Cancer Letters, 28 (1985) 35-42 Elsevier Scientific Publishers Ireland Ltd.

35

MALIC ENZYME AND MALATE DEHYDROGENASE ACTIVITIES IN RAT TRACHEAL EPITHELIAL CELLS DURING THE PROGRESSION OF NEOPLASIA

WILLIAM J. WASILENKOa** and ANN C. MARCHOKb***

aUniversity of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and bBiology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (U.S.A.)

(Received 14 February 1985) (Revised version received 9 May 1985) (Accepted 16 May 1985)

SUMMARY

Malic enzyme and malate dehydrogenase (MDH) activities were radio- metrically assayed in digitonin fractionated normal primary cultures (NPC), preneoplastic selected primary cultures (SPC) and tumor-derived primary cultures (TPC) of rat tracheal epithelial cells. Carcinogen-altered SPC and TPC selectively grow in the absence of pyruvate, which is required by NPC for survival. Mitochondrial-containing particulate fractions from TPC and especially SPC had markedly higher levels of NADPdependent malic enzyme than NPC in the presence or absence of pyruvate. This suggests that induction of mitochondrial malic enzyme activity occurs early in the progression of neoplasia. Malic enzyme activities in the soluble fractions from the various populations were not distinctly different. In contrast, particulate-bound MDH activity was higher in NPC and SPC than TPC in most cases, indicating a decrease in this enzyme late in tumorigenesis.

INTRODUCTION

We recently showed that carcinogen-altered rat tracheal epithelial cells from all stages of neoplasia can be selected-out from normal cells and identified in vitro by their ability to survive and grow in culture medium lacking a pyruvate supplement [3,4]. In contrast to altered cells, normal rat tracheal epithelial cells stringently require exogenous pyruvate for survival and growth [3,11,15]. The metabolic basis underlying this growth

*Present address: Department of Microbiology, University of Virginia School of Medi- cine, Charlottesville, VA 22908, U.S.A. **To whom reprint requests should be sent.

Q304-3835/85/$03.30 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

36

advantage by altered tracheal cells is unknown. Compared to normal tracheal cells, many of the altered tracheal cells exhibit lower hexose uptake [ 141 and glycolysis [16], suggesting that these cells may rely upon metabolic pathways other than glucose catabolism for the production of pyruvate as well as other growth-limiting metabolites. Several recent reports suggest that glutamine metabolism, malic enzyme and malate dehydrogenase (MDH) play important roles in cell proliferation and the growth advantage of tumor cells [5,6,8,9,10,12]. Therefore, we examined the activities of these enzymes in primary cell populations representing 3 stages of carcinogenesis, i.e. normal, preneoplastic and tumor cells. These measurements were made using a new and sensitive radiometric assay developed by McKeehan [ 5,7] with which enzyme activities in small primary cell cultures can be deter- mined.

MATERIALS AND METHODS

Culture conditions and establishment of primary cell cultures Cell cultures were routinely maintained as described previously [ 3,4] in

an enriched Waymouth MB 752/l medium (GIBCO, Grand Island, NY) containing 2 mM pyruvate. The primary cultures of carcinogen-altered cells were established as outgrowths from dimethylbenz[a]anthracene (DMBA)-preexposed tracheal implants before the development of tumors [ 41. The altered cells were selected out from the normal cells by culture for 2 weeks in medium lacking pyruvate in which the normal cells die [ 3,4] . These selected cultures of preneoplastic cells are designated SPC. Two separate populations of tumor primary cultures (TPC-1, TPC-2) were established as the SPC, but from pieces of carcinomas which formed in nude mice after innoculation of subcultured cells derived from DMBA- preexposed implants [ 41. Normal primary cell cultures (NPC) were obtained from pieces of non-exposed tracheas.

Cell fractionation Prior to fractionation, the number of cells in each culture was determined

using a nondestructive method recently described by Marchok et al. [ 41. Cells from 3-5 cultures were scraped, combined and fractionated into soluble and particulate fractions using digitonin as recently described by McKeehan et al. [ 51. The fractions were stored at -70°C until assay. Lactate dehydrogenase (LDH) and citrate synthase (CS) were assayed radiometrically [5] to assess cross-contamination between fractions. Soluble fractions contained 95% of the total LDH activity, whereas particulate fractions contained 85% of the total CS activity. For the assay of malic enzyme, fractions were first passed through a G-25 Sephadex column equilibrated with a 20 mM Hepes buffer (pH 7.2) containing 2 mM EDTA, 0.2 mM dithiothreitol, 50 mM KC1 and 0.02% NaN3. Protein in the fractions was determined using Coomassie Blue (BioRad assay kit, Richmond, CA).

