Int. J. Cancer: 76, 6847688 (1998)
`© 1998 Wiley-Liss, Inc.
`
`Publication of the International Union Against Cancer
`Publication de I’Union Internationale Contre le Cancer
`
`
`
`ESTROGENIC REGULATION OF CLUSTERIN mRNA IN NORMAL
`
`AND MALIGNANT ENDOMETRIAL TISSUE
`
`Winfried WUNSCHEl, Martin P. TENNISWOODZ, Martin R. SCHNEIDER3 and Gunter VOLL
`1| nstitut fUr Bi ochemi sche Endokri nol ogi e, Mediz' nische Universitat, LUbeck, Germany
`2WAlton Jones Cell Sci ence Center Inc, Lake Placi d, NY, USA
`3Research Laboratories, Scheri ng AG, Berlin, Germany
`
`
`
`
`,Riss
`
`Clusterin is a heterodimeric, 80kDa, glycoprotein that is
`synthesized in a wide variety of tissues in response to a
`number of diverse stimuli, including hormone ablation. We
`have investigated the regulation of clusterin expression by
`estradiol and anti-estrogensin RU CA-I rat endometrial adeno-
`carcinoma cells in vitro and in vivo. We have also compared
`clusterin expression in endometrial tumors and in normal
`uterine tissue. Estradiol treatment significantly increasesthe
`steady state mRNA levels of clusterin in RUCA-I cells cul-
`tured on a reconstituted basement membrane, with a maxi-
`mal induction 24 hr after estradiol treatment. The inductive
`effects of estrogen on clusterin mRNA steady state levels in
`vitro are significantly more pronounced than the effects on
`fibronectin mRNA levels, an estrogen-repressed gene in
`RUCA-I. In vivo, induction of clusterin expression in primary
`and metastatic endometrial adenocarcinoma is also depen-
`dent on the presence of estradiol,
`in marked contrast to
`expression of clusterin in the normal endometrium of the
`same animals. These data suggest
`that clusterin mRNA
`expression in rat endometrial adenocarcinoma cellsistightly
`regulated by estrogens and anti-estrogensin vitro and in vivo,
`and that there is a complex mechanism of regulation of
`clusterin expression in the normal and cancerous endome-
`trium. Int. J Cancer 76:684—688, 1998.
`© 1998 WIey—Lisi Inc.
`
`Clusterin, also known as testosterone-repressed message-2
`(TRPM-2), sulphated glycoprotein-2 (SGP-2) and apolipoprotein J
`(ApoJ), was first isolated from the ram rete testis fluid (Blaschuk et
`al., 1983) The 80 kDa, heterodimeric glycoprotein and its mRNA
`have been identified in many tissues and has been identified in a
`variety of species (Jenne and Tschopp, 1992). In many hormone-
`dependent tissues, such as prostate and mammary gland, clusterin
`expression is induced after hormone ablation and appears to be
`involved in the apoptotic processes associated with tissue remodel-
`ling (Tenniswood et al ., 1992). For example, clusterin expression is
`induced following hormone ablation in regressing, androgen-
`responsive, Shionogi mouse mammary tumors (Akakura et al.,
`1996). In this model system, the expression of clusterin remains
`elevated when the tumors become hormone-independent and fail to
`initiate apoptosis after hormone deprivation (Akakura et al ., 1996).
`In MCF-7 breast cancer cells it has been shown that estrogen
`withdrawal
`in vivo (Kyprianou et al., 1991 and treatment with
`vitamin D3 analogs (Simboli-Campbell et al., 1996)
`induce
`apoptosis and increase clusterin expression. In the uterus, hormone
`ablation induces apoptosis of the epithelial tissue compartment and
`the expression of clusterin in the luminal and glandular epithelial
`cells (Brown et al ., 1995). The expression of clusterin in the mouse
`uterus, while it requires estrogens, appears to be induced primarily
`in response to the fall
`in progesterone levels, which is usually
`associated with the tissue remodeling that occurs after regression
`(Brown et al ., 1995).
