Post on 22-Feb-2023
Endocrine-Related Cancer (2008) 15 635–648
Targeted therapy for adrenocortical tumorsin transgenic mice through their LH receptorby Hecate-human chorionic gonadotropin bconjugate
Susanna Vuorenoja1,2, Adolfo Rivero-Muller1, Adam J Ziecik3,Ilpo Huhtaniemi1,4, Jorma Toppari1,2 and Nafis A Rahman1
1Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland2Department of Pediatrics, University of Turku, 20520 Turku, Finland3Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-714 Olsztyn, Poland4Department of Reproductive Biology, Imperial College London, London W12 ONN, UK
(Correspondence should be addressed to N A Rahman; Email: nafis.rahman@utu.fi)
Abstract
Novel strategies are needed for the treatment of adrenocortical tumors that are usually resistantto chemotherapy. Hecate, a 23-amino acid lytic peptide, was conjugated to the 15-amino acid(81–95) fragment of the human chorionic gonadotropin b (CGb) chain, which would selectivelykill cancer cells expressing the LH receptor (LHR) sparing the normal ones with LHR. To provethe principle that Hecate-CGb conjugate may eradicate tumors ectopically expressing plasmamembrane receptors, transgenic (TG) inhibin a-subunit promoter (inha)/Simian Virus 40 T-antigen mice, expressing LHR in their adrenal gland tumors, were used as the experimentalmodel. Wild-type control littermates and TG mice with adrenal tumors were treated with eitherHecate or Hecate-CGb conjugate at the age of 6.5 months for 3 weeks and killed 7 days afterthe last treatment. The Hecate-CGb conjugate reduced the adrenal tumor burden significantly inTG male but not in female mice, in comparison with Hecate-treated mice. Hecate-CGb
conjugate treatment did not affect normal adrenocortical function as the serum corticosteronelevel between Hecate and Hecate-CGb conjugate groups were similar. The mRNA and proteinexpressions of GATA-4 and LHR colocalized only in tumor area, and a significantdownregulation of gene expression was found after the Hecate-CGb conjugate in comparisonwith Hecate- and/or non-treated adrenal tumors by western blotting. This finding providesevidence for a selective destruction of the tumor cells by the Hecate-CGb conjugate. Hereby,our findings support the principle that Hecate-CGb conjugate is able to specifically destroytumor cells that ectopically express LHR.
Endocrine-Related Cancer (2008) 15 635–648
Introduction
Adrenocortical carcinomas (ACCs) are rare and aggres-
sive types of humanmalignancies with poor prognosis as
they often are diagnosed late and the survival rate
remains quite low (Schulick & Brennan 1999b). ACCs
tend to occur in the first and fifth decades of life and are
relativelymore common inwomen thanmen (Schulick&
Brennan 1999a,b). There are still no efficient forms of
therapy for adrenal tumors. The only effective treatment
Endocrine-Related Cancer (2008) 15 635–648
1351–0088/08/015–635 q 2008 Society for Endocrinology Printed in Great
at this moment is complete adrenalectomy since
with partial adrenalectomies the survival rate remains
quite poor. However, even in patients undergoing
complete surgical resection, recurrent and metastatic
disease is common (Reincke et al. 1994, Bornstein et al.
1999). Chemotherapy with mitotane (o,p0-DDD) or
cisplatin is indicated for advanced stages of disease,
although the outcome remains often poor with plenty of
side effects including adrenal insufficiency (Ahlman
et al. 2001). Thus, there is a great need for new curative
Britain
DOI: 10.1677/ERC-08-0015
Online version via http://www.endocrinology-journals.org
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
methods as well as novel markers for early detection
of these rare ACCs. Besides the normal gonadal
expression of luteinizing hormone receptor (LHR),
there are reports about malignant androgen-producing
LH/human chorionic gonadotropin (hCG)-dependent
adrenal tumors (de Lange et al. 1980, Leinonen et al.
1991, Lacroix et al. 1999). The LHR is expressed at low
levels in normal human adrenal cortex (Pabon et al.
1996), and hCG stimulates dehydroepiandrosterone
sulfate synthesis of human fetal adrenals (Jaffe et al.
1981). LHRexpression has been shown in different kinds
of human adrenal pathologies i.e., virilizing or Cushing’s
syndrome (CS)-related adrenocorticotropin-independent
macronodular adrenal hyperplasias (AIMAH; Lacroix
et al. 1999, Bourdeau et al. 2001, Miyamura et al. 2002,
Feelders et al. 2003, Goodarzi et al. 2003,Mijnhout et al.
2004). Pregnancy-associated CS is also a known
condition (Sheeler 1994). LHR expression has also
been shown in aldosterone- (Saner-Amigh et al. 2006) or
androgen-producing LH-dependent adrenal adenomas
(Werk et al. 1973, Givens et al. 1975, Larson et al. 1976,
Smith et al. 1978, Takahashi et al. 1978, de Lange et al.
