| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Departments of Anatomy [Y. W., A. A. D., P. Y., G. R. C.], Urology [S. W. H., R. D., G. R. C.], University of California, San Francisco, California 94143-0452; Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 [T. J., J. S.]; University of California Davis Center for Comparative Medicine, University of California, Davis, California 95616 [R. D. C.]; and Department of Surgery, Division of Urology, University of Michigan Comprehensive Cancer Center, University of Michigan Ann Arbor, Michigan 48109 [M. L. D.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
13 days of gestation. In this study, we
have rescued Rb-/- prostates by grafting pelvic organ
rudiments from Rb-/- mouse embryos under the renal
capsule of adult male nude mouse hosts. Grafts of embryonic pelvic
organs developed into functional prostatic tissue. Some of the
prostatic tissue generated was further used to construct chimeric
prostatic tissue recombinants by combining wild-type rat urogenital
mesenchyme (rUGM) with Rb-/- and Rb+/+
prostatic epithelium (PRE). The tissue recombinants were grown as
subcapsular renal grafts and treated from the time of grafting with
Silastic capsules containing 25 mg of testosterone plus 2.5 mg of
estradiol. During 5–8 weeks of hormone treatment,
rUGM+Rb+/+PRE tissue recombinants developed prostatic
hyperplasia, whereas PRE in rUGM+Rb-/-PRE tissue
recombinants developed hyperplasia, atypical hyperplasia, and
carcinoma. During carcinogenesis in rUGM+Rb-/-PRE tissue
recombinants, prostatic epithelial cells of the basal lineage
disappeared, whereas the luminal cells underwent carcinogenesis.
Epithelial E-cadherin almost totally disappeared. In all cases,
epithelial PCNA labeling was elevated in tissue recombinants containing
Rb-/- versus Rb+/+ epithelium.
These epithelial changes were associated with almost total loss of
smooth muscle cells in the stroma. In contrast, in untreated hosts
rUGM+Rb+/+PRE tissue recombinants developed normally, and
rUGM+Rb-/-PRE tissue recombinants developed mild
epithelial hyperplasia. The results of this study demonstrate that
Rb-/- prostatic tissue can be rescued from embryonic
lethal mice and used to test its susceptibility to hormonal
carcinogenesis. Deletion of the Rb gene predisposes
prostatic epithelium to hyperplasia and increases proliferative
activity. Susceptibility to hormonal carcinogenesis in response to
exogenous testosterone + estradiol is manifested in the
progression from atypical hyperplasia to carcinoma. Thus, these
findings demonstrate that the absence of the Rb tumor
suppressor gene may predispose prostatic epithelial cells to
carcinogenesis. Rescue of organs from Rb-/- embryos not
only provides an opportunity to analyze the Rb gene
pathway in the development and progression of prostate cancer but also
provides an opportunity for specifically evaluating the role of the Rb
pathway in development and carcinogenesis in other organs, such as the
mammary gland and colon. Because rUGM greatly stimulates prostatic
epithelial proliferation, the tissue recombinant model is a
particularly useful tool for assessing the functional role of other
genes in prostatic carcinogenesis through use of the appropriate
transgenic or gene knockout mice. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The major in vivo models of prostatic carcinogenesis are: (a) those models in which prostate cancer can be induced, or its incidence increased, by chemical and/or hormonal carcinogens. Examples include the Noble and Lobund-Wistar rat prostatic carcinoma models. These models replicate many of the early phases of carcinogenesis. Their main drawbacks are long latency and low incidence of cancer (1, 2, 3, 4, 5) ; (b) transgenic mouse models of prostate cancer. These include the TRAMP (6) and the LPB-Tag or "LADY" series (7) , which use the probasin promoter to target expression of SV40-T antigen to the luminal prostatic epithelial cells. The basal prostatic epithelial cells apparently do not express SV40-T antigen in these models. These transgenic mice develop prostatic cancer with very high efficiency. Their tumors are androgen responsive and they metastasize; (c) prostatic reconstruction models of prostatic cancer. The prostatic reconstruction model (8) uses the use of tissue recombinants composed of UGM and UGE grafted beneath the renal capsule of athymic mouse hosts. Thompson et al. (8) used viruses to introduce activated oncogenes into UGM and UGE of the developing prostate to generate prostatic tissue expressing oncogenes in the prostatic epithelium or stroma.
Each of the above models is contrived and uses highly abnormal approaches. Each model has, however, significantly advanced the field of prostatic carcinogenesis, despite this inherent artificiality. The newly established model described in the present communication also has it own degree of artificiality but is valuable because prostatic carcinogenesis is elicited at high efficiency and with low latency in response to treatment with T and E2. This model uses aspects of the hormone-induced tumor models, genetic modification, and tissue recombination models.
