New Facets of the Biochemistry of Hormone Actio&

H. G. WILLIAMS-ASHMAN

(The James Buchanan Brady Urologicat In.@titute, The John.@ Hopkins Ho.@pi1ul, and 7'he Department of I'harmacology and Experimental Therapeu1ic.@, The JOhnS Hopkins (Tniver.sity School of .tfedicine, Baltiniore, Maryland 21205) SUMMAR ‘@: Some aspects of i-ecent experimental appi-oaches to the molecular l)aS)s of steroid hormone action are reviewed. Among the topics considered are: (1) the bearing of structure-activity relationships, and of pathways for the metabolism of steroid hor mones, on the I)roblem of the nature of the I)rimary cellular receptors for these hor mones; (2) coenzyme-like actions of sex hormones in isolated enzyme systems; (3) steroid hormones as allosteric effectors ; (4) steroid hormones u.s regulators of gene cx pres.sion and of intermediate reactions involved in nucleic acid and protein synthesis.

There has beeii a prodigious increase iii the volume and bioassay systems are just. as striking as their specificities. variety of recent experiment at attacks on the problem of Indeed, the very concept of “targetorgans― for certain the molecular basis of stei-oid hormone action. This is of doubtful significance. There are abundant. paper will not attempt aiiy coniprehetisive coverage of this examples of a single steroid inducing dissimilar or even per@)1exingfield, but is rather restricted to consideration of completely opposing effects on various tissues in the same a few arbitrarily selected researches that have excited much animal. The variations in the response of many neo effort and controversy and that attest to the truth that. i)lasrns to different steroids (or combinations thereof) am-c the “difficultyiii most scientific work lies in framing the particularly baffling, especially in those all too common (luestiolls rather than iii finding the aiiswers― (18). A cases where the tumors may eventually lose their hormone great deal of interesting @voi-kon interactions of steroid dependence (20, 95, 96, 101). It is obvious that changes hormones with biologic membranes (86, 262, 268, 277) rep iii the biologic effects of steroids resulting from alterations resents but one of the many inmpoi-t.ant areas of contem in subst.ituent groups or molecular geonietry may reflect [email protected]@Yinvestigation which are largely omitted from the not only differences in the reactivity of these substances Pi@eSe11t discussion. toward their primary hormonal receptors, but also the operation of other factors, such as the nature and extent of STRUCTURE-ACTIVITY RELATIONSHI PS, the metabolic transformations of the hormones (in their STEROID METABOLISM, AND THE target tissues or elsewhere) ; the relative distribution of the RECEPTOR PI{OBLEM steroids and their metabolites among various organs, cells, A vast heap of information is now extant concerning and organdIes; and so forth. For it is well established relat ions of the primary chemical structure of steroids and that exogenous steroids can be converted in vivo into sub related substances to their activities in various bioassay stances with enhanced or altered types (cf. Refs. 13, 26, systemns (7, 25, 26, 46, 193, 203). Appelations such as 114) of biologic activity, in addition to being “inactivated―

‘‘androgen,' ‘ ‘‘ , ‘‘‘‘gestageii, ‘‘‘‘mineralocorticoid,― (13, 26, 236, 239). Indeed, we cannot even be sure at and “glucocorticoid― have, of course, a purely operational present as to the nature of the “activeforms―of steroid inemuming, and it is practically impossible to overestimate hormones in susceptible cells, although there are yet no the (.ofliplexit.y of the biologic respotises to which they coiiipelling reasons to believe that , refer. This is strikingly illustiated in the realm of female 17@, and progesterone do imot combine as such with the reproductive physiology by the studies of Huggins and receptors responsible for their sex hormonal actions.2 .Jensen (97—100) on the so-called impeded estrogens amid on For these amidother reasons, reflections on the electronic the repertoire of vaginal responses to different steroids, as structure and molecular geometry of steroids and synthetic well as by the dependence of estrogenic effects on local estrogens have contributed little solid insight into the na V(iSUS systemic adniinistrat ion of aromatic substances ture of the hormonal receptors. There may be limitations which led Emmens et a!. (52) to enunciate the concepts of to the heuristic value of rather general concepts such that “@)roestrogen―and“trueestrogen.― So often, the over the attachment of steroids t.o their receptors occurs at mul @ im@l)s the biologic activities of various steroids in many tiple points (203) ; that electrostatic and London dispersion

@ Original work from the authors laboratory was supported by 2 Jensen aIRI Jacobsoti's (ill) hypothesis that estroiie must Grant HJ)-0l266 from the National Institute of Child Health and he converted into estradiol-17fl before it. can exert uterotropic Hurn:tir l)evelopment and (ra,il CA-tE545 from the National action has l)eeli criticized on experimental grounds by Stone arid (‘nilcer Institute. Martin (230).

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLIAMs-AsHMAN—Steroid Hormone Action 1097 forces, hydrogen, and hydrophobic bonds may all be in (224, 225, 232, 233) have obtained evidence—albeit some volved in their emigagement with the receptors (26, 203); what tenuous (34)—that many of the actions of estrogens and that when the latter occurs, restrictions may be im oil the uterus may be due to liberation of histamine and posed on the translational degrees of freedom of the ste perhaps serotonin, possibly from mast cells. Naturally, roids (26). This is not to detract from the utility of any comprehensive account of hormone action must spec certain purely empirical geiieralizations—such as Fried (56, ify the chronology of the induced biochemical events. But 57) proposed with respect to the activities of halogenated here one must be on guard against the pitfalls of post hoc, steroids—in sem-ving as fruitful guides for synthesis of ergo propter hoc fallacies. highly active novel compounds. Scrutiny of 2-dimen The idea that the cellular receptoms for most drugs amid sional structural formulae may certainly be misleading in hormones must be proteins, rather than any other class of consideration of structure-activity relationships, amid the mnacromolecule, is firmly entrenched in contemporary bio importance of the exact. conformations that the steroids logic thought. Because they are coml)rised of so niaiiy can assume in solution has been emphasized repeatedly more types of building blocks thaim are either polysacelia (26, 203, 236). Especially iii the field of the aronmt.ic rides or polynucleotides, the proteins are clearly capable of estrogens—where the geometrical requirements are less a much greater variety of localized and sterospecific dOffi stringent thami with the androgens, gestagens, and corti plememitary imiteractions with sniall molecules, and also of coids—perhaps not enough att.entioim has been paid to their subtle modifications of their tertiary structures. It is mr-electronic emiergy levels, now that these are becoming only natural that, of the host of proteins present in cells, more tractable to coml)utatioli (150). Here we may men various emizymes and structural membrane proteins should tion some recent l)ointers (40, 102) to analogies between have been considered as candidates for hormone receptor the molecular geometries of hormuonally active sten-oids (as functions. The overwhelming emphasis on enzynies iii well as of some carcinogenic aromatic hydrocarbons) aiid their cytoarchitectural context as the all-importanit utmits the base pairs of DNA. determining cell structure and function, so ably elaborated Today it remains an entirely open question as to whethem on by Mueller (162) at a comparable symposium 7 yeai@s t he effects of any one steroid hormone on any particular ago, is very characteristic of the Zeitgeist. But the ye cell are set in motion via a number of different types of markable progress imithe biochenmistry of protein synthesis l)rimary biochemical interaction (perhaps at varyimig intra and gene expressiomi over the intervening years has now cellular locations), or rather as a result of a single basic pointed to various types of 1)olymmucleotideas the primary physicochenucal event which triggers off all subsequent targets for some physiologically active trace substances. physiologic responses. That a given steroid affects all This seems to be the case, for example, with respect to the susceptible normal and mieoplastic cells by the same basic action of certain alkylating agents (48, 87) and of ant ibi niechamiism is also purely a matter of conjecture. Ingle's otics such as actimiomyciri D (84, 188, 190). And it. is well (106) incisive experiments pointing to the “permissive― established that sonic purinie and j)yrimidine analogs nitty ttct iOU of steroids show how careful one must be iii talking inifluemicebiologic i@roces@esasa result of being incorporat ed about the “regulatory―action of certain hormones. As into “fraudulent―polymiucleotide structures (48, 87, 104). Bush (26) neatly puts it : “Everyresponse that was cx The melancholy failure of so niany attempts to “explain― mtmined which had previously been supposed to be subject. homiuone action imiternis of priniary classical chemnical in to the regulatory action of the glucocorticoids of the adrenal teractions with macromolecules in solution has eveim led cortex (cortisol amidcorticost.erone) was shown by this type some authors to imagine that. hoimones may act accordimig of experiment to depend upon the presence of this type of to entirely different pm'imiciples,which could only be cx steroid but to be under the regulation of some other sys pressed in the language of the quantum theory of solids. 1cm.― Bush (26) also draws attention to the neglect. of Thus, arguing from the effects of trace levels of douoi- or investigations on the circulatomy failure iii adrenocortical acceptor impurities on the propert ies of semi-conductors, insufficiency, and to the danger of attributing to metabolic Szemit-Gyorgyi (234) suggested that certain drugs and hor abnormalities events which may actually result from dy momies might, through charge-tramisfer imiteractions, in - culatoi-y disturbances. The lag period of hours or even fluence the conducting properties of fused energy bands in days which is associated with many of the biologic effects biologic macromolecules. Unfort unately, molecular or of steroid hormones is another enigma. Such appan-emit bital calculations show that it is energetically very expen delays iii hormonal response probably reflect neither a sive to excite electrons into the empty common energy slow rate of combination of the hormones with theim- i@e bands of proteins or DNA, and there seems little likelihood ceptoms, nor the tinie required for their distributioni among that. conductiomi bands iii molecular systems play an im various tissues or their conversioii to “activeforms,― but rort.ammt.role in living ln@ocesses outside the territory of merely our ignoramice of the antecedent biochemical pro @)h0t0bi0l0gy (148, 150). ceedings iii the tam-get cells. Recent studies on the actions With the recemit availability of labeled steroids of very of a variety of hormones whose metabolic actions seem to high specific radioactivity, some insight has been gained be comitimigent upon theii enhancing the conversion of ATP into the levels of steroids and their metabolic products iii to adenosine 3' ,5' cyclophosphate (231, 246) make it clear various tissues after- their administration in truly physio how very intricate may be the trains of enzymatic events logic doses. Estradiol-17$ causes the rat uterus to grow which are essent ial for observable hormonal responses, whemi its concentration thereimi, assuming equal dist.rihu eveii though only a few of the reactions are directly in tion between cells and extracellular fluid, is about. 3 X fluenced by the hon-moimes. Again, Szego amid Spaziani 1O@ @t(109—11). Similarly, the male accessory reproduc

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tive glands are profoundly affected by androgens at levels to be responsible for the observation of Villee et at. (255) of less than 10@ @i(180). These values emphasize the that low levels of estradiol-17@3 or accelerated the caution required in interpretation of effects of sex hormones reduction of NAD catalyzed by crude placental extracts on isolated systems in concentrations of 10@ M or greater. incubated with isocitrate. It was proved (238—240)that Experiments with labeled steroids have also revealed the apparent activation by steroidal estrogens of what some degree of selective concentration of androgens and Hagerman and Villee (78) believed to be an “estrogensen est.rogens by some accessory reproductive organs (29, sitive isocitric dehydrogenase― required catalytic amounts 10@3—11,180). Injected amidrogens seem to be converted of NADP, and was in reality due to a coupled reaction into a variety of products in the prostate (82, 180). But between a NADP-specific isocitric dehydrogenase and a in the uterus of at least 2 species, estradiol-17$ does not specific steroid-sensitive tramtshydrogenating system. The appear to be comiverted imiany great extent into other sub latter reaction was showmi to be catalyzed by a placental stances (109—11). 17@3-hydroxysteroid dehydrogenase, originally discovered 1mman excellent review on transformation of adrenocor by Langer et al. (139), according to the following equations: tical steroids by peripheral tissues, Berliner and Dougherty Estrone + NADPH + H@ @=±Estr&Iiol-17$ + NADP@ (13) argue that the “metabolism of these hormones is re @ Estradiol-17$ + NAD+ Estrone + NADH + H@ lated riot to mechanism of action but to their turnover, which imposes requirements for steroid renewal.― Some NADPH + NAD@ @::±NADP@+ NADH deliberations on structure-activity relationships have ar rived at a similar point of view (26—28,193, 203). Yet in The placental hydroxysteroid dehydrogenase was purified recent years there has come to light a number of well more than 2500-fold, and in high yield, by Jarabak et al. authenticated examples of physiologic concentrations of (108). In addition to catalyzing dehydrogenase and trans steroid hormones exertimig a coenzyme-like role in reactions hydrogenase reactions involving NAD and NADP, the catalyzed by refined enzymes in the test tube. Some oxi same protein also promotes the dehydrogenation of estra dizing enzymes that catalyze sloichiometric oxidations or diol-17@9by a number of pyridine nucleotide analogs, as reductions of sex hormones (which actually occur during well as transhydrogenations between NADH and the var the metabolism of the hormones in vivo) may, under some ious analogs in the presence of catalytic levels of estradiol what. different experimental conditions, promote extensive 17$ or estrone. The many lines of evidence supporting hydrogen transfers between biologically important mole the view that the same protein catalyzes both the dehydro cules iii which cakilytic levels of the hormones act as hydro genase and transhydrogenase reactions have been reviewed gen earners by undergoing cyclic oxidoreductions. We in extenso (108, 239, 269, 271) and wifi not be detailed here. shall now consider the miature and physiologic significance But it may be mentioned that Villee and his co-workers of these coenzyme-like actions of sex hormones. (1, 255) still maintain that human placenta contains, in addition to the 17/3-hydroxysteroid dehydrogenase (which COENZYME-LIKE ACTIONS OF SEX HORMONES they now (1) admit does catalyze steroid-mediated trans IN ISOLATED ENZYME SYSTEMS hydrogenation), an entirely separate “estrogen-sensitive Transhydrogenations catalyzed by hydroxysteroid dehydro transhydrogen&se,― which is activated by steroidal estro genases.—It is miow well established that certain mamma gemis in some mysterious manner not involving alternate han hydroxysteroid dehydrogenases catalyze not only the oxidation and reduction of their 17$-hydroxyl groups. reversible oxidation of hydroxysteroids to ketosteroids, Such an enzyme is also said to be present in the human but also the efficient transfer of large quantities of hydro myometrium (1). This putative “estrogensensitive trans gen between NADPH3 and NAD in the presence of cata hydrogenase― has not been purified extensively, and its lytic levels of their steroid substrates. The pioneer stud existence is controversial (108, 239, 269). A 17-ketone ies of Talalay (153, 236, 237) with inducible bacterial en or an oxidizable 17$-hydroxyl group is clearly required zynies established that hydroxysteroid dehydrogenases for any steroid to serve as a mediator of transhydrogena catalyze pyridine nucleotide-linked oxidations of the fol tions catalyzed by the placental 17@3-hydroxysteroid dehy drogenase (2, 85, 239). lowing type: Hurlock and Talalay (103) demonstrated that the 3cr Hydroxysteroid + NAD(P)@ hydroxysteroid dehydrogenase of rat liver, which also @:±Ketosteroid + NAD(P)H + H@ reacts with both NAD and NADP, promotes hydrogen ex change between the 2 natural forms of pyridine nucleotide Some of the hydroxysteroid dehydrogenases in mammalian in the presence of trace levels of its steroid substrates. tissues, unlike their bacterial counterparts, can function This was confirmed by Koide (132). It was originally about equally well with both NAD and NADP as hydro proposed (238) that many other hydroxysteroid dehydro gen acceptors. An enzyme of this class in human placental genases in mammalian tissues might catalyze steroid-de cell sap was found by Talalay and Wffliams-Ashman (240) pendent transhydrogenations between NADPH and NAD. 3 The following abbreviations are used: NADPH, reduced nico This hypothesis became improbable, however, following tinamide adenine dinucleotide phosphate; NAD, the later demonstration (158, 256, 267) that a number of adenine dinucleotide; ATP, adenosine triphosphate; ADP, adeno mammalian hydroxysteroid dehydrogenases in fact react sine diphosphate; TJDP, uridine diphosphate; RNA, ribonucleic acid; PP1, inorganic pyrophosphate; DNA, deoxyribonucleic acid; at significant rates with only NAD or NADP, but not with GTP, guanosine triphosphate; polv UG, copolymer of uridylic and both coenzymes. Hydroxysteroid dehydrogenases of the guanylic acids. latter category obviously could not catalyze steroid-medi