37

Enzyme assay Prior to assay, cell cultures were pre-incubated for 48 h in growth medium

with or without pyruvate (2 mM) plus 10% dialyzed fetal bovine serum. Media were changed daily. Malic enzyme and malate dehydrogenase were radiometrically assayed according to procedures recently described in detail by McKeehan and McKeehan [5,7]. NAD’- and NADP’dependent malic enzyme activities were assayed in the malate pyruvate direction in an assay mixture consisting pf 100 mM Tris-HCl (pH 7.6), 5 mM MnCl?, 0.2 mM dithiothreitol, 20 mM malate, 50 mM (0.4 Ci/ml) [l-‘“Cl alanine (52 mCi/ mmol; Amex&am, Arlington Heights, IL), 1 mM NAD’ or NADP’ (Sigma, St. Louis, MO), 2 IU per ml of glutamic-pyruvic transaminase (Sigma G9880) and the cell extract. In the reaction, malic enzyme catalyzes the formation of [ l-"Cl pyruvate from malate. [ 1-“Cl Pyruvate then undergoes an alanine aminotransferase catalyzed isotope exchange with [ l-‘4C] alanine (H202 stable). The [ 1-14C] pyruvate formed from the exchange is decarboxylated by H202. The quantity of pyruvate formed from malate is calculated from the amount of [ 1-‘4C] alanine lost as a consequence of its conversion to [1-‘4C]pyruvate (H202 labile). Malate dehydrogenase was assayed in the oxaloacetate malate direction by following the conversion of [ 4-14C] oxalo- acetic acid (acid-labile) to [ 4-14C] malic acid (acid-stable). [ 4-14 C] Oxalo- acetate was first generated from [ 4-14C] aspartate in a reaction mixture (50 ~1) containing 200 mM Tris-HCI (pH 7.6), 2 mM EDTA, 4 mM 2- oxoglutarate, 4 IU per ml of glutamic-oxaloacetic transaminase (Sigma G2751) and 20 mM (4 Ci/ml) [4-14C]aspartate (5.5 mCi/mmol; Amersham). After 15 min of incubation, the cell extract and 0.2 mM NADH (Sigma) were added and final volume was brought up to 100 ~1. Incubation was extended for another 15 min. All the enzyme assays were carried out in duplicate or triplicate and generally varied less than 10% of the mean.

RESULTS AND DISCUSSION

The NADP’-linked catalysis of pyruvate formation by the particulate and soluble fractions from normal, preneoplastic and tumor cells cultured in the presence and absence of pyruvate is shown in Fig. 1. It can be seen that pyruvate formation from malate (malic enzyme activity) was linear with time in all the fractions. The most prominent difference among the fractions from the various stages of carcinogenesis was the low amount of pyruvate formation catalyzed by the particulate fraction from normal cells cultured in the presence or absence of pyruvate (Fig. 1A). This was most striking when compared to the large amount of pyruvate generated (110-140 nmol in 30 min) by the particulate fraction from the preneoplastic SPC cultured in either medium (Fig. 1B). This suggests that the induction of particulate NADPdependent malic enzyme occurs early in the progression of neoplasia in tracheal epithelium, and that it is not regulated by exogenous pyruvate. Other studies [8,12], have shown low mitochondrial NADP’dependent

38

160 A

1

10 30 10 30 10 30 10 30

MINUTES

Fig. 1. NADP-dependent malic enzyme activity in particulate and soluble fractions from normal, preneoplastic and tumor-derived rat tracheal epithelial cells. Primary cultures of NPC (A,E), SPC (B,F), TPC-1 (G,C) and TPC-2 (H,D) cells were cultured for 48 h in growth medium supplemented with ( t-e) or without (0 - - -0) pyruvate, and then fractionated into particulate (A-D) and soluble fractions (E-H). Malic enzyme was assayed in these fractions by isotope exchange as described in the Materials and Methods. 2.5 pg of protein from each fraction was used in the assays.

malic enzyme activity in normal and regenerating liver, and an increase in the enzyme correlated with the degree of dedifferentiation and malignancy of visible tumors as the other extreme.

Since isozymes of malic enzyme with specific requirements for either NAD’ or NADP’ exist in other types of mammalian cells [ 5,131, the amount of NADP’- and NAD’dependent malic enzyme activities in fractions obtained from the 3 types of tracheal cell cultures were measured (Table 1). The results again demonstrate very low NADP’dependent malic enzyme activity in the particulate fraction of NPC, particularly when compared to SPC. These differences were not compensated by higher or lower soluble NADP’-linked malic enzyme activity, indicating separate regulation of the soluble and particulate-bound enzymes. The particulate NADP’-dependent malic enzyme activities in TPC-1 and TPC-2, cultured in pyruvate-supple- mented medium, were 2X higher than in normal cells, but still 4-fold less than that found in SPC. Also, TPC-1 responded differently than TPC-2 to the removal of pyruvate. These changes late in carcinogenesis probably reflect varying degrees of differentiation and malignancy acquired by different tumor populations. We have reported marked differences in other