`We have established an estrogen receptor positive, rat endome-
`trial adenocarcinoma cell line, RUCA-I (Schiitze et al., 1992). In
`vitro, despite relatively high levels of estrogen receptor, this cell
`line only responds to treatment with estradiol if cultured on a
`reconstituted basement membrane (Vollmer et al., 1995a). When
`grown on the appropriate substratum these cells exhibit estrogenic
`inducibility of proliferation and complement C3 expression and
`repression of fibronectin expression by estrogens (Vollmer et al.,
`1995b). When injected into the flank of syngeneic animals, the
`
`RUCA-l cell line gives rise to estrogen sensitive, metastasizing
`endometrial adenocarcinoma, which responds to pure antiestro-
`gens, such as ZK 119,010, ICI 164,384, and ICI 182780, and to
`antiestrogens exhibiting partially agonistic activities, such as
`tamoxifen (Vollmer and Schneider, 1997). RUCA-I cells, cultured
`on a reconstituted basement membrane or grown in ViVO, therefore
`represent an excellent endometrial derived cell culture model to
`study estrogenic regulation of gene expression in ViVO and in vitro.
`Since the clusterin promoter contains a complex array of
`potentially important hormone response elements (Wong et al.,
`1993), and since increased clusterin expression has been found in
`various normal and malignant processes, we have investigated the
`estrogen sensitivity of clusterin expression in the RUCA-l
`rat
`endometrial tumor model. We have demonstrated that clusterin
`expression in this model system is estrogen-sensitive, both in vitro
`and in ViVO, and we provide evidence that regulation of clusterin
`expression in the tumors in vivo is diametrically reversed when
`compared to the expression in the normal uterus of the host animal.
`
`MATERIAL AND METHODS
`
`Hormones
`
`The 17B-estradiol and tamoxifen were obtained from Sigma
`(Deissenhofen, Germany), ICI 164,384 was provided by Dr. A.E.
`Wakeling (Zeneca, Macclesfield, UK), ICI 182,780 was synthe-
`sized at Schering (Berlin, Germany).
`
`Cell culture
`
`Prior to experimental use, RUCA-I cells (from frozen stocks of
`passage 30) were cultured for one passage in DIVIEM/F12 medium
`without phenol red containing 10% fetal calf serum, and for 2
`passages in the above medium containing 5% dextran-coated
`charcoal treated FCS. For routine experiments 2.5730 X 105 cells
`were seeded on 300 pl of ECM-substrate (Harbour matrix; Cell
`Systems, Remagen, Germany) per well of a 24-well plate in the
`presence of 2 ml serum-free defined medium (DMEM/F 12 supple-
`mented with 2 ug/ml insulin, 4 mM glutamine, 40 ug/ml transfer-
`rin, 10'8 M hydrocortisone, 2 X 10'8 M sodium selenite, and 1
`ug/ml putrescine). The cells were maintained in a humidified 5%
`C02 atmosphere at 370C and medium was changed daily.
`
`Hormonal treatment of RUCA—I cellsin vitro
`
`Prior to hormonal treatment, 2.5730 X 105 RUCA-l cells were
`seeded on ECM-substrate and cultured in serum free defined
`medium for 2448 hr. The cells were then incubated for 2748 hr
`
`with 10'7 M estradiol in the absence or presence of 5 X 10'7M ICI
`164,384', 5 X 10'7M ICI 182,780', or 10'6 M tamoxifen. These
`
`Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers: V0410/
`5-1 and V0410/5-2; Grant sponsor: NATO; Grant number: CRG 971593.
`
`*Correspondence to: Institut fiir Biochemische Endokrinologie, Mediz-
`inische Universitat, Ratzeburger Allee 160, D-23538 Lubeck, Germany.
`Fax: (49)451-500-2729. E-mail: vollmer@imm.mu-luebeck.de
`
`Received 18 August 1997; Revised 10 December 1997
`
`InnoPharma Exhibit 1088.0001
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`

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`CLUSTERIN mRNA IN ENDO
`
`
`
`
` « TRIAL TUMORS
`
`68 5
`
`the estrogen and
`concentrations were chosen to ensure that
`anti-estrogens were present in approximately equipotent concentra-
`tions based on their relative binding affinities to the estrogen
`receptor (Wakeling et al., 1991). Control cultures received ethanol,
`which was used as vehicle. Medium was changed daily and
`hormonal treatment was repeated.
`
`Complement C3 and fibronectin cDNA probes were also radiola-
`beled as described previously. In all cases agarose gel purified
`restriction cDNA fragments were radiolabeled with 32P—dCTP
`using a multi-prime labeling kit (GIBCO/BRL, Eggenstein, Ger-
`many). Fibronectin was used as a comparative probe for in vitro
`studies, complement C3 was used for the in ViVO studies.
`
`In vivo growth of RUCA—1 cells
`RUCA-I cells (from frozen stocks of passage 30) were cultured
`for one passage on plastic in DMEM/F12 medium without phenol
`red containing 10% charcoal stripped FCS, and harvested by
`trypsinization. Seven to eight week-old female DA/Han rats were
`ovariectomized under ether anesthesia 48 hr prior to the s.c.