Prostatic carcinogenesis is a multistep process involving both genetic alterations to the epithelial cells such as activation of oncogenes (9, 10, 11, 12) , inactivation of tumor suppressor genes (13 , 14) , as well as perturbation of stromal-epithelial interactions (15, 16, 17, 18) . The involvement of multiple oncogenes and tumor suppressor genes in carcinogenesis has been demonstrated for many types of carcinomas (19 , 20) . Alterations in tumor suppressor genes such as the RB gene have been suggested to play a role in the development of prostate cancer (13 , 21, 22, 23) . The RB gene is located on human chromosome 13 and encodes a Mr 110,000 nuclear protein involved in cell cycle control and other processes (24) . The importance of the RB gene in tumorigenesis was originally recognized in familial retinoblastoma and subsequently the involvement of RB has been described in a number of other human neoplasias including bladder (25) , breast (26, 27, 28) , and lung cancer (29, 30, 31) . In human prostate cancer, RB gene mutations have been reported in 16.4% of primary human prostatic cancers, which suggested that inactivation of RB may be an important event in at least a subset of prostatic carcinomas (32, 33, 34) . Moreover, it was reported that 27% of human prostatic adenocarcinomas have lost one RB allele (35) . The relatively high frequency of this occurrence suggests that RB may be involved in the development of prostatic lesions.
To study Rb function, gene targeting has been used to inactivate Rb in mice (36, 37, 38) . Mice homozygous for Rb disruption (Rb-/-) die at about 13 days of gestation, several days before the prostate forms. Thus, it appears problematic to study prostatic carcinogenesis in Rb-/- mice. To solve this problem, we have generated Rb-/- prostatic tissues by grafting embryonic pelvic viscera beneath the renal capsule of male nude mice. Embryonic prostatic rudiments grafted under the renal capsule grow substantially, undergo prostatic morphogenesis, and produce prostatic secretory proteins (39) .
It is well established that UGM can induce prostatic epithelial growth, ductal branching morphogenesis, and cytodifferentiation in a variety of embryonic and adult epithelia (39, 40, 41, 42) . In particular, we have shown that embryonic rat or mouse UGM can induce small (100–300 µm) segments of prostatic ducts (PRE) to form as much as 50–60 mg wet weight of prostatic tissue after 1 month of growth in nude mouse hosts (42 , 43) . The method involves grafting UGM + PRE tissue recombinants under the renal capsule of intact male nude mice (42) . An important feature of the UGM + PRE tissue recombinant system is the marked stimulation of epithelial proliferation, because the original 300-µm ductal segment containing about 5,000 epithelial cells yields about 20,000,000 prostatic epithelial cells during 1 month of in vivo growth (42) .
E2 in combination with testosterone is an effective method of inducing prostatic cancer in adult Noble rats (2 , 44, 45, 46, 47, 48) . The starting point of the carcinogenic process in the model described here is a single genetic defect, the homozygous deletion of Rb in combination with an established model of hormonal carcinogenesis. Because the RB gene is perturbed in only a relatively small percentage of human prostate cancers, it should be stressed that the present study uses Rb-knockout prostatic tissue, not because of a role of RB in human prostate cancer but rather because of the enhanced susceptibility of this tissue to undergo further genetic change leading to prostatic carcinogenesis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Characterization of in Vivo Rescued Prostatic
Tissue.
The prostatic phenotype of tissues within pelvic visceral grafts was
confirmed by histological and immunohistochemical staining using a
panel of antibodies against known prostatic markers as outlined below.
Tissue Separation, Recombination, and Grafting.
Pregnant Sprague Dawley rats were obtained from Charles River
(Wilmington, MA). rUGM was prepared from 18-day embryonic fetuses. For
this purpose, urogenital sinuses were dissected from fetuses and
separated into epithelial and mesenchymal components after tryptic
digestion and mechanical separation, as described previously
(49)
. Rb+/+ and
Rb-/- prostatic ductal segments were cut into
small (200–500 µm) pieces and placed on top of rUGM in dishes
containing a nutrient agar medium, as described previously (43
, 50)
. After 24 h, the tissue recombinants were grafted
underneath the renal capsule.
Induction of Carcinogenesis.
Athymic male mouse hosts (Charles River) bearing rUGM + Rb+/+PRE and rUGM + Rb-/-PRE tissue recombinants were treated
hormonally at the time of grafting by surgical implantation of a 1-cm
Silastic capsule filled with 25 mg of T and a 0.4-cm Silastic capsule
filled with 2.5 mg of 17ß-estradiol. The Silastic tubing (Dow-Corning
Co., Midland, MI) had an inside diameter of 1.54 mm and an outside
diameter of 3.18 mm. E2 and T were obtained from Sigma Chemical Co.
