Biochemical alterations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors from rats subjected to caloric restriction

Biochimica et BiophysicaActa 929 (1987) 239-246 Elsevier

239

BBA 12054

B i o c h e m i c a l a l t e r a t i o n s in 7 , 1 2 - d i m e t h y l b e n z l a ] a n t h r a c e n e - i n d u c e d m a m m a r y t u m o r s f r o m rats s u b j e c t e d to caloric r e s t r i c t i o n B r u c e A. R u g g e r i , D a v i d M. K l u r f e l d a n d D a v i d K_ritchevsky The Wistar Institute of Anatomy and Biology, Philadelphia, PA (U.S.A.) (Received 8 December 1986)

Key words: Calorie restriction; Mammary tumor; Glycolytic enzyme; Malic enzyme; Fructose-l,6 bisphosphatase

Caloric restriction reduces the incidence and progression of a broad spectrum of neoplastic diseases, yet little is known about the biochemical and molecular mechanisms involved. Profiles of enzyme activities of importance in cellular energy utilization were examined in 7,12-dimethylbenz[a ]anthracene-induced (DMBA) mammary adenocarcinomas from rats fed ad libitum or calorically restricted diets. The diets provided equal nutrients except for fewer carbohydrate-derived calories; graded caloric restriction was 10, 20, 30 and 40%. The specific activities of hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme and fructose-l,6-bisphosphatase were all elevated to varying degrees in both large palpable and small, non-palpable tumors from calorically restricted hosts compared to activities in tumors from ad libitum-fed rats. Phosphofructokinase activity was increased in palpable tumors from calorically restricted hosts but markedly reduced in non-palpable tumors. These results suggest adaptive or compensatory alterations in tumor enzyme profiles in response to the altered nutritional state of the host.

Introduction It has been observed that caloric restriction of rodents upon weaning or up to and beyond middle age increases life span [1-3], prolongs and heightens cell-mediated immune responsiveness concomitant with a reduction of autoimmune processes [4-7], and reduces the onset and severity of a spectrum of chronic degenerative disease processes [1,8]. In this regard, among the most dramatic biologic effects of caloric restriction are those influencing the incidence and progression of a variety of neoplastic diseases. Beginning with the

Abbreviations: MCA, 3-methylcholanthrene; DMBA, 7,12-dimethylbenz[a]anthracene; NMU, N-methylnitrosurea, MAM, methylazoxymethanol; DMH, 1,2-dimethylhydrazine. Correspondence: D. Kritchevsky, The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104, U.S.A.

early studies of Moreschi [9] on transplantable murine sarcomas and subsequent studies by Rous [10] on mammary tumorigenesis in mice, it was demonstrated that caloric restriction reduced tumor incidence and growth. Tannenbaum [11-13] pioneered the systematic study of nutritional influences on tumorigenesis, demonstrating the effects of degree of caloric restriction and the composition of the diet on reducing spontaneous and carcinogen-induced (MCA) epitheliomas and sarcomas in various strains of mice. Lavik and Baumann [14] and Boutwell et al. [15,16] extended these observations and provided further evidence for a role of caloric restriction per se in neoplastic progression distinct from the promotional effects of the level and type of fat in the diet. In the early 1970's, Ross and Bras [17], in examining 25 different taxonomic tumor types, demonstrated that caloric restriction even for 1 month post-weaning was effective in reducing the onset of a variety of

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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neoplasms, with tumors of endocrine and epithelial origin being more responsive than neoplasms of lymphorecticular and hematopoietic origin. Since that time, a number of studies have demonstrated the influence of caloric restriction in reducing the incidence and growth of spontaneous [18,19] and DMBA- and NMU-induced [20-24] m a m m a r y tumors in rats and mice, MAM-induced intestinal tumors [25,26] and DMH-induced colonic tumors [27] in rats and azaserine-induced pancreatic carcinomas in rats [28]. Despite these extensive observations, relatively little progress has been made in elucidating the mechanism(s) at the cellular and molecular level to explain the p r o n o u n c e d effects of caloric restriction on neoplastic growth. The role of estrogens and prolactin in mediating the effects of caloric restriction on m a m m a r y tumor development has been addressed by a number of investigators [19-21]. Although suggestive, these findings are neither conclusive nor do they explain the influence of caloric restriction on such a range of neoplasms of varying origin. A more limited and indirect body of experimental evidence has implicated reductions in circulating insulin a n d / o r somatomedin activity as mediating the effects of caloric restriction in reducing chondrosarcoma growth [29] and DMBA-induced m a m m a r y tumorigenesis [30] in rats. The studies reported here are the first to attempt any type of biochemical characterization of neoplasms developing within a calorically re-

stricted host in comparison to weight-matched tumors induced in ad libitum-fed animals. Materials and Methods

