Enzymes in Tissues Responsive to Corticosteroids*

FRED ROSEN

(Departmeni of Experimental Therapeuiies, Roawell Park Memorial institute. New York State Departmentof Health, Buffalo, New York)

SUMMARY The biochemical reactions which mediate the physiological effects exerted by the adrenal cortical hormones remain to be determined. It has been euggested that the @ hormonal control of cellular functions is accomplished by altering the activity of specific enzymes. This report concerns the induction of several enzymes by cortisol in tissues (liver, thymus, transplantable tumors) which are responsive to the glucocorti coids. Also, some factors involved in studies on the actions of hormones are considered. The problem of resistance to comtisol and the technics being used to evaluate the physiological significance of enzyme induction by cortisol in target tissues is dis cussed. The various dietary and physiological conditions which influence the level of the inducible enzymes are considered in regard to an explanation of the manner in which glucocorticoids alter the activity of the adaptive enzymes.

Several recent articles on the adrenal corticoids action of the adrenal corticoids. This is a relative have emphasized the disparity between the de ly new area of investigation that has revealed tailed knowledge available on the chemistry and interesting facts concerning glucocorticoid action the biosynthesis or degradation of these hormones at the biochemical level. and the relative lack of information about bio The difficulties in determining the biochemical chemical events which underlie their modes of basis for certain physiological effects exerted by action (15, 925,46). Although the physiological ef the adrenal cortical hormones are apparent from fects elicited by large doses of the glucocorticoids the many different methods which have been used are well known, it is not yet clear whether such to study this subject in recent years (7, 41). Most effects are related to the major role these hormones of the studies are predicated on the basis that the fulfill under normal physiological conditions. hormonal control of cellular functions is mediated These gaps in our knowledge are not limited to the by altering the activity of specific enzymes (920, glucocorticoids. The same difficulty has been en 31). Attractive as this concept may be, it has not countered in elucidating the action at the molecu yet provided us with a clear-cut relationship be lam level of many other hormones (60). tween a biochemical event and a physiological ef Clinically the glucocorticoids have been shown feet produced by the glucocorticoids. There has to be of value in the treatment of leukemias and been renewed interest in the possibility that lymphomas (492). In this regard, the comticosteroids hormones, in general, may act to facilitate the deserve study with respect to the changes they entry of metabolites into cells, mainly because of produce in the metabolism of responsive tumors. the recent work on insulin (59) and antidiuretic In keeping with the objectives of this symposium, hormone(1,9). I do not intend to review in this paper the various Although much of the content of this paper is an mechanisms which have been proposed to explain account of studies on the action of corticosteroids the action of the glucocorticoids. Emphasis will be on intracellular enzymes, there is at present no placed almost entirely on the effects of the definitive evidence that this type of effect will glucocorticoids on the induction of certain en turn out to be the basis of their cellular actions. zymes in those tissues which are targets of the More important at this time is that such studies S This investigation was supported in part by research grants (AM-04389 and CA-05671) from the U.S. Public Health reveal the need for new information and indicate Service. areas of investigation that deserve attention. 1447

