[CANCER RESEARCH 38, 4204-4211,
0008-5472/0038-0000$02.00
November
1978]
Receptor and Centriole Pathways of Steroid Action in Normal and
Neoplastic Cells1
Italo Nenci
Istituto di Anatomia
e Istologia
Patologica,
Università di Ferrara, 44100 Ferrara, Italy
Abstract
An experimental system designed for tracing the estra
dici kinetics in target cells by specific antibodies has been
applied to human breast cancer. Several major defects of
the estradici receptor mechanism have been demon
strated. The detected changes (lack of cytoplasmic recep
tors, impaired nuclear transfer of estradiohreceptor com
plexes, and abnormal nuclear retention of these com
plexes) have been demonstrated in most human breast
cancers that appear to be composed of hormone-depend
ent and autonomous mixed-cell populations. These ab
normalities could be the biological background for the
overall or partial unresponsiveness of breast cancer to
endocrine management.
The participation of steroids in the regulation of centriole activities is taken into account since immunoreactive steroids are traceable by UV and electron microscopy
at the level of this cell organelle by steroid antibodies.
Moreover, the presence of steroids in the pericentriolar
material correlates well with the modulating appearance
and activity of the centriole throughout the cell cycle. A
new centriole pathway is suggested by which steroid
hormones can regulate cell proliferation.
Introduction
Immunocytochemistry investigators recently succeeded
in demonstrating sex steroid hormones in both source and
target tissues (4, 30, 33, 34, 36, 39, 40, 52) (Figs. 1 to 4).
This approach allowed the tracing of steroid hormones at
different intracellular sites and provided some new insight
into the biology and pathophysiology of steroid action. In
regard to human breast cancer, the immunocytochemical
approach led to the demonstration of some abnormal
events in the steroid action mechanism that are relevant to
the tumor hormone responsiveness (31, 32, 34, 36, 39, 40).
The unpredictable response of human breast cancer to
endocrine therapy is axiomatic, and the search for reliable
markers of hormone responsiveness has been a major
concern in recent years. The realization that most of the
effects of steroid hormones are mediated by receptor pro
teins (2, 12, 22) has prompted attempts to evaluate the
relationship between the presence of receptors in breast
tumors and the response to endocrine therapy (15, 18, 24,
25, 27, 28). Thus, the best current index of the hormonal
sensitivity of a breast cancer is the concentration of estro
gen receptor proteins in the tumor tissue. Nevertheless,
and contrary to expectation, the mere presence of estrogen
1 Presented at the John E. Fogarty International Center Conference
Hormones and Cancer, March 29 to 31. 1978. Bethesda. Md.
4204
on
receptors is not an absolute indicator of hormone depend
ency.
This report deals chiefly with the problem of cell hetero
geneity in human breast cancer as far as hormone depend
ency is concerned, with special attention to some kinetic
defects that can be detected in the steroid action mecha
nism by the fluorescent antibody technique.
Methodology
Three main cytochemical approaches can be envisaged
for localizing steroid hormones in target tissues: (a) the use
of labeled hormone, exploited already by Stumpf and Sar
(46) and currently tried again as fluorescent hormone by
Dandlikeref al. (7); (o) the antibody to the purified receptor,
obtained recently by Greene ef al. (14) and expected to be
a very efficient tool for precise localization of receptors; and
(c) antibodies to steroid hormones, which we (33, 34, 36)
and other groups (39, 40) presently use. This approach
exploits the main property of the steroid receptor, i.e., highaffinity steroid binding, and appears to be a powerful
technique for tracing the actual distribution of steroids in
large populations of target cells. Moreover, not only the
presence of the receptor, but also its working state can be
appraised by this approach; this is an advantage especially
remarkable with regard to human breast cancer.
The experimental system for monitoring the estradiol
intracellular kinetics in human breast cancer cells has
already been extensively described elsewhere (33, 34) and
is only schematized in Table 1. It is important to note that,
although the specificity of the fluorescent staining ulti
mately depends on the quality of the antibody, the reliability
of the results depends heavily on the accuracy of control
experiments.
Steroid Receptor Pathway
Cytoplasmic Receptor. The currently accepted model for
the mechanism of action of steroid hormones includes
interaction with cytoplasmic receptor proteins as an initial
and essential intracellular event (2, 12, 22).
Under our experimental conditions the well-known cold
immobilization of est rad ioI:recepto r complexes in the cyto
plasmic compartment allowed a selective fluorescent visu
alization of the estradiol bound at the cytoplasmic level
(Figs. 5 and 6). Specificity for receptor binding was sur
mised from the results of the competitive inhibition by
nonsteroid estrogenic (diethylstilbestrol) and antiestrogenic (nafoxidine, tamoxifen) drugs. Marked differences in
the cytoplasmic uptake were apparent among cells belong
ing to the same tumor (Fig. 7); it may be that this varying
positivity can be related to some extent to the cell cycle (5).