Experimental design and dietary regimens. Virgin female Sprague-Dawley rats were received at 21 days of age and housed 1 / c a g e in an air-conditioned room maintained at 21oC with a 12 h light-dark cycle. Animals were maintained on standard commercial laboratory rat ration. At 50 days of age, each rat received by gavage 5 mg of D M B A (Eastman Kodak, Rochester, NY) dissolved in 0.5 ml of corn oil. Rats were maintained on standard diet for 1 additional week before being placed randomly on the specially formulated semipur.ified diets (Dyets Inc., Bethelehem, PA) listed in Table I. 20 rats (n = 20) were fed the control diet ad libitum and the four calorically-restricted groups (10, 20, 30 and 40%; n = 20 r a t s / g r o u p ) were pair-fed to the control animals [22,27]. Absolute intakes of all micro- and macronutrients, except for carbohydrates, were similar for each group creating a controlled calorically restricted state and not a malnourished or starved condition. Groups of rats not administered carcinogen and fed the ad libitum and 40% restricted regimens were also maintained. Food consumption was measured daily, while body weight and palpable tumors were assessed weekly. The study was terminated at 23 weeks from the start of feeding experimental diets, at which time rats were

TABLE ! C O M P O S I T I O N OF C O N T R O L (AD LIBITUM) A N D C A L O R I C A L L Y R E S T R I C T E D DIETS a ( g / 1 0 0 g) Ingredient

Sucrose Casein DL-Methionine Corn oil Cellulose Mineral mix b Vitamin mix ~ Choline H 2 citrate

Control

58.0 21.6 0.3 5.0 10.1 3.8 1.0 0.2

Kritchevsky, D., et al. [22]. b Bernhart-Tomarelli formula [62]. c AIN-76A [63].

Restriction 10%

20%

30%

40%

53.4 24.0 0.3 5.6 11.6 4.2 1.1 0.2

47.3 27.0 0.4 6.3 12.6 4.8 1.3 0.3

40.9 30.1 0.4 7.1 14.4 5.4 1.4 0.3

30.1 36.0 0.5 8.3 16.8 6.3 1.7 0.3

241 killed between 8 : 3 0 - 1 1 : 0 0 a.m. (after overnight fast) by an overdose of sodium pentobarbital. It is impossible to kill all groups in a caloric restriction study following food consumption; hence food was removed from all animals at the same time. Livers and mammary tissues from control animals and non-necrotic mammary tumors from DMBAtreated rats were washed with 0.9% sodium chloride and stored at - 70 ° C. Sample preparation. Cytosolic fractions of tissues were prepared in 0.25 M sucrose, 10 mM mercaptoethanol and 1 mM EDTA [31,32]. The filtered 100 000 × g supernatants were utilized for enzyme assays and protein determination. Protein was quantitated by the method of Bradford [33] using bovine serum albumin as a standard. Tumors were categorized as large, palpable or small, nonpalpable. Tumors weighing no more than 100 mg were classified as small, non-palpable; these tumors were not palpable through the skin even at necropsy. Enzymatic assays of tumors and organs. Enzyme assays were performed under zero-order conditions and specific activities were calculated under first-order conditions [35]. The oxidation or reduction of pyridine nucleotide coenzymes (extinction coefficient= 6.22 cmZ//~mol at 340 nm) was monitored spectrophotometrically in a thermostatically controlled Gilford 2400 spectrophotometer. Hexokinase (EC 27.1.1) and glucokinase (EC 27.1.1) were assayed by a coupled enzyme system [36,37] with a glucose concentration of 0.5 mM (hexokinase assay) and 100 mM (glucokinase assay). Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was determined by a modification of the method of Kaplan and Fried [38]. Phosphofructokinase (EC 2.7.1.11) was quantitated by the method of Ling et al. [39]. Pyruvate kinase (EC 2.7.1.40) activity was monitored in the presence of 1 mM fructose 1,6-diphosphate by the coupled enzyme assay detailed previously [40]. Lactate dehydrogenase (EC 1.1.1.27) was assayed according to Lee et al. [41] and malic enzyme (EC 1.1.1.40) activity was assessed by the method of Ochoa [42]. Fructose-l,6-bisphosphatase (EC 3.1.3.11) was measured by slight modifications of coupled enzyme methods published previously [32,43]. Each sample was analyzed in triplicate along with a blank in which substrate a n d / o r ATP (for kinase