SoME FA@rORS To BE CONSIDERED IN STUDIES glucocorticoids has not been cleanly demonstrated ON THE Ac'nIoNS OF Hoit@&o@zs in liver slices (19) but does occur in liver perfused Target tiastses.â€”Mostof the experiments under with cortisol (3, 19). taken to gain information concerning metabolic Amount of hormone and it. specr@ficttyof action.â€” changes which underlie the physiological effects Bush (7) has recently commented in detail on one of the glucocorticoids have been carried out in of the difficulties encountered in studies in vitro liver. There are only a few reports on changes in namely, the choice of a reasonable concentration adaptive enzymes produced by adrenal corticoids of adrenal hormone. Frequently, in such studies, in lymphoid tissues and certain neoplasms that are amounts of the steroid far in excess of a reasonable so remarkably responsive to these steroids. How physiological concentration are required to pro ever, there are many studies on the metabolism of duce a significant effect. For the same reason, the glucocorticoids by target cells (11). Tissues of this high dosage used in many in vivo studies has been type which show such a rapid and demonstrable subject to criticism. Probably undue criticism has response to corticosteroid therapy are capable of been given to the use of large amounts of a hom yielding important biochemical information re mone in these different systems. Many examples lated to the effects exerted by these hormones. A can be cited in which the use of â€œunphysiologicalâ€• major objective of our work is to determine the amounts of a compound has contributed sig basis for the selective action of glucocorticoids on nificantly to an understanding of its mechanism of lymphoid neoplasms. The manner whereby ad action and pathways of metabolism. renal corticoids impair the growth of lymphoid Other criteria must also be kept in mind when tumors may bear certain similarities to the assessing hormone experiments. The intenpreta processes underlying thymic involution or the tion of the data is difficult when the effect pro catabolism of protein. duced is not specific for a given class of hormones In vitro vs. in vivo 8tUdies.â€”The choice be or when an effect is obtained with an analog that tween in vitro and in vivo experiments is often is inactive in the animal. Numerous studies can determined by the problem under investigation be cited in which a variety of hormones with dif and by the technics familiar to the investigatOr. ferent structures and physiological properties act Engel (15), Villee (63), and Bush (7) have recently. similarly in their effect on a biochemical reaction emphasized the importance of the in vitro expeni (5, 14, 923, 68). Also, to be of significance in an ment to obtain unequivocal evidence for a hor in vitro system, the effects observed should bear monal effect. This approach is often justified on some discernible relationship to the effects of the the basis that it permits adequate control of many hormone in vivo, with allowances being made for of the variables that make it difficult to interpret differences in metabolism in the two systems. studies in the whole animal. However, it is not yet ENZYMES RESPONSIVE TO GLUCOCORTICOIDS clear whether the physiological or pharmacological effects of a hormone are entirely mediated by a di It is probable that alterations in the activity of certain enzymes underlie the pronounced effects rect effect of the hormone on responsive tissues. In view of our limited knowledge about hor of glucocorticoids on carbohydrate and protein mone action, it would seem that the experiment in metabolism. In this regard, it is of interest that which the test substance is administered to the most enzymes which show an increase in activity animal and the tissues subsequently examined for after glucocorticoid treatment are involved in the certain biochemical changes be given preference metabolism of glucose or amino acids (592). at this time oven the experiments involving addi Enzyme studies in different tissues of adrenal tion of the hormone to an in vitro system. The in ectomized animals have been carried out in an effort to reveal metabolic functions which are in vivo experiment is likely to provide us initially fluenced, at least in part, by adrenal hormones with the important leads concerning metabolic (30). A drop in enzyme activity following adrenal changes related to glucocorticoid action. There ap ectomy has in many instances provided cimcum pear to be few examples of any substantial effect of stantial evidence that the enzyme is under the glucocorticoids in low concentration on slices or control of the adrenal hormones. Whereas many homogenates of liver. The findings of Chiu and of the adaptive enzymes involved in amino acid Needham (8) that adrenal cortical extract in metabolism show a decrease in level after adrenal creased liver glycogen of surviving liver slices in ectomy, a number of the carbohydnate-metaboliz vitro have only recently been confirmed (924). The ing enzymes that respond to glucocorticoids do not induction of certain enzymes that can be readily undergo any significant change in activity (65). shown to occur in the liver of rats treated with The factor(s) which maintain normal hepatic

