Estrogen responsive creatine kinase in normal and neoplastic cells

Journal 01 Steroid Biochrmiwy Vol. IS. pp. 69 10 75. 1981 Printed in Great Britain. All rights reserved

ESTROGEN

0022-1731/81/010069~7~2.~~ Copyright 0 1981 Pergamon Press Ltd

RESPONSIVE CREATINE KINASE IN NORMAL AND NEOPLASTIC CELLS

A. M. KAYE*,N. REISS*,A. SHAER*,M. SLuYstzat, S. IACOBELLI$, D. AMROCHS: and Y. SOFFER/~ *Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel, tDivision of Endocrinology, The Netherlands Cancer Institute. Amsterdam, The Netherlands. $Laboratory of Molecular Endocrinology, Catholic University, Rome, Italy, §Department of Surgery A., Kaplan Hospital, Rehovot, Israel, l/Department of Obstetrics and Gynecology, Asaf Harofe Hospital, Zerifin, Israel SUMMARY

Estrogen-responsive creatine kinase (uterine estrogen-induced protein, CK-BB) activity was compared during ontogeny of the rat uterus, in the human menstrual cycle and in mouse and human mammary tumors. Between days 2 and 26 of post-natal development of rat uterus, the specific activity of creatine kinase increases 4.5-fold. The glycolytic enzymes, phosphoglycerate kinase and phosphoglycerate mutase, show no increase and pyruvate kinase activity increases 1.5-fold, during this period. Only creatine kinase BB activity was increased by estrogen administration. In human endometrium, the specific activity of creatine kinase increases in the late-secretory stage of the menstrual cycle. During the progression from hormonal dependence to independence shown by the GR mouse mammary tumor in the course of successive transplantations, the total activity of creatine kinase increases. The increase in the MM (muscle type, non-estrogen responsive) isozyme activity of creatine kinase exceeds the increase in crcatine kinase BB activity. Normal human breast and breast tumors display both creatine kinase isozvmes. Preliminary evidence for in virro estrogen responsiveness of creatine kinase. in human breast explants, raises the possibility that creatine kinase BB may be a suitable marker for assessing the hormonal dependena of human tumors.

INTRODUCTION

responsive creatine kinase as a marker for estrogen action.

Sina its description in 1966 by Notides and Gorski[l], the “estrogen induced protein” (IP) of the rat uterus has been a favorite marker for studies on the mechanism of action of estrogens and anti-estrogens. Its utility is derived from two properties; an increased rate of IP synthesis is detectable within 40 min of estrogen treatment [2], and the stimulation of IP synthesis by estrogen is demonstrable in surviving uteri in vitro as well as in the intact rat [33. Recently, we have found two further advantages to the use of IP as a marker protein. Translatable mRNA for IP was shown to double 1 h after estrogen treatment [4] and the overwhelming component of IP in rat uterus was identified as the brain type (BB) isozyme of creatine kinase [SJ. The role of creatine kinase appears to be the maintenance of optimal intracellular concentrations of ATP through what has been termed an ATP “buffering” action. The finding of an enzymic activity for IP, which provided a rapid, sensitive and reproducible assay procedure, made it possible to begin to evaluate how unique is the stimulation of creatine kinase (CK) compared to other enzymes of energy metabolism. Moreover, the suggestion that IP might make a useful clinical marker for tumor responsiveness to estrogens [5-73 could now be followed up using creatine kinase assays. This paper is a first survey of systems which are being developed to utilize the advantages of estrogen 69

METHODS

Animals and animal treatment Rats of the Hormone Research Department colony, derived from Wistar stock, were housed and fed as previously described [8]. The day on which pups were first found with their mother was considered as day 1. Estradiol-17/l (Organon, Oss, The Netherlands, in l”/‘,ethanol), or the ethanol vehicle alone, was injected intraperitoneally at a dose of 1 pgj7g body weight. The rats were killed at the specified time by cervical dislocation followed by decapitation; uterus and brain were removed, frozen in liquid nitrogen and stored at - 70°C. Induction and serial transplantation of tumors in CR mice Mammary tumors were induced by estrone and progesterone treatment in ovariectomized GR mice [9]. Estrone was dissolved in ethanol (2 mgiml) and the solution was added to the drinking water to give a final concentration of 0.5 pg/ml. Three progesterone pellets (2.7 mg progesterone/pellet) were introduced per week. subcutaneously, in the neck region of the mouse. Mammary tumors were usually found after 3-4 months. For serial transplantation. tumor tissue was minced with scissors and suspended in 0.14 M NaCI. Portions of this suspension were