assays) was omitted. Activities are expressed as units of activity/min per mg cytosolic protein. Statistical analyses. Tumor incidence and the percentage of large or small tumors were evaluated by contingency table analysis using the chi-square statistic. Tumors per tumor-bearing rat were compared with the Kurskal-Wallis test. Total tumor yield was assessed using the chi-square test. Comparisons of enzyme activities between tissues from rats fed ad libitum or restricted calories was made with the Student's t-test for paired data. All analyses were performed using NWA Statpak software (Northwest Analytical, Portland, OR). Results

Effects of caloric restriction on host and tumor growth The terminal body weight of rats fed ad libitum was 364_+ 11 g ( m e a n + S.E.). The weight of animals subjected to 10% caloric restriction was 327 ± 10 g; 20% restriction, 277 + 7 g; 30% restriction, 239 ± 6 g; and, 40% restriction, 219 ± 7 g. These weights of the restricted groups represent 10, 24, 34 and 40% reductions, respectively, from the ad libitum group. At necropsy, organ weights (except adrenals) from calorically restricted animals (40%) were proportionally reduced compared to ad libitum-fed controls (data not shown). Adrenal weights were increased slightly relative to carcass weight, although this increase was not statistically significant. Table II summarizes data on mammary tumor development in the various groups given DMBA. Caloric restriction not only reduced overall tumor incidence, but tumors that developed in restricted animals were of a smaller size compared to those in ad libitum-fed rats. In the calorically restricted animals, a high proportion of tumors are very small (no larger than 100 rag); it was decided to investigate whether these small non-palpable tumors differed metabolically from large, palpable mammary tumors. Enzyme data in liver and mammary tissues Tables III and IV give the specific activities of several representative enzymes of cellular energy metabolism and reductive biosynthesis examined in fiver and mammary tissues of ad libitum-fed

242 T A B L E II MAMMARY

TUMORS IN AD LIBITUM-FED AND CALORICALLY RESTRICTED FEMALE SPRAGUE-DAWLEY

Dietary regimen

Tumor incidence

Tumors/ tumor-bearing rat

Total tumor yield

Percent of LP a tumors

Percent of S N P ~' tumors

Ad libitum 10% restriction 20% restriction 30% restriction 40% restriction Statistical significance

60% 60% 40% 35% 5%

4.7 + 1.3 b 3.0 -+ 0.8 2.8 _+0.7 1.3 -+ 0.3 1.0 c

59 36 22 10 1

83 79 77 40

17 21 23 60

P < 0.005

n.s.

P < 0.001

RATS

P = 0.0006

~' LP. large, p a l p a b l e ; SNP, small, n o n p a l p a b l e . b Mean-+ S.E. ~" This single t u m o r was necrotic and not used for s u b s e q u e n t e n z y m a t i c studies.

and 40% calorically restricted rats not exposed to DMBA. The findings observed for hepatic hexokinase and glucokinase activities are in agreement with earlier studies by Vinuela et al. [44] and DiPietro and Weinhouse [45]. These results demonstrate that hepatic hexokinase is relatively unresponsive to nutritional modulation in its induction and activity, while glucokinase increases in response to the well-fed state, but shows little or no activity during caloric restriction. These findings may be related to the differences in K m value for glucose exhibited by hexokinase (low K m) and glucokinase (high Kin). M a m m a r y tissue T A B L E III SPECIFIC ACTIVITY a OF VARIOUS ENZYMES IN LIVER FROM FEMALE SPRAGUE-DAWLEY RATS FED A D L I B I T U M O R 40% C A L O R I C A L L Y R E S T R I C T E D DIETS Hepatic enzyme

Ad libitum

40% restricted

Hexokinase c Glucokinase c M a l i c enzyme d Glucose-6-phosphate dehydrogenase d Fructose 1,6-bisphosphatase ~

3.0 -+ 0.2 2.7 + 0.6 17.0 + 1.0

4.0 + 0.1 n.d. b 10.0 _+ 1.0

74.0 -+ 5.0

41.0 _+4.0

63.0 -+ 7.0

32.0 _+4.0

Specific activity ~ u n i t s . m i n b n.d., not detectable. Significant at P < 0.001. d Significant at P < 0.005. " Significant at P < 0.02.

