endogenous concentration and subcellular distribution of ......the concentration of estrone in...

(CANCER RESEARCH 45, 2900-2906, June 1985]

Endogenous Concentration and Subcellular Distribution of Estrogens in Normaland Malignant Human Breast Tissue1

A. A. J. van Landeghem,2 J. Poortman, M. Nabuurs, and J. H. H. Thijssen3

Department of Endocrinology, University Hospital, State University of Utrecht, 101 Catharijnesingel, 3500 GV Utrecht, The Netherlands

ABSTRACT

The endogenous concentrations and subcellular distribution ofestrone and estradiol were measured in malignant and nonmalig-nant human breast tissue from pre- and postmenopausal women.

The most striking finding was the significantly higher concentration of estradiol per g of tissue in the malignant tissues than inthe nonmalignant tissues. The tissue concentrations of estradiolin pre- and postmenopausal women were similar despite the

large differences in the peripheral plasma levels. No correlationwas found between the estradiol receptor content and endogenous concentration and subcellular distribution of estradiol. Nodifference in the estrone tissue concentration was found betweenmalignant and nonmalignant tissues. In comparison with humanuterine tissues, which we have reported previously, humanbreast tissue "handles" estrogenic hormones differently from

human uterine tissue. At equal concentrations of the estradiolreceptor, concentrations and subcellular distribution of the estrogens are different in both tissues. It is concluded that themechanism of action of estradiol via its receptor, a mechanismmainly based on studies in animal uterine tissue, applies onlyqualitatively to human breast cancer tissue.

INTRODUCTION

In view of the differences in urinary excretion, plasma levels,and metabolism of estrogens and androgens in breast cancerpatients (10, 30, 46), we have investigated the possibility that adiminished production of androgens is related to the development or to the growth rate of mammary tumors (37). 17/3-

Estradiol (estradiol) exerts its biological activity by binding to aspecific receptor protein in the cytosol prior to entering into thenucleus as a steroid:receptor complex (16) where it binds to an"acceptor site" on the chromatin (32), resulting in an increased

synthesis of mRNA (22). Poortman ef al. (29) reported thatADIOL,4 a metabolite of DHEA, is able to inhibit in vitro the

binding of estradiol to its specific receptor, at a concentrationwithin the physiological range. It was suggested that the lowerplasma levels of ADIOL, as a result of a diminished productionrate of DHEA 3-sulfate and DHEA, at unaltered levels of estradiol

(i.e., an imbalance between estrogens and androgens) mightfavor an enhanced estrogenic stimulus. However, plasma levelsof estradiol and ADIOL in mammary cancer patients were notdifferent from women without breast cancer. Present evidenceindicated that there is no simple relation between plasma levels

1Supported by the Koningin Wilhelmina Fonds, The Dutch Organisation for

Cancer Research.2To whom requests for reprints should be addressed.'Present address: St. Elisabeth Hospital, Hilvarenbeekseweg 60, 5000 LC

Tilburg, The Netherlands.'The abbreviations used are: ADIOL, 5-androstene-3fÂ¡,17fÂ¡-dÂ¡ol;ER, estrogen

receptor; DHEA, dehydroepiandrosterone.Received 5/3/83; revised 5/29/84, 2/27/85; accepted 3/4/85.

and tissue concentrations of steroid hormones (8,9,26,35, 39).Consequently, to determine the ratio between androgens andestrogens at the cellular level in the target organ in vivo, it isnecessary to measure their tissue concentration. Measurementswithin the cell, in cytosol and nucleus, give additional informationabout the mechanism of action of estrogens and androgens. Inorder to test the hypothesis of an imbalance between androgensand estrogens occurring at the cellular level, we have measuredthe concentrations of estrone, estradiol, DHEA, DHEA 3-sulfate,and ADIOL in the cytosol and nuclear fraction of human malignantprimary breast tumor tissues and of nonmalignant breast tissuesof pre- and postmenopausal women.

This paper reports data on estrogens. The data on androgensand on the ratio between androgens and estrogens are reportedseparately (45).

MATERIALS AND METHODS

Tissue Specimens. Breast tissue specimens were obtained from 113women. The tissues were classified in several subgroups, depending onthe histology of the tissue as judged by the pathologist and on themenopausal status of the patient. "Normal" nonmalignant tissue was

obtained from 3 groups of patients. It was excised from: Group a,mammary specimens from premenopausal women which contained onlybenign lesions at histological examination (n = 19); Group b, mammary

specimens from postmenopausal women which contained a malignanttumor (n = 6); and Group c, specimens from premenopausal womenwhich contained a benign tumor at histological examination (n = 5).