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ated hydrogen transfers between the 2 natural forms of strable in human placenta (239, 255), anterior pituitary pyridine nucleotide. (255), and mammary gland (88), but not in rat uterus McGuire and Pesch (157) have described a particulate (163). Baron et al. (11) claimed that androsterone-de transhydrogenase system from beef anterior pituitary pendent transfer of hydrogen from NADPH to NAD is which is stimulated in &ro by a number of hormones, in catalyzed by soluble extracts of the rat ventral prostate. eluding epinephrine, norepinephrine, estradio1-17@3, and But in 2 other laboratories (202, 270), androgen-mediated serotonin. This hypophyseal transhydrogenase has not transhydrogenase reactions could not be detected in the been purified extensively, and until this is achieved, the rat prostate under a variety of experimental conditions. mechanism by which it is stimulated by phenolic hormones b) Hormonally potent substances fail to serve as trans will doubtless remain a mystery. hydrogenating coenzymes in the isolated enzyme systems, The division of metabolic labor between NAD(H) and and conversely, certain hormonally inactive compounds NADP(H) is now recognized to be of fundamental impor are excellent stimulators of the in vitro transhydrogenase tance in the metabolism of virtually all cells (93, 113, 238). reactions. Substances like ethynylestradiol-17@3 and di For NAD(H) is predominantly involved in ATP genera ethylstilbestrol, whose estrogenic actions are qualitatively t.ion, by serving as the specific hydrogen acceptor in the and quantitatively very similar to those of ovarian steroids, oxidative step of glycolysis, and as the principal hydrogen fail utterly to mediate transhydrogenations by the placen donor for the phosphorylating electron transport system tal enzyme (2, 239, 269). Furthermore, some steroids of mitochondria, whereas NADPH is the preferred hydro which are virtually devoid of estrogenic activity are excel gen donor for many reductive biosyntheses. The mito lent activators in this system (85, 239). Again, Hurlock chondria of many mammalian cells contain firmly bound and Talalay (103) found that the hormonally inert etio pyridine nucleotide transhydrogenases which do not re. cholan-3cr-ol-17-one is an even better mediator than an quire steroids or any other low molecular weight substances drosterone of the NADPH-NAD exchange catalyzed by as obligatory cofactors,4 and there is evidence for an im rat liver 3a-hydroxysteroid dehydrogenase. portant regulatory function of such mitochondrial trans c) In comparison with the tissue levels of many soluble hydrogenases, particularly in making hydrogens from pyridine nucleotide-linked dehydrogenases, and also of the NADPH available to the phosphorylating respiratory non-steroid-requiring mitochondrial transhydrogenases, chain (113). The 2 mammalian hydroxysteroid dehydro the transhydrogenating capacity of the above mentioned genases mentioned above, for which direct evidence of hydroxysteroid dehydrogenases is rather feeble (16, 103, their transhydrogenase functions has been documented, are 227, 239, 255). localized in the cytoplasmic cell sap. It can thus be im d) In experiments with tritium-labeled steroids Jensen agined (239, 240, 255) that steroids might regulate a flow (109—11)has shown that estradiol-17j3 in the immature or of hydrogen between a number of metabolic systems by estrogen-primed uterus is not converted into estrone (or serving as coenzymes of pyridine nucleotide transhydro any other products) in yields ot more than a few percent. genase reactions in the soluble portion of the cytoplasm of A greater degree of transformation might be expected if certain cells. This, in theory, would permit the generation the hormone transports hydrogen in the transhydrogenase of biologically useful energy (ATP) from the oxidation of reactions by undergoing alternate oxidation and reduction NADPH, and might also drive NADP-dependent path at carbon 17. ways, such as the hexose monophosphate shunt, by making Interaction of phenolic estrogens with metalloprotein en available NADP. Certainly the transhydrogenations zymes.—Some natural and synthetic phenolic estrogens catalyzed by the placental 17@3-and the hepatic 3cr-hydrox can also function in a coenzyme-like manner in oxidations ysteroid dehydrogenases are mediated by levels of their catalyzed by refined metalloenzymes. In these isolated steroid substrates which lie within the physiologic range. enzyme systems, the aerobic oxidation of donors such as And the small increase in the respiration, as well as incor reduced pyridine nucleotides or ferrocytochrome c is me poration of radioactive precursors into proteins, lipids, and diated by catalytic concentrations of the phenolic hor nucleic acids, found by Villee et al. (112, 255) to be induced mones. Two distinct types of such model reaction have by addition of estradiol-17@3 to isolated placental prepara been described. In the presence of various plant pheno tions, may conceivably have resulted from a stimulation lases, estrogenic phenols appear to be first hydroxylated to by the hormone of hydrogen transfer from NADPH to o-diphenolic derivatives, which then transport hydrogen by @ NAD. But the hypothesis that steroid-mediated trans the reaction o-diphenol o-quinone, the quinone form hydrogenations are of central importance for hormonal being reduced back to the diphenol nonenzymatically by action is militated against by the following facts: the hydrogen donor (120, 272). In a different set of reac a) The requisite enzymes appear to be lacking in certain tions catalyzed by certain hemeprotein peroxidases, phe tissues which are extremely responsive to steroids. Thus, nols in the presence of Mn@ and oxygen transport hydro @ estradiol-mediated transhydrogenase reactions are demon germsor electrons by undergoing the cyclic change phenol

4 Pesch et al. (182) recently found that transhydrogenation be phenoxy radical (3, 272). The specificity of the metallo tween NADH and the 3-acetylpyridine analog of NAD catalyzed enzymes toward various phenols in the 2 model reactions by a particulate fraction from rat liver mitochondria is stimulated depends somewhat on the source of the enzyme and on the by some steroidal estrogens and by . The estro nature of the hydrogen or electron donors employed (3, gens had no effect on the NADPH-acetylpyridine-AD hydrogen exchanges catalyzed by the same mitochondrial extracts, which 120). Although numerous hormonally inactive phenols did not contain hydroxysteroid dehydrogenases. The mechanism are excellent carriers in both systems (as are some aromatic of these effects of estrogens in vitro is completely unclear. amines in the peroxidase-catalyzed reactions (273)), many