`injection of 106 RUCA-I cells into the right flank. The animals were
`then randomized into 5 groups of 4 animals: untreated control
`females; ovariectomized females; ovariectomized females receiv-
`ing a single depot injection of estradiol undecylate, 50 ug/rat in 100
`pl (Progynon Depot 100, Schering), a dose known to be sufficient
`to stimulate EnDA-tumor growth in ovariectomized DA/Han rats
`for at least 6 weeks (Horn et al., 1994)‘, intact females receiving 5
`mg/kg/day ICI 182,780 dissolved in 20% benzyl benzoate in castor
`oil', and intact females receiving 20 mg/kg/day tamoxifen dissolved
`in 20% benzyl benzoate in castor oil. The anti-estrogens were
`administered 6 days a week for 5 weeks starting one day after
`tumor cell
`injection. Tumor growth was monitored weekly by
`caliper measurement. After 5 weeks the animals were killed, the
`tumors, ipsilateral lymph nodes and uteri were excised and the wet
`weights were determined.
`In the control group, animals were
`selected at random and not at specific times in the estrus cycle. The
`statistical analysis of tumor and tissue wet weights was performed
`using Student’s t—test.
`
`RNA preparation and Northern blot hybridization
`RNA was extracted from tissues (2007500 mg), after pulveriza-
`tion in liquid nitrogen or from cell cultures (pools of cells from 6
`wells) using guanidinium isothiocyanate and centrifugation through
`cesium chloride (Chirgwin et al., 1979.). After determining the
`RNA concentration by UV absorption at 280 nm,
`total RNA
`(10720 ug) were electrophoresed on 2.2 M formaldehyde gels,
`and transferred to nylon membranes by vacuum blotting (Pharma-
`cia, Freiburg, Germany).
`After prehybridization the membrane was first hybridized at
`420C overnight with the 32P-labeled clusterin probe (4 X 108712 X
`108 cpm/ug plasmid, 12 X 106 cpm total). The membranes were
`washed once at room temperature for 5 min, once at 420C for 5 min
`and 3 times at 500C for 15 min. The membranes were then exposed
`for 2448 hr to Kodak XOmat film using intensifying screens.
`After stripping with 0.1 X SSC for 15 min at 700C, the membranes
`were was rehybridized with a 32P-labeled control probe recognizing
`the 18S ribosomal RNA (4 X 10871.2 >< 108cpm/ug plasmid,
`3.2 X 105 cpm total).
`The 350 bp Sst I/Sac I fragment (representing positions 1577
`506 of the rat clusterin cDNA) or the 316 bp Xba fragment
`(representing positions 17316 of the rat clusterin cDNA) or the
`entire rat clusterin cDNA were used as hybridization probes.
`
`RESULTS
`
`Estrogenic regulation of CI usteri n apr on in vitro
`To investigate the potential estrogenic regulation of clusterin,
`RUCA-I cells were cultured on a reconstituted basement mem-
`brane and treated with estradiol or the pure anti-estrogen ICI
`164,384 for 248 hr (Fig. 1). The steady state level of clusterin
`mRNA does not alter significantly over the 48 hr time course when
`incubated in the presence of ethanol alone. Estradiol treatment of
`RUCA-I cells induces a time-dependent increase of steady state
`level of clusterin mRNA, reaching a maximum at around 24 hr.
`Treatment of cells with the pure anti-estrogen ICI 164,384 substan-
`tially reduces the steady state level of clusterin mRNA to those seen
`in the untreated control cultures. The effects of estradiol and the
`antiestrogen, ICI 164,384, on clusterin mRNA expression were
`more pronounced than the effects on the expression of fibronectin
`mRNA, an estradiol repressed protein in RUCA-I cells (Vollmer et
`al., 1995b)
`(Fig. 1). The expression of fibronectin mRNA is
`transiently repressed by estradiol, reaching a nadir at approxi-
`mately 20 hr (Fig. 1). ICI 164,384 does not appear to significantly
`affect the steady state fibronectin mRNA, although there is a slight
`increase 48 hr following treatment (Fig. 1). In summary, at these
`doses of estradiol (10'7M) and ICI 164,384 (5 X 10'7M) clusterin
`gene expression appears to be significantly more responsive to
`these hormones than fibronectin in RUCA-I cells.