Primary mammary tumor tissue was obtained from premenopausal(Group d, n = 21) as well as from postmenopausal (Group e, n = 34)

patients. Hypertrophie tissue was obtained from premenopausal womenundergoing a mamma correction (Group f, n = 16). In addition, benigntumor tissue (Group g, n = 6) and tissue containing fibrocystic lesions(Group h, n = 6), both from premenopausal women, were included in

our study. After collection of all data and statistical analysis, we foundno differences between Subgroups a and c. The individual data for thespecimens of Subgroup c were normally distributed over Group a. Inaddition, we have combined the 3 subgroups of nonmalignant tissuefrom premenopausal women as Subgroup f (hypertrophie tissue),Subgroup g (benign tumor tissue), and Subgroup h (fibrocystic tissue).In the final analysis, we have used 5 groups: Group 1, Subgroups a plusc, premenopausal, normal tissue; Group 2, Subgroup d, premenopausal,malignant tissue; Group 3, Subgroup b, postmenopausal, normal tissue;Group 4, Subgroup e, postmenopausal, malignant tissue; Group 5,Subgroups f, g, and h, premenopausal, nonmalignant tissue. A summaryof data on tissue specimens is given in Table 1.

Preparation of Subcellular Fractions. All tissues were put directly onice in the operation room. They were freed from surrounding fat andconnective tissue by the pathologist. Aliquots were taken for histologicalexamination, and a representative part of the tissue was stored at -80Â°C

until analysis. In general, 0.8 to 1.0 g of tissue was minced, pulverizedin a Micro-Dismembrator (Braun, Melsungen, West Germany) and homogenized in 4 ml of cold Tris buffer (0.01 M Tris-HCI:0.001 M

EDTA:0.003 M NaN3, pH 7.5). The cytosol and nuclear fraction were

CANCER RESEARCH VOL. 45 JUNE 1985

2900

on March 17, 2016. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

ENDOGENOUS ESTROGENS IN MAMMARY CANCER TISSUE

Table 1Age and receptor status in pre- and postmenopausal women and breast cancer

patients

Tissue groupsNo. of Mean age Range of

patients (yr) ages (yr)

Receptor status

ER+ ER-

1. Normal tissue,premen-opausal2.

Malignant tissue,pre-menopausal3.

Normal tissue, postmenopausal4.

Malignant tissue, postmenopausal5.

Nonmalignanttissue,premenopausal242163428384665693215-5530-5856-8249-9516-5014

527

5

prepared by centrifugation of the homogenate at 4Â°C for 30 min at

100,000 x g in a Beckman Model L5-65 ultracentrifuge. Aliquote of this

cytosol were used for quantification of receptor sites and protein content.The remainder of the cytosol and the nuclear fractions were stored at-20Â°C until further analysis of estrogens and androgens.

Quantitation of Receptor Sites. Receptor sites were measured according to a modified European Organization for Research on the Treatment of Cancer procedure (1973) as described earlier (31, 43). Aliquotsof cytosol were incubated overnight at 4Â°Cwith increasing concentrations of [3H]estradiol (0.3 to 2 x 10~9 M). Values were corrected for

nonspecific binding by carrying out control incubations containing a 1000-

fold molar excess of antiestrogen nafoxidine (Upjohn; U 11.100). Boundand free steroids were separated by dextran-coated charcoal. Results

were calculated from Scatchard plot analysis from the binding datacorrected for nonspecific binding. The results are expressed as fmol/mgof cytosol protein. Tumors were classified as positive (ER+), borderline(ERÂ±),and negative (ER-) according to the following limits: ER+ > 10,ERÂ±3 to 10, and ER- <3 fmol/mg of cytosol protein (21). In addition,the K,, should be<10~9M.

Extraction of Estrogens from Cytosol and Nuclear Fraction. Estroneand estradici were extracted from the cytosol and the nuclear fractionas described earlier (44). In brief, to both subcellular fractions, 3H tracers

of the individual hormones were added to enable correction for procedural losses. Estrogens were extracted from the cytosol by precipitationin ethanohacetone (1:1, v/v); the nuclear fraction was sonicated inethanohacetone (1:1, v/v). The ethanol:acetone extracts were defattedwith 70% methanol and, after evaporation of the methanol, the residueswere subsequently extracted with ether after resolution in Tris buffer.The extracts were purified on LH-20 columns on which both estrogens

were separated. In a previous paper, we have demonstrated that thisextraction procedure removes more than 95% of the endogenous estrogens (44).

Measurement of Estrogens. After separation of estrone and estradicion LH-20 columns, both steroids were measured, in duplicate, by ra-

dioimmunoassay using highly specific antisera. All the assays met therequirements of specificity, accuracy, and precision as we describedearlier (42, 44). Results for both cytosol and nuclear fraction are expressed as pmol/g, wet weight, of tissue.

Miscellaneous Techniques. Cytosol protein concentration was measured according to the method of Lowry ef a/. (18) using bovine serumalbumin as the standard. Radioactivity was measured by adding 10 mlof a scintillator (Instagel; Packard Becker BV-Chemical Operations, Gron

ingen, The Netherlands) to the samples. Liquid scintillation counting wasperformed with a Packard Model 2660 liquid scintillation counter withautomatic correction for quenching.