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, provided they contain at least 1 free emihanced sulfamate synthesis by serving as sulfate-traits hydroxyl group, are active imiexceptiomially small amounts. fering agents by a mechanism involving the intermediate Klebanoff (124, 125) discovered that. thyroxine acts cata formation of steroid 17-enol sulfates. However, further lytically in a maniier similar to estradiol-17@ in various analysis by Roy (199, 200) of the effects of ketosteroids on oxidations catalyzed by peroxidases. rat amidguinea pig liver arylamine sulfokinase showed that Whether ammyof the phenolase- or peroxidase-catalyzed this was not the case. Rather, the apparent affinity of reactiomis itt which est.rogenic phenols act as hydrogen car the sulfokimiases for their' substrates is altered when the riers are of @)hysiologicsignificance is a moot point. Cer steroids combine with the enzymes. The activation by taimily these enzymes, acting umider rather different exper 17-ketosteroids of sulfamate synthesis by rat liver aryla iment.al conditions, can convert phenolic estrogens to a mine sulfokinase, then, cannot be regarded as another cx variety of hormomially inactive products. Phenolases are ample of a coemizymatic function of steroids, but instead doubtless among the emizymes responsible for the “inactiv probably represents a case of allosteric control of enzyme ation―of phenolic estrogens which has long been known to function (see below). occur when these hormones are incubated with plant cx Comments on coenzyme-like properties of steroid hor,,,one,-. tracts (cf. Ref. 239). The carrier function of the diphe —It is debatable whether widespread physiologic signif miolicderivatives of certain estrogens in the presence of phe icance can be attributed to the aforementioned examples miolases may conceivably have some bearing on Mueller's of steroids participating catalytically in group transfer re (161) report that 2-hydroxy- amid 4-hydroxyestradiol-17$ actiomis. The coenzyme-like actions of phenolic horn@onies stimulate the incorporation of forniate into proteins of in oxidations catalyzed by pheniolases amid peroxidases isolated uterine hormis, umider conditions where the parent. show that low levels of certain estrogens can transport hy estradiol-17@3 is inactive. The dist.ributiomi of phenola.ses drogen according to the mechanisms defined. But time among mammaliami tissue is, however, rather miarrow (cf. proteins catalyzing these reactions are very unspecific in Refs. 120, 239). 2-Methoxy derivatives of estrone amid their ability to recognize phenols, amid anyway it is difficult are found as major metabolic products of estradiol to see how such phenol-mediated oxidations of reduced 17f3 (134). But although these substances are formed in respiratory carriers could be of much energetic or other vivo by enzymatic met.hylation of the corresponding 2-hy value to amiy cell. The tramishydrogenase functions of droxyestrogens, the latter seem to be synthesized by mi sonic mammalian hydroxysteroid dehydrogena.ses are of crosomal enzymes requiring NADH (or NADPH) amid greater potential significance because of the exquisite ster oxygen (123), rather thami by phemiolase action. The eospecificity of their bindimig sites with respect to their uterus of some species contains an active peroxidase, the steroid substrates, and because of the extraordinarily low activity of which is strictly dependent in invo on estrogenic concemitratiomis at which steroids serve as mediators of such hormones (151, 154). There is circumstantial, but not hydrogen transfers. In the case investigated so far, how conclusive, evidence that. this uterine peroxidase is respon ever, it is evident that their tianishydrogenating capabil ruble for the phenol- amid Mmi@-depenident oxidation of it.ies probably do riot. represent tt n@ajor function of by reduced pyridinie nucleotides in uterus that was first de droxysteroid dehydrogenases in viVa. Of the various scribed by Hollander and Stephens (92). The latter work transformations undergone by steroid hormones in the ers also showed (245) that certaimi 1)hemlolicestrogens could living animal, only the hydroxysteroid dehydrogenase-cat act in vitro as mediators for the oxidation of NADH by this alyzed conversions of steroid secondary alcohols to steroid uterine system. But. recent imivestigations (8, 201) suggest ketones are freely reversible (236, 239). Other metabolic that the high peroxidase content of the estrogen-@)m-irned reactions of steroid hormones—such as saturation or isom uterus is due to an increased populatiomi of eosinophi1i(@ erizat ion of double bonds, hydroxylat ions, conj ugat ions gramiulocytes that cont aiim peroxidase-rich cytoplasmic with sulfate5 or glucuronide residues, arid methylat.ion of particles. Moieover, it is difficult. to see how the phenol pheniolic groups—are very l)oorly reversible (236, 239). stimulated oxidations of reduced respiratory carriers could Thus these transformations do not afford opportunit ies be of much importance in the nietabolic economy of living for the steroids to funct.iomi in a cyclic catalytic fashion. It cells, since these are energetically completely wasteful re cannot be excluded that the hitherto described coenzy actions which, unlike the oxidations of the mitochondrial mat.ic functions of steroids will serve a,s useful models for respiratomy chain, are not coupled with the generation of other, more j)hysiologically nmeaningful, catalytic actions ATP. of the hormones. But in the Opinion of the author, this is Steroid-dependent sulfate transfers. —Roy (198) reported improbable. And it is worth miotimigthat some other the that. the transfer of sulfate from adeiiosine 3'-phosphate ones of steroid hormone action invoking chemically nieces 5'-sulfatophosphate to 2-naphthylanmine as catalyzed by sary carrier functions of steroids now seem equally unten rat liver arylamine sulfokinase is enhanced about 400 % by able—as, for example, Wilbrandt's (268) hypothesis that cert an i 17 -ketosteroids imicor icentrations of approximately sodium-retainimig steroids fumnetionias lipid-soluble carl-icr's @ 10-i The steroidal effects were clearly catalytic in mitt for metal ions (cf. Ref. 26). t.ure, because the steroids increased the net synthesis of Hypotheses that steroid hormones regulate biologic more than 5 times their own molar concentration of su.lf 5 Until recently, it was believed that steroid sulfates were syn aniat e. The synthesis of 2-naphthylamiiine sulfaniat.e by thesized only in the liver, arid that these water-soluble derivatives amianalagous sulfokinase from guinea pig liver was, on the were essentially excretory products (cf. Ref. 238). Recent work contrary, depressed by 17-ketosteroids in vitro. Roy (198) suggests, however, that sulfated derivatives may be involved in steroid synthesis in the adrenal arid ovary (47, 257). But there is originally hypothesized that. imithe rat. liver system, the no evidence that steroid sulfates are of importance from the steroids acted in a coenzyme-like fashion, arid that they standpoint of hormonal action.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLIAMs-AsHMAN—Steroid Hormone Action 1101 lrocesses by serving as obligatory group-transferring coen ALLOSTERIC PROTEINS AND STEROID zyrnes in conjunction with specific activating proteins have HORMONE ACTION eertai ni iml)lications from an evolutionary standpoint. In a recent essay, Monod et at. (159) have codified the The integrative regulation of metabolic, reproductive, amid comicept of so-called allosteric proteins, which play a pivotal behavioral functions by steroids seems to have developed role in the regulation of many metabolic processes by small relatively late in the evolutiomiary process. For even molecules. “Theseproteins are assumed to possess 2, or though certain insects use steroids and polyisoprenoid com at least 2, st.ereospecifically different., mionoverlapping bi rid pounds as hormones (116, 207), there is no evidence for ing sites. One of these, the active site, binds the substrate steroid hormones in many lower invertebrates (despite the amid is responsible for the biologic activity of the protein. prediction of Schneidermami arid Gilbert (207) that this The other site, or allosteric site, is complementary to the may be the case). Amid steroids seem to be totally absent structure of another metabolite, the allosleric effector, which from certaimi primitive forms of life, such as bacteria and it binds specifically arid reversibly. The formation of the blue-green algae (236), although they may be abundant. in cnrzyme-allost.eric effector complex does not activate a re some fungi. As the Scharrers discuss in their admirable action irivolvimig the effector itself; it is assumed only to hook (205), mi lower forms such as crustaceans arid amine bring about a discrete reversible alteration of the structure lids, integrative functions are in the maimi the preroga of the l)rot..ein or ailosteric transition, which modifies the tive of the nervous system, either as a result of nervous structure of the active site, changing 1 or several of the ttctioii as such, or via the production of hormone symithe kinetic parameters which characterize the biologic activity sized by cells of a neural character. “Asone proceeds of the 1)roteini―(159). Allosteric transitions are envisaged from lower to higher orders of animals the ratio between by Monod et al. to consist essentially of conformational at hormone-producing arid purely nervous mechanisms terations in protein structure, which may or may not result changes, since progressive evolution of sensory centers and in aggregations or dissociations of subunits. Phenomena motor systems is not matched by a correspomiding expami of this type have, of course, been known in biochemistry sioni of the endocrine functions of nervous tissue. Instead, for a long time, one of the mos.t thoroughly investigated tliP latter l)ecome increasingly delegated to glands outside being the imicrease of oxygen dissociatiomi of hemoglobin t he central nervous system. These organs serve as ampli which accompanies a lowering of pH (Bohr effect). Allo tiers of blood-borne sigmials which the nervous system con steric effectors by definition mieed bear no chemical resem t inues to emit., even in the most. highly developed mam blance to the substrates of catalytic proteins, as opposed mals― (205). The idea that steroid hormones act. as to “isost.eric―effectorsof the type responsible for compet. coerizymes iniplies that, parallel with their relatively late itive inhibitions. The concept of allosteric binding sites (‘volutionary selectiomi as humoral regulators, there occur is of especial importance iii uniderstamiding the mechanism red niut.ationis permitting the synthesis of miew classes of of “endproduct― or “feedback―inhibitions that are in si eroid-dependent enzymes which would presumably be creasingly being found to be important in the control of bio rate-limiting for various metabolic pathways. On the synthetic pathways. Usually in such feedback inhibitions, contrary, the coenzymatic role of hemes, trace metals, amid the 1st enzyme involved in a biosynthetic chain is cx water-soluble vitamin derivatives seems to have been man t.remely sensitive to inhibition of the product. of the fimial ifest in the most primitive cells. reactions, e. g., the inhibition of aspartic traniscarbamyla.se ‘rheinamn@alianmhydroxysteroiddehydrogemmasesthat. (the 1st. enzyme required for j)yrimidine synthesis) by (‘@ttrtlyze steroid-mediat ed t.ranishydrogenase reactions have cytidine triphosphat.e (253). Allost.eric proteins cart lose been studied extensively from the stamidpoint of steroid their sensitivity to allosteric inhibitors after treatments, binding and sl)ecificity (2, 85, 238, 239), as have other pu such as mild heating or exposure to urea, which do not rihdd enzymes that catalyze transformations of steroids, affect their actual catalytic activity. Amid “desensitiza like the NAD-specific hydroxysteroid dehydrogenases tioni―to allosteric effectors without. loss of catalytic iower (153, 237, 239) amid the steroid isonTlerase (258) of Pseudo may occur after the structure of an allosteric protein is ))lOfl@LS leslosteronj. These i ivestigat.ions have delineated modified as a result of genie mutation (159, 253). Ilianmy of the factors which determine the fit of steroids to The theory of allosteric binding sites on regulatory pro till' proteins, and competitions between various steroids t.eins is related to Koshland's (133) “induced fit―theory of amid related substances for' the active sites. (Measure enzyme action, which postulates that substrates also in mnents of the free energy changes involved in noncovalent. duce configurational changes iii catalytic proteins. interactions of steroid hormones with various plasma pro Moniod et a!. (159) consider the possibility that low mo leinis (264) are of much interest from the stamidpoinit of lecular weight hormones may function as allosteric effect hormimonetransport in blood, hut are probably largely ii-- 01-s. As we shall see, this is an attractive idea, especially relevant to the problem of the nature of hormone receptors as the allosteric proteins with which the hormones react in tissues, as are studies (231) on rather nonspecific inter might. be not only enzymes of intermediary metabolism a.s @tdtiOlis of steroids amid diethylstilbestrol with polynucleo classically defined, but. also genetic repressor-s. However, I ides arid crude histomie preparations.) But combinatiomis this hypothesis is obviously of little value unless the pro of various steroids with these refined steroid-metabolizing teins with allosteric binding sites which specifically recog enzymes are largely if not exclusively due to “isosteric― nize hormones can be identified rigorously, amid until it is initeractiomis with the active sites of the proteins that. are proved that such l)roteimis are indeed key determinants of respomisible for their catalytic activity. These may be less the hormonal responses. Allosteric phenomena are wide ielevant to the problem of hormonal action than are ‘ailo spread, and steroids can induce changes in the conforma steric' transitions of the type niom@'to be discussed. tion, amid hence catalytic activity, of a number of enzymes

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that are probably not of major importance to the action of protein concentrations, whereas determinations of enzyme the hormones. As Monod et a!. themselves state (159): activity were by necessity monitored at low enzyme con “Themost serious objection to the concept of allosteric centrations. Frieden (cf. Ref. 59) provided evidence that control is that it could be used to ‘explainaway' almost any at the high dilutions required for estimation of catalytic mysterious physiologic mechanism.― activities, glutamic dehydrogenase remains dissociated To begin with, we may mention a number of in vitro ac into subunits. But even though the extent of association tioiis of steroids and synthetic estrogens on purified en per se does not seem to affect enzyme activity, relationships zymes which indeed may turn out to be examples of hor between inhibition of glutamic dehydrogenase function mone-induced allosteric transitions. These will not be and extent of dissociation (measured at high protein con considered in extenso, as they are the subject of reviews by centration) may be meaningful because of the ability of the Tomkins et al. (248, 249) and by others (271, 275, 280). steroids to induce conformational changes in the enzyme Suffice it to say that inhibitions by direct addition of ste (59). It is noteworthy that thyroxine is also a powerful roid hormones of enzymes such as glucose 6-phosphate inhibitor of glutamic dehydrogenase and causes the protein dehydrogenase (143), succinic oxidase (33), and NADH to dissociate into subunits (cf. Refs. 248, 280). That these cytochrome c reductase (282) may well be due to allosteric effects of steroids are allosteric in nature is corroborated by alterations in conformation of the activating proteins. It the fact that the sensitivity of the enzyme to steroids is is conceivable that these phenomena may be related to the abolished after treatment of the protein with p-hydroxy depression by certain steroids of respiration, glycolysis, mercuribenzoate sulfonate. Yielding and Tomkimis (281) and synthetic reactions in some cells, which may in turn suggest that certain steroids and diethyistilbestrol alter underlie such biologic events as the lymphocytolytic ac the conformation (and hence catalytic activities) of glu tionis of adremial steroids, as White el at. suggest (265). tamic dehydrogenase by interfering with noncovalent The inhibition of various pyridoxal phosphate-dependent (probably hydrophobic) bonding between the subunits. enzymes by disulfate (155) and phosphate (204) esters of The steroid-sensitive liver aldehyde dehydrogenase estrogenis may also represent allosteric phenomena, al which is activated or inhibited by various steroids, arid though in these situations there may be direct competition which has been studied so carefully by Maxwell (156) arid between the estrogen esters and the pyridoxal phosphate Topper et at. (250) is another example of an enzyme that is binding sites. (It must be remembered that there is no subject to allosteric regulation by hormones. Here again, evidence for any hormonal activity of the estrogen esters). there is evidence (156) suggesting that steroids modify the Other effects of progesterone and deoxycorticosterone on tertiary structure of the protein via noncovalent interac mitochondrial ATPase arid oxidative phosphorylation (cf. tions. But in contrast to the findings with glutamic dehy Refs. 15, 248) are probably more complex and may be re drogenase, the steroidal effects on the hepatic aldehyde lated to changes in permeability of the mitchondrial mem dehydrogenase are not accompanied by changes in the branes. sedimentation characteristics of the protein (156). These An important series of investigations by Tomkins and allosteric effects probably account for the stimulation of his co-workers (249, 281) has shown that some steroids can galactose oxidation in liver by progesterone, since by imi induce unequivocal allosteric effects on crystalline liver hibiting the aldehyde dehydrogenase, the latter hormone glutamic dehydrogenase, a protein whose molecular weight diminishes the formation of NADH, which is in turn an is about 1 million and which can dissociate into 4 appar inhibitor of the UDP-galactose-4-epimerase reaction, one ently identical subunits of 250,000 daltons. The dissocia of the initial steps in galactose oxidation (250). Again, tiomi of the tetramer is accompanied by loss of ability of the Kimberg and Yielding (122) have suggested that the in protein to catalyze the reductive amination of cr-ketoglu hibitions of crystalline pyruvic kinase by diethyistilbestrol tarate. Frieden (58) discovered that NADH, 1 , 10-o- and some steroid hormones are allosteric phenomena. phenanthroline, and high pH all cause dissociation of the Yet it is improbable that widespread physiologic sig enzyme into subunits, whereas association into the tetra nificance can be attributed to the aforementioned allosteric meric form is favored by NAD and also ADP, both of effects of steroids on purified enzymes. This is because which counteract the NADH-induced dissociation. Tom (a) the concentration of hormones necessary to elicit these kins et at. (249, 281) showed that sex hormones such as actions in vitro is extraordinarily high, (b) there is little estradiol-17f3, diethyistilbestrol, and progesterone also en correlation between the ability of these hormones to serve hance dissociation of glutamic dehydrogenase. The en as allosteric effectors in vitro and their major biologic po zyme dissociated by these or other means exhibited a tencies in vivo, (c) similar conformational cum catalytic diminished glutamic dehydrogenase activity, whereas its changes are induced in the enzymes by hormonally inactive alanine dehydrogenase function was enhanced. The hor substances such as o-phenanthroline and detergents. mones thus affected the catalytic properties of glutamic Nevertheless, these model investigations have established dehydrogenase in a qualitative as well as quantitative that steroids are capable of inducing profound modifica manner. It was shown (281) that NAD, ADP, and leucine tions in the tertiary structure of enzymes, by combining (which enhance association of subunits) prevent dissocia with sites on the proteins other than those involved in the tions induced by NADH or diethyistilbestrol. It must be binding and activation of the substrates. emphasized, however, that the changes in molecular weight The foregoing examples of coenzymatic and allosteric of glut.amic dehydrogenase resulting from various treat effectsof steroid hormones have dealt with the regulation ments were determined by ultracentrifugation and light of enzyme activities, which, likefeedback controls (253), can scattering measurements conducted at relatively high be regarded as “finecontrol―mechanisms and are manifest