1. r a g 1 cytosolic protein.

hexokinase was increased in calorically restricted animals, although this increase was not significant. As shown in Tables III and IV, respectively, a 40% and 80% reduction in hepatic and mammary ME, respectively, and a 45% and 70% decrease in hepatic and mammary glucose-6-phosphate dehydrogenase, respectively, were observed in 40% calorically restricted rats. The 50% reduction of fructose-l,6-bisphosphatase activity in rats subjected to chronic caloric restriction suggests that the metabolic effects of caloric restriction differ from those observed during acute fasting. This change in chronic calorically restricted animals suggests an overall reduction in glycolytic activity to a new steady state in these animals. Carcass analysis of total body protein (microkjeldahl) [46] and total body lipid (Soxhlet extraction) in the ad libitum-fed and 40%

T A B L E IV SPECIFIC ACTIVITY OF VARIOUS ENZYMES IN NORMAL MAMMARY TISSUE FROM FEMALE SPRAGUED A W L E Y R A T S F E D A D L I B I T U M O R 40% C A L O R I CALLY RESTRICTED DIETS M a m m a r y tissue e n z y m e

Ad libitum

40% restricted

Hexokinase a Malic enzyme b Glucose-6-phosphate dehydrogenase a

2.0_+ 0.7 19.0--+ 5.0

6.0_+ 1.0 4.0_+0.1

30.0 _+ 10.0

9.0 +_ 3.0

a N o t significant. b Significant at P < 0.01.

243

restricted animals [47] revealed a 62% decline in total body lipid (as percent of carcass weight) in 40% restricted rats, while total protein was actually 3% greater (as percent of carcass weight) in these animals. This would suggest that hepatic gluconeogenesis from body protein was not occurring at an accelerated rate as in early starvation, but that body fat deposition was reduced, and possibly utilized as available energy substrate, during adaptation to reduced caloric intake [48].

Mammary tumor enzymology The enzymatic profiles of the large, palpable and small, non-palpable DMBA-induced mam-

mary tumors obtained from rats on the ad libitum-fed and restricted regimens are shown in Table V. As the dietary regimens employed were subjecting the host animals to energy restriction, the activity of a spectrum of enzymes of importance in cellular energy metabolism was evaluated. The results in Table V illustrate the varying degrees of difference in tumor enzyme profiles both between dietary treatment groups and large, palpable and small, non-palpable tumors within each group. As discussed below, the tumor tissues demonstrated 'progression-linked' [52] increases in the activity of glycolytic enzymes. It is noteworthy that, with the important exception of reduced

TABLE V E N Z Y M E PROFILES OF D M B A - I N D U C E D M A M M A R Y T U M O R S F R O M A D L I B I T U M - F E D A N D C A L O R I C A L L Y R E S T R I C T E D F E M A L E S P R A G U E - D A W L E Y RATS A L and 10%, 20% and 30% refer to ad libitum-fed and 10%, 20%, 30% caloric restriction, respectively, according to the regimens described in Table I. Enzyme activities are expressed as units, min -1. m g - 1 cytosolic protein and represent the mean +_S.E. of five samples or more at three replicates/sample. LP, large, palpable tumor; SNP, small, non-palpable tumor (no more than 100 mg); n.d., not detectable. Cytosolic

Dietary regimens

enzyme

AL

Hexokinase LP SNP

8.0 +_ 1.0 n.d.

25.0+_ 43.0 +_

4.0 2.0

16.0+_ 28.0+_

3.0 2.0

25.0+_ 40.0+_

0.5 1.5

Phosphofructokinase LP 27.0 +_ 5.0 SNP n.d.

45.0_+ 5.0+_

8.0 0.1

46.0+12.0+-

8.0 0.8

77.0+20.0+_

5.0 3.0

Pyruvate kinase LP SNP

234 n.d.

Lactate dehydrogenase LP 401 SNP n.d.