(St. Louis, MO). Hosts were hormonally treated for 5 or 8 weeks,
beginning at the time of grafting. The control group received empty
Silastic capsules. All animals were housed in the University of
California San Francisco Animal Care Facility with food and drinking
water ad libitum under controlled lighting conditions (12 h
light, 12 h dark).
Histopathological Grading and Hoechst 33258 Dye Staining.
Host animals were sacrificed at 5 or 8 weeks after implantation of
T + E2 capsules by cervical dislocation. The tissue
recombinants were carefully dissected from the host kidney, fixed in
10% neutral buffered formalin, and embedded in paraffin. Six-µm
sections were stained with H&E to determine the histopathology of the
prostatic tissue recombinants, which were also stained with Hoechst dye
33258 (CalBiochem, La Jolla, CA) to confirm that the epithelium was of
mouse origin and the stroma of rat origin (51)
. Slides
were examined histologically from serially sectioned tissue
recombinants to determine the incidence of prostatic lesions. Every
15th section was examined (a separation of 90 µm). Approximately five
sections per tissue recombinant were examined, dependent upon the size
of the harvested graft. Slides were scored to determine the presence of
normal, hyperplasia, atypical hyperplasia, or carcinoma.
Immunohistochemical Staining.
Tissue sections were deparaffinized in Histoclear (National
Diagnostic, Atlanta, GA) and hydrated in graded alcoholic solutions and
PBS. Endogenous peroxidase activity was blocked with 0.5% hydrogen
peroxide in methanol for 30 min, followed by washing in PBS. Normal
goat serum was applied to the sections for 30 min to bind nonspecific
sites. The sections were then incubated with the primary antibodies
overnight at 4°C or with nonimmune mouse IgG. In these experiments,
rabbit polyclonal anti-androgen receptor antibody was purchased from
Affinity BioReagents (Golden, CO). Mouse monoclonal anti-cytokeratin 14
(CK14, LL001) and monoclonal anti-cytokeratin 8 (CK8, LE41) were
generously provided by Dr. E. B. Lane (University of Dundee, Dundee,
Scotland). Mouse anti-PCNA monoclonal antibody was purchased from
PharMingen (San Diego, CA), and the mouse anti-smooth muscle 
-actin
monoclonal antibody was purchased from Sigma Chemical Co. (St. Louis,
MO). Rabbit anti-mouse dorsolateral prostate secretion (mDLP; Ref.
52
) was used at a dilution of 1:5000. Rabbit anti-mouse
seminal vesicle secretion (53)
was also used for
immunohistochemical staining at a dilution of 1:5000, as described
previously (52)
. Mouse anti-E-cadherin monoclonal antibody
was purchased from Transduction Laboratories (San Diego, CA).
Antibodies against Rb and related family members (P107 and P130) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). After careful
washing, the sections were incubated in biotinylated secondary
immunoglobulin of appropriate species specificity (Sigma; diluted with
PBS at 1:200) for 30 min at room temperature. After incubation with the
secondary antibody, sections were washed in PBS (three 10-min washes)
and then incubated with avidin-biotin complex for 30 min at room
temperature. After the last PBS wash, the sections were developed for
about 1–5 min using 3,3-diaminobenzidine in PBS and 0.03%
H2O2. Sections were
counterstained with hematoxylin and dehydrated in alcohol. Control
sections were processed in parallel with mouse nonimmune IgG at the
same concentration as the primary antibodies.
Generation of PCNA Labeling Indices.
After sectioning and staining, multiple grafts (range, 6–12) were
examined to determine the percentage of epithelial cells showing
positive immunoreactivity against PCNA. For each individual graft, the
percentage of expressing cells was counted in six separate, low
magnification microscopic fields. Mean and SE were calculated for each
group. Differences were analyzed statistically by ANOVA.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Histologically, the ductal tissue from both wild-type and
knockout sources resembled prostate and stained with anti-mDLP antisera
(Fig. 1b)
, which reacts with mouse prostatic secretory
proteins (52)
. Cytoplasmic and luminal staining of
prostatic secretion was observed. Cytokeratin 14 was visualized in
basal epithelial cells, whereas luminal cells expressed cytokeratin 8.
Androgen receptors were detected by immunohistochemistry in both
epithelial and stromal cells of the Rb+/+ and
Rb-/- rescued prostatic tissue (Fig. 1c)
. Histological analysis of the grafted embryonic pelvic
viscera also revealed bladder, colonic, and seminal vesicle tissue. The
seminal vesicle tissue did not stain with mDLP antisera but stained
with anti-mSV antisera (not illustrated). As expected, Rb protein could
not be detected in either wild type or Rb-knockout tissue. P130 was
expressed in nuclei of all epithelial cells. P107 was detected in
nuclei of a subpopulation of epithelial cells. Both
Rb-/- and Rb+/+ grafts
gave similar results. Stromal expression of P130 was extremely low in
the wild-type grafts, whereas in the Rb-knockout tissue, 40% of the
stromal nuclei were stained. Expression of P107 and P130 was not
affected by hormonal treatment or type of histopathological lesion. On
the basis of immunocytochemical observations, the absence of Rb was not
compensated by increased expression of P107 and P130.