70

A. M. KAYE cr al.

grafted S.C. into the right Bank of (020 x GR)Fl hybrid mice (0.5 ml/mouse), which had been ovariectomized or orchiectomized about 1 week previously. For testing of hormone dependence. tumor tissue was inoculated into two or more castrated female or male mice that received no hormone treatment. Two or more castrated animals, treated with estrone and progesterone as described above. served as controls. If within 3 months no tumor appeared in the animals that did not receive hormones, but tumors grew in one or both of the hormone treated animals, the tumor was considered hormone dependent. If the tumor grew in the nontreated animals as well. and the time of its appearance co-incided with that in the hormone treated group, the tumor was considered hormone independent (autonomous). Tumors that were transplantable into nontreated animals but which appeared more than one week earlier in the hormonetreated animals were called hormone responsive. Hormone-dependent tumors were transplanted every 4-6 weeks; hormone-independent tumors were transplanted every 2-4 weeks. The tumors were stored at -70°C before being assayed for creatine kinase activity. Preparation of enzyme extracts

For the assay of creatine kinase activity, organs were homogenized in 3-6 vol. of homogenization buffer consisting of; 50 mM Tris HCl pH 6.8, 5 mM MgS04, 0.4 mM EDTA, 5.5 mM 2-mercaptoethanol and 250mM sucrose, using a glass-glass homogenizer; cytosols were obtained by centrifugation at 4°C at a minimum of 1.5 x lo6 g.min/cm sedimentation path.

0.25 mM NADH, 5 mM ADP, and 7.0 units/ml of lactate dehydrogenase. The phosphoglycerate kinase assay mixture contained: 50 mM Tris HCI pH 7.5, 2 mM MgSO,. 0.8 mM EDTA, 5 mM glycerate-3-phosphate, 0.25 mM NADH, 1 mM ATP. and 2.5 units/ml of glyceraldehyde-3-phosphate dehydrogenase. The phosphoglycerate mutase assay mixture contained: 50mM Tris HCl pH 7.5, 1 mM MgSOb, 0.2 mM EDTA, 5 mM glycerate-3-phosphate, 0.6 mM ADP, 0.12 mM glycerate-2,3-phosphate, 0.25 mM NADH, 1.4 units/ml of both enolase and pyruvate kinase. and 7.0 units/ml of lactate dehydrogenase. Separution oj’creatine kinase enzymes by step gradient chromatography

Column chromatography was performed as described previously for enolase isozyme separation [ 10, 111. with slight modifications. Cytosol fractions were applied to a DEAE cellulose column (1 ml) at pH 7.9 in 20 mM NaCI, after aggregates were removed. The MM isozyme was not absorbed to the column; the MB isozyme was eluted with 40mM NaCI, 100 mM Tris HCl (pH 6.4), 5 mM MgSO* and 0.4mM EDTA; the BB isozyme was eluted with 250 mM NaCl in the same buffer. Protein cleterminution

Protein was determined by the Bradford method [12] using bovine serum albumin (Sigma) as the protein standard. RESULTS AND DISCUSSION

Enzyme assays

Preferential synthesis of creatine kinase BB in rat uterus

For all enzymes, the unit of activity was defined as that amount of enzyme yielding 1 pmole of substrate per minute at 30°C. All assays were performed in a Gilford 250 automatic recording spectrophotometer in a volume of 0.5 or 1 ml total assay mixture. Enolase was assayed at 240 nm in a solution contaming 0.8 mM 2-phosphoglycerate, 50 mM Tris HCl pH 6.8, 60 mM KCl, 1 mM MgSO* and 0.4mM EDTA. Creatine kinase, phosphoglycerate kinase. phosphoglycerate mutase and pyruvate kinase were measured in coupled assays at 340 nm. The creatine kinase assay was either as previously described [S] or in an assay mixture containing in 1.0 ml vol., 50 mM imidazole acetate pH 6.7, 25 mM creatine phosphate, 2 mM ADP, 10 mM Mg acetate, 20mM D-glucose, 2 mM NAD, 5 mM EDTA, 10 pg/ml bovine serum albumin, 50 PM diadenosinepentaphosphate (myokinase inhibitor), 20 mM N-acetyl cysteine. 2 mM DTT, 2.4 units of glucose-6-phosphate dehydrogenase and 1.6 units of hexokinase. The pyruvate kinase assay mixture contained: 50mM Tris HCI pH 7.5, 12.5 mM MgS04, 0.2 mM EDTA. 60 mM KCI. 1 mM phosphoenolpyruvate,