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLIAMs-AsHM@—Steroid Hormone Action 1103 quickly. But evidence is beginning to accumulate that zymes were detected after treatment with corticosteroids many steroids primarily interfere with more sluggish that increased the activity of tryptophan pyrrolase (222). “coarsecontrol― mechanisms involved in the synthesis of Although the control of amino acid transport and activa enzymes and other proteins. This leads to considerations tion by steroid hormones merits much further investiga of the hormonal control of gene expression. For the bio tion, many effects of sex and adrenocortical hormones on chemistry of specific protein biosynthesis can no longer, protein biosynthesis do not seem to be operative at this even in the most highly differentiated of nondividing cells, level. be divorced from gene-determined processes in which var Rok of ribonucleic aci@1sin gene expression ari4 the operon ious types of polyribonucleotide play a central role (146, concept.—The participation of nucleic acids in protein bio 208). synthesis is the subject of many reviews (see Refs. 12, 104, 146, 208, 276 for references), and only a few very recent REGULATION OF GENE EXPRESSION BY developments need be considered here. It must be em STEROID HORMONES phasized that contemporary insight into the biochemistry Recent studies involving inhibitors of RNA and protein of gene expression rests largely on investigations with bac synthesis (68, 70, 73, 172), and of a kinetic (196, 211, 212) teria, and that some concepts which have emerged from and immunochemical (55, 117, 210) nature have estab microbiologic experiments may not be strictly applicable lished that many steroids can govern the rate of de novo to the problems of gene action and protein biosynthesis in synthesis of many enzymes in various tissues—a conclusion mammalian tissues. Considerations of the fabrication in previously suspected from innumerable observations on the nucleus of messenger RNA's and their utilization by variations in tissue enzyme activities induced by adminis ribosomal systems have featured most prominently in tration or deprivation of sex and adrenocortical hormones studies on the steroid hormonal control of protein synthe (102,126—28,164,186,195,246,270,275).Themajoritysis, and it is noteworthy that little attention has been given of recent investigations on the mechanism of steroid hor in this respect to the mitochondrial protein-synthesizing monal control of the biosynthesis of various proteins have machinery. The requirements for incorporation of labeled centered around the production and utilization of ribo amino acids into the proteins of isolated mitochondria (142) nucleic acid intermediates. But first we may briefly con are remarkably different from those manifest by ribo sider the possible hormonal control of other possible rate nucleoprotein particles associated with the endoplasmic limiting steps in protein synthesis. reticulum (208). This may have bearing on hormonal Amino acid transport and activatüm.—Two ways in control of protein synthesis in certain cells, now that evi which steroids might regulate the formation of proteins dence is accumulating for the genuine existence of DNA in are, of course, to govern either the penetration of amino mitochondria, and for regulation of the self-duplication of acids into cells, or their activation (at the expense of these organelles by their own (DNA) constitutive and regu ATP) to form transfer RNA-amino acids. These topics lator genes (65). (An increased tissue oxygen consump have been recently reviewed by Tomkins and Maxwell tion and epithelial cell mitochondrial population density (248) and Segal (213). Treatment with steroids in vivo (177, 186, 270) are striking early concomitants of androgen can indeed result in large changes in the passage of labeled action on male accessory genital organs.) amino acids into various tissues, as studied in living It is commonly held that there exist 3 major types of animals (cf. Ref. 213) of after removal therefrom (175). ribonucleic acid—transfer, ribosomal, and messenger But in most instances, comparable effects of steroids in and that, with the possible exception of the pCpCpA ter vitro on the active or passive entry of amino acids into mini of transfer RNA's, all of their base sequences are isolated intact cell preparations have not been demonstra complementary to specific cistrons located on the nuclear ted, although Christensen et at. (35) reported that estrogen DNA. Suggestions (189) that in animal tissues the bulk sulfates directly accelerate the concentration of amino ribosomal RNA's may not be readily distinguishable from acids by ascites tumor cells. In many experiments on the messenger RNA copies of DNA structural gene cistrons influence of steroid hormones on these processes in vivo, it are based on rather flimsy evidence, and there are reasons is not altogether clear whether the measured uptake or to believe (146) that the messenger and ribosomal RNA's intracellular accumulation of amino acids is really due to do not simply “melt―intoone another. However, avail their active transport, because the former represents the able methods for separation and identification of discrete summation of movements of amino acids both into and out RNA species leave much to be desired. A clear-cut de of cells (213). The biochemistry of amino acid accumula generacy with regard to certain transfer RNA's cognate tion in tissues is poorly understood, and the nature of the to the 20 amino acids commonly found in proteins is firmly effects of steroid hormones on these processes remains to be established, and the different transfer RNA's for some in clarified.6 Gonadal hormones increase the activity of var dividual amino acids can be separated chromatographically ious amino acid-activating enzymes (as determined by (208). But fractionation and determination of the tern amino acid-dependent ATP-PP1 exchanges—procedures plate activity of various messenger RNA's with respect to somewhat open to criticism) in certain male (131) and incorporation of amino acids into specific proteins (as op female (163, 164) accessory sexual organs. But no varia posed to ill-defined mixtures of polypeptide material) is tions in the levels of hepatic amino acid-activating en still hard to achieve (cf. Refs. 69, 75). The extraordinary complexity of the genetic coding problem (12, 141, 173) is 6 Riggs (192) has given a comprehensive account of the influ ence of steroid and other hormones on the transport of substances emphasized by recent demonstrations that the template across cell membranes. activity of polyribonucleotides in ribosomal systems is

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extremely dependent upon their ordered secondary struc broadened by the additionmal idea that low molecular weight ture in solution (221, 223, 235), as well as upon their base effectors combimie with allost.eric sites on the repressors and sequeni('es (cf. Rcfs. 141, 173). Criteria such as rapid thereby induce conformationial chamiges which would, de met ai)Olic turnover, DNA-like base composition, ability pending on the type of repressor, activate or inhibit the to hybridize with homologous DNA's, sedimentation char ability of other sites on the repressor proteins to react with acteristics, etc., are riot absolutely diagnostic of messenger appropriate operator genies. RNA. Arid the nature of the nomiimiformnational bulk The essence of the operon model of Jacob amid Moniod ribosonmal RNA's—with sedinienitat ion constants of about is that repressors act negatively to prevent. RNA transcrip 18 5 arid 28 S—in various animal tissues is miot.well under tion, and hence protein synthesis under the direction of stood. It seems that pulse-labeled RNA in mammalian various structural genies. According to this hypothesis, 1issues is coml)rised of 2 maui components : (a) a high mo “induction = derepressioni.― In this way, it might be lecular weight material (approximately 45 S )—apparenitly possible to comprehend how low molecular weight hor of nmucicolar origin—which can break down by an act.ino mones (such as steroids) could regulate expression of in mycin D-insensit.ive pn-ocess into 28 S arid 18 S ribosomal dividual genies—or, as is more likely, of whole operons—by RNA (206, 242), arid (b) a fractiomi which preferentially serving as allosteric effect.ors of certain repressors. Karl hybmidizes with DNA (181), is heterogeneous in size (64, son (115, 116) and Zalokar (284) have promulgated the 206, @42),and exhibits a DNA-like base composition (64). revolutionary hypothesis that some hormones (especially The latter RNA probably is synthesized in noriniucleolar those that have a lonmglatemit period of action and control l)arts of the miucleus amid rutty be largely messemiger RNA, the functional differentiation of various tissues) may ef although this has not been proved rigorously. The mecenit fectively serve as gene regulators. Vulgar misconceptions developniemit by Kirby (cf Ref. 121) of countercurrent of these notions invoking the improbable idea that hor methods for separation of messenger-like RNA's holds monies must, therefore, recognize and engage with discrete great promise, but studies on messenger RNA synthesis loci omi DNA or complementary RNA's are rendered mm amidutilization will 1Wiflil)eded until precise determination necessary by application of the operon concept, if it is as of their imidividual teniplat.e activities is possible. sumed that the repressors are proteins rather than poiy The question as to the for-rn in which RNA copies of nucleotides. It may be added that although the operont structural genie DNA templates are fabricated amid utilized model emerged from considerations of regulation of en in protein syiithesis is of central importamice to uniderstanid zyme synthesis in microorgammisms, it has had fruitful ap ing the regulation of genie expression. 1mmthis conimiectioni, plication to problems in the genetic control of the synthesis the “operomi―modelof Jacob and Monod (107) has had of mammalian proteins, notably hemoglobin (9). The widespread influence, especially on some speculations on value of the operon concept will remain limited, however, steroid hormone actioni. Genetic amid enizymological cx until time repressors are isolated as discrete entities arid l)cn'in@enmtsoniinducible annd repressible erizymites in viral their chemical properties ascertained. This will require arid bacterial systemlis led Jacob amid Monod (107) to pos development of in vitro test. systems for repressor functions. t.ulate that groups of (Onitiguous structural genie cistrons Over the last 2 years, enmeridatiomis of the operon model on the DNA may for-rimdiscrete functional units or operons, have been proposed which are not. without interest to the the expression of which may be governed by a sinigle linked problem of hormone action. These advances are reviewed operator gene. It ivas enivisagtXl that if the operator is by Stent. (229) amid Ames arid Martini (6). The original “open,―thermniesseniger I@NA copies of every structural theory of .Jacob arid Monod (107) envisaged messenger genie cist.ron of the operoni can be synthesized. But if the RNA copies of each structural genie as individual entities operator is “closed―theirnionie of the structural genes on arid placed control of specific proteimi biosynthesis at the the operon (armserve as templates for contplememitary RNA level of messenger RNA production rather than utilization. synit hesis. The theory further assumed that specific “re Indeed, there is evidence (cf Refs. 6, 229) that. low molec l)ress@rs―(themselves products of other, so-called regulator ular weight enzyme inmducersenhance the accumulation in genies) dttfl combine with arid inactivate the operators and growing bacteria of the corresponding messenmger RNA's. hence silence whole operonis. The ability of small mole Amid it has become evident. that polycist.ronic messenger citIes to serve as inducers or repressors of enzyme formatiomi RNA copies of whole operonis, rather than of individual \@‘aspostulatedto result front their interactioni with 2 types structural genies, may be formed in vivo, i. e., there being of repressor. If the repressor is inactivated by the low “1operomm-1messenger― rather than “1gene-i messenger.― molecular @@eight.effector,then the effector serves as ami The discovery of polysomes (cf. Ref. 259 for references) elizynie inducer by preventing fruitful engagement of the provided a mechanism for the readimig of such polycistronic repressor with the oj)erntor. Contrariwise, other types of messengers, which earn be in simultaneous combination mepressor may exist. which are activated by small molecules, with nmamiyribosornes. But it now appears that control of which in this instance would suppress enzyme formation. protein synthesis may operate at the level of polycistronmic Although it was originally believed by Jacob amid Momiod messenger RNA function as well as of transcription of these (107) that the repressor pmoducts of regulator genies were ribonucleic acids off the DNA template. Oht.aka arid polyribonucleotides, t his hypothesis was abamidommedfol Spiegelmani (i78) have imideed obtained evidence for- regu lowing inter alia the discovery of temperature-semisitive and lationi ill a microbial cell-free system of the reading of also effect.or-insensitive repressor mutations (ci. Refs. 6, polycitronmic viral RNA messages. 229). It. was then suggested that repressors are proteins Control of protein synthesis at a translational rather rrtthei thafl riboriucleic acids (159). This theory was than traricriptiorial level might ‘iccountfor the i)hellomenia