|
, as
determined by nuclear staining patterns with Hoechst dye 33258, whereas
the stroma was predominantly rat in origin (except for host mouse
vasculature). The ductal epithelium of these tissue recombinants
exhibited immunostaining with anti-mDLP and androgen receptors as
described above (not illustrated). Epithelial PCNA labeling, a measure
of proliferation, was consistently and significantly elevated in all
tissue recombinants containing Rb-/-
versus Rb+/+ epithelium, irrespective
of hormone treatment (Table 1)
. Average epithelial PCNA labeling values within the
Rb-/- versus
Rb+/+ groups did not differ significantly at the
time of harvest between different hormone treatment groups.
|
. In such epithelial ducts, the cytokeratin 14-positive
basal epithelial cells formed a discontinuous layer underneath the tall
columnar luminal epithelial cells (Fig. 2b)
. Ductal lumina
were lined by tall columnar luminal cells expressing cytokeratin 8
(Fig. 2c)
. E-cadherin was expressed along adjacent
epithelial cell membranes (Fig. 2d)
, and -actin-positive
smooth muscle cells (Fig. 2e)
surrounded the epithelial
ducts in rUGM+Rb+/+PRE tissue recombinants. The
smooth muscle layer was in intimate association with the epithelial
basement membrane. Occasional foci of epithelial hyperplasia were also
seen in these grafts (Table 2)
. The epithelial proliferation rate was low as judged by PCNA staining
(Fig. 2f)
. Rb was not detected by immunohistochemistry, P130
was detected in all epithelial cells and a very low number of stromal
cells, and P107 was detected in a minor population of epithelial cells
and in a low number of stromal cells.
|
|
. Such hyperplastic epithelial ducts exhibited an increased
density of basal epithelial cells expressing cytokeratin 14 (Fig. 3b)
in comparison with the untreated control group (compare
with Fig. 2b
). The ductal lumina were lined by tall columnar
luminal cells expressing cytokeratin 8 (Fig. 3c)
. E-cadherin
was expressed along adjacent epithelial cell membranes (Fig. 3d)
. The induced prostatic tissue contained
-actin-positive smooth muscle cells in intimate association with the
ductal epithelium (Fig. 3e)
, similar to that of
rUGM+Rb+/+PRE tissue recombinants grown in
untreated hosts. Foci of epithelial hyperplasia were also seen in these
grafts (see Table 2
). The cribiform pattern in the ducts of the
rUGM+Rb+/+PRE tissue recombinants suggests a
rather extensive hyperplasia. Epithelial proliferation as judged by
PCNA staining is shown in Fig. 3f
. Expression of Rb, P130,
and P107 was identical to the untreated group.
|
40% of grafts, regions of
hyperplasia were also seen; these were characterized by a more or less
continuous layer of cytokeratin 14-positive basal cells (Fig. 4, a–c)
. E-cadherin was expressed along adjacent epithelial
cell membranes (Fig. 4d)
. The stroma contained
-actin-positive smooth muscle cells in intimate association with the
prostatic ducts (Fig. 4e)
. PCNA labeling was elevated in the
Rb-/- epithelium (Fig. 4f)
relative
to that of wildtype epithelium in
rUGM+Rb+/+PRE tissue recombinants grown in
control hosts. Thus, the two features unique to the untreated
rUGM+Rb-/-PRE tissue recombinants were
expansion of the basal epithelial cell compartment to a continuous
layer and a distinct elevation in PCNA labeling in PRE.
|
).
In rUGM+Rb-/-PRE tissue recombinants
treated with T + E2, focal areas of prostatic hyperplasia
were observed as ducts lined with multiple layers of polarized
epithelial cells. Numerous papillary projections were present in the
hyperplastic ducts (Fig. 5a)
. In addition, there were minor pleomorphic nuclear changes
with occasional mitotic figures in the epithelial ducts and in the
stroma. Luminal epithelial cells continued to express cytokeratin 8
(not illustrated). The continuous layer of cytokeratin 14-positive
basal cells showed focal areas of stratification (Fig. 5b)
.
The hyperplastic epithelium retained a normal cellular polarity, and
E-cadherin was expressed along adjacent epithelial cell membranes (Fig. 5c)
. The stroma surrounding the hyperplastic epithelium
expressed smooth muscle -actin (Fig. 5d)
. Epithelial
proliferation rate was high as judged by PCNA labeling (not
illustrated).
|
. The Rb-/- epithelial cells of
rUGM+Rb-/-PRE tissue recombinants showed
nuclear crowding, enlargement, and pleomorphism (Fig. 6a)
with a high proliferation rate as judged by PCNA staining (not
illustrated). Some mitotic figures were observed. In large ducts,
several atypical hyperplastic foci were sometimes seen within a single
duct, although not all ducts exhibited atypical hyperplastic changes.