`
`Hormonal regulation of CI usteri n mRNA expression in vivo
`To determine whether the steady state level of clusterin mRNA is
`regulated by estrogens in ViVO, syngeneic DA/Han rats were
`injected s.c. with 106 RUCA-I cells and randomized in 5 experimen-
`tal treatment groups. Primary tumors developed within 30 days in
`all groups at the site of injection. These tumors subsequently
`metastasized to the axillary lymph nodes and lung. Tumors grown
`in ovariectomized animals or in intact animals treated with ICI
`182,780 grew significantly more slowly than untreated controls
`demonstrating significantly reduced tumor weight (p < 0.01) (Fig.
`28) when compared to untreated controls. In these groups, the
`uterine weight of the host animals (Fig. 2b) and the weight of the
`ipsilateral axillary lymph nodes (Fig. 20) were also reduced when
`compared to untreated controls (p < 0.01). Administration of
`estradiol to these animals restores the growth parameters such that
`they are not significantly different from those seen in untreated
`controls (Fig 2a, b, C), demonstrating the importance of estradiol
`for the growth of RUCA-I cells in ViVO. Treatment with tamoxifen
`had no significant effect on the weight of the RUCA-I primary
`tumors (Fig. 2a) compared to controls, and while tamoxifen also
`clearly reduced uterine weight (p < 0.01) (Fig. 2b), it demonstrated
`a distinctly agonistic effect on the weight of the axillary lymph
`
`Ethanol
`
`Estradiol
`
`ICI 164,384
`
`Fibronectin
`
`Clusterin
`
`
`
`
`
`-.
`
`185 r RNAEWIIIIII
`
`
`2
`
`6
`
`2O 24 48
`Time (h)
`
`2
`
`If
`20 24 48
`6
`Time (h)
`
`2
`
`I"!
`6
`20 24 48
`Time(h)
`
`8000 nt
`
`1900 nt
`
`FIGURE 1 7Steady state levels of clusterin
`and fibronectin mRNAs in RUCA-I cells.
`RUCA-I cells were cultured on a reconsti-
`tuted basement membrane and treated with
`estradiol (10'7M), ICI 164,384 (5 X 10'7M) or
`ethanol (control) for 248 hr. RNA was
`extracted, electrophoresed on formaldehyde
`gels,
`transferred to nylon membranes and
`hybridized with clusterin or fibronectin cDNA
`probes as described in Material and Methods.
`To correct for loading the blots were stripped
`and re-probed with a cDNA probe specific for
`18S rRNA.
`
`InnoPharma Exhibit 1088.0002
`
`

`

`1000
`
`500
`
`400:
`
`EDD:
`
`200;
`
`Weight
`
`(mg) 100}
`
`
`
`Axillary Lymph Nodes
`
`FIGURE 2 7 Effect of hormone deprivation and anti-hormone treatment
`on primary and metastatic tumor growth of RUCA-I cells. RUCA-I
`cells (105) were injected s.c. into the right flank of intact syngeneic
`female DA/Han rats, ovariectomized DA/Han rats, ovariectomized
`DA/Han rats substituted with estradiol-undecylate, intact DA/Han rats
`treated with ICI 182,780 or intact DA/Han rats treated with tamoxifen.
`Panel a: Primary tumor weight (mean i SD, n : 4); Panel b: Weight of
`the normal uterus (mean i SD, n : 4); Panel 0: Weight of ipsilateral
`lymph nodes (mean i SD, n : 4). Stars above individual bars indicate
`values that differ significantly from the control at p < 0.01. The wet
`weight of axillary lymph nodes in animals treated with tamoxifen
`differed significantly from controls at p < 0.05.
`
`nodes, promoting an axillary weight almost twice as high as that
`measured in untreated control animals (p < 0.05) (Fig. 2C).
`The steady state clusterin mRNA level
`in the tumor tissue
`correlates with tumor weight at the end of the experiment, with
`high levels of clusterin expression in the tumors excised from
`untreated controls animals, from ovariectomized animals adminis-
`
`686
`
`WIHTNSCHE ETAL.
`
`F’rimar‘yr Tumor
`
`tered estradiol, and from intact animals administered tamoxifen
`(Fig. 3a). Almost undetectable levels of clusterin expression were
`found in tumors grown in ovariectomized animals and in intact
`animals given 1C1 182,780.The effects of estrogens on the steady
`state levels of complement C3 mRNA in the primary tumors was
`considerably less pronounced than the effects on clusterin (Fig. 3a).