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Receptor and Centriolo
Table 1
Basic procedure for tracing the estradiol kinetics in target cells by
estradio/ antibodies
Experimental system
1. Isolated cell suspension is ob
tained by tissue mincing and
squeezing.
2. Cells (3 x 10" to 5 x 106/ml) are
incubated with 10 nw estradiol for
1 hr at 4°.
3. Cells are carefully washed for 1 hr
at 4°.Some aliquots of cells are
then spread on slides by a cytocentrifuge and air dried at 4°.
4. Other aliquots of cells are postincubated at 37°for 1 to 24 hr in
TC199 plus 10% fetal calf serum.
Aliquots are then treated as in Step
3.
5. Indirect immunofluorescence is
performed with rabbit anti-estradiol-6-BSA antiserum (final dilu
tion 1:100) and goat fluoresceinconjugated anti-rabbit immunoglobulin antiserum (final dilution,
1:25). The temperature is kept at 4°
throughout the procedure.
Control tests
Cell viability: dye exclu
sion
Binding specificity:
competitive inhibition
by diethylstilbestrol
(10 MM),nafoxidine
(0.1 HIM), or tamoxifen (0.1 mw)
Immune specificity:
preimmune serum;
estradiol-absorbed
antiserum.
More frequently, 2 different cell populations were observed
in the same tumor, one endowed with cytoplasmic estradiol
receptor and the other devoid of binding capacity (Fig. 8). A
quantitative evaluation could be made; yet a mere survey
assessment supports the suggestion that almost all breast
cancers are composed of hormone-dependent and autono
mous cells as far as the cytoplasmic estradiol uptake is
concerned.
Estrogen Receptor Movement to the Nucleus. After the
binding of the hormone to its own receptor, the subsequent
movement of the hormone-receptor complex from the cyto
plasm to the nucleus is fundamental to the action of steroid
hormones (2, 11, 12,55). Previous studies from our labora
tory monitored this temperature-dependent intracellular re
distribution of estradiol following the initial uptake. A pro
gressive increase in nuclear estradiol concomitant with the
depletion of the cytoplasmic hormone was observed (Fig.
9), like that occurring in vivo (Fig. 15). Many tumors ap
peared to contain a varying number of cells with an evident
cytoplasmic uptake but a failing nuclear transfer of the
bound estradiol (Figs. 10 and 11). Two main aspects of the
faulty movement to the nucleus have been observed : (a) fail
ure of the bound hormone to move toward the nucleus, as if
it were fixed at the cytoplasmic level; and (b) failure of the
moving estradiol to penetrate the nucleus, around which
the hormone appeared to concentrate in a perinuclear ring
outside the nuclear membrane (Fig. 12). Data implicating a
defective receptor, the receptor binding to the nuclear
membrane, and/or a membrane-controlled access to the
nuclear compartment have already been presented (16, 21,
29, 34, 42, 47, 49, 55). It may be that, in such neoplastic
cells lacking the nuclear filling, a physiological nuclear
membrane step no longer masked by the usual nuclear
positivity can emerge.
Nuclear Binding and Retention. It is generally accepted
Pathways of Steroid Action
(6, 12, 55), despite some conflicting opinion (51), that a key
event in the regulation of gene expression by steroid hor
mones is the interaction of steroid:receptor complexes with
a limited number of specific nuclear acceptor sites. More
over, a prolonged nuclear retention, rather than just the
nuclear binding, of complexes seems to be required to
determine appropriate biological actions (6, 11, 12, 21). In
this respect, some differential releasing rates of the estra
diol accumulated in the nucleus were apparent during the
following time course. Two main pools of nuclear-associ
ated estradiol were identified: (a) a larger, fast-clearing
one, which leaves the nucleus soon after; and (b) a finite
number of long-lasting retention sites associated with the
chromatin network (Fig. 13).
It seems noteworthy that in some tumors there were
several cells that, although retaining the normal nuclear
transfer, lacked the prolonged nuclear retention of the
estradiol (Fig. 14); cell independence of endocrine control
may be ascribed also to reduced or absent "true" nuclear
acceptor sites. The observed defects (Fig. 16) in the nuclear
translocation and binding times strengthen the previous
assessment (19, 23) that nuclear-bound receptors may pro
vide a better indicator of tumor responsiveness than does
the assay of cytoplasmic receptors only.