The atypical hyperplastic epithelial cells were cytokeratin 8 positive
(not illustrated). Cytokeratin 14-positive basal epithelial cells were
rarely detected (Fig. 6b)
. E-cadherin was abnormal and
variable. In some areas, E-cadherin was expressed weakly along adjacent
epithelial cell membranes, whereas in other areas, E-cadherin was
undetectable or cytoplasmic (Fig. 6c)
. Atypical hyperplastic
prostatic ducts were surrounded by a disorganized incomplete
-actin-positive smooth muscle layer, which was separated by variable
distances from the epithelial basement membrane by an unstained
connective tissue containing extracellular matrix and unstained cells
(presumably fibroblasts; Fig. 6d
).
|
. Disorganized cribriform patterns were observed containing
tumor cells having large pleomorphic nuclei with prominent nucleoli.
Neoplastic cells filled entire ducts and invaded the interacinar
connective tissue or invaded adjacent acini, resulting in the formation
of tumor masses composed of small back-to-back glands (Fig. 7a)
. Mitotic figures were detected in some areas, and
abnormal mitoses were occasionally observed. Cytokeratin 14-positive
basal cells were not detected or were present in reduced numbers (Fig. 7b)
and instead the carcinoma cells expressed the luminal
cell marker, cytokeratin 8 (Fig. 7c)
. E-cadherin expression
was abnormal, and in most areas E-cadherin was undetectable (Fig. 7d)
. In carcinomatous areas, few -actin-positive smooth
muscle cells were observed (Fig. 7e)
. Epithelial
proliferation rate was high in carcinomas based upon PCNA staining
(Fig. 7f)
. Thus, areas interpreted as malignant exhibited
nuclear pleomorphism, elevated epithelial proliferation, increased
mitoses and abnormal mitoses, loss of basal epithelial cells, loss of
prostatic smooth muscle, reduction or loss of E-cadherin staining, and
perturbation in ductal organization and epithelial polarity. Rb was not
detected in any Rb-/- tissue. P130 and P 107 expression mirrored that
seen in the tissue recombinants that used wild-type epithelium. It was
noted that the expression patterns of these proteins were consistent
across the whole range of histological phenotypes from normal to
cancer. Thus, at least at the level of immunohistochemical detection,
there was no apparent regulation of these proteins in relation to tumor
progression.
|
. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and/or
ER-ß (55)
. Estrogen appears to play an important role in
the pathobiology of the prostate, although normal development of the
prostate occurs in mice null for ER- or ER-ß
(56, 57, 58)
. Exogenous estrogens given during the perinatal
period elicit permanent abnormalities in prostatic growth
(59, 60, 61, 62)
, differentiation (63)
, function
(64)
, androgen metabolism (65)
, and
expression of androgen receptors (66
, 67)
and may lead to
prostatic cancer (68, 69, 70)
. In adulthood, chronic treatment
with estrogen acts synergistically with androgen to induce benign
prostatic hyperplasia in dogs (71)
. E2 in combination with T given at a pharmacological level is an effective method of inducing prostatic cancer in adult Noble rats (46, 47, 48) . This rat model of prostatic cancer generally has a long latency and low tumor incidence, although new hormonal treatment protocols have partially addressed these problems (2) . The model of prostate cancer presented in this report combines hormonal induction of prostate cancer (2) , enhanced epithelial proliferation elicited by tissue recombination with rUGM (42) , and use of Rb-/- mouse prostatic epithelium generated through rescue of embryonic tissues from Rb-/- embryos. In this way, the starting point of the carcinogenic process is a single genetic defect, homozygous deletion of Rb.
The rescue of Rb-/- prostatic epithelium from embryonic lethal embryos was achieved by grafting embryonic pelvic organs at 12 days of gestation, 1 day before the expected demise of Rb-/- embryos (37) . The grafts develop a range of tissues (prostate, bladder, colon, and seminal vesicle tissue), from which the prostatic tissue can be easily recognized grossly using a dissecting microscope because of its ductal morphology. Such "rescued" prostatic tissue exhibits prostatic ductal morphology and expresses androgen receptors and mouse prostatic secretory proteins and is organized in a manner characteristic of wild-type prostate.