10%

20%

30%

+_36

361 360

+_ 28 +_ 15

396 363

+_ 34 +_ 6

411 400

+_ 68 _+ 43

+32

1025 1300

+_ 97 +_113

1040 1090

+_120 +_190

1570 1700

+_100 +250

Glucose-6-phosphate dehydrogenase LP 26.0 _+ 5.0 SNP n.d.

81.0_+ 129.0_+

7.0 2.0

60.0_+ 96.0_+

6.0 3.0

19.0_+ 24.0+

2.0 1.0

16.0+_ 14.0+_

2.0 0.5

Fructose-l,6-bisphosphatase LP 0.93_+ 0.17 SNP n.d.

2.4_+ 2.5-+

0.47 0.49

2.2-+ 1.4-+

0.10 0.17

Cytosolic protein ( m g / g w.wt.) LP 18.5 SNP n.d.

8.2 3.8

Malic enzyme LP SNP

5.0 _+ 0.6 n.d.

9.1 8.3

100.0_+ 12 125.0_+ 7.0

21.0_+ 19.0+_ 2.6+_ 2.0-+ 7.0 9.5

2.0 3.0 0.20 0.15

244

phosphofructokinase activity observed in small, non-palpable tumors, the specific activities of other enzymes in tumors from calorically restricted rats demonstrated increases to varying degrees compared with mammary tumors from ad libitum-fed animals. These differences were most notable for hexokinase, lactate dehydrogenase and glucose-6phosphate dehydrogenase. The elevation of phosphofructokinase activity in large, palpable tumors from calorically restricted rats as well as the marked reduction in activity observed in small, non-palpable tumors compared to large, palpable tumors within each level of caloric restriction suggests an alteration in this key rate-controlling step in glycolysis. The significance of this observation and its possible implications in altering the influx of substrates into the glycolytic pathway in these tissues awaits more detailed investigation. Discussion

The findings reported here describe alterations of enzyme activities in carcinogen-induced mammary tumors in rats subjected to caloric restriction. Despite the widespread use of the DMBA-induced mammary carcinoma model (see Ref. 49), there have been few reports on the biochemical characterization of these tumors with regard to enzymes of energy metabolism [34,50,51]. Consequently, there are few reports available for direct data comparison. There are a number of anomalies in the metabolism of a proliferating tumor that aid its rapid growth and survival. Among the most notable are the alterations in tumor carbohydrate and energy metabolism, a subject reviewed extensively by Weinhouse [55] and Weber [52,54,58]. An increase in glucose uptake is a frequent feature associated with cell transformation from a normal to neoplastic state [59], although this is neither a universal nor necessary feature of neoplastic transformation [60]. The observed alterations in carbohydrate metabolism in neoplastic cells are significant. In his extensive studies of chemically induced and transplantable hepatoams and renal carcinomas, Weber [52,54,58] demonstrated that the activities of key glycolytic enzymes are markedly increased and those of gluconeogenesis diminished in these neoplasms. These altered enzymic activities are classified as progres-

sion linked, as they bear no relationship to the degree of malignancy or differentiation, but serve to discriminate normal from neoplastic tissues [52]. These aberations result in enhanced glycolytic utilization of substrates and a reduction in gluconeogenesis to support the proliferative capacity of the transformed cell. Moreover, it has been observed [55,58] that there is an overall replacement of high K m, hormonally and nutritionally responsive enzymes of carbohydrate metabolism with low K m fetal isozymes, normally repressed in the mature tissue. The result is an escape from normal host cellular regulatory mechanisms facilitating proliferation of neoplastic cells. A high glycolytic rate is normally reduced under aerobic conditions, a phenomenon known as the Pasteur effect first described by Warburg, who observed that an increase in aerobic glycolysis (Warburg effect) was a common feature of tumor energy metabolism [58]. It is now known [52,54] that this phenomenon is not a universal feature of neoplasia; rather, it is often observed in moderately and rapidly growing tumors, but not in slower-growing, well-differentiated ones. Weinhouse has postulated that replacement of normal liver type to fetal isozyme pyruvate kinase in rapidly growing, highly glycolyzing hepatomas may help explain the Warburg effect in these tumors, i.e., enabling the fetal isozyme pyruvate kinase to compete better with the mitochondrial respiratory chain for ADP under aerobic conditions [55]. Phosphofructokinase is a key rate-determining enzyme in glycolysis and plays a critical role in its regulation in both normal and neoplastic tissues [53]. In addition, phosphofructokinase mediates the Pasteur effect [52,54,55] and thus, plays a role in coordinating the two modes of cellular energy production, i.e., glycolysis and respiration [56]. Vora et al. [56] demonstrated both quantitative increases and isozymic alterations of phosphofructokinase during neoplastic transformation in rapidly growing lymphomas and leukemias. These changes were correlated with rates of DNA replication and overall metabolism of these neoplastic cells both in vivo and in vitro [56]. In the present study, the enhanced glycolytic enzyme activities observed in mammary tumors from calorically restricted rats, and the alterations in phosphofruktocinase activity observed in small,