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. ROSENâ€”Tissue Enzymes Responsive to Corticosteroids 1449 levels of certain glucocorticoid-inducible enzymes those previously noted for tryptophan pyrrolase, in the liver of adrenalectomized animals are not there were also certain notable differences. Ad known. renalectomy alone did not lower the level of this Carbohydrate-metabolizing enzymes.â€”The ad enzyme in liven, and when tyrosine was ad ministration of cortisol to rats for 5â€”7days in ministered to adrenalectomized rats, the activity creased the hepatic activity of glucose-6-phos of the enzyme was not increased. Cortisol proved phatase to values 50â€”100per cent above normal to be an effective inducer of tyrosine transaminase (64). The activity of this enzyme is increased in both intact and adrenalectomized rats. More under conditions which stimulate gluconeogenesis, recent studies by Kenney and Flora (927) have suggesting that it may have a role in the produc indicated that tyrosine is not specific as an in tion of glucose in liver. Evidence for the hormonal ducer of this tnansaminase. The administration of and physiological regulation of the level of glucose tryptophan, D,L,-5-OH-tryptOphan, on serotonin 6-phosphatase in liver has been thoroughly dis has been found to produce significant increases in cussed in a review by Ashmore and Webem (92). the level of tyrosine transaminase in the liver of in Fmuctose-1,6-diphosphatase also responds to corti tact and admenalectomized rats (50). Thus, these sone in intact (87) and adrenalectomized mats (892, indoles would appear to be acting in some specific 65), whereas other enzymes involved in glycolysis manner, and not via a stress-mediated adrenal (phosphohexoseisomerase,phosphoglucomatase,response, to induce the activity of this enzyme. lactic dehydrogenase) were found to be stimulated It has long been considered an attractive possi by comtisol in the liver of adrenalectomized mats bility that cortisone could increase gluconeogenesis (65). and impose a negative nitrogen balance by enhanc Amino acid-metabolizing enzymes.â€”Various en ing transamination reactions (36). In an earlier zymes that function in the metabolism of amino study, Beaton and co-workers (4) found that acids have been shown to be responsive to the daily treatment of adult rats with cortisone did not glucocorticoids. These include tryptophan pynro affect aspartic transaminase in liver but doubled lase, tymosine-, alanine-, and tryptophan-a-keto the activity of alanine transaminase. In 1957 glut.arate transaminase, and the urea cycle Gavosto et al (17) reported that almost toxic doses enzymes (592). Significant increases in the activity of cortisol (1920mg/kg) administered to rats daily of each of these enzymes have been demonstrated for S days resulted in a 67 per cent increase in in the liver of rats treated with cortisone or aspartic transaminase activity in liver and an 81 contisol. Other transaminases such as aspartic-a pen cent rise in the level of alanine transaminase. ketoglutarate and histidine- and phenylalanine Studies in our laboratory revealed as much as a pyruvate do not respond to cortisol. No answer is 500 pen cent increase in alanine transaminase yet available as to why some transaminases re activity in livers of rats treated with cortisol for 7 spond to cortisol and others do not. However, days, whereas, under the same conditions, the there is no reason to believe that the list of values for aspartic transaminase were not sig enzymes which act within this area of metabolism nificantly different from those in the untreated and which respond to contisol is complete. control animals (53). Only those steroids which Tryptophan pynrolase has been studied exten are active as glycogenic and anti-inflammatory sively by Knox and co-workers (80) and by others agents induce an increase in the activity of (18, 61). This enzyme can be induced in the liver alanine transaminase in liver (54). A direct rela of the intact mat by the injection of many different tionship between the alanine transaminase me types of compounds (928). Indoles, induding sponse and the dose of cortisol administered was tryptophan, and glucocorticoids are the most observed (55). The initial response to treatment potent inducing agents and are the only com with glucoconticoids involves a rapid increase in pounds effective in stimulating the activity of this the availability of glucose which can result in enzyme in adrenalectomized rats (50). The re measurable glycogen deposition within several sponse of tryptophan pynrolase to cortisol in rat hours. The slow response, over a period of several liver was dose-dependent, and it was suggested days, of alanine tmansaminase in the liver of rats that this method could serve as a bioassay for treated with cortisol correlates with the response glucocorticoids (692). to prolonged stress rather than with the initiation A marked increase in tyrosine transaminase of of gluconeogenesis. It would seem, however, that rat liver after administration of tyrosine or this transaminase is capable of regulating the cortisol was first observed by Lin and Knox in capacity of the liver for gluconeogenesis (89). 1957 (34). Although several aspects of the in Treatment with deoxycorticosterone lowered duced response of this enzyme were similar to the level of alanine transaminase in rat liver to 50

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. 1450 Cancer Research Vol. 23, September 1963 per cent of the normal values. Since deoxycorti Table 1. Recently, detailed information on this costerone was ineffective in this regard in hy enzyme has been reported (40), and the effects of pophysectomized on adrenalectomized mats, its various physiological conditions on the activity of inhibitory effect on this enzyme in intact animals many enzymes have been reviewed (592). has been attributed to interference with the release The conditions listed in Table 1 (fasting, high of corticotrophin from the pituitary (921). protein intake, and alloxan diabetes) known to me In other studies it was found that treatment of sult in an enhanced rate of gluconeogenesis cause rats with testosterone, progesterone, growth hor marked increases in the level of hepatic alanine mone, or insulin did not stimulate an increase in transaminase and have also been found to cause hepatic levels of alanine transaminase, whereas increases of about the same magnitude in the estrogen and thyroxine caused a slight rise in the activity of tmyosine tmansaminase' and tryptophan activity of this transaminase (54) pyrmolase (929). However, in many of these condi tions, marked differences in the response of several TABLE 1 of the cortisol-responsive enzymes have been oh DIFFERENT PirYsIoLoGIc@L CONDITIONS AFFECTING served. For example, although alanine transami ALANINE TRANSAMINASE ACTIVITY nase and tryptophan pymrolase are significantly lowered in activity following adrenalectomy, the