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLIAMs-AsHMAN—Steroid Hormone Action 1105 of polarity mutations (6, 229), in which the synthesis of 1 translation by availability of particular forms of degenerate erizynme on a particular operoni is lost., while the formation transfer RNA's, it may be mentioned that in a study on of other emizymes on the operorm is reduced. Genetic niap the influence of diethylstilbestrol omiphosphoproteini syni ping experiments show that in such polarity mutationms, thesis in chickens, Carlsen et at. (32) observed that the the cistromi of the deleted enzyme is always nearer to the production arid turnover of specific hepatic transfer RNA's operator genie than are the cistronms concerned with other for serine and phosphoserine were increased after adminis enzymes on the operori whose levels are diminished but not tration of this estrogen. obliterated. Furthermore, Zabin (283) found that the The foregoing considerations underscore the large gaps rate of synthesis of proteins corresponding to operator mmour understanding of the euzymology of complementary l)roxinimalcist.ronmsmay, on a molar basis, far exceed that of RNA and protein synthesis. Only very recently, for cx proteins whose cistrons are more distal to the operator ample, has it become apparent that biosynthesis of the genie, event though the operator control of formation of all minor nmethylated bases mmDNA and RNA involves t rans enzymes on the operon is invariably coordinated in the methylations at a polynueleotide level, probably iii the same fashion. These facts are umiderstandable if it is as nucleolus (14, 226); that selective transcription of RXA sumed (cf. Refs. 6, 229) that protein synthesis is initiated copies of only 1 strand of DNA duplexes (previously dent at the region of the polycistroniic messenger which corre onstrated in living cells) cart occur in vitro by arm as yet sponids to the operator-proximal cistroim, amid that the tem nTiysterious process dependent upon the continuity arid plate function of the polycistronic nimessemigerRNA cart be conformation of the DNA printer (63); arid that in isolated halted before every cistron has been deciphered. This ribosomal systems, small molecules such as streptomycini iml)iies that the farther away a cistroni is from the operator can cause specific alterations mi the coding properties of gene of the operon, the lower will be the rate of over-all synthetic polynucleotides, the code shifts being determined synthesis of the proteimi. This in turn could be explained by the genetics of the cells from which the ribosomes are if it is assumed that the trarislatiomi process could be stop derived (42). The rapid pace of discovery of new arid l)ed at a particular point (niucleotide sequence) on a poly often unsuspected reactions in this field will surely riot cistronic messenger, which could be determined by coding diminish in the near future, amid as each new enzymatic for a specific degenerate species of transfer RNA. As process is uncovered, the j)ermutations amid combinations Stent (229) says: “Ifthe intracellular availabilities of dif of possible I)Oiflts of hormonial control become more and fererit transfer RNA species cognate to the same anmirio niore formidable. acid are widely different, corresponding to ‘major' and Messenger RNA stability and cell dmfferentialzon.-—Be ‘minor'representations of that amino acid, therm the rate fore turning to specific details of the steroid hornional of synthesis of a polypeptide, and of all polypeptides of control of RNA and protein synthesis, it may be worth operator-distal cistroims carried by the same polycistronic while to consider the question of messenger RNA stability mimesseriger,depends on the relative occurrence of major amid in relation to cell differentiatiomi. This is particularly ger minor coding representations in the cistronuc message.― marie to the regulation by sex hormones of the funictionial Stent (229) suggests that repressors primarily affect differentiation of accessory reproductive organs. It is now messenger function arid that they nmay be enzymes which widely held that the differentiation process does riot in degrade particular transfer RNA species, perhaps by cat volve segregation of the geriot.ype, but rather the sequeni alyzing the hydrolysis or phosphorolysis of the terminal tial activation or depression of specific genie function along pCpCpA sequence. “Theessence of this notion is, there the time axis of development, which cant be influenced by fore, that the nucleotide sequence of one or nmore cistrons specific molecules in the cellular environment. Lit.tau et of an operon contains one or more niodulatimig codoris call al. (147) provided evidence that. in highly differentiated ing for transfer RNA molecules whose . . . pCpCpA thymocytes, as much as 80 % of time DNA (probably that terminus is subject. to both removal and restoration by portiomi appearing in a conidemised rather than in a diffuse sl)eciflc regulator enzymes.― In other words “regulator and extended state) may be repressed arid incapable of genes code for the t.ransfer-RNA-specific nucleases or serving as a template for complenmentary RNA synthesis. phosphorylases, arid the operator genes are . . . genies As adunibrated by the original niesseriger hypothesis responsible for synthesis of the corresponding species of (cf. Ref. 107), many bacterial RNA's holding informimationi modulating transfer RNA― (229). It was further postu for the synthesis of specific enzynmes are metabolically very lated that feedback loops may exist between messenger unstable. In exponentially growimig bacteria, the average formation and function and that. the faster the messenger half-life of RNA messengers is of the order of a few nun works in protein synthesis, the more rapidly it is syrithe (53). The bacterial messeniger RNA's seem to be pro sized. Noteworthy in this connection are the recent cx tected against degradationi as long as they are in conubinia periment.s of Byrne et at. (30) demonstrating a coupling tion with the ribosomes, but. are rapidly destroyed after between RNA transcription and ribosomal protein synthe their release from the ribomiucleoprot.eimi l)articles (53). sis iii E. coli, translation of the complementary RNA mes But in some animal cells, certain messenger RNA's nitty sage apparently beginning while the complementary RNA be very long lived, for the cells continue to synthesize is still bound to the DNA template arid continuing after proteins and DNA for protracted periods after exposun-e to its release therefrom. This might. represent a mechanism concentrations of actinonmycini D which virtually halt all for the stabilization of messenger RXA against digestion DNA-directed RNA synthesis (i90, 191, 209). Davidson by nucleases or other enzymes. et al. (41) observed that the biosynthesis of some structural In the light. of the possible control of messenger RNA cell components iii cultures of differentiated mammalian

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cells declined rapidly after addition of actinomycin D, teran polytene chromosomes changes precisely during whereas the levels of succinic dehydrogenase remained un development of the insects, and ecdysone administration changed, suggesting large differences in the stability of induces puffing patterns which are identical with those oc messenger RNA's cognate to the various proteins. The curring during pupation. It was shown (36, 37, 116) that lack of RNA synthesis in reticulocytes which actively the “puffs―(whichseem to result from unfolding of the manufacture hemoglobin is well known (208), pointing to genetic material) are active sites of RNA synthesis. Front a high degree of stability of the globin messenger. The the studies of Clever (36) and Karlson (1 15, 116) the con studies of Gross et at. (76, 77) on early development in cept emerged that ecdysone can differentially activate Arbacia punctulata provided an interesting sidelight on the specific genes at key times during development of the stability of template RNA's in relation to cell growth and creatures. The effects of ecdysone on the puffing pattern differemutiation. It was shown that the increase of amino are manifest within 15 mm after injection of as little as acid incorporation into proteins which occurs after fertiiz 10--s @gofthe steroid. Experiments in living insects with ation is quite refractory to actinomycin D. The antibiotic various inhibitors of RNA and protein synthesis (215) are has little effect on protein synthesis or cell division until consistent with Karlson's hypothesis (1 15, 116) that ec the blastula stage. This suggests that after fertilization, dysone acts in the nucleus to accelerate the fabrication of pre-exist.ing RNA templates that were somehow seques specific messenger RNA's, and hence of specific proteins. tered become ‘unmasked', and can function in ribosomal Sekeris and Lang (217) reported that RNA isolated by protein synthesis. In line with thisconclusionis the finding Georgiev's hot phenol method (cf. Ref. 64) from white of Nerner (170) that the postfertiization increase in the prepupae of the blowfly Calliphora erythrocephata stimula capacity of isolated sea urchin egg ribosornes to incorporate ted the formation of dopa decarboxylase by an in vitro rat amino acids into protein is accompanied by a decrease in liver microsomal system. In this species, ecdysone secre the response of these particles to synthetic polyribonucle tion occurs only a few hr before pupation (8th day of larval otides that code for the appropriate amino acids. Thus, life), and 7-day-old larvae were used as naive controls. after fertilization an increasing number of ribosomes ap (Pupal nuolting begins with sclerotization, and ecdysone pear to be pre-empted with messenger RNA's of high sta induces the formation of dopa decarboxylase in the larvae, bility, which exist in unavailable form in the unfertilized thus enabling synthesis of N-acetyldopamine, the sciero egg amid only become functional after activation. How tizing substance.) RNA extracted from the 7-day-old lar ever, prevention of new RNA synthesis by actinornycin D vae did not induce dopa decarboxylase in the liver riboso completely stops all true differentiation, so that the anti mal system and was stated to be much less effective than biotic halts development at the gastrula stage, which in RNA prepared from the (ecdysone-stimulated) white pre this instance appears as an inert multicellular mass. pupae in enhancing leucine incorporation into acid-insolu Scott and Bell (209) have concluded that “utilizationof ble proteins by liver ribosomes. These preliminary find long-lived messenger RNA appears to be the exception ings were considered by Sekeris and Lang (217) as evidence rather than the rule in cells which are differentiating and that ecdysone induces the formation in the blowflies of the synthesizing large amounts of specialized product at the messenger RNA for dopa decarboxylase. same time.― However, evidence was obtained for long An important question is whether the effects of ecdysone lived messengers in ocular lens, down feather, and reticu of RNA synthesis at discrete loci on the polytene chromo locyte cells, which make large quantities of a very limited somes are direct or indirect. Clever (36) and Karlson number of proteins. “Inallthree cell types the nucleus is (116) proposed that ecdysone regulates complementary effectively ‘turnedoff' as differentiation of the cells pro RNA formation in the cell nucleus, perhaps by acting as an gresses toward its terminal state. By the time this occurs, anti-repressor. In line with this view is the fact that in differentiation of these cell types is a one way street paved jection of actinomycin D or mitomycin simultaneously with messenger RNA molecules which have a long half with ecdysone inhibits the puffs induced by the latter hor life―(209). mones, whereas puromycin does not affect these chromo At present, very little is known about the selective sta somal responses (36, 37). However, puffing patterns in bilization of specific RNA copies of structural gene cistrons giant chromosomes are also induced by rather nonspecific in nuammalian cells, or about the biochemistry of the deg agents such as narcotics, zinc and cadmium ions (136), radation of these RNA messengers. The close associa temperature shock (36), ribonuclease (194), and so on. tion of (poly)ribosomes with the lipoprotein nuembranes of Kroeger (136) believes that ecdysone works indirectly, by the endoplasmic reticulum of many mammalian cells may changing the permeability of the nuclear membrane to be particularly germane to the problem of nuessenger R.NA sodium and potassium ions. He claims that by altering stability. the sodium and potassium levels of the fluid bathimug sali Action of insect steroid hormones.—A series of investi vary gland explants he can change the puffing pattern of gations on the mechanism of action of ecdysone—a the giant chromosomes. But other workers (cf. Refs. 36, steroid secreted by the prothoracic gland of certain in 37) find that the puffing patterns resulting from these non sects amid which initiates larval growth and molting—has specific treatments are not really comparable with those had considerable impact on recent thinking about hor induced by ecdysone, and Kroeger's (135, 136) view that mone action. This is because injection of ecdysone in hormone's effects on polytene chromosomal RNA synthe duces “puffing―at specific locations on the giant chromo sis are mediated via changes in the intranuclear ionic en somes present in the salivary glands of Diptera such as vironment have not found general acceptance (37). The Chironomus (36, 116, 207). The puffing pattern of dip possibility that the insect juvenile hormone (the action of

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLI@s-AsHMAN—Steroid Hormone Action 1107 which is mimicked by terpenes such as farnesol and far effects of the inhibitors are often dependent on the nuode nesyl-O-methyl ether) also acts on RNA synthesis in the and time of their administration. nucleus is considered by Schneiderman and Gilbert (207). Many of the early biochemical changes in uteri of juve Burdette et at. (21—24)clainu that growth and protein nile animals induced by natural or synthetic estrogens are synthesis in certain mammalian cells, including some tu blocked by prior or simultaneous treatment of the animals rumors,can be influenced by ecydsone. It would be interest with puromycin or actinomycin D. Thus Mueller (165) irig to examine in detail the effects of nuammalian steroid showed that injection of puromycin nullified the early ef hormones on developmental processes and polyterme chro fects (164) of estradiol pretreatment on the entry of radio mosome puffing patterns in insects. active precursors into proteins, phospholipids, and RNA Rzlxmucleic acid and protein synthesis in relation to ster by uterine horns front spayed rats. Puromycin also in oid hormone action.—One approach to the mecharmism of hibited the rapid water imbibition in the uterus in response action of steroid hormones has been to study interference to estrogen. (Recently, Jensen (1 10) showed that neither with their biologic actions by inhibitors of discrete meta puromycin nor actinomycin D impede the uptake and re bolic events. Perhaps the first experiments of this nature tention of radioactive estradiol-i7@3 by the rat uterus.) were carried out by Hertz and Tuliner (89), who discovered Cycloheximide (actidione)—another substance which in that administration of some antagonists of purmnes arid of hibits protein synthesis without affecting RNA or phos folic acid obstructs the growth response of accessory re pholipid synthesis—depresses the elevation in uterine wet productive organs to exogenous and endogenous estrogens. weight and in RNA and phospholipid synthesis, which oc Following the pioneer investigations of Mueller et al. (164, cur a few hr after treatment of immature rats with estrogen 165, 252), much attention has been given of late to inhib (68). Ui and Mueller (252) showed that actinonmycin itors of RNA and protein synthesis as antagonists of sex treatnuent counteracted estradiol-stimulated uterine pro and adrenocortical hormones. In this connection, ethio tein and lipid synthesis, but did not depress these proc nine and puromycin have found favor as inhibitors of eases in ovariectomized controls that did not receive the protein synthesis, and blockade of various steroid effects hormone. Mueller's experiments on inhibition of the early by actinomycin D has been cited as evidence that new biochemical actions of estrogens on rat uterus were cor DNA-directed RNA synthesis (84, 188, 190) is involved in roborated by Hamilton (79, 80). Taiwar and Segal (241) the hormonal responses. Again, purine and pyrimidine reported that local application of actinomycin D prevented analogs that are capable, among other things, of being in the vaginal cornification induced by estradiol-17fl; they corporated into “fraudulent―polynucleotides (87) were also observed that actinomycin D inhibited the action of found to depress some effects of steroid hormones. Ob some other hormones, including the response of inunuature viously such inhibition experimmuemitsareopen to all sorts of rat testis to gonadotropins. Effects of actinomycin and artifactual complications. Many of the inhibitors used puromycin on the response of isolated toad bladder to are extremely toxic substances, and in some instances their are discussed below. It should be emphasized influence on steroid actions in vivo might be due largely to that other actions of various mammalian hormones on a nonspecific nutritional debilitation, or even, in vitro, to complex physiologic events are not blocked by puromycin cell death. Again, it may be significant, in the light of or actinomycin D. This is true, for example, of the action well known antagonisms between various types of steroid of insulin on glucose uptake by isolated rat diaphragm (50) hormones, that Planelles et al. (184) found an increase in and on blood sugar levels in vivo (228). And although, as the 17-hydroxycorticosteroid level of guinea pig blood we shall consider shortly, actinomycin D prevents the in plasma after single injections of small doses of actinonmycin. duction of many hepatic enzymes by hydrocortisone, it And many metabolic inhibitors may have multiple sites of does not seem to retard other effects of glucocorticoids in action. For example, ethionine has long been regarded as vivo. Ray et at. (187) reported, for example, that hydro an inhibitor of protein synthesis, but recent evidemuce (54) cortisone stimulates gluconeogenesis (as reflected by blood points to this amino acid analog being a rather general sugar and liver glycogen levels) in both normal and adre “ATPtrap― which preferentially bleeds off ATP in the nalectomized rats even though the creatures were pre form of S-adenosylethionine. Ethionine can therefore treated with actinomycin D in sufficient amounts t.o de affect a host of metabolic processes in a rather unspecific press RNA synthesis, and niutatis mutandis, to supress fashion. Inspection of the structural formula of puromy completely the hydrocortisone-induced induction of he cm might lead one to suspect that this antibiotic may inter patic pyruvate carboxykinase7 (cf Ref. 140). fere with enzymatic reactions (cf. Ref. 68) other than those Another inhibitor which interferes with certain action of involved in aminoacyl transfers from transfer RNA-amino steroid hormones is the pyrimidine analog 5-fluorouracil. acids in ribosomal systems (168). Moreover, dernonstra Cantarow and Zagerman (31) observed that fluorouracil tion that a particular metabolic inhibitor blocks a response inhibits the testosterone-stimulated growth of seminal yes to any steroid does not necessarily imply that the reactions ides in orchiectomized rats. The extent of the inhibition that are sensitive to the inhibitor are the primary targets was dependent upon the amount of hormone administered of the hormone ; it simply nmeans that intactness of the and could be overcome completely by massive (100 mg/ relevant enzyme systems is required at some stage(s) in the train of biochemical events set in motion by the hor 7 Munck and Koritz (166) found that the deposition of liver nuone. But all of these caveats notwithstanding, recent glycogen in the fasted adrenalectomized rat induced by either cortisol or glucose isprevented by ethionine, but also by methionine inhibitor studies have provided considerable insight into and other agents. They concluded that enzyme induction is not the actions of sonic steroids. As might be expected, the involved in the response of liver glycogen to cortisol.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. 1108 Cancer Research Vol. 25, August 1965