In the uterus of the 25-day-old rat, stimulation by a single injection of estradiol leads to a rapid increase in the specific activity of creatine kinase but not in the specific activity of a triad of reference glycolytic enzymes: phosphoglycerate kinase, phosphoglycerate mutase and pyruvate kinase (Fig. 1). The increase in creatine kinase specific activity at 1 and 2 h after estrogen injection, has been shown to be due, at least in part, to an increased rate of synthesis of CK-BB as revealed by PAGE fluorography of immuno-precipitates of “S-methionine labelled uterine cytosols [S]. The biphasic nature of the rise in creatine kinase activity shown in the experiment may be due, in part, to the biphasic nature of the increase in uterine wet weight. shown in Fig. 1. The fact, that the three reference enzymes also show a dip in specific activity at 4 h after estrogen injection, fits this explanation. However, the possibility that different cell types in the uterus show different time courses for their responses [cf 133 requires further investigation. When prolonged estrogen stimulation is applied to immature rats. as by the stimulation of estrogen secretion by injection of pregnant mare serum gonadotropin. the increase in creatine kinase specific activity

71

Estrogen responsive creatine kinase

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8 Time

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(h)

Fig. I. Preferential stimulation of creatine kinase activity in rat uterus by a single injection of estrogen. Rats received 5pg of estradiol-17g intraperitoneally (except for control, 0 time, rats) and were killed at the specified times. Uteri were removed and weighed; the cytosol fraction was prepared and the activity of each enzyme was assayed spectrophotometrically in a coupled assay as described in Methods. CK, creatine kinase; PGM, phosphoglycerate mutase; PK, pyruvate kinase; PGK, phosphoglycerate kinase; wt, wet weight. Each point represents the mean of duplicate values of pools of 5 uteri. continues beyond 24 h and can reach more than looo/, within 48 h [SJ. The induction of creatine kinase BB activity by estrogen in rat uterus is not confined to rats of 25 days or older in which all the known uterine responses to estrogen can be demonstrated [14]. Beginning as early as 2 days after birth, in the first stage of the sequential post-natal acquisition of uterine responsiveness to estrogen. stimulation of creatine kinase activity can be observed (Fig. 2). This response to estrogen takes place over and above the constitutive increase in creatine kinase activity, which occurs between days 2 and 25 after birth. The parallel increase in the specific activity of creatine kinase BB in rat brain during this period (Fig. 2) is unaccompanied by any indication of stimulation by estrogen. However, in both uterus and brain during postnatal development, there is a preferential constitutive synthesis of creatine kinase BB, compared with other enzymes concerned with energy metabolism such as pyruvate kinase, enolase, phosphoglycerate mutase, and phosphoglycerate kinase (Fig. 3). While uterine creatine kinase increases in specific activity by 4.5-fold between 2 and 26 days after birth, a decrease in the specific activity to 73-93% of the value at day 2 was found, at day 26, for phosphoglycerate kinase, phosphoglycerate mutase and enolase; the specific activity of pyruvate kinase increased by approximately 50%. In the rat brain during this period of rapid postnatal development, all of the five enzymes assayed show an increase in specific activity (Fig. 3). However, the increase displayed by creatine kinase (4.2-fold) is greater than that shown by the other four enzymes (3.1 to 3.6-fold). The increase in the specific activity of CK-BB during the post-natal development of both uterus and