`In this, and other Northern analyses of clusterin mRNA levels in
`RUCA-I cells grown in ViVO and in normal uteri, the clusterin
`cDNA probes hybridized to a sequence of between 354.0 kb that
`showed the same expression pattern in vivo as clusterin. This
`sequence was not detected in Northern analyses of clusterin mRNA
`from RUCA-I cells grown in vitro. The identity of this sequence,
`and its relationship to clusterin mRNA have not been determined.
`The regulation of clusterin steady state mRNA levels in lymphog-
`enous metastases of RUCA-I exhibited the same expression pattern
`as that seen in the primary tumor. Clusterin mRNA was signifi-
`cantly expressed in axillary lymph nodes of untreated control
`animals, ovariectomized animals that were substituted with estra-
`diol and intact animals treated with tamoxifen (Fig. 3b). Clusterin
`mRNA expression was barely detectable in lymph nodes of
`ovariectomized animals and were undetectable following treatment
`ofintact animals with 1C1 182,780 (Fig. 3b).
`
`A.
`complement 0-39E5800 nt
`Unidentified RNA —-)
`4000 nt
`
`Clusterin -)
`
`1900 nt
`
`B.
`Unidentified RNA -)
`
`_
`Clusterin —) -
`
`
`4000 nt
`
`1900 nt
`
`C.
`
`Complement C-3 —)
`
`Unidentified RNA —>
`
`Clusterinu)
`
`5800 nt
`
`4000 nt
`
`1900 nt
`
`0
`
`\o‘
`
`o
`
`l
`
`0+
`
`Kg»
`32'
`x
`\
`«0
`c}
`0
`‘
`xx
`0"
`(be
`\0‘
`\°
`
`FIGURE 3 7 Steady state mRNA levels of estrogen-sensitive genes in
`primary tumors, axillary lymph nodes and normal uterus. RUCA-I cells
`(105) were injected s.c. into the right flank of intact syngeneic female
`DA/Han rats, ovariectomized DA/Han rats, ovariectomized DA/Han
`rats substituted with estradiol-undecylate, intact DA/Han rats treated
`with ICI 182,780 or intact DA/Han rats treated with tamoxifen. RNA
`was extracted, electrophoresed on formaldehyde gels, transferred to
`nylon membranes and hybridized with cDNA probes specific for
`clusterin, or complement C3. To correct for loading the blots were
`stripped and re-probed with a probe specific for 18S rRNA. Panel a:
`steady state levels of clusterin and complement C3 mRNAs in primary
`tumors; Panel b: steady state level of clusterin mRNA in lymph node
`metastases; Panel C: steady state level of clusterin and complement C3
`mRNAs in uterus.
`
`InnoPharma Exhibit 1088.0003
`
`

`

`CLUSTERIN mRNA IN ENDO
`
`
`
`
` * TRIAL TUIVIORS
`
`687
`
`In marked contrast, the effects of estrogens on clusterin mRNA
`expression in the uteri of host animals was very different from that
`seen in the primary tumors derived from RUCA-I cells or their
`lymph nodes metastases. In the normal uterus, estradiol suppressed
`clusterin expression, whereas clusterin mRNA levels were signifi-
`cantly induced after ovariectomy or treatment with the pure
`anti-estrogen ICI 182,780 (Fig. 3C). Tamoxifen, which has both
`agonistic and antagonistic effects, also suppressed clusterin expres-
`sion. This conspicuous inversion of estrogenic regulation of gene
`expression in normal and tumorigenic tissue was not detected for
`complement C3 expression, which exhibited increased steady state
`mRNA levels in ovariectomized animals supplemented with estra-
`diol and in intact animals treated with tamoxifen (Fig. 30).
`
`DISCUSSION
`
`Our data demonstrate that clusterin is an estrogen-regulated gene
`in RUCA-I, rat endometrial adenocarcinoma cells, both i n vitro and
`in ViVO. Furthermore, the expression of clusterin mRNA in vitro
`appears to accurately reflect the expression of the gene in primary
`and metastatic tumors derived from RUCA-I cells in ViVO. Al-
`though the estrus state of the control animals was not analyzed,
`there appears to be a marked switch in the regulation of clusterin by
`estrogens during the process of endometrial carcinogenesis, since
`the expression of clusterin in the normal uterus is repressed by
`estrogens, but is upregulated in RUCA-I primary adenocarcinomas
`and in lymph node metastases. In this respect, clusterin is the first
`gene described which has diametrically opposed hormonal control
`in a normal tissue and in tumors derived from that tissue. As such,
`clusterin may represent a valuable marker gene in the search for
`transcription factors that contribute to the malignant transformation
`of the endometrium and possibly of other hormone-dependent
`tumors, such as the prostate and breast. In this context, it would be
`interesting to determine how clusterin expression is modulated
`during the estrus cycle in normal uterine tissue and in malignant
`tissue.