day) doses of testosterone propionate. Dorfman (45) has amino acids with the aid of hepatic amino acid-activating provided other evidence for the ant.i-androgenic activity of enzymes; the charged transfer RNA's were then isolated fluorouracil. This fluorinated pyrirnidine is readily in and added back as aminoacyl donors to the prostatic ribo corporated into RNA, blocks some hornione-induced en somal systems. The reaction mixtures were replet.e with zyirie imiductions in animal tissues (171), and perhaps allows GTP, a high energy phosphate generating system, Mg2@ the formation of fraudulent messenger RNA's in micro ions, sulfhydryl compounds, and prostatic soluble proteins, organisms (107). As Cantarow and Zagerman (31) point all of which were required for maximal rates of aminoacyl out , however, the principal effect of fluorouracil in animal transfer to the protein fraction. Either L@valine@m4Cor tissues is inhibition of DNA synthesis (probably due in L-phenylalanine-'4C were employed as the labeled amino large measure to depression of thymidylate synthetase by acids. Conditions were established under which the rates fluorodeoxyuridylate), and the precise biochemical locus of and extents of anminoacyl transfers were proportional to the the anti-androgenic action of fluorouracil remains to be total quantity of ribosonmal material added to the systents. elucidated. Here we may draw attention to the important The amino acid incorporations, nueasured in the absence or unsolved puzzle as to whether the effects of steroid hor presence of various synthetic polyribonucleotides, were monies on DNA replication arid cell division are due to virtually abolished by low concentrations of puromycin. their primary interference with 1 or more reactions in When valine-'4C was used as the labeled amino acid, it was volved in the final stages of DNA synthesis, or are perhaps found that the ability of prostatic ribosomes to promote more in the nature of epiphenomena resulting from steroi its entry into protein from transfer RNA was markedly dal control of the level of DNA or RNA precursors, or of dinminished if the particles were isolated from animals that the synthesis of complementary polyribonucleotides off had been orchiectomized 2—3days previously. This effect the DNA template. of withdrawal of testicular hormones was completely re Recent investigations are consistent with the view that versed by treatment of the castrates with testosterone. the stimulatory action of testosterone on protein synthesis The prostatic ribosonmal valine-incorporating system re in rodent accessory genital organs (129, 274, 276, 278) and sponded to poly UG, which contains codons for valine. mouse kidney (128, 130) are contingent on new RNA syn The transfer of valine to the protein fraction by prost.atic thesis. Wilson (278) showed that testosterone administra ribosomes isolated from castrate was enhanced as nmuch as tiomTrhadlittle influemuceon the penetration of labeled anuino 3-fold by poly UG (U/G = 5—7). But poly UG had a much acids, or on the formation of transfer RNA-amino acids by smaller effect on valine incorporation by prostatic ribo rat seminal vesicle slices, whereas the entry of labeled somes from testosteronme-treated castrates. In these ex anuino acids into acid-insoluble proteins was greatly en periments, the soluble proteins required for the incorpora hamiced by the androgen. He concluded that testosterone tions were always derived from the prostates of the primarily governed the transfer of aminoacyl groups from androgeri-treated animals amid were added to the reaction transfer RNA-amino acids to nuicrosomal ribonucleopro mixtures in excess. A possible interpretation of these teiri. It is well known that cytoplasmic basophilia and findings is that prostatic ribosomes prepared from recently total area of the ergastoplasnuic nuembranes in epithelial orchiectomized rats are relatively deficient in tenuplate cells of the prostate and senuirmalvesicles are very depend RNA, if it is assumed that such template RNA constitutes ent on testicular hormones (44, 83, 186). Moreover, in only a small fraction of the total polyribonucleotide bound these organs the levels of total RNA, arid especially of to the ribosornes (145). ribosomal RNA, decrease after castration, are normalized Similar conclusions were drawn from experiments in by physiologic doses of testosterone, and cart be even fur which transfer RNA charged with L-phenylalaflifle-'4C and ther elevated by excessive amounts of androgen (129, 149, 19 other nmC4abeled amino acids was used as the aminoacyl 276). Kochakian et al. (128, 130) observed large effects donmor (276). The entry of phenylalanine into peptide of testosterone on the RNA/DNA ratio in mouse kidmmey linkage catalyzed by prostatic ribosomes front rats with and accessory glands, and suggested that androgens affect every evidence of normal testicular function was increased growth and protein synthesis in these tissues by regulating 4- to 10-fold by saturating levels of poly U. In the absence synthesis of both ribosonual arid messenger RNA's. It is of synthetic polyribomiucleotides, the phemmylalanine-in interesting that Butenandt et at. (29) noted that most of a corporating activity of prostatic ribosomes isolated from single dose of labeled testosterone was eliminated fronimrat. rats which were castrated 72 hr previously was less than seniiinal vesicles within 6 hr after its administration, half of that exhibited by prostatic ribosomes from castrates whereas a 4-fold stiniulatiomi by the androgeri of leucine imi injected with testosterone. However, in the presence of corporation into seminal vesicle protein was largely niani an excess of poly U, the differences in the activities of the fest after this time. 2 types of ribosomal preparation were no longer apparent. Experiments on the incorporation of labeled amino acids It roust be emphasized, however, that testosterone in into polypeptide linkage by isolated prostatic ribosonmes creased the yield of ribosomes that could be isolated front revealed that these processes are profoundly affected by the prostate as well as timecapacity of the ribommucleoproteinm testosterone in vivo and that the hormonal effects on amni particles to promote anninoacyl transfers. rioacyl transfers may be mediated through alterations in Direct measurements of the template activity of various the levels of functional template RNA associated with the prostat i( polyribonucleotide fractions were recently carried ribosonnes (145, 274, 276). The prostatic riboriucleopro out by Liao (144), employing Nirenberg's (173) Escherichia tein particles were isolated by treatnuerut. of cell particulate coli ribosomal system, which is deprived of functional fractions with deoxycholate. Rat liver transfer RNA was messenger RNA by suitable preincubationi. RNA cx charged with 1 ‘4C-labeledarid 19 other ‘2C-labeledL tracted front purified rat ventral prostate cell nuclei

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLIAMS-ASHMAN-—St€rOid Hormone Action 1109 increased the entry of a number of amino acids into pro of immatureaccessorygenitalorgans,then, is oneof rapid teimmsby this system ; normal prostatic ribosomal RNA but probably relatively unspecific immcreaseof nuclear RNA was niuch less effective in this respect, and prostatic cyto synthesis which may well explain nuammyifnot all later hor plasnuic soluble RNA was totally inert. (Prostatic nuclear mortal effects on other synthetic reactions. The data hint RNA also enhanced amino acid incorporation by prostatic that the gonadal hormones may remove various blocks on ribosomes from castrated rats, but was hardly active when DNA-directed RNA synthesis, but other points of act ion, tested with prostatic ribosornes fromn testosterone-treated such as at the ribosonmal level, are by no means excluded. animals.) Determimmations of the relative template activ As yet, no meaningful in vitro effects of steroids on these ities of prostatic nmuclearand ribosomal RNA's in the E. coli RNA- or protein-synthesizing systenus in isolated accessory systenu using 4 different radioactive amino acids strongly organ preparations have been demonstrated. And it is implied that the prostates of testosterone-treated animals impossible to tell whether the sex hormones specifically are richer in messenger RNA's tharm are glands obtained recognize systems involved in expression of particular from control castrates. The androgen given to recently genes or operons, or if the specificity of response is inherent castrated rats over periods of 40—60hr did not alter either in the already partially differentiated cells of the sex ac the base conupositionis of the prostatic nuclear and ribo cessory organs of juvenile or castrated animals. But the somal RNA fractions, or the sedimentatiort profiles of the findings do imply that the sex hormone-induced functional nuclear RNA, mi a sucrose density gradient. These find differentiation of these tissues entails inter alia the elabora ings stand in accord with the previously discussed data on tion of relatively unstable messenger RNA's, which are aminoacyl transfers by prostatic ribonucleoprotein parti speedily inactivated or destroyed when the sustaining hor des. atones are removed. Since the report of Hertz and Tullner Much nuore rapid effects of testosterone injection on (90), surprisingly little attentioru has been paid to the RNA turnover in the seminal vesicles of rats castrated chenuical physiology of postca.strate regressions of sex hor 12—15hr previously were documented by Wicks and Ken monte-dependent tissues, which appear to be far from pas ney (266). Incorporation of 32@after a 50-nun pulse was sive phenomena. The recent finding of MacLeod et at. used as a measure of RNA synthesis. Within 1 hr after (152) that 9 a-fluoroprednisolone induces an increase in the intraperitoneal injection of testosterone, the specific activ ribommuclease activity in sensitive strains of lymphosarconma ity of the vesicular RNA was about double that of un P1798 (but not in liver or in corticoid-resistant strainis of treated controls. Effects of the androgen on RNA labeling the tumor) is of interest in this connection. Although the were observed within as little as 20 mm after hornuone in effect of the steroid mumightconceivably be due to release of jectiomi, but not before. Examination of the distribution RNase from lysosornes (cf. Ref. 262), MacLeod et at. (152) of isotope in the nmucleotides of the newly synthesized RNA suggest that it is more likely that the actual synthesis of suggested that testosterone increased the entry of 32@into RNase is stiniulated by the corticoid. In such a fashion, a nuixture of both ribosomal and DNA-like RNA's. it is possible that a hormone could cause regression of a Rather sinmilar conclusions have emerged from studies tissue by influencing synthesis of a hydrolytic enzyme. Oh the early effects of estrogens on RNA and protein syn Noteworthy in this respect is the suggestion of Brandes thesis mu the femmualegenital tract. As nienitioned previ and Groth (19) that the “electrondense bodies―which ap ously, the experiments of Mueller et al. (162—165) point to pear in the prostates of orchiectomized and senile rats may the importance of early changes 1mmuterine nuclear RNA be lysosomes. synthesis following estradiol stimulation. Ui and Mueller Analysis of regulation of hepatic enzyme levels by glu (252) found that a simigle dose of est.radiol-i7@ to spayed cocorticoids has provided considerable informationu about rats increased the incorporation of injected tritiated uridine the phenornenology of emuzynie induction in manmnialiani and 32P-labeled imuorganic phosphate into RNA. Density cells. In many species, large changes in the content of gradient centrifugation of the isolated uterine RNA showed certain liver enzymes are brought about by factors such as that estrogen increased the entry of these precursors into diet (70, 126, 127, 183, 246), hormommes(61, 70—73,102, 117, “alltypes―of R.NA, no specificity being observed in regard 1i8, 126, 127, 246, 255), arid coenizymes or their precursors to any particular fraction. Noteboomn and Gorski (176) (vitaniins) (70). Studies ommhepatic enzymes such as concluded that one of the earliest effects of estrogen is the tryptophan pyrrolase and tyrosinie tranmsanuinase show that induced synthesis of a small group of proteins that are es 2 independent mechanisms can elevate the levels of these sential for armincreased nuclear RNA production, which mu enzymes; the “substrate―type and the “hormone―type turn underwrite a later generalized increase in amino acid (126). In the cases of tryptophami pyrrolase and t.yrosine incorporation into proteins. On the commtrar-y, Wilsonm tranisaniminiase, for exaniple, iniductions of the 1st category (279) has comicluded that early eruhanicement of uterimme are effected by substrates (tryptophani, tyrosine), inhibi template amid transfer RNA synthesis is an early effect. of tors (serotonini, a-nmethyltryptophani), prosthetic groups estradiol arid that armincrease in ribosonimal RNA is a later (hematin), and coenzynie precursors (pyridoxine) (70, event. However, rapid alterations in uterine ribosonmal 126). These “substrate―inductions are inhibited by population density following estrogen injections have beeru treatnnenit of the aninmals with puromycin, but not by ac reported by 1\Ioore and Hamiltomi (160). And Greenmami tinomycin D. The time courses of these “substrate― and Kenney (74) concluded that estrogens increased both inductions, which occur in adrenalectomized animals, are the number arid template RNA-lirnited amino acid-imicor quite different from those of the “hormone―type evoked porating capacity of rat uterine ribonucleoprotein particles. by hydrocortisone, arid the “substrate―type inducers al The picture provided by these studies on the biochemical most certainly act by preventing degradation of the enm conconmitants of male amudfenuale sex hormonal stimulation zymes, the steady-state concemit.rationi of these proteins iii