Experimental level of tyrosine transaminase in liver is unchanged .. enceCortisol-treated5-ConditionsResponse5Refer after removal of the adrenals. Also, although alanine transaminase and to a lesser extent tryp week(42)Aging to 7-fold increase in 1 increase in 1 year (so) tophan pynmolase show a rise in specific activity in Adrenalectomy 60% decrease in mature ani (50) liver with age, tyrosine (58) and aspartate trans mals only aminase (9292)levels are not affected by aging. Hypophysectomy 2- to S-fold increase (44) Fasting 5-fold increase after 4 days (44) Hypophysectomy, which raises the activity of 50%(44)Alloxan-diabetesectomized) Protein diet Increase after one week, 4-fold alanine transaminase, is without significant effect (intact), 2.5-fold (adrenalec tomized) on the hepatic levels of tryptophan pymrolase and â€˜15%Proteindiet6-foldIncrease after one week, 7.5- tyrosine transaminase. fold (intact), 4-fold (adrenal In confirmation of the earlier observation by S. to 5-fold increase within 1 Curry and Beaton (10), the level of alanine trans week(44)X-ray aminase in the liver of pregnant mats is decreased, treated increase within 2 days and it cannot be induced by doses of contisol as Pregnancy50%(53)Pregnancy+cor 50% decrease between days 15 to 21(51) large as 100 mg/kg injected during the last several days of pregnancy. In contrast, pregnancy did not tisolt No significant increase 53) depress the activities of tymosine transaminase or Partial(53)tomy hepatec 70% decrease within 48 hours(52, tryptophan pymrolase in mat liver or impair the in Fetal animalsValues 50% lower than in new duction of these enzymes by cortisol.2 Although born(50) several explanations have been proposed to ac count for the failure of hepatic alanine transami S In reference to the level in liver of unoperated control rats about 6 weeks of age fed a stock diet. nase in pregnant rats to respond to cortisol (10, t 20â€”100mgof cortisol/kgdailyadministeredforseveral 592), none of these can satisfactorily explain why days after the 10th day of pregnancy. two other inducible enzymes are not similarly affected. EFFECTS OF CERTAIN PHYSIOLOGICAL It is of interest that the activity of alanine CoicmTIoNS ON INDUCIBLE transaminase can be increased in intact or adrenal ENZYMES ectomized mats by feeding diets containing a high Glucocorticoids are thought to play a dominant content of protein, whereas the substrates of this role in the regulation of enzyme activity, but there enzyme when injected or fed in the diet do not is much evidence that dietary modifications and stimulate an increase in its activity.' When cortisol certain altered physiological states often can exert was administered to adrenalectomized rats main parallel and equally pronounced effects on enzyme tamed on different levels of dietary protein, a sig systems. These findings provide insight concern I Rosen F.; Harding, H. R.; Milholland, R. J.; and Nichol, ing possible mechanisms of corticostemoid action C. A. Glucocorticosteroids and Transaminase Activity. VI. and suggest new experimental approaches to this Comparison of the Adaptive Increases of Alanine- and Tyro sine-a-Ketoglutarate Transaminases. problem. Conditions which alter the level of al 2 Harding, H.; Rosen, F.; and Nichol, C. A. Changes in anine transaminase in liver, or change the in Alanine Transaminase Activity and Response to Corticos ducibility of this enzyme by cortisol, are given in teroids, Pregnancy and Partial Hepatectomy.

nificant but less than maximum increment in Response of Walker carcinoma 9@56to corti alanine tmansaminase activity was observed with costeroids.â€”The growth of the Walker tumor was the 0 and 925per cent protein diets, whereas the 50 suppressed, and the activity of alanine transami and 75 per cent protein diets permitted a maximal nase was increased significantly in animals receiv response to contisol. Thus, the maximal alanine ing cortisol (10â€”SOmg/kg daily for 14 days). The transaminase response in the liver of adrenal effect of cortisol on this tumor was not related to ectomized rats appears to be attributable to an reduced dietary intake and loss of body weight. additive effect of both dietary protein and ad When tumor-bearing rats were treated with 30 ministered cortisol. mg deoxycorticosterone/kg for 92weeks, the trans aminase activity in the tumor was reduced by 50 ENZYME INDUCTION IN TARGET TISSUES per cent and, concomitantly, the rate of growth of AND IN EXPERIMENTAL TUMORS the tumor was increased consistently (Table 92). It was of considerable interest to find that the Thus, an inverse relationship between the activity activity of alanine transaminase could be marked of alanine transaminase and the growth of the