Centriole Pathway
Steroid antibodies detected by immunocytochemical
techniques strongly decorate the cell centrioles (Fig. 17) in
steroid-target and nontarget cells (31, 35). The immunoperoxidase technique in electron microscopy has been instru
mental in establishing the actual presence of immunoreactive steroids at the level of this cell organelle (Figs. 21 to 23).
Moreover, after treatment with digitonin (a well-known
reactant for 3/3-hydroxysteroids), centrioles of cultured
cells turn out highly biréfringentby polarized-light micros
copy (Fig. 20); 3j8-hydroxysteroids are thus expected to be
present in cell centrioles. In mitotic cells only one of the 2
centrioles present at each pole of the mitotic spindle is
reactive to steroid antibodies (Figs. 18 and 19). This feature
can be related to the known cycling activity of centrioles
(10, 45); in fact the 2 parent centrioles normally separate at
prophase, each accompanied by its still immature daughter,
which will attain functional and morphological maturity
only in the following interphase. Therefore, the presence of
steroids at the centriole level seems to reflect the modulat
ing activity of this organelle throughout the cell cycle.
The precise identity of the immunoreactive steroid(s)
traced at this level is not yet established, and whatever
relationship may exist between the centriolar and the recep
tor pathways is not clear. Only some suggestions encom
passing steroid action and centriole function can be put
forward at present. Centrioles are notoriously involved in
the regulation of cell duplication and differentiation. The
biology and biochemistry of centrioles are unexplored fields
(10,13, 43, 45); the centrioles are probably governed chiefly
by local control, of which hardly anything is known. Never
theless, it has already been suggested that steroid hor
mones can directly affect the structure and/or function of
these organelles (3, 48) and influence cell mitotic behavior
by interfering with the functional role of these microtubule-
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4205
/. Nenci
organizing centers (37). It seems unlikely that tubulin, the
major constituent
of microtubule
organelles and a wellknown binder for many ligands (43, 44), is involved in this
steroid localization.
Instead of microtubules
(20), steroid
antibodies heavily stain the pericentriolar material, drawing
a "negative" picture of the microtubule framework (Fig. 22).
Centrioles themselves are not displayed by specific antitubulin antibody by UV light and electron microscopy (8, 38);
the lack of access of the tubulin antibody has been attrib
uted to the presence of the pericentriolar material to which
we trace immunoreactive
steroids.
The pericentriolar
amorphous cloud, which seems to be
endowed with nucleic acids and specific centrosomic prop
erties (10, 13), fits well as the repository of the interplay
between steroids and centriolar activity. In this respect it
seems remarkable that, in mitotic cells, steroid antibodies
do not detect the immature daughter centriole, which lacks
only the pericentriolar
amorphous material. The concomi
tant modulation
of the centriole activity and its steroid
supply strongly suggests a new pathway by which steroid
hormones can affect the cell duplication,
growth, and
differentiation.
This observation represents a most important question to
be answered in the future, although an arduous task to be
accomplished,
since it is directly related to the regulation
of cell proliferation.
Conclusion
In light of present data, the biological background for cell
control by steroid hormones does not look as simplistic as
previously thought. With regard to the current receptor
pathway, the labeled antibody approach indicates that sev
eral pathological variants of the tumor cell receptor pattern
can influence endocrine
responsiveness
and the clinical
course. Moreover, transcriptional
or posttranscriptional
de
fects downstream
in the sequence of biochemical events
may conceivably lead to unaffected tumor behavior, in spite
of plentiful and working receptors. In fact, the investigation
of some phenotypic effects (1) has been prompted (27, 28)
in pursuit of a reliable postreceptor
marker of hormone
dependence. In this respect our present course concerns
the evaluation of the predictive meaning of some steroidinduced enzyme activities in breast cancer cells, such as:
(a) endogenous peroxidase, already proposed as a reliable
marker for estrogen action (9, 17, 26, 31, 32); and (b) 17/3hydroxysteroid
dehydrogenase,
which seems to be regu
lated via progesterone receptors (41).
In this regard preliminary
results have confirmed
the
assumption of the constant cellular heterogeneity of almost
all breast cancers, while examining cell responsiveness to
hormones. The existence of such cellular heterogeneity is a
major concern in establishing the optimal therapeutic ap
proach to breast cancer. We and others (15, 24, 32, 50)
firmly believe that a correct approach of combined endo
crine and cytotoxic treatments could be envisaged in a
majority of cases. The definition of the optimal combination
for customized therapy should take into account the chance
offered by recruitment and synchronization
of responsive
cells (53, 54) attainable due to the regulation of growth rate
parameters of breast tumors by estrogen.