Multifocal areas of prostatic hyperplasia, atypical hyperplasia, and carcinoma were observed in all rUGM+Rb-/-PRE tissue recombinants grown in T + E2-treated hosts, whereas in untreated rUGM+Rb-/-PRE tissue recombinants, the most severe lesion observed was simple basal cell hyperplasia. Carcinomatous areas in T + E2-treated rUGM+Rb-/-PRE tissue recombinants exhibited nuclear pleomorphism, elevated epithelial proliferation, increased mitoses and abnormal mitoses, loss of basal epithelial cells, complete loss of prostatic smooth muscle, loss of membrane staining for E-cadherin, and perturbation in ductal organization and epithelial polarity. Epithelial proliferation was generally elevated in rUGM+Rb-/-PRE versus rUGM+Rb+/+PRE tissue recombinants, especially in hyperplastic, atypical hyperplastic, and carcinomatous regions. This elevation in PCNA staining is consistent with the role of Rb as a cell cycle check point modulator (72 , 73) . Studies in progress indicate that the carcinomas described are transplantable, and moreover, the atypical hyperplasias progress to cancer when serially grafted to new hosts.
The present model resembles human prostate cancer in that it involves change in the histodifferentiation of both the epithelium and the stroma. Normal prostatic epithelium, rescued Rb-/- and Rb+/+ prostatic epithelium, and prostatic epithelium of untreated rUGM+Rb-/-PRE and rUGM+Rb+/+PRE tissue recombinants contains a discontinuous layer of cytokeratin 14-positive basal cells underlying the cytokeratin 8-positive luminal epithelial cells. The prostatic hyperplasias that developed in a response to T + E2 treatment contained epithelium having a nearly continuous layer of basal cells, which in some cases was stratified. Prostatic carcinomas in T + E2-treated rUGM+Rb-/-PRE tissue recombinants were composed exclusively of cytokeratin 8-positive epithelial cells as is also the case for most human prostate cancers (74) . The cytokeratin 14-positive basal cells almost completely disappeared during the carcinogenic process in T + E2-treated rUGM+Rb-/-PRE tissue recombinants. This means that carcinomatous clones probably arose from the cytokeratin 8-positive luminal epithelial cells, although other interpretations are possible.
The progressive architectural derangements in the prostatic epithelium of T + E2-treated rUGM+Rb-/-PRE tissue recombinants were manifested in loss of epithelial polarity, epithelial crowding, stratification, and invasiveness. Such changes were associated with perturbation or loss in the membrane expression of E-cadherin. E-cadherin is a Mr 120,000 transmembrane glycoprotein involved in epithelial cell adhesion (75 , 76) . A functional E-cadherin system is required for maintenance of normal epithelial morphology. E-cadherin is localized on adjacent cell membranes of normal epithelial cells (including PRE) and in various highly differentiated "noninvasive" carcinoma cells (including prostate cancer). In contrast, E-cadherin expression is lower or entirely lacking in poorly differentiated invasive carcinoma cells (75, 76, 77, 78, 79) . Patients with prostatic tumors negative for E-cadherin or having abnormal patterns of E-cadherin expression have a worse prognosis than patients having prostatic tumors with normal expression of E-cadherin (80) . The loss of portions of chromosome 16, which is fairly common in prostate cancer, may result in deletion of all or part of the E-cadherin gene, which maps to 16q21 (81) . The loss of membrane expression of E-cadherin in carcinomas observed in T + E2-treated rUGM+Rb-/-PRE tissue recombinants is presumably related to the disorganization of ductal architecture.
Stromal histodifferentiation was coordinately perturbed in T + E2-treated rUGM+Rb-/-PRE tissue recombinants with the loss of the smooth muscle layer that is normally in intimate contact with the ductal basement membrane. Thus, during the carcinogenic process induced by T + E2, the normal homeostatic interaction of prostatic epithelium with smooth muscle has become an interaction of abnormal prostatic epithelium with a predominantly fibroblastic stroma. This abnormal fibroblastic stroma, which emerged during the carcinogenic process, has been shown to profoundly affect carcinogenic progression through altering epithelial differentiation, proliferation, and apoptosis as described recently (18 , 82) . Stromal alteration has been reported during carcinogenesis in many organs. Carcinoma-associated fibroblasts show a spectrum of differences from their normal counterparts (83, 84, 85, 86, 87, 88, 89, 90, 91) . The importance of the stromal reaction in malignant breast tumors has been stressed recently (92) . Likewise, the importance of stromal change and the role of carcinoma-associated fibroblasts have been demonstrated in prostatic carcinogenesis and tumorigenesis (8 , 10 , 18 , 93 , 94) . Thus, the pathway to prostatic carcinogenesis appears to entail progressive and concomitant changes in both epithelial and stromal elements with resultant abnormalities in homeostatic communication between abnormal stromal and a genetically altered PRE. Such abnormal cell-cell interactions promote malignant progression (18) .