TISSUECORTISOLRELATIVETRANSAMINASEACTIVITYLIVER*:T@

@L 4V @I + + ASPARTATETRANSAM@4ASE TRANSAMINASETHYMUS* â€”ALANINE

WALKER** :j: CARQNOSARCOMA -@-.. 256:@: for Tdoys I*3.OU U N N P N U N 14

CHART 1.â€”Specificity of transaminase response in tissues sensitive to corticosteroids

ly increased in tissues other than liver which are TABLE 2 responsive to treatment with the glucocorticoids EFFECT OF CORTICOSTEROIDS ON THE GROWTH AND (54). Comparative data for induced enzyme activ ALANINE TRANSAMENASE AcTIvITY OF ity in liver, thymus gland, and the cortisol-sensi WALKER CARCINOMA 256 tive tumor Walker carcinoma 9256 are shown in Chart 1. A thirteen-fold increase in alanine trans TRANLLMI TREATMRNT5TUMOR OROWTRtAwmiz aminase activity was observed in thymus tissue ACTIVITYCompound(mg/ RARE following daily injections of cortisol for 1 week. This treatment resulted in a 95 per cent reduction sub cent strate utilized! in the size of the thymus gland. Similarly, treat kg! controlNone control)(mMolesgm protein/hr day)(Per ment with cortisol inhibited the growth of the at 58' C.)Treated! Walker tumor by about 90 per cent and increased the activity of alanine transaminase in this tissue .O6@ Cortisol 1.4 Â±1.0 fourteen-fold. In the same tissues, the activity of 30 8 4.7 Â±1.6 14.8 aspartate transaminase was only slightly in Deoxycorticos terone ace creased, indicating that the rise in alanine trans tate10 3036 148 (P>O05)0.32Â±0.15Â±0.064.4 0.47 aminase activity is not due to concentration of this enzyme in residual tissue. In other studies, the S Compounds were injected subcutaneously for 14 days; treatment began 24 hours after the tumor was transplanted. level of tymosine tmansaminase in thymus and t Theanimalsweresacrificedandthe tumorsweighedlb Walker tumor was unaffected by treatment with days after transplantation. @ contisol.' Average value Â±standard deviation.