1980, Leinonen et al. 1991) and inACCs (Wy et al. 2002,
Barbosa et al. 2004). Thus, this abundant/upregulated
LHR expression in the adrenal hyperplasia/adenomas
and adenocarcinomas makes them a susceptible target to
ligandas a strategy for tumor eradication throughHecate-
human chorionic gonadotropin b (CGb) conjugate.During the last few years, a novel targeted
treatment approach for hormonally active, LHR-
bearing tumors has been developed using the
Hecate-CGb conjugate (Leuschner et al. 2001).
Hecate is a synthetic lytic peptide based on the
structure of melittin, the principal toxin of honeybee
venom. It rapidly destroys the outer membrane shell
of only negatively charged cells such as bacteria and
cancer cells by making small pores to the membrane
(Leuschner & Hansel 2004, Rivero-Muller et al.
2007). Cancer cells have been shown to be negatively
charged due to the changed distribution of phospha-
tidylserines in the outer cell membrane and thus, in
comparison with the healthy cells, only negatively
charged cells are destroyed by this lytic peptide
(Utsugi et al. 1991). To target the action of this lytic
peptide directly to the cancer cells through a
membrane receptor, Hecate can be conjugated to a
ligand or its receptor-binding domain, such as, in our
case, to the 15-amino acid fragment (81–95) of the
human CGb chain (Hecate-CGb conjugate). This
allows the Hecate-CGb conjugate to bind specifically
and disrupt cells bearing the LHR. The same principle
should apply to the plasma membrane receptors
ectopically expressing in cancer cells. The mechanism
636
of destruction is necrosis without activation of
apoptosis (Bodek et al. 2005b) and Hecate-CGbconjugate appears to selectively kill only cancer
cells with LHR and spare all other gonadal/non-
gonadal healthy cells (even with LHR) due to
negative/positive charge changes in their membrane
potential (Leuschner & Hansel 2004, Bodek et al.
2005b). Hecate-CGb conjugate has previously been
studied in specific cancer cell lines and xenografts of
LHR-bearing carcinomas of prostate (Leuschner et al.
2001, 2003b, Bodek et al. 2005a), mammary gland
(Bodek et al. 2003, Leuschner et al. 2003a), and ovary
(Gawronska et al. 2002). In vivo studies have so
far been carried out in transgenic (TG) mice bearing
ovarian and testicular tumors (Bodek et al. 2005b).
TG mice expressing the inhibin a promoter/Simian
virus 40 (SV40) T-antigen (inha/Tag) were
originally found to produce gonadal tumors with
100% penetrance at the age of 6 months, and if
gonadectomized prepubertally, they produced dis-
cernible adrenal tumors at the same age, but never in
intact mice (Kananen et al. 1996a, Rilianawati et al.
1998, Rahman et al. 2004). The adrenal tumors and a
tumor-derived cell line (Ca1) express very high
levels of LHR (Kananen et al. 1996a, Rilianawati
et al. 1998, Rahman et al. 2004), and the growth and
steroidogenesis of the adrenal tumors were shown to
be dependent on LH stimulation (Mikola et al. 2003).
The post-castration elevation of LH levels apparently
induced the ectopic LHR expression, which together
with the potent oncogene Tag expression triggered
adrenocortical tumorigenesis (Mikola et al. 2003).
LH dependence of the tumors was proven by findings
that they failed to appear if the post-castration
increase in gonadotropins was blocked by either
treating the mice with a gonadotropin-releasing
hormone antagonist or crossbreeding them to the
gonadotropin-deficient hpg (hypergonadotropic)
genetic background (Cattanach et al. 1977, Kananen
et al. 1997). The adrenocortical tumors tend to grow
in prepubertally gonadectomized mice by hyperpla-
sia–adenoma–carcinoma sequence, as hyperplasia of
the adrenal cortex/adrenal adenoma could be
observed at the age of 4 months, whereas discernible
gonadal tumors were seen only at 6 months with low
metastatic incidence (Rahman et al. 2004). In this
study, we took the advantage of this inha/Tag TG
adrenocortical tumor model in order to prove the
principle that adrenocortical tumor cells expressing
ectopically a hormone receptor are sensitive to the
Hecate-CGb conjugate treatment in vivo, sparing
simultaneously healthy cells.
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Endocrine-Related Cancer (2008) 15 635–648
Materials and methods
Experimental animals
In order to induce adrenal tumors, we gonadectomized
inha/Tag TG mice prepubertally as described pre-
viously (Kananen et al. 1996a). As these mice have
earlier been extensively characterized (Kananen et al.