0LLZI 0 1020x) Aga (days) Fig. 2. The postnatal development of creatine kinase BB and its responsiveness to estradiol in rat uterus’ (squares) and brain (circles). Animals received a single intraperitoneal injection of estradiol (I pg/7 g body weight: filled symbols) or 1% ethanol (open symbols) at the indicated ages, and were killed 24 h later. Preparation of ctyosol and determination of creatine kinase activity are described in Methods.

brain is directly correlated with the increase in the concentration of CK-BB in these organs as revealed by protein staining following SDS-PAGE (Fig. 4). Thus the estrogen-responsive creatine kinase of the rat uterus is apparently under dual control during post-natal development, with endocrine stimulation superimposed on a constitutive increase in activity. It will be of interest to determine whether in ovariectomize.d rats, the same constitutive increase in CK activity indeed occurs, between 0 and 10 days, in an experiment parallel to that by which Clark and Gorski[lS] showed that the presence of ovarian estrogen was not necessary for uterine growth or the increase in uterine estrogen receptors during this period. Uterus

Braln

PGM

PGK

CK

Fig. 3. Preferential post-natal development of creatine kinase in rat uterus and brain. Assays were performed as described in Methods on pools of organs. Unfilled bars, 2day-old rats; cross-hatched bars, 26-day-old rats, PK. pyruvate kinase; E. total enolase: PGM, phosphoglycerate mutase; PGK. phosphoglycerate kinase. N.B. Enolase is designated total enolase as a reminder that a very small fraction of uterine enolase (the r; isozyme) is a minor constituent of IP and shows increased activity after estrogen stimulation.

72

A.

M. KAVEet ul.

RS4 tubulln

actln

2

6

11 16

21 26

31 2

(Age

6

11

16

21 26 31

days)

Fig. 4. The postnatat~~~~~prnen~ of creatine kinase in rat uterus and brain as revealed by SDS-PAGE and Coomasaie Brifhant Blue staining of proteins. Cytosol samples (15pig of protein) of the same extracts prepared from the uteri of untreated rats, presented in Fig. 2 were subjected ta electrophoresis in the modified buffer system which resolves CKB and enolase y subunits [5]. Lanes 1-8 uterine cytosoIs: lanes 9-16 brain cytosols; RSA, rat serum albumin.

Creatine kinase actiairy luring the estroouscycle in the rut and the menstruai fyrfe in womer4

responsiveness to physioio~~l luteal phase endometria [19].

The specific activity of creatine kinase in adult rat uterus was found to be at a nadir on the day of diestrus (4.2 units/mg protein), to rise to nearly peak value by proestrus and to reach a maximum value of nearly 6 units/mg protein on the day of estrus. Total enolase specific activity in rat uterus remained constant during the estrous cycle [ 1I]. In human endometrial curettage samples a fairly constant average activity (0.9 units/mg protein) of CK w~.rnain?~~ untii the Iate lute& phase at which time an increase in speciiic activity was observed (f.6 units/min/mg protein). A sample from the late luteal phase was found to have an isozyme composition of 93% BB and 7dr;;MM. Once again, this increase took place against a background of constant enolase specific activity in human endometrium during the menstrual cycie [I I]. During pregnancy, a doubling of human creatine kinase in myometrium was observed as early as 1965 [16]. Recently, similar data was obtained for bovine myometrial creatine kinase activity during gestation [17,18). The finding of physiolo~ca1 regulation of human endometrial CK with particular sensitivity in the htteal phase should be considered in retation to the search for human estrogen-induced proteins as exemplified by our recent report of in &o and in vitro

Creutine k&se activities of CR motlse ~mury twnars during progression from hormone dependence to independence

doses of estrogen by

In the well defined GR mouse mammary tumor system [ZO,211, hormone dependent tumors have been found to have higher concentrations of estrogen receptor C9.221 progestin receptor [23] and prolactin receptor than jnde~nden~ tumors. In this study we found that the total CK specific activity of GR tumors was higher in the hormone-independent, autonomous tumors (1.5 rtr 0.4 SEM flmol/min/mg protein) than in the hormone dependent and hormone-responsive tumors (0.7 rf: 0.1 SEM pmol/min/ mg protein), a statistically significant difference (P = 0.046, Student’s t-test). When rest&s of assays on 40 tumor samples were examined as a function of tumor generation (Fig. Sk the average CK specific activity was seen to remain at a value near 0.6 gmoll min/mg protein in the first four tumor generations, to rise steeply between generations 4 and 6 and to reach a value of approximately t.5 ~mo~/min/mg protein in transplant generations 6-16. Some tumor lines showed a peak in CK activity at the transition from hormone dependence to inde~nden~. The activity of creatine kinase in GR mouse mammary tumors during serial transplantation was not