39

TABLE 1

COMPARISON OF NADP+- AND NAD+-DEPENDENT MALIC ENZYME ACTIVITIES IN NPC, SPC AND TPC OF TRACHEAL CELLS

Cell Medium culture pyruvate

Malic enzyme (units per lo6 cells (X 10-‘))a --~

NADP’ NADC

Ptb Solb % total Pt in Pt

NPC SPC TPC-1 TPC-2

NPC SPC TPC-1 TPC-2

(+I 7.6 78.0 8.8 24.8 54.2 72.4 42.2 26.6 13.1 40.7 24.3 28.8 13.8 42.5 24.5 42.5

t-1 4.7 76.9 5.7 16.2 32.8 45.0 42.2 42.3 45.4 51.6 46.8 29.2 12.0 60.1 16.6 39.7

aOne unit of malic enzyme activity is that amount of enzyme which catalyzes the produc- tion of 1 pmol pyruvate/min. Specific activities were obtained from the slope of a plot of activity generally using 3 protein concentrations. Reaction time was 30 min.

bPt = particulate fraction; sol = soluble fraction. ‘No soluble activity detected.

metabolic parameters correlated with growth and differentiation of tumor cells from trachea [ 4,141.

NAD’dependent malic enzyme activity was detected exclusively in the particulate fractions of the cells (Table 1). Although some differences in activities among the cell types were noted, no progression-linked changes were evident. The data suggest that an NAD(P)‘dependent isoenzyme is present in normal and carcinogen-altered cells. It cannot be distinguished whether the particulate NADP’-dependent activities observed in this study are due to a single NAD(P)‘dependent isozyme acting alone or in concert with a strictly NADP’dependent enzyme.

Malate dehydrogenase, a purported competitor of malic enzyme for malic acid in cells [ 51, was assayed to determine if its activity was also progression-linked. Contrary to conventional spectrophotometric methods based on oxidation-reduction of NAD’-NADH, the isotope exchange method permits assay of MDH in the same crude cell preparations assayed for malic enzyme. Compared to malic enzyme, the activities of MDH in the particulate and soluble fractions from cells at all stages of neoplasia were markedly higher (Table 2). A similar trend between malic enzyme and MDH levels has been observed in human fibroblast [ 51, even though the reported levels of each enzyme in these cells appear to be much lower (>lO-fold) than the

40

TABLE 2

MALATE DEHYDROGENASE ACTIVITY IN NORMAL, PRENEOPLASTIC AND TUMOR-DERIVED TRACHEAL CELLS

Ceil culturea

Medium pyruvate

MDH (units per 10” Cells (X lo-‘))

Pt Sol

NPC (+I 389 SPC 325 TPC-1 98 TPC-2 92

NPC SPC TPC-1 TPC-2

t-1 316 269 600 184 270 309

69 199

220 179 153 175

aThe same cultures shown in Table 1 were assayed for MDH. A unit of maiate dehydro- genase activity is that amount of enzyme which stabilizes 1 pmol oxaloacetate/min. Reaction time was 15 min.

levels observed here for rat tracheal epithelial cells. Particulate-bound MDH activity was similar in NPC and SPC and showed a reduction of at least 3-fold in both TPC-1 and TPC-2 cultured in pyruvate supplemented medium. Interestingly, particulate MDH from TPC-1, just as the particulate NADP’- dependent malic enzyme from this tumor population, responded to the removal of pyruvate from the medium by increasing activity 3-fold. The particulate-bound MDH from SPC also showed an increase in the absence of pyruvate. The high particulate MDH activity in SPC along with a high particulate NADP’dependent malic enzyme do not support simple competi- tion for malate as a regulator for these enzyme activities. The lower particu- late MDH activities in both tumor populations suggest that a decrease in mitochondrial MDH may be a progression-related change which occurs at a late stage of neoplasia in rat tracheal epithelium. This is in direct contrast to reported increases in the mitochondrial isozyme of MDH in human breast tumors [l] and human lung tumors [ 21. However, it is clear from our findings with TPC-1 and TPC-2 that different tumor cell populations, in particular, can respond quite differently to modulators of enzyme activities. As we intimated earlier, this is probably a reflection of the state of differentiation and malignancy finally assumed by the frankly neoplastic cells.