Statistical Analysis. Because the individual data per group werefound to be not normally distributed, differences between the groupswere tested for significance by the Wilcoxon rank sum test for 2 samples.

RESULTS

The subcellular concentrations of estrone and estradiol weremeasured in 113 breast tissue specimens, divided into 5 gropus

based on the histology of the tissue and the menopausal statusof the patient. Data on receptor status, menopausal status, meanage of the patients, range of ages, and cytosol protein contentare given for each group in Table 1. Data on tissue concentrationsof estrone and estradiol are given in pmol/g, wet weight, of tissuewithout correction for cellularity based on DNA or protein contentdue to different ploidy of tumor cells and because we found nosignificant differences in protein content between malignant andnonmalignant tissues.

Intracellular Concentrations in Relation to Normal PlasmaLevels. The concentrations of estrone in the cytosol, in thenuclear fraction, and in total tissue (calculated as cytosol plusnuclear fraction) are shown in Chart 1. Tissue concentrationsshowed a large variation; 95% of the values ranged from about0.2 to 3.3 pmol/g tissue. For each individual group, medianvalues are indicated. In approximately one-half of the tissues,

the concentration of estrone in cytosol and total tissue (pmol/g)is much higher than in peripheral plasma (pmol/ml); the range ofplasma levels of normal healthy pre- and postmenopausal vol

unteers is indicated in Chart 1. Tissue levels in Groups 3 and 4should be compared with the plasma levels in postmenopausalwomen and in Groups 1 and 2 with plasma levels in premenopausal women. Lower concentrations for estrone were found inthe nuclear fraction. In the majority of tissues studied, the nuclearlevels were within the range of normal plasma levels.

A large concentration range of the tissue concentrations ofestradiol was found (Chart 2); 95% of the values ranges fromabout 0.2 to 2.6 pmol/g tissue. In the majority of the premenopausal nonmalignant specimens, the concentration of estradiolin cytosol and in total tissue is within the range of normal plasmalevels. In most of these specimens, the concentration in thenuclear fraction is below the peripheral plasma level. The tissue-

plasma gradient for estradiol, especially in the postmenopausalmalignant group (Group 4), is very high. This gradient was foundboth in cytosol and in total tissue and, to a lesser extent, in thenuclear fraction. For both estrogens, the concentrations in thenuclear fraction are lower than in the cytosol.

Comparison of Estrogen Concentrations in the DifferentGroups. In premenopausal women (Group 2), the concentrationof estradiol in cytosol, nuclear fraction, and total tissue is significantly higher in malignant than in normal tissue of premenopausal women (Group 1) (P <0.05, P <0.05, and P <0.01, respectively). No significant differences were found in the concentrations of estrone in malignant tissue (Group 2) and normal tissue(Group 1) of premenopausal women. For the comparison ofnormal and malignant tissue of postmenopausal women, normaltissue from 6 patients was available (Group 3). The concentrationof estradiol was significantly higher in malignant than in normaltissue of postmenopausal women, in the cytosol and nuclearfractions (P <0.01, P <0.05, respectively). No significant difference was found in the tissue concentrations of estrone in pre-

and postmenopausal women with breast cancer. Nevertheless,the tissue concentration of estrone tended to be higher in thepremenopausal patients. Because of the considerable differencein peripheral plasma levels, especially of estradiol, between pre-

and postmenopausal women, it is very striking that the concentration of estradiol in the mammary tissues were similar in pre-

and postmenopausal breast cancer patients.In premenopausal tissue samples (Group 1), the concentra

tions of estrone tended to be higher than in the postmenopausal


2901




cytosol Ei nuclear fraction Ej total tissue

Chart 1. Estrone (Â£i)levels in the cytosol,the nuclear fraction, and total tissue in the 5groups studied. For each group, median valuesare shown. The range of plasma levels of normal healthy pre- and postmenopausal volunteers is indicated; F. L, and O, upper level in thefollicular. luteal, and ovulatory phases, respectively, of the menstrual cycle. Tissue levels ofGroups 3 and 4 are to be compared with theplasma levels in postmenopausal women; tissue levels of Groups 1 and 2 should be compared with plasma levels of premenopausalwomen. Group 5 represents 2 differentsubgroups: hypertrophie tissue (â€¢);benign tumor and fibrocystic tissue (D).

3.50- p mol/gram tissue

3.00

2.50

ZOO-

1.50

1.00

0.50

group 1

T i

3A

3B

<tÂ°

3C

plasma

â€”lO

pre post

Â£2cytosol E-2nuclear fraction E2 total tissueâ€¢g. -SÂ» Â«â€¢ -ÃŽÂ» 'W

Chart 2. Estradici (Â£2)levels in the cytosol,in the nuclear fraction, and in total tissue in the5 groups studied. Median values are shown foreach group. The range of plasma levels ofnormal healthy pre- and postmenopausal volunteers is indicated. F, L, and O, upper level inthe follicular, luteal, and ovulatory phases, respectively, of the menstrual cycle. Tissue levelsof Groups 3 and 4 are to be compared with theplasma levels in postmenopausal women; tissue levels of the other groups should be compared with those of premenopausal women.Group 5 represents 2 different subgroups: hypertrophie tissue (â€¢);benign tumor tissue andfibrocystic tissue (O).

