dividing normal cells as thymocytes [128].

Williams and Manson [129] firstly described in mitochondria from HeLa cells a glutaminase which con- verts glutamine into equimolecular quantities of glutamic acid and ammonia; inorganic phosphate, sulfate, or arsenate was necessary for activity of glutaminase at pH values below 8. Kovacevid [130] reported a similar glutaminase activity in isolated mitochondria of Ehrlich ascites cells. Huang and Knox partially purified this enzyme from a mammary carcinoma, taking advantage of the enzyme polymerization in the presence of phosphate-borate buffer [127]. Ouesada et al. [131] firstly purified the phosphate-activated glutaminase from a highly malignant Ehrlich ascites carcinoma. The enzyme has a M, of 135 kDa and consists of two different subunits of 64 and 56 kDa. The activity of purified tumor glutaminase was maximal at pH 9 and undetectable at pH 7 or acidic pH. The concentration dependences for both glutamine and inorganic phosphate were sig- moidal in the solubilized protein, with apparent S0~ values of 7.6 and 48 raM, and Hill coefficients of 1.5 and

1.6, respectively; but isolated mitochondria showed hy- perbolic kinetics for both glutamine and phosphate. Using the digitonin permeabilization technique for metabolic compartmentation [132], Medina et al. [133] report that cytosolic Atkinson's energy charge re- mained with slight changes during incubations of Ehr- lich cells in the presence of 5 mM glucose, 0.5 mM glutamine, or in the presence of both substrates. On the contrary, it can be observed an initial decrease of the mitochondrial energy charge; the initial value was reco- vered after 5 min, when glucose was present, but not in the presence of glutamine. The initial values of inorganic phosphate concentrations in the cytosolic compart- ment decreased; mitochondrial inorganic phosphate concentration diminished in incubations with glucose, and increased in incubations with only glutamine [133]. The high concentrations of inorganic phosphate found in the mitochondria of tumor cells [133] could explain the high activity of glutaminase 'in vivo'. In Ehrlich cells incubated with glutamine the mitochondrial phosphate concentration reached values of 100 mM [133], concentration at which glutaminase activity is approximately 70% that of V .... [131]. The rate of glutamine utilization seems to be controlled by the activity of phosphate- activated glutaminase [134].

In this context, Matsuno [47] suggests that both processes, transport through the inner mitochondrial membrane and hydrolysis of amide group, could be intercon- nected.

Glutaminase is the first enzyme in the glutaminolytic process. According to McKeehan [33], glutaminolysis can occur completely inside the mitochondria, though it can also take place as a compartmentalized process. It has been proposed that the mitochondrial malic enzyme might play a major role in the production of pyruvate from glutamine in some fast growing cells, so that carbon skeletons and nitrogen from glutamine could be incorporated into citrate and alanine during glucose and glutamine metabolisms, if the malate-aspartate and malate-citrate shuttles were operative [21,135]. However, at least in Lettr6 cells, these shuttles are not operative [9, 1361.

In Ehrlich cells, the main products in the metabolism of glutamine are NH4 +, CO2, glutamate and aspartate [137]. Actually, glutamine is a good respiratory substrate for tumor cells. As a matter of fact, when Ehrlich cells were incubated in the presence of only 0.5 mM glutamine, 20% of the total consumed glutamine was oxidized to CO~_ [138].

Many reports agree in stating that glutamine is oxi-

dized in mitochondria exclusively via a pathway in- volving glutamate-oxaloacetate transaminase, because glutamate dehydrogenase activity was undetectable or very decreased in tumor cells. Kallinowski et al. [139] claim that the generally held opinion that glutamine is a major substrate for energy metabolism of rapidly growing tumor cells should be reconsidered since evidence for this hypothesis has been derived from 'in vitro'

experiments under aerobic conditions. The results with isolated mitochondria should not be extrapolated to intact cells. Glucose and long chain fatty acids decreased the rates of glutamine utilization indicating that tumor cells prefer these metabolites as energy substrates, if they are present [10]. Very recently, Gauthier et al. [140] reversely correlate the degree of differentia- tion and mitochondria capacity to respire glutamine; the difference in ability to oxidize glutamine is due to a decrease in succinate dehydrogenase and glutamate dehydrogenase activities in the mitochondria of undifferentiated cells.

Interactions with the metabolism of other energy

substrates

Glucose and glutamine have been shown to be the major energy sources for mammalian cells in culture [34]. Recently, glutaminolysis and glycolysis interactions in proliferant cells have been reviewed [1]. In HeLa cells 10 mM glucose produced a 64% decrease in the rate of glutamine utilization compared to its rate in the presence of galactose and fructose [141], and in Ehrlich cells, glucose decreased the rate of glutamine utilization by 31% [138]. In the same way, Olavarrfa et

al. [142] show that glutamine can not induce a depolarization of the inner mitochondrial membrane, but this depolarization occurs in the presence of glucose. In immobilized HeLa, myeloma, and hybridoma cells perifused with glutamine, the intracellular phosphate concentration was significantly higher than in those cells perifused with glucose [134]. A similar result was obtained with Ehrlich cells, where the intramitochondrial phosphate concentration is much higher with glutamine as a substrate than with glucose [133].