245 non-palpable tumors, lead to speculation that compensatory changes in glycolytic activities are occurring in these tumors compared to those in ad libitum-fed hosts. These observed increases, particularly in smaller, slower-growing tumors, may represent a compensatory mechanism for more efficient utilization of available substrates under conditions of reduced substrate availability, i.e., caloric restriction of the tumor-beating host. Such adaptive alterations may represent a biochemical effort to uphold the 'biochemical commitment to replication' described by Weber [52,54,58] for successful tumor proliferation and survival. Such adaptive alterations in enzymic activity are not without precedent in normal tissues as well. In studies of controlled caloric restriction and its effects on the aging process, Weindruch et al. [2,61] observed that liver mitochondria from restricted mice demonstrated increased state-3 rates of oxygen utilization, but normal state-4 rates, compared to fiver mitochondria from ad libitumfed controls. These alterations resulted in an elevated respiratory control index in restricted animals, suggesting more effective coupling of oxidative phosphorylation to electron transport [2]. Similarly, it has also been demonstrated that caloric restriction is capable of maintaining transcriptional and translational enzyme activities that normally exhibit a progressive decline with aging in vivo [1,6]. Hilf et al. [34,50] observed that neither morphological nor biochemical parameters, i.e., glycolytic, glycogenic, glycogenolytic and lipogenic enzymes demonstrated good correlation with tumor size and growth rate in DMBA- and MCA-induced m a m m a r y tumors. The notable exception, hexokinase, doubled in activity in more rapidly growing tumors [34]. The data of Hill et al. [34] are in contrast with those observed here, as slower growing tumors in calorically restricted animals exhibited increased hexokinase activity compared to their larger, more rapidly growing counterparts (Table V). The pyruvate kinase activity exhibited no demonstrable differences between tumors of different size and growth rate [34] in agreement with the data presented here. A study by Cohen and Hilf [51] reported that insulin-dependent DMBA-induced mammary tumors which regressed in streptozotocin-induced diabetic rats

showed a reduction in glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase and phosphofructokinase activities. While these studies found alterations in tumor enzyme profiles in response to, and as a consequence of, alterations in host metabolic state, strict comparisons with the present findings cannot be made, as the previous study [54] employed tumorbearing diabetic animals maintained on a commercial grain-based diet, whereas semipurified diets were used in the present study. Our data and those of Hill et al. [51] refer to total pyruvate kinase activity in tumor extracts. Susor and Rutter [57] showed that isozymes of pyruvate kinase (i.e., fetal, liver and muscle types) differ not only in their kinetic, immunologic and electrophoretic properties, but also in their responsiveness to positive allosteric effectors (i.e., fructose 1,6-diphosphate, phosphoenol pyruvate), inhibition by ATP and nutritional and endocrine modulation. Elucidation of the isozymic forms of pyruvate kinase present in these carcinogen-induced mammary tumors may reveal differences in the activity of specific isozymic forms as a function of caloric restriction of the tumor-bearing host. The mechanisms by which caloric restriction exerts its effects on neoplastic progression are not understood. Calorically restricted diets require the sacrifice of some macronutrient and in these studies the diets have been devised to reduce the carbohydrate (sucrose) content. We recognize, then, that the results could be viewed as due to carbohydrate restriction rather than caloric restriction. Studies designed to clarify this question are in progress. Further experiments relating to cell bioenergetics, substrate uptake capabilities and hormonal- and growth-factor influences in tumor development may help clarify both the observations reported here and the mechanisms by which caloric restriction reduces tumor incidence and proliferation in experimental animals.

Acknowledgments This work was supported by a training grant (CA-09485) and a Research Career Award (HL00734) from the NIH and a grant from the A m e r i c a n Institute for Cancer Research (83B13C84B) and funds from the Commonwealth of Pennsylvania.

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