3.50- p mol /gram tissue

3.00-

2.50-

2.00-

1.50-

1.00-

0.50-

group 12345A

*..Â«f. V v ^v* * â€” *XT- - T

plasma

â€”iO

3

C

5 pre post


2902




tissue samples (Group 3) (P <0.1 for the cytosol, the nuclearfraction, and total tissue). In normal tissue of pre- and postmen-

opausal women, similar estradici levels were observed. AlthoughGroup 5 represents a heterogeneous group, analysis showed nodifference between the 3 subgroups. Comparison of Group 5with Group 1 (premenopausal normal) showed no statisticaldifference for estrone or for estradici. The subgroups in Group5 more closely resemble the normal tissues in Group 1 than themalignant tissues in Group 2.

Estrone:Estradiol Ratio. Significantly different estrone:es-

tradiol ratios were found in normal and malignant tissues ofpremenopausal women (Chart 3) (P <0.001 for both subcellularfractions and total tissue). These differences are greater in thecytosol and in total tissue than in the nuclear fraction. Malignanttissues especially show ratios less than one. This can be explained by the higher estradici concentrations in malignant tissueand not by a lower concentration of estrone. For all tissues, theratio is lower in the nuclear than in the cytosol fraction, causedby higher nuclear concentrations of estradici than of estrone. Nodifferences between the subgroups in Group 5 were found. Theestrone:estradiol ratios in the tissues in Group 5 were morecomparable to those in the normal tissues of Group 1 than tothose of the malignant tissues of Group 2.

ER and Estradici Tissue Concentration. The receptor content was measured in the tumors of 19 premenopausal and 32postmenopausal patients. From the premenopausal patients, 14were classified receptor positive (>10 fmol/mg protein); in the

postmenopausal group, 27 tumors were positive. The meanreceptor concentrations in the pre- and postmenopausal patients

were 67 Â±12.5 (SE) and 134 Â±17.5 fmol/mg protein, respec

tively. The difference in receptor content between pre- and

postmenopausal tumor tissue was highly significant (P <0.01).There was no significant correlation between the receptor content and the concentration of estradiol in the cytosol, expressedin fmol/mg protein (Chart 4). The concentration of estradiol inthe cytosol of receptor-positive tumors is significantly higher (P<0.05) than in the receptor-negative tumors (Chart 5). Also, in

the nuclear fraction and in total tissue, the estradiol concentrations in the receptor-positive tissues are significantly higher (P<0.02) than in receptor-negative tissues.

Subcellular Distribution. Calculations of the ratio of the concentration in the nuclear fraction to the concentration in thecytosol for all groups revealed that the concentration of theestrogens was the highest in the cytosol. The following meanvalues were found: estrone, 0.31 Â±0.29 (SD; n = 105); estradiol,0.62 Â±0.39 (n = 108). For estrone, 95% of the values ranged

between zero and 1.18, and the median values of the 5 groupsranged from 0.16 to 0.30. For estradiol, 95% of the valuesranged from 0.02 to 1.52, and the median values ranged between0.46 and 0.69. No significant differences were found betweenthe groups. Although the mean receptor content in the receptor-

positive postmenopausal patients was found to be twice thereceptor content in premenopausal receptor-positive tissues, no

significant difference was found in the ratio of estradiol in thenuclear fraction to estradiol in the cytosol in pre- and postmenopausal tumor tissues. The mean ratios were 0.80 Â±0.52 (n =14) and 0.62 Â±0.35 (n = 27) for pre- and postmenopausalreceptor-positive tumor tissues at median values of 0.65 and0.53, respectively. This difference holds also for receptor-negative tumors with mean values of 0.61 Â±0.43 (n = 5) for premen-

/E2 cytosol Ej /E2 nuclear fraction Ej /Â£>total tissue

Charts. Estrone (Â£,):estradiol(Â£2)ratio inthe subcellular fractions and in total tissue inthe 5 groups studied. Groups 3 and 4, postmenopausalwomen; Groups 2 and4, malignantbreast tumor tissues of pre- and postmenopausal women, respectively; Group 5, 2 different subgroups: hypertrophie tissue (â€¢);benigntumor and fibrocystic tissue (O)- The rangeof plasma ratios of normal healthy pre- andpostmenopausal volunteers is indicated. Tissue ratios of Groups 3 and 4 are to be compared with the plasma ratios in postmenopausal women; tissue ratios of the other groupsshouldbe comparedwith those of premenopausal women.