Neither energy nor biosynthetic requirements can explain totally the very high glycolytic and glutaminolytic fluxes found in tumor and proliferant cells, which behave as powerful energy dissipative systems [1,143]. This apparent wasteful spreading of energy has been theoretically justified on the basis of the quantitative

HO 0 HO 0 \ / / \ / /

C C I I

H2N ~ C - - H H 2 N - - C - - H I I

CH CH 2 / \ ]

CH 2 0 CH 2 I I L

C - - N / /c% C1 HC O

II N + II N

ACIVICIN DON

Fig. 4. Glutamine analogues used in chemotherapy.

principles of metabolic control [14]. It has been suggest- ed that, in rapidly dividing cells, high rates of glutaminolysis and glycolysis are needed nor for energy or precursor provision, but for high sensitivity of the pathways involved in the use of precursors for macromole- cule synthesis to specific regulators to permit high rates of proliferation when required [14].

Fatty acids also interfere with glutamine metabolism. In studies carried out in Ehrlich cells, it was shown that there was a significant decrease in glutamine utilization in incubations with 1 mM palmitate added [10]. Further- more, there was a cumulative effect of palmitate and glucose on the decrease of glutamine uptake.

G l u t a m i n e - r e l a t e d c h e m o t h e r a p y

Several glutamine analogs competing with glutamine have been studied as possible chemotherapy agents in experimental oncology and in human patients (see 144 for a review). Figure 4 shows the chemical structure of these compounds. Three of these analogues contain a diazo group: azaserine, azotomycin, and DON [145-146]. DON (6-diazo-5-oxo-L-norleucine) and azotomycin (N-(N-gammaglutamyl-6-diazo-5-oxo-- norleucinyl)-6-diazo-5-oxonorleucine) are glutamine antagonists that were earlier tested in human malignant tumors in the 1950s [147]. DON is probably the active

HO O \ / /

C I

H2N - - C - - H

I CH 2 I

O I

HC O [I N + II N"

AZASERINE

HO O \ / /

C I

H2N - - C - - H I

CH2 I

OH2 I

HN / % 0

OH

7-GLUTAMYL HYDROXAMATE

form of azotomycin. Results in human xenografts (es- pecially, the CX-2 colon tumor) have stimulated renew- ed clinical interest in DON [147]. The drug uptake was studied in mouse P388 leukemia cells; DON utilizes the transport system L, inhibited by glutamine but not by glutamate, and strongly inhibited by p-chloromercuribenzene sulfonate and sodium ions [104]. The drug seems to be extensively metabolized by the patients, and produces cytotoxic activity manifested as transient mild leukopenia and, rarely, thrombocytopenia [148]. DON interrupts cellular nucleotide synthesis and there- by halts the synthesis of DNA and/or RNA in the tumor cell [144].

Acivicin ((alpha,S,5S)-alpha-amino-3-chloro--4,5- dihydro-5-isoxazoleacetic acid) is an antitumor antibiotic obtained from the fermentation product of Strep- tomyces sviceus which is active in a variety of mouse tumour models and human tumor xenografts [149]. The drug is not metabolized by the host, since it is excreted in the urine in unchanged form. An interesting phenom- enon in mice is the sex and age differences in suscepti- bility to acivicin toxicity; no sex differences in toxicity were noted in dog or monkeys [149]. The antitumor activity is probably due to the inhibition of enzymes catalyzing amido transfer in the biosynthesis of purines and pyrimidines, mainly CTP synthetase, GMP synthetase, XMP aminase, carbamoyl-phosphate synthetase II, and amido-phosphoribosyl transferase [144,

10

149-153]. The drop in enzyme activities is accompanied by a decline in the pools of GTP and CTP [150]; but the nucleotide pools returned to normal levels within 2 or 3 days after a single acivicin injection in experimental hepatoma [153]. In hepatoma cells in culture, acivicin inhibited the growth, showing a LCs0 of 1.4/xM; a combination of the nucleosides cytidine, deoxycytidine and guanosine completely protected the hepatoma cells against the cytotoxic action of acivicin [154]. 'In vitro',

acivicin selectively inactivated the glutamine-dependent carbamoyl phosphate synthetase II from human colon carcinoma, and it did not affect the ammonia dependent activity [152]. As DON, acivicin also inhibits the phosphoribosyl-pyrophosphate amidotransferase [155], simultaneously with an increase in the intracellular 5-phosphoribosyl-l-pyrophosphate levels [150].

In addition to the acivicin action as competitive inhibitor for glutamine-utilizing enzymes, the drug exerts a direct inactivating effect on enzymes, probably by alky- lation [151]. This alkylating effect is very similar to that described for DON in glutaminase [156].

Experiments carried out in rats bearing methylcho- lanthrene-induced sarcoma showed that acivicin significantly reduced tumor weight by 65%, 34 days after tumor induction [157].