4206
Acknowledgments
The author thanks all those with whom he collaborated on various aspects
of this work, particularly Dr. M. Donatella Beccati. Dr. Guido A. Fabris. Dr.
Elisabetta Marchetti. Dr. Andrea Marzola, Dr. Carla Arslan-Pagnini, and
Professor Adriano Piffanelli.
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Fig. 1. Estradici crystals treated by the labeled antibody technique brightly fluoresce under UV.
Fig. 2. Fluorescent antibodies display immunoreactive estradici in follicles of a rat ovary, c/, corpora lutea.
Fig. 3. An evident nuclear localization of estradici is traced by the fluorescent antibody technique applied to the myometrium obtained from a 20-day-old
rat, 1 hr after the i.p. injection of estradici (100 ng/g body weight).
Fig. 4. Endometrium from a rat treated as described in the legend to Fig. 3. Immunoperoxidase technique displays estradici chiefly in nuclei of epithelial
and stremai cells.
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Figs. 5 to 8. Dispersed cells incubated with estradici at 4°for 1 hr, washed, and stained with antiestradiol antibody.
Fig. 5. Hyperplastic breast cells. The hormone has been taken up at cytoplasmic level, where it is demonstrated by the fluorescent antibody.
Fig. 6. Human breast cancer cells exhibiting an homogeneous cytoplasmic uptake.
Fig. 7. Human breast cancer cells. The variable cytoplasmic staining seems related to cytological differences.
Fig. 8. Mixed populations of receptor-positive and receptor-negative cells obtained from the same breast cancer.
Figs. 9 to 12. Dispersed cells incubated with estradici at 4°for 1 hr, washed, and postincubated fori hrat 37°.Immunofluorescence technique.
Fig. 9. A predominant nuclear localization of estradiol is clearly traced by fluorescent antibodies in these hyperplastic epithelial cells from human breast
tissue.
Fig. 10. Une of these clustered breast cancer cells fails to display any nuclear incorporation of estradiol.
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Fig. 13. Hyperplastic cells from human breast tissue, postincubated at 37°for 5 hr. Long-lasting nuclear retention sites of estradiol appear asa fluorescent
dotting of the chromatin network.
Fig. 14. Human breast cancer cells displaying scanty or no (O) retention of estradiol in the nucleus.
Fig. 15. Cryostatic section of an intraductal breast carcinoma. The predominant in vivo nuclear localization of the estradiol is clearly traced by the
fluorescent antibody also, when applied on tissue sections.
Fig. 16. Cryostatic sections of 2 infiltrating breast cancers exhibiting a variable mixture of estradiol binding as displayed by the fluorescent antibody.
Fig. 11. Breast cancer cells. Impaired nuclear transfer of the estradiol is evident in some cells, whereas in others the usual nuclear filling by translocated
estradici receptor complexes is exhibited.
Fig. 12. Human breast cancer cells failing the nuclear transfer in spite of the cytoplasmic uptake. An evident perinuclear concentration of estradiol is
shown by these cells; the nuclear transfer behaved properly only in the central cell of the cluster shown in a.
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Receptor and Ceninole Pathways of Steroid Action
Figs. 17 to 19. HEp-2 cells grown in monolayer and stained with antiestradiol antibody (see Ref. 35).
Fig. 17. Two prominent, brightly fluorescent centrioles stand out close to the nucleus. Immunofluorescence technique.
Fig. 18. In this mitotic cell, only 1 of the 2 centrioles present at each pole of the mitotic spindle is detected by the immunofluorescence technique.
Fig. 19. Mitotic cells at metaphase. a: horseradish peroxidase-labeled antibody decorates only the parent centrioles located at each side of the equatorial
plate, b: the peroxidase reaction product draws an evident pericentriolar ring in the same cell by phase contrast.
Fig. 20. The treatment by digitonin brings out centrioles sharply defined when viewed by phase contrast (a) and highly biréfringentby polarized light (b).
Fig. 21. Electron micrograph of a pair of interphase centrioles heavily stained by the immunoperoxidase reaction with antiestradiol antibody.
Fig. 22. The immunoperoxidase technique heavily stains the immunoreactive steroids of the pericentriolar material, drawing a negative picture of the
centriole microtubule triplets.
Fig. 23. Architecture of the microtubule framework of the interphase diplosome of a cell exposed to preimmune rabbit serum.
NOVEMBER
1978
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Receptor and Centriole Pathways of Steroid Action in Normal
and Neoplastic Cells
Italo Nenci
Cancer Res 1978;38:4204-4211.
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