9-.7-

.â€¢â€¢5-

1â€¢^3-â€¢

â€¢â€¢Aâ€¢

â€¢1group!.9

D*"*

JÂ£Ã¬;

Ãœfâ€¢.TtA.;.1

Ã• Â°.â€¢

â€¢GI

*Â°'1

j. Â«i-Â¡flit1â€¢*JtÂ«â€¢f . .,-4^1i

423~T~512345 123""

4 8 C5 pre post


2903




oi

Â¿00-

2 300-

ol. 200

8tuu 100-

â€¢o.uÃ ,â€” ESTRADICI.

10 30 50 70 90Chart 4. Estradiol levels in the cytosol and the number of estradiol receptors in

prÃ©-and postmenopausalbreast cancer patients.

3.50- p mol/ gram tissue

3.00-

2.50-

2.00-

1.50-

1.00-

0.50-

â€¢o

o

ER * - Â« - * -cytosol nuclear total' fraction tissue

Chart 5. Estradiol levels in the subcellular fractions and in total tissue of pre-and postmenopausal breast cancer patients in relation to receptor status, ERpositive (EW+)or ER negative (ER-). E2,estradiol.

opausal and 0.31 Â±0.32 (n = 5) for postmenopausal breasttumor tissue at median values of 0.53 and 0.32, respectively.

The nucleus:cytosol ratio for estradiol was higher in receptor-positive (0.68 Â±0.42, n = 41) than in receptor-negative tumors(0.46 Â± 0.39, n = 10) at median values of 0.58 and 0.41,

respectively, but the difference was not significant. No correlationwas observed between the receptor and the nucleus:cytosolratio for estradiol, either in premenopausal or in postmenopausalpatients.

DISCUSSION

In all groups, the tissue-plasma gradients for estrone were >1.

The gradient was most pronounced in the cytosol and the totaltissue in the postmenopausal breast cancer patients. In contrast,a distinct tissue-plasma gradient for estradiol was present only

in the postmenopausal breast cancer group, and this gradientwas much higher for estradiol than for estrone. The ratio ofestrone to estradiol, calculated for each tissue specimen, clearlydemonstrated the differences between tissue and plasma forthese steroids. Peripheral plasma levels were therefore not reflected in endogenous tissue concentrations. This supports thefindings of others who measured simultaneously plasma levelsand breast tumor tissue concentrations (26, 39) or tissue concentrations in the human reproductive tract (8, 9, 35).

It should be noted that the steroid determinations were madeafter Chromatographie purification using highly specific antiserafor radioimmunoassay. The validation and reliability criteria ofthese assays were described previously (42, 44). In comparisonwith published data, the intracellular concentrations of estradioland estrone in human breast tumor tissue are in good agreementwith the literature (3, 13, 14, 19, 20, 26). Millington ef al. (25),however, reported higher tissue concentrations of estrone andestradiol, while Valient ef al. (39) reported lower estrogen concentrations in tumor tissue but much higher concentrations inbenign tumors. Only Maynard ef al. (19, 20) measured estradiolin the nuclear fraction of breast tumor tissue; they reportedconcentrations similar to ours. In this study, the tissue concentrations have been compared to plasma levels of normal healthyvolunteers. In agreement with the literature, the plasma levels inhealthy women were not significantly different from plasma levelsof breast cancer patients.

The most striking difference in tissue-plasma gradients wasthat between estrone and estradiol for pre- and postmenopausal

breast cancer patients, while the gradient for estradiol wasdifferent between malignant and nonmalignant tissue. Severalfactors may affect the tissue concentrations and therefore thetissue-plasma gradients: (a) uptake of steroid hormones from

the circulation; (b) tissue levels of specific binding proteins (receptors) or nonspecific binding proteins with low affinity but highcapacity; (c) metabolic conversion from precursors to othersteroids, not only in the tumor cell but also in the surroundingnormal cells and fat cells. As far as we know, there are nodifferences in permeability for steroid hormones between normaland malignant breast cell membranes.

Whether steroid hormones enter the cell by passive diffusionor by a membrane-mediated process is still unknown. In a recent

review on this subject, Rao (33) concluded that passive diffusionalone was unlikely.

Our data confirm the higher estradiol concentrations in the


2904




cytosol of receptor-positive tumors than in the cytosol of receptor-negative tumors (13, 14, 19, 20, 38). Also, the significantlyhigher estradiol concentration in the nuclear fraction of receptor-positive tumors is in agreement with the data of Maynard ef al.(20). Furthermore, no differences in tissue estradiol concentrations occurred between pre- and postmenopausal patients, although the receptor content in postmenopausal patients wastwice the concentration in premenopausal women. These resultsare in agreement with published data (13, 19, 20, 26, 34, 36,38). It is suggested that the differences in estradiol tissue concentrations cannot be explained solely by the levelof the receptorproteins because: (a)no correlation existed between the estradioland the receptor concentrations; (b) with the exception of aboutone-half of the breast tumor tissues, the concentration of estronewas higher than the estradiol concentration and showed norelation with the receptor content; (c) the estradiol concentrationin human endometrium and myometrium has been reported tobe about 10 times higher than in breast tumors (7) at similarreceptor concentrations (40). Thus, besides the involvement ofthe estrogen receptor, it is possible that proteins with a lowerbinding affinity and higher capacity, so-called nonspecific bindingproteins, play a role in the uptake and concentrating mechanismfor both estrone and estradiol.