Walker tumor could be demonstrated with respect ant lines of a lympho8arcoma.â€”The development of to both inhibition and stimulation of tumor growth cortisone-sensitive and -resistant lines of lympho in rats treated with cortisol or deoxycorticosterone sarcoma P1798 was described in detail by Lamp (49). kin and Potter (38). When F, mice (BALB/c X Another aspect of this study was concerned with DBA/92) bearing established P1798 tumors are the growth and transaminase activity of the tumor given subcutaneous injections of 1 mg. of cortisol in animals treated with cortisol by three different daily for 4 days, the sensitive line undergoes al routes of administration (49). Tumor growth was most complete regression, but the growth of the most effectively suppressed when the steroid was resistant tumor is not impaired. It was of interest given subcutaneously. Maximum increases in the to determine whether the sensitive line of this tu alanine transaminase activity of liver and tumor mom would respond in the same way as the Walker were also associated with this mode of administra tumor with respect to stimulation of alanine tion. The inhibition of tumor growth (about 50 transaminase activity and whether the refractory per cent) observed in animals given cortisol orally tumor would not show a change in the activity of or intraperitoneally was not associated with a this enzyme (51). The effects of cortisol on growth change in tumor alanine transaminase activity, and alanine transaminase activity of the cortisol although the level of this enzyme in liver was ele sensitive and -resistant lines of lymphosarcoma vated. Slow absorption of cortisol from a subcu P1798 are shown in Table 3. In animals treated taneous depot appears to be necessary for maximal with 50 mg comtisol/kg daily for 4 days, the inhibition of the growth of the Walker tumor and growth of the contisol-sensitive tumor was me for the greatest increase in liver and tumor alanine duced to one-fifth the weight of the control tu transaminase activity. moms, and transaminase activity was increased The activity of alanine transaminase was also three-fold. A significantly greater reduction in tu examined in the Walker tumor of animals fed mom size and a nine-fold increase in alanine trans high protein diets, fasted,or made diabetic by al aminase activity resulted when 100 mg cortisol/ loxan (49). Each of these conditions was previous kg was administered. The growth of the contisol ly shown to stimulate the activity of this enzyme resistant tumor was not affected by treatment with in liver to an extent comparable to that obtained the smaller dose of cortisol, whereas the 100 mg/kg by injection of cortisol. In comparison to rats fed dose reduced tumor growth by 50 per cent. In each a purified diet containing 18 per cent casein, the case, the changes observed in alanine transaminase growth of the tumors in the animals maintained activity were not considered significant. on the 50, 75, and 95 per cent protein diets was me Table 4 shows the effects of cortisol treatment duced significantly. Maximum inhibition of tumor on the tymosine- and aspartic- a-ketoglutamate growth was obtained in the rats fed the 75 per tnansaminase activity of the cortisol-sensitive cent protein diet. Associated with reduced tumor and -resistant lines of P1798. The tyrosine trans growth, theme was a three-fold increase in the aminase activity of the sensitive tumor was in alaÃ±ine transaminase activity in the tumors of the creased several-fold by treatment with contisol. In rats fed the 75 and 95 per cent protein diets. the resistant tumors, the administration of cortisol The several-fold increase in tumor alanine trans resulted in a 60 per cent rise in the activity of this aminase activity observed in animals fed high transaminase. protein diets or in rajs injected with cortisol was In previous studies, aspartate transaminase was not observed in the diabetic or fasted animals, found to be refractory to treatment with cortisol even though marked inhibition of tumor growth in liver, thymus gland, and Walker tumor. How was observed in each case. Suppression of tumor ever, in the cortisol-sensitive lymphosarcoma, the growth in diabetic or fasted animals may result activity of this transaminase was observed to be from impairment of a number of different meta significantly lower than the control value follow bolic functions, each important for growth. The ing treatment of the animals with cortisol. The increase in alanine transaminase activity in both significance of this finding is not clear but is of liver and tumor in rats treated with cortisol or fed interest insofar as it provides a different example a high protein diet might be associated with higher of a change in transaminase activity induced by a levels of circulating amino acids than would occur glucocorticoid. in animals in which the liver is converting amino In contrast to the action of deoxyconticos acids to carbohydrates that are required as a terone in stimulating growth and lowering the source of energy by the diabetic on fasted animal. alanine transaminase level in the Walker tumor, Comparison of glucocorticoid-sensitive and -reaiit neither the growth nor the activity of alanine or

aspartate tmansaminase in the cortisol-sensitive with cortisol (Chart 92). Additional results on other and -resistant lines of P1798 was affected by tumors are needed before this relationship is clear treatment@with this mineralocorticoid. ly established. Altered alanine transaminase activity related to the sensitivity of tumors to cortisol.â€”Oun work to MODE OF AcTION OF GLUCOCORTICOIDS date has indicated a possible relationship between It is the purpose of studies on the mechanism of changes in the levels of alanine transaminase and action of the glucocorticoids to determine the rate the responsiveness of several tumors to treatment of interaction of the steroid with the receptor in

TABLE S EFFECT OF CORTISOL ON THE GROWTH AND ALANINE TRANSAMINASE ACTIVITY OF THE CORTICOSTEROID-SENSITIVE AND -RESISTANT TUMOR, LYMPHOSARCOMA P1798

GROWTHAi@simis ACTIVITY(Grams)(Per

TRRATMHNT5No. MICETUMOR cent substrate controlNone control)(jiMolesutilized/gm. protein/br)Treated!

2.9 0.31 bomgcortisol/kg 4 0.6 21 0.95 3.1 100 mg cortisol/kg 4 0 .28 8 3 .0 9.7

CORTISOL-RThISTANT

@ None 9 3.4 0.28 50 mg cortisol/kg 2 3 .4 100 0 . 14 0.60 loomgcortisol/kg7 [email protected] 52 0.42 1.5

a The amount of cortisol indicated was injected subcutaneously daily for a period of 4 days. TABLE 4 EFFECT OF CORTISOL ON THE TYROSINE AND ASPARTATE TRANSAMINASE AcTwmrY OF THE CORTICOSTEROID-SENSITIVE AND -RESISTANT TUMOR, LYMPHOSARCOMA P1798

LLVZLSTyrosineAspartateActivityfTreated!

TREATMENT5No. MIcETIIANSAMINA$@

controlNone controlActivitytTreated!