Glutamine is a major source of energy in tumors and many cell cultures. Several investigators have suggested that NAD(P)‘dependent malic enzyme may aid in the efficient metabolism of glutamine to pyruvate in these cells through a relatively simple pathway called glutaminolysis [6,123. Recently, a more complex pathway has been proposed, that is highly compartmen-

41

talized in the cell, and requires the integrated action of mitochondrial NAD(P)‘-linked malic enzyme and malate dehydrogenase [8,9]. In this pathway, mitochondrial NAD(P)‘-linked malic enzyme still has the impor- tant role of converting malate to pyruvate. However, this malate does not originate from glutamine but from other metabolic pathways. We found that [ 14C] glutamine is metabolized by NPC and SPC at the same rate, while the rate of incorporation of exogenous [‘“Cl pyruvate is at least 3 times higher in cellular fractions of-NPC compared to SPC [16]. The loss of a need for exogenous pyruvate for survival by carcinogen-altered tracheal cells may be related to the increase in activity of mitochondrial malic enzyme and/or malate dehydrogenase and the ability to synthesize intramitochon- drial pyruvate and/or oxaloacetate. Further investigation of factors control- ling these enzyme activities, and the corresponding changes in metabolic pathways and synthesis of metabolic products in different cell compartments should elucidate fundamental changes in regulation of cell growth and differentiation during the progression of neoplasia in tracheal epithelium.

ACKNOWLEDGEMENTS

The advice and sharing of unpublished data by Dr. Wallace McKeehan is gratefully acknowledged. Research supported by NC1 Grant CA-30529 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-AC05-840 R21400 with the Martin Marietta Energy Systems, Inc. W.J.W. was supported by NIHGM Predoctoral Training Grant 7431.

REFERENCES

1 Balinsky, D., Platz, C.E. and Lewis, J.W. (1983) Isozyme patterns of normal, benign, and maiignant human breast tissues. Cancer Res., 43, 5895-5901.

2 Balinsky, D., Greengard, O., Cayanis, E. and Head, J.F. (1984) Enzyme activities and isozyme patterns in human lung tumors. Cancer Res., 44,1058-1062.

3 Marchok, A.C. (1984) In vitro models for the study of carcinogenesis in rat tracheal epithelium. In: In Vitro Models for Cancer Research. Editors: M. Webber and I. Sekely. CRC Press, Boca Raton, in press.

4 Marchok, A.C., Huang, SF. and Martin, D.H. (1984) Selection of carcinogen-altered rat tracheal epithelial cells preexposed to 7 ,l P-dimethylbenz( a)anthracene by their loss of a need for pyruvate to survive in culture. Carcinogenesis, 5, 789-796.

5 McKeehan, W.L. and McKeehan, K.A. (1982) Changes in NAD(P)‘-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation. J. Cell Physiol., 110,142-148.

6 McKeehan, W.L. (1982) Glycolysis, glutaminolysis and cell proliferation. Cell Biol. Int. Rep., 6,635-650.

7 McKeehan, W.L. and McKeehaq, K.A. (1981) Simple radiometric assay for amino acid : P-oxoacid aminotransferases and P-hydroxyacid : 2-oxoacid oxidoreductases. Biosci. Rep,, 1,661-668.

8 Moreadith, R.W. and Lehninger, A.L. (1984) The pathways of glutamate and gluta- mine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)‘- dependent malic enzyme. J. Biol. Chem., 259,6215-6221.

42

9 Moreadith, R.W. and Lehninger, A.L. (1984) Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria. J. Biol. Chem., 259, 6222-6227.

10 NageI, W.O., Dauchy, R.T. and Sauer, L.A. (1980) Mitochondrial malic enzymes: an association between NAD(P)+-dependent malic enzyme and cell renewal in Sprague-Dawley rat tissues. J. Biol. Chem., 255, 3849-3854.

11 Rice, R. and Marchok, A.C. (1980) Nutritional requirements for growth of normal rat tracheal epithelial primary cultures. in Vitro, 16, 215.

12 Sauer, L.A., Dauchy, R.T., Nagle, W.O. and Morris, H.P. (1980) Mitochondrial malic enzymes: mitochondrial NAD(P)‘-dependent malic enzyme activity and malate- dependent pyruvate formation are progression-linked in Morris hepatomas. J. Biol. Chem., 265,3844-3848.

13 Sauer, L.A. and Dachy, R.T. (1978) Identification and properties of the nicotinamide adenine dinucleotide (phosphate)+-dependent malic enzyme in mouse ascite tumor mitoehondria. Cancer Res., 38, 1751-1756.

14 Wasilenko, W.J. and Marchok, A.C. (1984) Hexose uptake in 7,12-dimethylbenz(a)- anthracene-preexposed rat tracheal epithelial cells during the progression of neo- plasia. Cancer Res., 44, 3081-3089.

15 Wasilenko, W.J. and Marchok, A.C. (1984) Pyruvate regulation of growth and differ- entiation in primary cultures of rat tracheal epithelial cells. Exp. Cell Res., 155, 507-517.

16 Wasilenko, W.J. and Marchok, A.C. (1984) Utilization of ‘%metabolic substrates by 7,12dimethylbenz(a)anthracene-altered rat tracheal epithelial cells that grow without pyruvate. Proc. Am. Assoc. Cancer Res., 25, 1.