73

Estrogen responsive creatine kinase

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4

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GENERATION

Fig. 5. Creatine kinase activity in successive transplant generations of GR mouse mammary tumors. Preparation of cytosol fractions from frozen tumor samples and their assay is described in Methods. Average values f SEM per generation are presented except for generations 7-16 which were pooled to provide a single group with sufficient samples for statistical comparison.

increased by treating the tumor-bearing mice with estrone and progesterone. This contrasts with the situation in the rat in which ovariectomy generally results in a decrease in enzyme activity in ~mmary tumors and treatment with estrogens in an increase [24]. Since this rise of CK specific activity with autonomy was in the opposite direction to the loss of hormone responsiveness and hormone receptors during

Transplant

Generation

Froct~on number

Fig. 6. Creatine kinase isozyme distribution in GR mammary tumors. The isozymes of creatine kinase were separated on a column (1 ml vol.) of DEAE cellulose and assayed as described in Methods. Untilled columns, MM isozyme; solid black, BB isozyme; lightly stippled column (generation 6, series A). MB isozyme. A and B indicate individual tumor lines that were analyzed in several successive transplant generations.

Fig. 7. The ratio of creatine kinase BB to MM in successive transplant generations of two lines of GR mouse mammary tumor. Squares, hormone independent tumors; filled symbols, tumors from ovariectomized animals which received estrone and progesterone treatment as described in Methods. Open symbols, tumors from ovariectomized mice which were not treated with hormones.

progression, it did not seem likely that a hormone responsive isozyme of CK was responsible for the increased activity. It was therefore essential to determine the proportions of the estrogen-responsive BB (brain type) and estrogen-independent MM (muscle type) creatine kinase as a function of transplant generation in GR tumors. When different transplant generations (Fig. 6) or, preferably, series of generations of the same tumor lines were analyzed (Fig. 7), tumors were found to contain pr~ominantly CK-BE (estrogen responsive) in early transplant generations. The tumors were observed to undergo a switch in proportions of BB to MM isozymes in intermediate generations, resulting in equal proportions of the two isozymes or a preponderance of the MM isozyme in later generations. Note that in generation 6 (A) in Fig. 6. a small amount of the CK-MB enzyme was detected. This hybrid form of CK was detected in 10% of the tumors analyzed for their isozyme composition. The switch in proportions of isozymes, which seems to take place at the same generation. or a generation or two earlier than the increase in total CK activity and the transition from hormone dependence or responsiveness to autonomy (Fig. 7), may be correlated with other changes in energy metabolism that may accompany tumor progression from hormonal dependence to independence. Briand and DaehnfeldCZSJ have found an increase in the hexokinase content of GR mouse mammary tumors in late transplant generations accompanied by a decline in glucosed-phosphate dehydrogenase. Rees and Huggins[26] found that while there is no significant change in glucose& phosphate dehydrogenase, in hormone independent rat mammary tumors compared with hormone dependent tumors, lactate dehydrogenase activity is higher in independent tumors. In BR6 mice, pregnancy-independent mammary tumors have higher lactate dehydrogenase and nicotinamide adenine dinucleotide contents than pregnancy dependent tumors [27].

A. M.

74

KAYE et al.

Our finding (above) that. at the transition from hormone-dependence to independence. some tumor lines show a peak in CK activity also has precedents. Smith and King[27] have reported that hormoneindependent mammary tumors in BR6 mice have a transient increase in NAD + content which is greatest shortly after the tumors become independent, but declines again afterwards. They propose that the energy requirements for the tumors is greatest at, or just after, attainment of the independent state. We have previously reported that ConA mediated agglutination of GR mammary tumors also peaks at the transition from hormone dependence to independence [28], and that the tumors are especially sensitive to chemotherapy at this stage [29]. The increased CK activity, coupled with the higher rate of glycolysis in hormone independent GR tumors, may contribute to their higher rate of survival as measured by their lower number of cells required to kill 50% of host mice [30]. Estrogen-responsice breast