@ 0.023 21 0 Somgcortisol/kg 8 0.061 I 2.7 12 I 0.57 @ 100 mg cortisol/kg 4 14 0.67

CORTISOL-RRHISTANT

None 8 0.023 25 Somgcortisol/kg10 6CoRTIsofrs0.036 1.6ENB@@E 27 1.1

S The amount of cortisol indicated was injected subcutaneously daily for a period of 4 days. t isMolessubstrateutilized/gmprotein/hrat38Â°C.

the target cell and the subsequent metabolic to stimulate gluconeogenesis, such as high protein changes that account for the physiological and intake, diabetes, and fasting (55). morphological effects noted in the responsive tis Other evidence has been presented that this sue. Although there is a large body of information enzyme might be rate-limiting for glycogen syn on this subject, the actions of this class of hor thesis initiated by cortisol treatment. In the pyni mones at the molecular level remain unexplained. doxine-deficient animal, pyridoxal phosphate, the There is now considerable evidence that one of the co-factor of alanine transaminase, would be ex biochemical effects elicited by the glucocorticoids pected to be limiting, and thus the response of this involves the synthesis of enzymes. Therefore, it enzyme to cortisol would be impaired. Utilizing seems appropriate in this concluding section to this technic, Eisenstein (13) demonstrated a consider the manner in which glucocorticoids diminished glycogenic and alanine transaminase might influence the level of certain intracellular response to cortisol in â€¢pyridoxine-deficient rats. enzymes. Attention will also be given to the prob These results have been confirmed in somewhat tern of resistance and the technics we are using in similar studies in our laboratory (48) . The increase in activity of alanine transaminase after treatment A with cortisol was almost totally blocked in liver and inhibited markedly in the thymus of mats de

IO'\ CCRT@OL pleted of pynidoxine. However, whereas the failure \. of the glycogenic response paralleled the impaired â€˜ [email protected] enzyme response in liver, cortisol exerted its usual @@ â€˜4- a . 10 .@â€˜ 0 LYMPHOSARCOMA P1798/SEN. 50 thymolytic action despite the reduced response of â€˜ . U U /RE$.50 â€˜,.. 0 NOVIKOFF HEPATOMA 30 the transaminase observed in this tissue. These @\ I KREBS/2 00 data do not unequivocally demonstrate a correla A' tion between glycogen synthesis in liver and levels of alanine transaminase activity as influenced by

S-I cortisol. Since pynidoxal phosphate is a co-factor I 0 for many enzymes, the possibility exists that pyni 2 doxine deficiency may affect some other system I that is involved in glycogen synthesis. This technic was also used in studies concerned with a possible correlation between the growth

20 40 50 50 100 of cortisol-sensitive tumors and their alanine

TUMOR GROWTH (PERCENT OF CONTROL) transaminase response to cortisol. In experiments to date, the inhibition of growth of the Walker CHART2.â€”Relationship between changes in the level of alanine transaminase and the responsiveness of several tumors tumor and of lymphosarcoma P1798 in pymidoxine to cortisol. deficient animals treated with cortisol was equal to that obtained in animals bearing these tumors an attempt to evaluate the physiological sig but receiving a complete diet. However, since the nificance of enzyme induction by comtisol in target alanine transaminase response to cortisol in the tissues tumors was not significantly impaired by the B6 The major physiological effects produced by the deficiency, no definite conclusions can be drawn glucoconticoids include an accelerated rate of from these findings. Until the stimulation of this gluconeogenesis, the synthesis and deposition of enzyme by comtisol can be completely inhibited, as liver glycogen, enhanced prot.ein catabolism, and it is in the liver of the Be-deficient rat, it will not be a cytolytic action on lymphoid tissue. The hon possible to cleanly interpret the data. monal specificity of the alanine transaminase me Another approach to this problem involves the sponse to contisol, the magnitude of this effect, combined use of a compound which inhibits pro as well as the gluconeogenic potency of the sub tein synthesis, such as pumomycin or ethionine, stnates (pyruvate, alanine, glutamate) of this with cortisol in an attempt to block the effects of transaminase suggested that the cortisol-regulated the steroid on tumor growth. Mueller (38) has hepatic levels of this enzyme could serve as part of presented evidence that many of the biochemical a mechanism which regulates the capacity for effects that occur in the uterus subsequent to gluconeogenesis (53). Added support for this hy estrogen treatment are mediated by the synthesis pothesis was obtained when it was found that the of new protein. It would also appear that the syn levels of this enzyme in liver could be readily in thesis of enzyme protein is responsible for most of creased in three other conditions which are known the increase in the activity of several enzymes in