Many in vitro studies have demonstrated that breast tissuecontains a variety of enzymes capable of converting precursorsto biologically active steroid hormones (41). Some differences inthese conversions between pre- and postmenopausal patientsand between receptor-positive and receptor-negative tumorshave been reported (1-6,17, 23, 24, 28, 46). No specific metabolic pathway for either estrone or estradiol biosynthesis orassociation between receptor status and aromatization has beenreported for in vitro studies in human breast tumors (4, 17, 23,46). These investigations therefore add little to our understandingof the tissue-plasma gradients of the estrogens. No consensusof opinion on whether normal tissue is able to convert androgensto estrogens is available (4, 24, 27). Our findings of a higherestradiol concentration in the tumors than that in normal tissuesuggest that local aromatization might be involved. Humanbreast tumor tissue is able to metabolize steroid hormones Invivo (11,12,15, 42); however, data on differences in metabolicpathways between pre- and postmenopausal women and between receptor-positive and receptor-negative tumors are notavailable.

Consequently, it is concluded that the tissue concentrationsof estrone and estradiol in tumors, compared to malignant tissues from both pre- and postmenopausal women, cannot beexplained by selective uptake of estrone and estradiol or bydifferences in receptor status.

Previously, after a 12-h infusion of estrogens, we reported theuptake, metabolism, and subcellular distribution of estrogens inhuman endometrium, myometrium, and vagina (48). From thatstudy, we concluded that these tissues preferentially accumulateestradiol and that in these tissues the concentration of estronelargely results from intracellular conversion from estradiol. Afterinfusion of [3H]estradiol, most of the radioactivity was localizedin the nuclear fraction, while after infusion of [3H]estrone, the

majority of the activity was present in the cytosol, and only aminor amount was present in the nuclear fraction. These tracerstudies were confirmed by measurement of the subcellular distribution of endogenous estrogens in human uterine tissues using

the same technique as in the present study (7). In contrast tothe observed subcellular distribution of both estrogens in humanuterine tissue, the concentration of estradiol in human breasttissue was highest in the cytosol at a receptor content equivalentto that in uterine tissue. Moreover, a considerable amount ofestrone was present in the nuclear fraction. Although the nu-clearcytosol ratio of estradiol was higher in receptor-positivethan in receptor-negative tumors, no correlation was found between receptor content and estradiol concentration. It is therefore concluded that in human breast tumor tissue the receptorplays only a minor role in the subcellular distribution and that, inaddition to an active transport of estradiol into the nucleus, thereis a free distribution between cytosol and nucleus. Whether theintracellular concentrations and subcellular distribution of androgens play an additional role, at the cellular level, in the etiologyof human breast cancer remains to be seen.

REFERENCES

1. Abul-Hajj, Y. J. Metbolism of dehydroepiandrosterone by hormone dependentand hormone independent human breast carcinoma. Steroids, 26: 488-500,1975.

2. Abul-Hajj, Y. J. Steroid A4-5j8-reductase in human mammary tumors. Steroids,33; 115-124,1979.

3. Abul-Hajj, Y. J. Relationship between estrogen receptors, 17/3-hydroxysteroiddehydrogenase and estrogen content in human breast cancer. Steroids, 34:217-225,1979.

4. Abul-Hajj, Y. J., Iverson, R., and Kiang, D. T. Aromatization of androgens byhuman breast cancer. Steroids, 33. 205-222,1979.

5. Abul-Hajj, Y. J., Iverson, R., and Kiang, D. T. Estradiol 17/3-<lehydrogenaseand estradiol binding in human mammary tumors. Steroids, 33: 477-483,1979.

6. Abul-Hajj, Y. J., Iverson, R., and Kiang, D. T. Metabolism of pregnenolone byhuman breast cancer. Evidence for 17o-hydroxylase and 17,20-lyase. Steroids,34:817-827,1979.

7. Alsbach, G. P. J., Franck, E. R., Poortman, J., and Thijssen, J. H. H. Estradioland estrone levels in subcellular fractions of the human endometrium andmyometrium during the normal menstrual cycle. Contraception, 27: 409-421,1983.

8. Batra, S., Bengtsson, L. P., and SjÃ¶berg, N. 0. Interrelation between plasmaand tissue concentrations of 17Â£-oestradiol and progesterone during humanpregnancy. Clin. Endocrinol., 11: 603-610,1979.

9. Cortes-Gallegos, V., Gallegos, A. J., Sanchez-Basurto, C., and Rivadeneyra,J. Estrogens peripheral levels vs estrogen tissue concentration in the humanfemale reproductive tract. J. Steroid Biochem., 6:15-20,1975.