creatine

kinase

in

normal

human

siveness of tumors will be correlated with their content of CK-BB. The stimulation by estrogen of creatine kinase activity in normal breast tissue suggests the possibility that an increase, induced in citro, in the activity of creatine kinase BB may provide a direct test for estrogen responsiveness of human breast tumor. Acknowledgements-This work has been supported in part by grants from the Ford Foundation and the Rockefeller Foundation to Professor H. R. Lindner, whom we thank for his advice and encouragement throughout the project. We are grateful to S. Malnick and Smadar Admom for CK isozyme analyses and 0th Lewysohn for estrogen and progesterone receptor assays.

REFERENCES

1. Notides A. and Gorski J.: Estrogen-induced

2. 3.

tumors

Prior to testing for in vitro responsiveness to estrogen, a survey of creatine kinase activity and concentrations of estrogen and progesterone receptors was made in a series of 18 human breast tumor samples obtained from the Kaplan Hospital. Rehovot through the courtesy of Drs Shani and Tchernobilsky. The CK activities showed no correlation with the concentration of either steroid receptor. In several cases, significant CK activities were recorded in the absence of any detectable receptors. This result is compatible with the presence of varying amounts of CK isozymes in human breast tumors (below). In order to ascertain whether creatine kinase is also estrogen responsive in normal human breast, fragments of tissue (less than 1 mm in the smallest dimension) were incubated in Dulbecco’s Minimum Essential Medium, with continuous shaking, at 37°C under an atmosphere of 95”, Oz. 55; CO1 in the presence or absence of 3 x lo-” M estradiol-178. In 7 samples tested, the specific activity of creatine kinase increased (by an average of 940/,) over the control value (0.4 units/min/mg protein) after 2 h of incubation. Increased creatine kinase specific activity in the estrogen treated samples was also observed after 6 h of incubation. We found that normal human breast contains significant amounts of the BB isozyme of CK. De Luca et ~I.[311 have reported very recently that human breast tumors carried in athymic mice contain pre-. dominantly the BB isozyme of CK with some CK-MM activity and a moderate amount of mitochondrial creatine kinase. an isozyme distinct from BB, MM or MB type of CK. We find that in contrast, intraductal carcinomas of human breast show patterns of creatine kinase ranging from 100’4 BB to lOO?h MM. It will be intriguing to see if the respon-

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24. Hilf R.. Goldenberg H., Bell C., Michel I., Orlando R. A. and Archer F. L.: Some biochemical characteristics of rodent and human mammary carcinomas. Enzym. biol. clin. 11 (1970) 162. 25. Briand P. and Daehnfeldt J. L.: Enzyme patterns of glucose catabolism in hormone-dependent and independent mammary tumors of GR mice. Europ. J. Cancer 9 (1973) 763-770. 26. Rees E. D. and Huggins C.: Steroid influences on respiration, glycolysis and levels of pyridine nucleotidelinked dehydrogenase of experimental mammary cancers. Cancer Res. 20 (1960) 963-971. 27. Smith J. A. and King R. J. B.: Biochemical studies on hormone-responsive mammary tumors in BR6 mice. Cancer Res. 30 (1970) 20552060. 28. Sluyser M., Van der Valk M. A. and Van Blitterswijk W. J.: Changes in Concanavalin A-mediated agglutination of hormone-dependent mouse mammary tumor cells during serial transplantation. Br. J. Cancer 41 (1980) 348-355. 29. Sluyser M., De Goeij C. C. J. and Evers S. G.: Changes in sensitivity to cyclophosphamide of mouse mammary tumors during serial transplantation. J. narln. Cancer Inst. 66 (1981) 327-330. 30. Briand P.. Thorpe S. A. and Daehnfeldt J. L.: Difference in growth of hormone dependent and hormone independent mammary tumors of GR mice in oiuo and in vitro. Acra path. microbial. stand. Sect. A. 87 (1979) 427-436. 31 De Luca M., Hall N., Rice R. and Kaplan N. 0.: Creatine kinase isozymes in human tumors. Biochem. Biophps. Res. Commun. 99 (1981) 189-195.