ducible by glucocorticoids (16, 926,57). Ethionine resistant cell line. A relevant question, the con or puromycin have effectively prevented the in sidemation of which is outside the scope of this duction in liver of alanine (56) and tyrosine trans paper, concerns the possibility that in certain aminase (35) and tryptophan pyrmolase (45) by malignant cells there is a defect in the capacity for cortisol. In one preliminary experiment, puro regulation of the level of enzymes which are mycin injected at 2-hour intervals over a period of normally mate-limiting in the control of metabo 6 hours, starting 1 hour before the administration lism. This hypothesis has been proposed by Potter of cortisol, failed to prevent the inhibition of (44) and Pitot (43) and will be considered else growth of lymphosarcoma P1798 and also did not where in this conference. abolish the response of alanine or tymosine tmans In microorganisms, the synthesis of specific aminase in the tumor. Additional studies of this enzymes involves complex control processes. Sub type are needed to reveal whether the synthesis of strates or closely related compounds can induce enzymes in lymphoid tissues following cortisol the synthesis of many enzymes. The counterpart therapy is of significance with respect to the action of induction is repression, by which specific sub of the glucocorticoids on these tissues. strates repress the synthesis of particular enzymes. Differences have been noted in the way in It is not known whether enzyme repression is a which target cells metabolize corticosteroids that general phenomenon in higher animals. If this are not only related to cell type but also to the should prove to be the case, is it possible that stage of differentiation and function of particular glucocorticoids which stimulate enzyme induction cells (11, 192). The basis for the response of lym phocytes appears to be related to the presence of LEVELOFAMINOADDS 11-$-hydroxydehydrogenase, an enzyme which INLIVER catalyzes the interconversion of cortisone and lNf@REA5EIN ALAM@ TRAN@ @@ cortisol. Factors which shift the equilibrium of this DECREASE IN @ enzyme toward the formation of cortisone tend to ALAMNE TRANS@Mt@ASEI NÂ°@ â€”@ I@ Â°@ @.JsThRvATKiN @ favor lymphocytopoiesis, whereas conditions which PREGNANCY ( ALL@CAN-OiA8E1ES @ permit the accumulation of cortisol favor a lym TUMORGROWTh@4 HVPCPHVSECTCMV phocytic action. The two lines of lymphosarcoma HE@TECTCMYj@______@ P1798 would be ideal tissues in which to study the L .+\@;TISSUE possible relationship of such an enzyme, and the PROTEINS reaction it mediates, to the sensitivity or resistance CHART 5.â€”Hypothetical relationship between amino acid of a malignant tissue to comtisol. pool size and changes in alanine transamina.se activity. What other factors might account for the me sistance of a lymphoid neoplasm to adrenal act in this respect by interfering with the forma corticoids? The lack of responsiveness of the tion or availability of particular repressors? malignant tissue may simply be due to the failure A hypothetical mechanism by which gluco of cortisol to penetrate the cell. Thus, a change in corticoids stimulate enzyme synthesis must take the structure of the cell membrane or of a system into account the various dietary and physiological which regulates the transport of the steroid could conditions which increase the activity of the conceivably alter sensitivity to the corticosteroids. inducible enzymes, as well as those conditions Wilimer (66) has suggested that the properties of which depress the level of these enzymes (Table 1). the cell membrane could be altered by steroids An attempt has been made to include all these combining with the phospholipide-cholesterol observations, as they apply to alanine tmansami layer. Since the resistant form of P1798 was de nase in liver, into a tentative explanation (Chart veloped by exposure of the sensitive line to corti 3) of enzyme induction. sone during numerous transplant generations, it The treatments or conditions which lead to an might have a modified membrane that impedes the increase in activity of alanine transaminase have entry of glucocorticoids into the cell. been associated with an increase in the concentra As compared with the significant changes in tion of amino acids on protein precursors in liven. the activities of alanine, tyrosine and aspartate On the other hand, pregnancy, tumor growth, and transaminase observed in the sensitive line of hepatectomy involve the growth of new tissues, P1798, the resistant tumor showed only slight which requires amino acids for protein synthesis. alterations in the levels of these enzymes. It is Thus, under these conditions, the free amino acid possible that the protein-forming systems which pool in liver would beexpected to be decreased. control the level of the adaptive enzymes, alanine Treatment with contisol has been found to im and tyrosine transaminase, are impaired in the pair the mobilization of albumin and globulin in

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Fred Rosen

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