10. Dao, T. L. Metabolism of estrogens in breast cancer. Biochim. Biophys. Acta,560:397-426,1979.

11. Deshpande, N., Carson, O., Di Martino, L, and Tarquini, A. Biogenesis ofsteroid hormones by human mammary gland in vivo and in vitro. Eur. J.Cancer, 12: 271 -?76,1976.

12. Duvivier, J. D., Colin, C., Hustin, J., Albert, A., Lavigne, J., Dive, G., andMontfort, F. Comparison of levels of cylosol estrogen receptors with "arterial"and "venous" concentration of gonadic steroids in mammary tumors. Clin.

Chim. Acta, 112: 21-32,1981.13. Edery, M., Goussard, J., Dehennin, L., Scholler, R., Reiffsteck, J., and Dros-

dowsky. M. A. Endogenous oestradiol-170 concentration in breast tumoursdetermined by mass fragmentography and by radioimmunoassay: relationshipto receptor content. Eur. J. Cancer, 77:115-120,1981.

14. Fishman, J., Nisselbaum, J. S., Menendez-Botet, C. J., and Schwartz, M. K.Estrone and estradiol content in human breast tumours: relationship to estradiol receptors. J. Steroid Biochem., 8: 893-896,1977.

15. Griffiths, K., Jones, D., Cameron, E. H. D., Gleave, E. N., and Forrest, A. P.M. Transformation of steroids by mammary cancer tissue. In: T. L. Dao (ed.),Estrogen Target Tissues and Neoplasia, pp. 151-162. Chicago: ChicagoUniversity Press, 1972.

16. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W., andDeSombre, E. R. A two-step mechanism for the interaction of estradiol withrat uterus. Proc. Nati. Acad. Sci. USA, 59: 632-638, 1968.

17. LJ,K., and Adams, J. B. Aromatization of testosterone and oestrogen receptorlevels in human breast cancer. J. Steroid Biochem., 14: 269-272,1981.

18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Proteinmeasurement with the Polin phenol reagent. J. Biol. Chem., 793: 265-2751951.

19. Maynard, P. V.. Brownsey, B. G., and Griffiths, K. Oestradiol levels in fractionsof human breast tumours. J. Endocrinol., 77: 62p-63p, 1978.


2905




20. Maynard, P. V., and Griffiths, K. Clinical, pathological and biochemical aspectsof the oestrogen receptor in primary human breast cancer, in: R. J. B. King(ed.), Steroid Receptor Assays in Human Breast Tumors: Methodological andClinical Aspects, pp. 86-99. Cardiff, Wales: Alpha Omega Publishing, Ltd.,

1979.21. McGuire, W. L., Pearson, O. H., and Segaloff, A. Predicting hormone respon

siveness in human breast cancer. In: W. L. McGuire, P. P. Carbone, and E. P.Vollmer (eds.), Estrogen Receptors in Human Breast Cancer, pp. 17-30. NewYork: Raven Press, 1975.

22. Means, A. R., and Hamilton, T. H. Early estrogen action: concomitant stimulations within two minutes of nuclear RNA synthesis and uptake of RNAprecursor by the uterus. Proc. Nati. Acad. Sci. USA, 56: 1594-1598,1966.

23. Miller, W. R.. Hawkins, R. A., and Forrest, A. P. M. Significance of aromataseactivity in human breast cancer. Cancer Res. (Suppl), 42: 3365s-3368s, 1982.

24. Miller, W. R., Shivas, A. A., and Forrest, A. P. M. Factors affecting testosteronemetabolism by human breast tissues. Clin. Oncol., 4: 77-85, 1978.

25. Millington, D., Jenner, D. A., Jones, T., and Griffiths, K. Endogenous steroidconcentrations in human breast tumours determined by high-resolution massfragmentography. Biochem. J., 739.-473-475,1974.

26. Nagai, R., Kataoka, M., Kobayashi, S., Ishihara, K., Tobioka, N., Nakashima,K., Naruse, M., Salto, K., and Sakuma, S. Estrogen and progesterone receptors in human breast cancer with concomitant assay of plasma 170-estradiol,progesterone and prolactin levels. Cancer Res., 39: 1835-1840, 1979.

27. Perel, E., Davis, S., and Killinger, D. W. Androgen metabolism in male andfemale breast tissue. Steroids, 37: 345-352,1981.

28. Pewnim, T., Adams, J. B., and Ho, K. P. Estrogen sulfurylation as an alternativeindicator of hormone dependence in human breast cancer. Steroids, 39: 47-

52, 1982.29. Poortman, J., PreÃ±en,J. A. C., Schwarz, F., and Thijssen, J. H. H. Interaction

of A5-androstene-3fi,17#-diol with estradici and dihydrotestosterone receptors

in human myometrial and mammary cancer tissues. J. Clin. Endocrinol. Metab.,40: 373-379,1975.

30. Poortman, J., Thijssen, J. H. H., and Schwarz, F. Androgen production andconversion to estrogens in normal postmenopausal women and in selectedbreast cancer patients. J. Clin. Endocrinol. Metab., 37: 101-109, 1973.