These drugs have been proposed in different combination therapies. For instance, actinomycin and dipyridamole have been shown to act synergistically with acivicin [158].

Concern exists about providing dietary glutamine to the host with cancer, since it may stimulate tumor growth. However, experiments carried out in rats with large sarcomas have shown that glutamine-enriched oral diets may replete host glutamine stores and support muscle glutamine metabolism without stimulating tumor growth [159]. On the other hand, total parenteral nutrition (TPN) could stimulate tumor growth rate, but the combination of acivicin with TPN produces increased carcass weights and decreased tumor/carcass ratios ]157, 160]. When acivicin and insulin are combined with TPN, tumor growth is stopped, carcass weight is gained, and muscle mass is saved [161].

Very recently, it has been reported that the acivicin effects on central nervous system which limit the acivicin dose, may be prevented by elevation of normal amino acid concentrations in circulating plasma [162]. Acivicin levels were 13% those of the control animals in brain tissues of treated rats with a mixture of 16 amino acids not containing glutamine, glutamate, aspartate or cysteine. Similar results were obtained with a solution

of large neutral amino acids (leucine, isoleucine, phen- ylalanine and valine).

The combination of acivicin and cis-diaminedichlo-

roplatinum markedly enhanced the inhibition of the activities of thymidylate synthase and thymidine kinase, the enzymes involved in the final steps of the de novo and salvage pathways of the pyrimidine metabolism in A549 lung cancer cells [163].

Other glutamine analog, L-glutamic acid gamma- mono-hydroxamate, has also demonstrated high toxicity against tumor cells in culture and 'in vivo' against leukemia and B16 melanoma, but its action seems to be due to the hydroxylamine released in the tumor cells [164].

The promising results of glutamine analogues on tumor models are disappointing in phase I and phase II trials in humans. In patients with advanced sarcomas and mesotheliomas, none of 36 patients treated with DON infusion of 50 mg/m2/day for 5 consecutive days every 4 weeks developed an objective tumor response [165]. Similar negative results have been obtained with acivicin. In 33 evaluable patients with non-small cell lung cancer treated with acivicin, only two partial re- missions were documented, and neurotoxicity was seen in 48% of the patients [166]. In 33 evaluable patients with advanced colorectal carcinoma treated with acivicin, only one partial response occurred [167]. In 24 evaluable patients with epithelial ovarian cancer treated with acivicin, only one patient achieved a partial response lasting five months [168]. As for azaserine, it may induce pancreatic carcinogenesis in the rat [169- 1701.

The usefulness of glutamine analogues in antitumor therapy is limited, as it occures with chemotherapy in general; other paths must be investigated to fight against cancer. Specific inhibition of tumor cell glutamine uptake could be one of the possible ways. In this context, inhibition of tumor cell glutamine uptake by isolated neutrophils has been demonstrated, and this inhibition resulted in a clear cytotoxic effect [171]. It is likely that the combination therapies and amino acid supply by parenteral nutrition could improve the potential action of glutamine analogues. Glutamine clearance with glutaminase could be another possible way for glutamine-related therapy [172-174]. Recently, Gal- lagher et al. reviewed the use of asparaginase as a drug for the treatment of acute lymphoblastic leukaemia [175]. To have potential theurapetic actions, an enzyme should present the following properties: high affinity for the substrate(s), slow rate of clearance from blood

circulation, lack of cofactor requirements, and low anti- genicity. The enzyme L-glutaminase/L-asparaginase isolated from Acinetobacter, when succinilated to increase 10-fold its plasma half-life, seems to accomplish these conditions, and consequently this enzyme may be of importance in the future. Rosenfeld and Roberts [174] demonstrated that the combined actions of succinylated Acinetobacter glutaminase/asparaginase produced synergistic inhibition of nucleic acid synthesis in P388 tumor cells. Nevertheless, DON could behave as an inhibitor of glutaminase/asparaginase [176]. Thus, in combined enzyme-inhibitor application in antitumor chemotherapy, a detailed study should be carried out on the half life of the active enzyme with different inhibitor dosages.

Concluding remarks

As Argilds and Azcdn-Bieto pointed out [13], although the study of the metabolic environment of the cancer cells is not necessarily the only way to fight against cancer, it seems clear that further study is needed in the next few years. In this context, the study of the role of glutamine in tumor proliferation is specially important.

Glutamine is required for tumor cell survival. This is the basis for the therapeutic use of glutaminase to retire glutamine from the environment of cancer cells. An- other approach to fight against tumor proliferation could be the selective inhibition of glutamine transport by tumor cells. Unfortunately, so far such a selective inhibitor has not been found yet. On the other hand, the chemical signals released by tumor cells that make possible early sharp changes in the host glutamine metabolism at the beginning of neoplastic transformation remain to be identified.

Acknowledgements

Authors thank Mr. M. Flores for the careful correction of the manuscript. Thanks are due to Mr. R. Cameselle for the drawings. This work is supported by Grant PB/88/0445 from the DGICYT.

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relevance of glutamine metabolism to tumor cell growth

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