The model described in this report of prostatic carcinogenesis can be extended to the analysis of carcinogenesis in other organs such as the mammary gland, salivary gland, pancreas, and colon. The rescue of organs from embryonic lethal mouse embryos can be achieved for virtually any organ rudiment, provided the embryo survives to at least 12 days of gestation. For prostate and mammary gland, small ductal fragments can be induced to proliferate extensively when combined with UGM in the case of the prostate (42 , 43) or grafted into a cleared fat pad in the case of mammary tissue (95) . Although not tested, it is likely that similar methods could be developed in which recombination of embryonic homologous mesenchyme with adult epithelium stimulates proliferation of homologous adult epithelial cells. Use of rescued organs from Rb-/- embryos in carcinogenic studies does not necessarily imply a role of the Rb gene in carcinogenesis of a particular organ. Instead, the use of Rb-/- tissues provides a well-defined genetic model that rapidly recapitulates the sorts of genetic and phenotypic changes seen in progression of carcinogenesis. Thus, it is quite likely that comparable new models of experimental carcinogenesis could be devised using tissues from embryonic lethal embryos with well-defined genetic defects, specifically those implicated in human prostate cancer. In this regard, it is also technically possible to use tissues from embryonic lethal embryos with either single or double mutants in this assay.
|
FOOTNOTES |
|---|
1 Supported by NIH Grants CA 59831, CA 64872, CA
84294, AG16870, DK 45861, DK 47517, DK 52708, CA84308, CA42063,
CA69568, and DK 52721 and Grant TPRN-98-111-01 CSM from the American
Cancer Society. 
2 To whom requests for reprints should be
addressed, at Department of Anatomy, University of California, San
Francisco, Box 0452, San Francisco, CA 94143-0452. Phone:
(415) 476-4140; Fax: (415) 502-2270; E-mail: grcunha{at}itsa.ucsf.edu
3 The abbreviations used are: PRE, prostatic
epithelium; Rb, retinoblastoma gene product; UGM, rat urogenital sinus
mesenchyme; rUGM, rat UGM; UGE, urogenital sinus epithelium; T,
testosterone propionate; E2, estradiol; PCNA, proliferating cell
nuclear antigen; ER, estrogen receptor.
4 See
http://mammary.nih.gov/tools/mousework/Cunha001/index.html.
Received 1/17/00. Accepted 8/23/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(ER-) and estrogen receptor-ß (ER-ß) messenger ribonucleic acid in the wild-type and ER--knockout mouse. Endocrinology, 138: 4613-4621, 1997.This article has been cited by other articles:
|
|
 |
|
  S. Smith, D. Sepkovic, H. L. Bradlow, and K. J. Auborn 3,3'-Diindolylmethane and Genistein Decrease the Adverse Effects of Estrogen in LNCaP and PC-3 Prostate Cancer Cells J. Nutr., December 1, 2008; 138(12): 2379 - 2385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Yao, A. Parwani, C. Maier, W. D. Heston, and D. J. Bacich Moderate Expression of Prostate-Specific Membrane Antigen, a Tissue Differentiation Antigen and Folate Hydrolase, Facilitates Prostate Carcinogenesis Cancer Res., November 1, 2008; 68(21): 9070 - 9077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. A. Ricke, S. J. McPherson, J. J. Bianco, G. R. Cunha, Y. Wang, and G. P. Risbridger Prostatic hormonal carcinogenesis is mediated by in situ estrogen production and estrogen receptor alpha signaling FASEB J, May 1, 2008; 22(5): 1512 - 1520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Chiaverotti, S. S. Couto, A. Donjacour, J.-H. Mao, H. Nagase, R. D. Cardiff, G. R. Cunha, and A. Balmain Dissociation of Epithelial and Neuroendocrine Carcinoma Lineages in the Transgenic Adenocarcinoma of Mouse Prostate Model of Prostate Cancer Am. J. Pathol., January 1, 2008; 172(1): 236 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Hydock, C.-Y. Lien, C. M. Schneider, and R. Hayward Effects of voluntary wheel running on cardiac function and myosin heavy chain in chemically gonadectomized rats Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3254 - H3264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. O. Kaseb, K. Chinnakannu, D. Chen, A. Sivanandam, S. Tejwani, M. Menon, Q. P. Dou, and G. P.