31. Poortman, J.. Thijssen, J. H. H., and Schwarz, F. Steroid receptors in humanreproductive tissues. In: J. L. Wittliff and O. Dapunt (eds.), Steroid Receptorsand Hormone Dependent Neoplasia, pp. 45-58. New York: Masson Publishing,

Inc., 1980.32. Puca, G. A., Sica, V., and Noia, E. Identification of a high affinity nuclear

acceptor site for estrogen receptor of calf uterus. Proc. Nati. Acad. Sci. USA,77:979-983,1974.

33. Rao, G. S. Mode of entry of steroid and thyroid hormones into cells. Mol. Cell.Endocrinol., 27: 97-108, 1981.

34. Saez, S., Martin, P. M., and Chouvet, C. D. Estradici and progesterone receptorlevels in human breast adenocarcinoma in relation to plasma oestrogen and

progesterone levels. Cancer Res., 38: 3468-3473,1978.35. Schmidt-Gollwitzer, M., Eiletz, J., Genz, T., and Pollow, K. Determination of

estradiol, estrone and progesterone in serum and myometrium: correlationwith the content of sex steroid receptors and 17/3-hydroxysteroid dehydrogen-

ase activity throughout the menstrual cycle. J. Clin. Endocrinol. Metab., 49:370-376, 1979.

36. Thevo, N. O., Caristrom, K., Gustafsson, J. A., Gustaffson, S., Nordeskjold,V., Skoldefors, H., and WrÃ¤nge,Ã–.Oestrogen receptors and peripheral serumlevels of oestradiol-17/3 in patients with mammary carcinoma. Eur. J. Cancer,14: 1337-1340,1978.

37. Thijssen, J. H. H., Poortman, J., and Schwarz, F. Androgens in postmenopausal breast cancer: excretion, production and interaction with estrogens. J.Steroid Biochem., 6: 729-734, 1975.

38. Thorsen, T., Tangen, M., and Stoa, K. F. Concentration of endogenousoestradiol as related to oestradiol receptor sites in breast tumor cytosol. Eur.J. Cancer Clin. Oncol., 78: 333-337, 1982.

39. Valient, K., FehÃ©r,T., Bodrogi, L., and Ribai, Z. Steroid-Hormone-Gehalt derBrustsubstanz bei Mammatumoren. Chirurg, 53: 34-36,1982.

40. van Haaften, M., Wiegerinck, M. H. A. M., Poortman, J., Haspels, A. A., andThijssen, J. H. H. Progesterone receptors in human oestrogen target tissues.Maturitas, 4: 57-66, 1982.

41. van Landeghem, A. A. J. Hormones and breast cancer: endogenous concentration and subcellular distribution of oestrogens and androgens in normal andmalignant human mammary tissue. Ph.D. Dissertation, University of Utrecht,1983.

42. van Landeghem, A. A. J., Poortman, J., Deshpande, N., Di Martino, L., Tarquini,A., Thijssen, J. H. H., and Schwarz, F. Plasma concentration gradient of steroidhormones across human mammary tumours in vivo. J. Steroid Biochem., 74:741-747, 1981.

43. van Landeghem, A. A. J., Poortman, J., Faber, J. A. J., and Thijssen, J. H. H.Absence of seasonal variation of estrogen receptor levels in human breastcancer tissue. Mol. Cell. Endocrinol., 23: 265-273,1981.

44. van Landeghem, A. A. J., Poortman, J., Helmond-Agema, A., and Thijssen, J.H. H. Measurement of endogenous subcellular concentration of steroids intissue. J. Steroid Biochem., 20: 639-644, 1984.

45. van Landeghem, A. A. J., Poortman, J., Nabuurs, M., and Thijssen, J. H. H.Endogenous concentration and subcellular distribution of androgens in normaland malignant breast tissue. Cancer Res., 45: 2907-2912,1985.

46. VÃ¡rela, R. M., and Dao, T. L. Estrogen synthesis and estradiol binding byhuman mammary tumors. Cancer Res., 38: 2429-2433, 1978.

47. Wang, D. Y. Androgens and breast cancer. Rev. Endocr.-related Cancer, 3:19-24,1979.

48. Wiegerinck, M. A. H. M., Poortman, J., Donker, T. H., and Thijssen, J. H. H.In vivo uptake and subcellular distribution of tritium labelled estrogens inhuman endometrium, myometrium, and vagina. J. Clin. Endocrinol. Metab.,56: 76-86, 1983.


2906



1985;45:2900-2906. Cancer Res A. A. J. van Landeghem, J. Poortman, M. Nabuurs, et al. Estrogens in Normal and Malignant Human Breast TissueEndogenous Concentration and Subcellular Distribution of

Updated version

http://cancerres.aacrjournals.org/content/45/6/2900

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications


http://cancerres.aacrjournals.org/content/45/6/2900

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


endogenous concentration and subcellular distribution of ......the concentration of estrone in...

Documents