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. 1110 Cancer Research Vol. 25, August 1965 liver being determined by a nice balance between their synthesis is the study of Olga Greengard and her co-work rates of synthesis and degradation (126). Induction of ens (71) on the diethylstilbestrol-induced formation of these hepatic enzynmes by hydrocortisone, on the contrary, plasma phosphoprotein in cockerels. After a lag period of is inhibited by treatment with actinomycin D as well as 1 day, injection of this synthetic estrogen causes the ap puronmycin (61, 70), which suggests that new RNA synthe pearance of phosphoprotein in the plasma of male chickens, sis is required for the hornuonal response. The same holds and by 48 hr its synthesis is as extensive as in laying hens. true for the hydrocortisone-stimulated formation of pyru If actinornycin D is given simultaneously with the estro vie carboxykinase in liver (140). The effects of actino gen, synthesis of the phosphoproteinis completely inhibited. mycin D on the hydrocortisone-induced induction of But the inhibition is only partial if actonomycin D is in tyrosine transaminase (126) and various enzymes involved jected 6—8hr after the diethylstilbestnol, and if given even in gluconeogenesis (260) in liver depend very much on the later, actinomycin hao no effect whatsoever. The effect tinme of administration of the antibiotic—the processes are of puromycin administration is just the opposite, since it inhibited if actinonmycin D is given together with the hor inhibits estrogen-induced phosphoprotein formation only nuone, but not if it is injected as little as 1 hr later. It during the later period. Thus the initial effect of diethyl should be mentioned, however, that in at least 1 case of stilbestrol appears to depend on an intact RNA-synthe elevation of the levels of a mammalian enzynme by admin sizing system; the RNA's fornued in the initial period seem istration of a bona fide substrate (apparently a rather rare to be to relatively stable and do not have to renewed dur phenomenon in animal as compared with bacterial cells) ing the later occurring synthesis of the phosphoprotein. —the induction of hepatic glucokinase by glucose—the Two recent publications have documented some rather process is inhibited by actinomycin D as well as puromycin paradoxical effects of actinomycin D on enzyme induction (183). And the conuplexity of the mechanism involved in in rat liver. Rosen et al. (197) showed that prolonged (5 “hornuone―and “substrate―inductions is shown by the days) treatment of young rats with actinomycin D resulted fact that 5-fluorouracil (171) and 8-azaguanine (137) in in marked elevations in the activities of 4 liver enzymes hibit induction of tryptophan pyrrolase by injection of known to be induced by cortisol: alanine tranisaminase, tryptophan (the latter inhibitor may, however, be a general tryptophan pynrolase, tyrosine transarninase, and serine inhibitor of protein synthesis in lieu of its metabolic deny dehydrase. Actinomycin D also induced responses of the atives' competing with GTP). Valuable reviews on sub 2 latter enzymes in adrenalectomized rats. These obser strate, cofactor, and hormone inductions are given by vations are of interest in the light of an important paper by Knox (126), Olga Greengard (70), Weber et at. (260), and Garren et at. (62), which describes the effects of early and Tomkins and Maxwell (248). late administration of puromycin, actinornycin D, and The experiments of Kenney et at. (118, 119), Feigelson 5-fluorouracil on the hydrocortisone-induced synthesis of and Greengard (55), Greengard et at. (70, 72), and Garren tryptophan pyrrolase and tyrosine tramisaminase in the et at. (61) showed that administration of hydrocontisone livers of adrenalectomized rats. Actinomycin D did not speeds up the rate of nuclear RNA synthesis in liver. It depress synthesis of the basal enzymes, but it inhibited in would thus appear that the sequence of events following duction of the enzymes when administered either sirnul hormone treatment is : (a) increased nuclear RNA syn taneously with, or i or 2 hr after injection of hydrocorti thesis, (b) transport of the newly manufactured RNA into sone. However, both actinomycin D and 5-fluorouracil the cytoplasm, and (c) utilization of this new RNA in en stimulated synthesis of tnyptophan pyrrolase and tyrosine zyme synthesis. Since hydrocortisone does not cause an transaminase when injected 5 hr or later after the steroid. increase in the synthesis of all enzymes, the question arises In the absence of inhibitors, tryptophan pyrrolase synthe as to where the specificity in these effects lies. Do glu sis induced by hydrocortisone reaches a miximum after cocorticoids, for example, influence the synthesis or utiliza about 5 hr. The maximal levels at this time are main tion of specific messenger RNA's for enzymes such as tamed for about 2 hr; the enzyme activities then fall to tryptophan pyrrolase and tyrosine transaminase? Stud basal levels over a period of some hr. The findings of ies by Kenney (119) and Garren et at. (61) on the frac Garren et at. (62) are consistent with the view that 4 hr tionation of newly labeled liver nuclear RNA after hydro after hydrocortisone administration a “repressor―appears cortisone treatment provide no evidence for this. The which inhibits further enzyme synthesis, probably by in production of both ribosomal and messenger-like RNA terfening with the translation of existing messenger RNA. (and probably transfer RNA as well) was stimulated by The appearance of this “repressor―is,like the initial hor the hormone, which rather seems to promote a general in mone induction of tryptophan pyrrolase, sensitive to ac crease in hepatic protein synthesis. It therefore seems tinomycin D, and thus requires new RNA synthesis. It possible that the selectivity of the hormone-induced eleva seems that the tryptophan pyrrolase messenger RNA is tions of enzymes such as tryptophan pyrrolase and tyrosine relatively stable, whereas the “repressor―hasa high turn transaminase is more apparent than real. It is interesting over rate. that the half-life of the latter 2 enzymes is of the order of a Kidson and Kirby (121) have undertaken a new ap few hr, whereas the half-life of other enzymes (such as proach to the question as to whether steroid and other glutamic-pyruvic transaniina.se), which undergo more mod hormones selectively influence the transcription of RNA est and slower changes after hydrocortisone administra copies of specific structural genes. They developed count tion, is very much longer (119). ercurrent distribution methods for separation of rapidly An important contribution to the role of new RNA syn labeled RNA's in rat liver. Complex and reproducible thesis in relation to hormonal control of specific protein patterns were found under well-controlled conditions. A

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. [email protected]@s-Asm@w.@—Steroid Hormone Action 1111 variety of hormones (including testosterone, hydrocorti High ionic strengths influence the physical state of these sone, and diethylstilbestrol) induced rapid, selective, and nucleoprotein gels and probably increase polymerase ac reversible changes in the RNA profiles. The hormone tivity either by preventing the action of associated inhibi effects varied with time and were both positive and nega tory substances (conceivably bistones) or of contaminating tive in character. Starvation also induced characteristic polynucleotide-degrading enzymes (cf Refs. 67, 94, 263, fluctuations in specific RNA labeling. Kidson and Kirby 276). As yet, it has not been feasible to isolate from non (121) are of the opinion that the various polyribonucleotide dividing mammalian cells the RNA polymerase protein in a fractions separated by their technics are largely messenger form that is active and free from DNA (10, 263). But an RNA's, and they consider seriously the possibility that RNA polymerase completely dependent on and directed by steroids influence selective transcription of certain genes. exogenous DNA's can be easily refined from organs con However, direct determinations of the template activity of taming many dividing cells, such as chicken embryo (60) any of these RNA's with respect to individual protein and rat testis (10). The activity of the purified testicular products were not performed. (The report of Sekenis and RNA polymerase (in marked contrast to that of “aggre Lang (216) that cortisol increases the template activity of gate― enzyme preparations from tissues such as liver, an RNA fraction extracted with warm phenol from rat uterus, and prostate) is inhibited by raising the ionic liver nuclei is questionable on technical grounds.) The strength of the reaction mixture. The heretofore reported procedures of Kidson and Kirby (121) permit the repro effects of steroid hormones on tissue RNA polymerase ducible separation of many more species of rapidly labeled activities, then, almost certainly do not reflect changes in RNA than do previously described density gradient cen the levels of the polymerase proteins, but are rather due trifugation methods, and their further application will to ill-defined perturbations in the priming properties of the doubtless be of great value in elucidating the mechanisms DNA bound to the crude nuclear extracts. of hormonal control of specific RNA and protein syntheses. Steroid hormones and isozymes.—Up to now we have con Some recent reports of steroid-induced elevations in the sidered some examples of steroid hormonal control of the RNA polymerase activity of crude nuclear extracts from synthesis of specific catalytic proteins. In recent years, it various tissues might seenu in agreement with certain early has been recognized that some enzymes that were previ effects of steroids on nuclear RNA synthesis. Thus, RNA ously considered as single entities are actually heterogene polymerase reactions (inhibited by DNase and requiring ous, and that various forms of the enzyme can be separated the simultaneous presence of all 4 major ribonucleoside by electrophoretic and other means. The lactic dehydro tniphosphates) catalyzed by nucleoprotein gels prepared genase of higher animals exhibits such a molecular heter from cell nuclei were reported to be increased in the pros ogeneity, and 5 forms of the enzyme can be distinguished tate of castrated rats following testosterone treatment (81, in most tissues. It turns out that these result from various 276), in rat liver after administration of cortisol (138), and combinations of 2 parent subunits—the heart (H) and in chicken liven (261) and immature rat uterus (67, 176) muscle (M) types (43). The active lactic dehydrogenases soon after injection of estradiol-17$. In the case of experi are tetramers, so that we find “pure―H(114) and “pure― ments with rat ventral prostate (81, 276) and chicken liver M (M4) species in addition to the molecular hybrids MH3, (261), the effects of steroid hormones were observed 24—72 M2H2, and M3H. The catalytic as well as physical prop hr after their administration. But increase in RNA po erties of the 5 isozymes are different. Goodfniend and lymerase activity in rat liver after cortisol injection (138) Kaplan (66) showedthat after treatment of immature rats or in uterus after treatment with estradiol-17fl (67) was and rabbits with estradiol, the synthesis of M subunits reported to occur within periods of 30—120mm. Note was stimulated to a greater extent than the synthesis of boom and Gonski (i76) observed that puromycin admin H subunits. The preferential stimulation of M subunits istered over the 1st 2 hr of estradiol treatment nullified the by estradiol was nullified by administration of actinomy effect of the estrogen on uterine polymerase activity. If cm D. Progesterone and testosterone, on the contrary, it is assumed that puromycin inhibits only protein synthe caused a parallel increase in both M and H subunits in the sis, then a possible explanation for this finding is that uterus. It is tempting to believe that estradiol may Se estradiol induced the formation of some protein that in lectively regulate expression of genes determining synthe turn permits an increased RNA polymerase activity. sis of M as opposed to H subunits, but Kaplan's expeni However, the action of male and female sex hormones on ments may reflect more an alteration by estradiol of the the RNA polymerase activity of crude nuclear extracts of relative proportion of myometrial vers'us endometrial cells prostate and uterus respectively are very dependent on the in the uterus. ionic composition of the reaction mixtures used to deter An even more striking effect of a steroid on tissue iso mine the nucleotide incorporations. The effects of sex zyme patterns was described by Shaw and Koen (220). hormone pretreatment were most pronounced in reaction They discovered an esterase isozyme in the kidneys of mixtures of low ionic strength, and tended to be nullified at mature male mice which was totally absent from female or high ionic strengths (67, 81, 276). Even though the RNA immature males. But if testosterone was injected for 7 polymnerase of such mammalian “aggregate―enzyme prep arations is inhibited by DNase, it is not unequivocally days, then the enzyme was readily detected in renal ex directed by exogenous DNA's, and the activities seem to tracts from both fentale and juvenile animals. Other be limited more by the printing ability of the DNA bound kidney esterases separable by starch-gel electrophoresis to the preparations than by the catalytic activity of the were not affected by sex hormones. Shaw and Keen (220) RNA polymerase-activating protein (10, 104, 263, 276). concluded that testosterone induced the manufacture of