`
`The regulation of clusterin steady state mRNA levels by
`estrogens is particularly interesting since no estrogen response
`elements have been identified in the rat or human clusterin
`promoter
`(Wong et al., 1993). While there are 8 half site
`androgemglucocorticoid responsive elements within the first intron
`of the structural gene,
`it is unlikely that they are used by the
`estrogen receptor. It is more likely that estrogens regulate clusterin
`expression through the 2 functional AP-1 sites located in the
`proximal promoter, since it is well established that estrogen rapidly
`induces the transcription of a number of immediate response genes
`including members of the foe and jun families (Webb et al ., 1993',
`Bigsby and Li, 1994) through the estrogen response elements in the
`promoters of these genes (Hyder et al., 1995). Since this mode of
`transcriptional
`regulation of the clusterin gene would require
`activation of the early response genes, clusterin expression most
`likely represents a relatively late response in this context. This may
`explain the relatively long time lag (approximately 15720 hr)
`before estradiol induction of clusterin expression in RUCA-1 is
`measurable in vitro. Whether the estrogen receptor in RUCA-I cells
`exerts its action viaAP-1 sites or there is an unidentified estrogen
`
`response element further upstream in the promoter, would require
`further investigation, particularly since estrogen action in a target
`cell apparently requires selective activation of different response
`elements.
`
`Clusterin has been identified as a gene that is expressed during
`the involution of glandular tissues such as the breast and prostate
`by apoptosis (Tenniswood et al., 1992). In these tissues, clusterin
`expression is usually transient since the gene is expressed in
`epithelial cells that die by apoptosis as the tissue regresses. In the
`rodent after hormone ablation, uterine regression is completed
`within 375 days (Moulton, 1994). Clusterin mRNA expression
`persists in endometrial tumors derived from RUCA-I cells for at
`least 30 days following ovariectomy or treatment with the anti-
`estrogen ICI 182,780, suggesting that it is unlikely to be associated
`solely with the apoptotic processes that is initially induced by these
`hormonal manipulations, and probably serves a cytoprotectant role,
`by mediating the clearance of redundant membranes and other
`cytolytic debris (Jenne and Tschopp, 1992).
`Clusterin is also known to inhibit complement mediated cytoly-
`sis (O’Bryan et al ., 1990', Jenne and Tschopp, 1992). The glandular
`surface of the normal rat uterus represents an interface of the body
`with the external surroundings and the regulation of mucosal
`immunity in the female reproductive tract via the endocrine system
`is well established (Wira and Stern, 1992). In response to estradiol,
`polymeric IgA and the cytoplasmic portion of its receptor, secretory
`component, as well as IgG, enter uterine secretions, as part of the
`immune defense system (Sullivan et al., 1983). Repression of
`clusterin expression, coupled with the upregulation of individual
`components of the complement cascade by gonadal steroids,
`probably ensures a competent immune defence system (Sullivan et
`al., 1983). Conversely, tumors derived from the RUCA-I cells are
`highly metastatic (Vollmer and Schneider, 1997), and should be
`subject to immune surveillance while entering the circulation or
`invading other tissue compartments.
`In this context, clusterin
`mediated inhibition of complement-induced cytolysis probably
`protects the RUCA-I adenocarcinoma cells from complement-
`mediated lysis and contributes to the highly metastatic phenotype
`ofthese cells.
`
`In summary, we have shown that regulation of the steady state
`mRNA level of clusterin in the rat uterus and in rat endometrial
`adenocarcinoma is controlled by estradiol, but is diametrically
`reversed in normal and malignant
`tissues, exhibiting estrogen
`repression of expression in normal tissue and inducible expression
`in tumor cells both in ViVO and in vitro. Clusterin may therefore
`represent a target gene that can be used to monitor changes in the
`transcription factors and signal
`transduction pathways that are
`responsible for the progression from normal
`to malignant
`to
`metastatic tissue.
`
`ACKNOWLEDGEMENTS
`
`This work was supported by the Deutsche Forschungsgmein—
`schaft Vo410/5-1 and Vo410/5-2 to GV, and NATO CRG 971593 to
`MT and GV.
`
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`
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`lnnoPharma Exhibit 1088.0004
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`WI'TNSCHE ETAL.
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