-V. Reddy Androgen Receptor and E2F-1 Targeted Thymoquinone Therapy for Hormone-Refractory Prostate Cancer Cancer Res., August 15, 2007; 67(16): 7782 - 7788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sharma, C. E.S. Comstock, E. S. Knudsen, K. H. Cao, J. K. Hess-Wilson, L. M. Morey, J. Barrera, and K. E. Knudsen Retinoblastoma Tumor Suppressor Status Is a Critical Determinant of Therapeutic Response in Prostate Cancer Cells Cancer Res., July 1, 2007; 67(13): 6192 - 6203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Davis, K. J. Wojno, S. Daignault, M. D. Hofer, R. Kuefer, M. A. Rubin, and M. L. Day Elevated E2F1 Inhibits Transcription of the Androgen Receptor in Metastatic Hormone-Resistant Prostate Cancer Cancer Res., December 15, 2006; 66(24): 11897 - 11906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhou, A. Flesken-Nikitin, D. C. Corney, W. Wang, D. W. Goodrich, P. Roy-Burman, and A. Yu. Nikitin Synergy of p53 and Rb Deficiency in a Conditional Mouse Model for Metastatic Prostate Cancer Cancer Res., August 15, 2006; 66(16): 7889 - 7898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Lamb and L. Zhang Challenges in Prostate Cancer Research: Animal Models for Nutritional Studies of Chemoprevention and Disease Progression J. Nutr., December 1, 2005; 135(12): 3009S - 3015S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Hill, Y. Song, R. D. Cardiff, and T. Van Dyke Heterogeneous Tumor Evolution Initiated by Loss of pRb Function in a Preclinical Prostate Cancer Model Cancer Res., November 15, 2005; 65(22): 10243 - 10254. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Cunha, N. J. Place, L. Baskin, A. Conley, M. Weldele, T. J. Cunha, Y.Z. Wang, M. Cao, and S. E. Glickman The Ontogeny of the Urogenital System of the Spotted Hyena (Crocuta crocuta Erxleben) Biol Reprod, September 1, 2005; 73(3): 554 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Gao, K. Ishii, J. Mirosevich, S. Kuwajima, S. R. Oppenheimer, R. L. Roberts, M. Jiang, X. Yu, S. B. Shappell, R. M. Caprioli, et al. Forkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation Development, August 1, 2005; 132(15): 3431 - 3443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. McCabe, J. N. Davis, and M. L. Day Regulation of DNA Methyltransferase 1 by the pRb/E2F1 Pathway Cancer Res., May 1, 2005; 65(9): 3624 - 3632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Davis, M. T. McCabe, S. W. Hayward, J. M. Park, and M. L. Day Disruption of Rb/E2F Pathway Results in Increased Cyclooxygenase-2 Expression and Activity in Prostate Epithelial Cells Cancer Res., May 1, 2005; 65(9): 3633 - 3642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kurita, R. T. Medina, A. A. Mills, and G. R. Cunha Role of p63 and basal cells in the prostate Development, October 15, 2004; 131(20): 4955 - 4964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Maddison, B. W. Sutherland, R. J. Barrios, and N. M. Greenberg Conditional Deletion of Rb Causes Early Stage Prostate Cancer Cancer Res., September 1, 2004; 64(17): 6018 - 6025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, H. Deng, I. Basu, and L. Zhu Induction of Androgen Receptor-Dependent Apoptosis in Prostate Cancer Cells by the Retinoblastoma Protein Cancer Res., February 15, 2004; 64(4): 1377 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lemjabbar, D. Li, M. Gallup, S. Sidhu, E. Drori, and C. Basbaum Tobacco Smoke-induced Lung Cell Proliferation Mediated by Tumor Necrosis Factor {alpha}-converting Enzyme and Amphiregulin J. Biol. Chem., July 3, 2003; 278(28): 26202 - 26207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Bianco, D. J. Handelsman, J. S. Pedersen, and G. P. Risbridger Direct Response of the Murine Prostate Gland and Seminal Vesicles to Estradiol Endocrinology, December 1, 2002; 143(12): 4922 - 4933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. C. Day, M. T. McCabe, X. Zhao, Y. Wang, J. N. Davis, J. Phillips, M. Von Geldern, T. Ried, M. A. KuKuruga, G. R. Cunha, et al. Rescue of Embryonic Epithelium Reveals That the Homozygous Deletion of the Retinoblastoma Gene Confers Growth Factor Independence and Immortality but Does Not Influence Epithelial Differentiation or Tissue Morphogenesis J. Biol. Chem., November 8, 2002; 277(46): 44475 - 44484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Park, J. E. Walls, J. J. Galvez, M. Kim, C. Abate-Shen, M. M. Shen, and R. D. Cardiff Prostatic Intraepithelial Neoplasia in Genetically Engineered Mice Am. J. Pathol., August 1, 2002; 161(2): 727 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shou, R. Soriano, S. W. Hayward, G. R. Cunha, P. M. Williams, and W.-Q. Gao Expression profiling of a human cell line model of prostatic cancer reveals a direct involvement of interferon signaling in prostate tumor progression PNAS, March 5, 2002; 99(5): 2830 - 2835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, D. Sudilovsky, B. Zhang, P. C. Haughney, M. A. Rosen, D. S. Wu, T. J. Cunha, R. Dahiya, G. R. Cunha, and S. W. Hayward A Human Prostatic Epithelial Model of Hormonal Carcinogenesis Cancer Res., August 1, 2001; 61(16): 6064 - 6072. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