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. 1112 Cancer Research Vol.25,August 1965 this nuouse kidney emizynie, which could not be detected in across these toad bladders is accelerated by treatment of other tissues. the ammimalswith aldosteromme (39), and also by addition of Histones and sleroidal control of gene activity.—The close the hormone in vitro (51, 218). As Edelman (51, 185) amud association of histories with DNA in cell nuclei, and the others have shown, the latter effects can be demmionstrated ability of histones to depress DNA-directed RNA polynu with concentrations of aldosterone in the range of i0@ to erase activity in vitro, has resulted in many speculations i0—@M,which approxintate to physiologic levels of aldos concermmingthe possible control of specific gene expression terone in man as computed front secretion rates (cf. Ref. by these basic proteins (17, 49). Different histones vary 218). The isolated toad bladder systenu exhibits a marked greatly in their ability to influence R.NA synthesis by iso specificity toward adrenal steroids. According to Sharp lated R.NA polynmerases (5, 94). And it appears that the and Leaf (218), deoxycorticosterone is 20 times less active degree and even specificity of histone-DNA interactions than aldosterone, cortisone is inert, and the aldosteronie can be influenced by relatively nuinuornuodifications in his antagonists SC 9420, SC 14266, and progesterone inhibit tone structure. Thus, Allfrey et at. (4) showed that his the response to aldosteronie. Somewhat different struc tones can be nuethylated and acetylated by enzynmatic ture-activity relationships were found in a thorough study group transfer reactions apparently occurring at the poly by Porter and Edelman (185). peptide level; the ability of sonmehistories to combine with A lag period of some hr occurs between the addition of DNA was hardly affected after acetylation, whereas acet aldosterone to the isolated bladders and an increase in ylated histones were less potent inhibitors of RNA synthe sodium transport, whereas aldosterone-3H uptake reaches sis in the test tube. The chemistry of histones rernaimusin a steady state within about armhr, and long before any a rather pristine state (167), but it seems that the hetero change in active transport of sodiunu can be detected (39, geneit.y of this class of prot.eimmsisrather narrow, and it is 51, 218). This suggests that the latent period for aldos very unlikely that there is a specific histone corresponding terone action on ion transport does not follow from a slow to every gene. I\Ioreover, among various organs of adults rate of penetration of the steroid, but rather that its actionu in a given species, the histone/DNA ratios (49, 169), and involves sluggish interniediate biochenuuical processes. also the patterns of histones separable by electrophoresis Pretreatnuent of the toad bladders with either puronmycinu on polyacrylanmide gels (169), are pretty niuch the sanme. or actinomycin D abolishes the effects of aldosterone in However, characteristic species differences are demonstra vitro (39, 51). That the effects of the antibiotic are miot ble (167), amid in the immature rat, the histones of brain conupletely unspecific is illustrated by the fact that ac and liver cant be distinguished electrophoretically (169). tinomycin D does not inhibit the response of the prepara It is interesting that no alterations occur in the histone/ tions to vasopressin or glucose (51), and that puromycin DNA ratio during the formation of RNA-synthesizing has much less influence on responsiveness to vasopressini as “puffs―ininsect polyt.ene chronmosonmes (cf Ref. 49). In compared with aldosterone (39, 51). Edelman et al. (51) general, there is little convincing evidence for gross quali found that aldosterone-3H is preferentially localized in the tative and quantitative changes in nuclear histones during mucosal epithelial cells, and inspection of radioautographs differentiation and development, although it must be ad at high magnification revealed selective localization of nutted that presently available analytic methods are crude. tritium over the nuclear arid perinuclear regions of the In a thoughtful essay, Zalokar (285) emphasizes the impor mucosal epithelium. Crabbe amid DeWeer (39) and Edel tant difference between commupetamiceamid induction in mart et at. (Si) concluded that aldosterone regulates sodiunu developmental processes amidsuggests that histones may be transport by increasing the synthesis of nuclear RNA, amuiong the factors which control the respomusivity of genes which therm passes into the cytoplasm and participates in to inducers. He inmuaginedthat “historieskeep genes in the synthesis either of enzymes involved in supplying en competent ; the renioval of histories does not. make genes ergy for the active ion transport or of protein(s) acting as active, but only niakes them susceptible to the actiomi of carriers for ion pumping. inducers― (285). Possible nuiechanisnis for the comitrol of Very recent experiments by Sharp and Leaf (219) con genie transcription by histories are discussed by Dulbecco firmed that puronmycin amid actiniomycin D abolish the (49). priniary stimulation of sodiuni t ransport by isolated un Very little is known at present about the role of histories nary bladder of Bufo marinus. Low levels of pyruvate in the response of cells to steroid horniones. Ts'o (251) induce a pronupt amid sustained stimulation of sodiummi has measured binding constants for the interactiomi of some t ransport in the presence of aldosterone, but not in the ab steroid hormones with calf thymus histone. In a careful sence of the steroid. This stiniulatory effect is confined to study, Cohen et al (38) were ummable to detect aimy changes pyruvate, or substances (glucose, lactate, oxalacetate) mmthe histone/DNA ratio following stimulation of imma which yield pyruvate durimig their metabolism. Sharp tune rabbit uterus with amounts of estradiol-17@ sufficient. and Leaf (219) suggest that aldosterone somehow enables to cause growth amid accelerated RNA synthesis in this the energy yielded by the decarboxylatiomm of pyruvate to organ. be more efficiently coupled to sodium transport. Their Action of atdosterone on the isolated toad btaddei.—An observations hint that the action of aldosteronie niay be inuiportammtexample of the need for intact protein and DNA nuore conuplex than a stinrrulation of synthesis of sonmecorn directed RNA-symmthesizing niachinery in cells responding poniemit(s) of the pyruvic decarboxylase system. to steroid hormones is provided by some recent experi Uterotropic actions of isolated uterine RNA .—Ifthe uter meats on sodium transport by urinary bladders excised otropic actions of estrogens are prinmanily mediated via an from Bnfo inarinus. The active transport of sodiunri ions increased production of specific RNA niessemigers, then at

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1965 American Association for Cancer Research. WILLIAMs-AsHMAN—-Steroid Hormone Action 1113 first sight it might be expected that RNA's isolated from sites but also with their true substrates at the active sites, estrogen-st.inmulated uteri could induce phenotypic estro tend to belie many naive “lockand key―ideas about the gen-like effects on the uterus of juvenile or spayed females. interaction of hormones with their receptors. There now Segal and his co-workers (2i4) applied crude uterine RNA seems little likelihood that sex or adrertocortical hormones preparations, via an indwelling polyethylene catheter in function as true coenzymes, that union of these hormones serted near the uterotubal junction, into uterine horns of with their receptors involves covalent linkages, or that oophorectomized rats. The RNA (0.i5 mg) was applied any of the metabolic transfornuations suffered by the hor unilaterally every 4 hr over a period of 2 days, the other nuones are primarily related to their mechanism of action. horns being injected with solvent and serving as controls. It. does seenu probable that the receptors for steroid hor RNA extracted from uteri of estrogen-treated castrates inones are proteinaceous in nature (although a receptor was reported to induce typical estrogenic hyperpla.sia of function for polynucleotides cannot be excluded). An the emidometnium. Treatment of the RNA preparations attractive hypothesis is that the hormones serve as allo with RNase reduced their activity, and liver RNA was steric effectors of these proteins, inducing conformational stated to be ineffective in this system. Segal et at. (214) alterations and thereby modifying their catalytic or other stated that the RNA extracts were devoid of contamnina properties. In this vein, it can be imagined that a given ting estrogens and regarded these experiments as support steroid could modify the immediate action of enzymes, the ing the view that “thestimulatory effect of estrogen on permeability of biologic membranes, arid biosynthesis of uterus is elicited by activatiomi of specific RNA synthesis, specific proteins by inducing allostenic transitions in ap and that the hormone is not necessarily involved in sub propriate proteins, including repressors. This viewpoint is sequent steps leading to secomidary changes in the target very well argued by Tomkins and Maxwell (248). organ.― @%!lantnmaliansteroid hormones serve as regulators of 3 Expeninmenmtal confirmation of these findings will be basic categories of biologic events : (a) homeostatic mech awaited with great interest. For the experiments of Segal anisms (ion transport, carbohydrate metabolism, etc.); et at. (214) imply that RNA harboring specific genetic in (b) areal cellular proliferations essential for reproduction fornmat.ion can enter uterine cells after its deposition into and developnment (especially the functional differentiation the uterine lumen, and that the RNA messengers can pre of accessory sexual organs, which often involves cyclical sumably find their way in correct proportions to the intra changes occurring over relatively short periods of the life cellular sites of protein synthesis and there serve as tem time of niammals) ; and (c) behavior. The paucity of our plates for formation of those proteins which are under present understanding of the effects of steroids on brain estrogenic control. This is redolent of the report of Niu centers such as the hypothalamus—with all their overtones et at. (174) that liver RNA can induce synthesis of such on higher nervous activity and on anterior pituitary func liver-specific proteins as serum albumin, glucose 6-phos tion (205)—precludes their comprehension at present in phatase, and tryptophan pyrrolase in isolated Ehrlich and molecular terms. But serious consideration must now be Nelson ascites, and Novikoff hepatoma, cells. In Niu's givert to the possibility that steroidal control of some if not experiments, an additiomial supply of liver RNA was not all horneostatic as well as developmental processes open necessary after induction to sustain synthesis of new liver ates at the level of production, intracellular translocation, specific proteins in the tumor cells. This suggested that or utilization of RNA copies of specific districts of the exogenous RNA added to the recipient cells could be rep DNA genome. Perhaps the nornual function of certain heated therein, either by affecting the DNA of the host receptors for sex and adrenocortical hormnonmesis to impede cells or by serving as its own template. (Conceivably either the transcription of specific messenger, ribosomal, this might hold for the analagous experiments of Segal or transfer RNA's, or the proper reading of genetic rues (214) on the uterotropic actions of uterine RNA.) Niu's sages encoded in complementary RNA's cognate to some (174) findings of induction by liver RNA of glucose 6- structural gene (poly)cistrons on the DNA, and that a phosphatase in ascites tumors could not, however, be basically inhibitory combination of steroids with these re confirmed in Ephrussi's laboratory (105). Moreover, ceptors can effectively initiate and maintain all sorts of other claimes by Hillman and Niu (91) that semi-purified biochemical processes, voiced through the nmouthpiece of RNA's can act as inducers in embryonic developmental specific protein synthesis. Alternatively expression of in processes have not been corroborated, and it seems that dividual structural genes or whole operons could be the active inducers are proteins rather than polynibonucle ‘switchedoff' if steroid-induced allostenic transitions per otides (cf. Ref. 247). mitted certain repressor proteins to inhibit synthesis or function of specific polynibonucleotides. These considera EN VOl tions will naturally remain of limited heuristic value until Insight into the role of various types of polyribonucleo in vitro, cell-free test systems can be devised to nieasure tide as internuediates in genie expression and protein bio these putative interactions of steroids with such repres synthesis, and the comicept of allosteric proteins, are 2 son mechanisms, and further progress along these lines will recent biochemical developnuments that have important undoubtedly be contingent upon a much better umider bearing on the problem of the molecular basis of steroid standing of the biochemistry of nucleic acid and protein hornuomie action. The amazing versatility of allostenic synthesis in mammalian cells. (Chemumical nuechamuisnis transitions, and realization that exquisite conformational involved in the coupling between complenmentary RNA changes in catalytic proteins can follow from their corn transcription and utilization (30) may be especially ger binat.ion not only with small molecules at their allostenic nuane to steroid action.) However, even if it turns out

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that the primary receptors for steroid hormones are rather uteri of untreated ovariectomized rats was reversed by in vitro remote from this biochemical realm, it seems that the addition of minute quantities of estradiol-17@9,butnot of cortisone. In somewhat similar experiments, Wacker et al. (297) obtained, steroid-receptor interactions somehow relay selective sig by chromatography on Sephadex G-100, a soluble protein fraction nals to the RNA-dependent nuachinery which dictates from P8eudomonas te8tosteroni that inhibits in vitro the activity of some genes to speak up and others to be silenced. Such E8cherichia coli RNA polymerase. The protein fraction isolated slow, coarse controls of enzyme synthesis are probably from cells grown in the absence of steroids inhibited RNA synthe more widespread determinants of the biologic actions of sis to a greater extent than protein from cells grown in the presence of testosterone (which induces a number of hydroxysteroid de steroid hormones than are swiftly induced alterations in the hydrogenases and steroid isomerase in this species). Moreover, properties of pre-existing enzymes or membranes. the inhibitory action of the protein fraction obtained from non Similar conclusions are very much in the air in regard to induced cells was partially overcome by addition of minute certain other hornuones—notably thyroxine (243, 244, 280) amounts of testosterone to the isolated RNA polymerase system. Both groups of workers suggest that these soluble protein fractions and those controlling germination (254) and flowering isolated by Sephadex chromatography may contain “repressors― (179) in plants—whose biologic effects often entail a pro of RNA synthesis whose action can be reversed by appropriate nounced lag period, and which regulate tissue differentia steroids both in vivo and in vitro. Although it is not clear from tion as well as being involved in homeostasis. In all these preliminary reports whether the inhibitory action of the events, focussing attention on biochemical genetic aspects protein fractions is directed toward the RNA polymerase protein or the DNA primer (or isdue toother actions), further experiments of steroid hormone action will surely advance research not along these lines may be enlightening. only in endocrinology, but also on what is perhaps the cen Mansour and Niu (294) have reported that uterine RNA (iso tral problem of cancer research and biology in general—the lated by the cold phenol procedure) when injected into the uterine molecular basis of cell differentiation. horns of oophorectomized mice induced the castrate uterus to assume its normal appearance and alkaline phosphatase activity. ADDENDUM Liver RNA, as well as RNase-treated or boiled uterine RNA, were inactive in this respect. RNA isolated from bull seminal vesicle Since this paper was submitted for publication, 2 excellent re was also found to increase alkaline phosphatase activity after views by Hechter and Halkerston (289, 290) that cover many its injection into uteri of castrated mice. The effects of uterine molecular and cellular aspects of steroid hormone action have RNA were sustained for 6 days, suggesting that either the func appeared. A recent volume edited by Karlson (292) also contains tional RNA is stable or that it somehow affects the DNA genome. many papers on these topics. Recent studies by Porter et at. (295) demonstrated that aldos Recent publications from Tomkins' laboratory (286, 287) pro terone enhances the entry of labeled uridine into epithelial cell vide evidence that changes in the structure and activity of liver RNA of isolated bladders from Bufo marinus. The effect of glutamic dehydrogena@e may follow from an equilibrium between aldosterone on uridine incorporation preceded or paralleled the 3 forms of the enzyme that may be represented as follows: effect on sodium transport. Autoradiographic experiments I II showed an increased labeling of the nuclei of the epithelial cells Polymer ;::@ Monomer x @±Monomery of the aldosterone-treated bladder. The chemical and autoradio graphic data support the concept that aldosterone increases the According to this scheme, monomer x exhibits glutamic dehydro rate of RNA synthesis, which was sustained for at least 6 hr after genase and also some alanine dehydrogenase activity and aggre introduction of the hormone into the serosal media. Progesterone gates to form higher molecular weight polymers as its concentra had only very small effects on RNA synthesis in toad bladder tion is elevated. Monomer y has increased alanine dehydrogenase under these conditions. activity (but little or no glutamic dehydrogenase activity) and Comprehensive investigations on the early effects of corticos does not readily aggregate at high protein concentration. Equi teroids on hepatic gluconeogenic enzymes, RNA metabolism, librium II is considered to be due to conformational alteration(s) and amino acid levels have been described by Weber et al. (298, in the monomers. The experiments suggest that allosteric 299). Further experiments on the diethylstilbestrol-induced effectors of glutamic dehydrogenase, including diethylstilbestrol synthesis of phosphoproteins in the livers of cockerels were re and some steroid hormones, primarily affect Equilibrium II. ported by Greengard and her co-workers (288). Two papers by King et at. (291,293) deal with the intracellular localization of highly radioactive estradiol-17fl in various rat REFERENCES tissues. 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H. G. Williams-Ashman

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