1996a, Rilianawati et al. 1998, 2000, Kero et al. 2000,
Rahman et al. 2001, 2004), the discernible adrenocor-
tical tumors appear at the age of 6 months with 100%
penetrance. We started all the treatments at the age of
6.5 months, in order to make sure that any treatment
effect will be only due to the anti-tumoral effect, but
not by prevention of the tumor development. Gona-
dectomy was performed under Avertin anesthesia and
postoperative buprenorphine analgesia was adminis-
tered on a routine basis. Seven to ten mice per
treatment group were selected for the experiments.
Wild-type (WT) control littermate mice (C57Bl/6N)
were used as controls. For routine genotyping, PCR
analysis was carried out using DNA extracts from ear
biopsies, as previously described (Kananen et al.
1995). After weaning at the age of 21 days, the mice
were housed two to four per cage, females and males in
separate cages, in a room of controlled light (12 h
light:12 h darkness) and temperature (21G1 8C). They
were fed with mouse chow SDS RM-3 (Whitham,
Essex, UK) and tap water ad libitum. The mice were
kept in a specific pathogen-free surrounding and were
routinely screened for common mouse pathogens. The
University of Turku Ethics Committee on Use and
Care of Animals approved the animal experiments.
Preparation of drugs
Hecate and the Hecate-CGb conjugate were syn-
thesized and purified in the Peptide and Protein
Laboratory,Department ofVirology,Hartman Institute,
University of Helsinki, as described previously
(Bodek et al. 2003).
Hecate and Hecate-CGb conjugate treatments
Male and female mice (inha/Tag or WT C57Bl control
littermates; nZ7–10 per group) were treated at the age
of 6.5 months with either Hecate-CGb conjugate
(12 mg/kg b.w.) or Hecate (12 mg/kg b.w.) by i.p.
injections. Since no substantial weight changes could
be observed between Hecate and Hecate-CGbconjugate by adding hCG to Hecate, both Hecate and
Hecate-CGb conjugate were given in the same dose
(Leuschner et al. 2001). The mice were injected once
per week for three consecutive weeks, according to an
earlier protocol for in vivo treatment for nude mice
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xenografts (Leuschner et al. 2001) and TG mice with
gonadal tumors (Bodek et al. 2005b). Seven days
following the last injection, mice were killed by
cervical dislocation and blood was collected by cardiac
puncture. Weights of body, tumor, and different organs
were recorded. Tissues were either snap-frozen in
liquid nitrogen, or fixed in Bouin’s solution or 4%
paraformaldehyde and embedded in paraffin. Paraffin
sections of 5 mm thickness were stained for further
histological analysis with hematoxylin–eosin or used
for immunohistological analysis. For each tissue, at
least five independent specimens were examined from
each group.
Northern hybridization analysis
Total RNAwas isolated from cells using the single-step
guanidinium thiocyanate–phenol–chloroform extrac-
tion method (Chomczynski & Sacchi 1987). Twenty
micrograms of RNA per lane were resolved on 1.2%
denaturing agarose gel and transferred ontoHybond-XL
nylon membranes (Amersham, Amersham Inter-
national). Membranes were prehybridized overnight at
65 8C in a solution containing 5! sodium chloride/
sodium citrate (SSC), 5! Denhardt’s solution, 0.5%
SDS, 50% formamide, and 5 g/l of denatured calf
thymus DNA. A complementary RNA probe for the rat
LHR generated from a fragment of the LHR cDNA,
spanning nucleotides 441–849 of its extracellular
domain, subcloned into the pGEM-4Z plasmid was
used for hybridization (LaPolt et al. 1990). The
[32P]-dUTP (800 Ci/mmol, Amersham) labeled probe
was generated using a Riboprobe system II kit
(Promega). The probes were purified with NickCol-
umns (Pharmacia Biotech). Hybridization was carried
out at 65 8C for 20 h in the same prehybridization
solution after an addition of labeled probe. After
hybridization, the membranes were washed twice in
2! SSC with 0.1% SDS at room temperature for
10 min, followed by two washes in 0.1! SSC with
0.1% SDS at 65 8C to remove most of the background.
For GATA-4, the membranes were prehybridized
overnight at 42 8C in a solution containing 5! SSC,
5! Denhardt’s, 0.5% SDS, 50% formamide, and 5 g/l
denatured calf thymus DNA. The GATA-4 cDNA
probes were cut out from pMT2-GATA4 (Arceci et al.
1993, Ketola et al. 1999), using EcoRI/PstI and SmaI
restriction enzymes respectively labeled with Prime-
a-Gene kit (Pharmacia) using [a-P32]-dCTP for 4 h at
37 8C and purified with NickColumns. Hybridization
was carried out at 42 8C for 20 h in the same
prehybridization solution after an addition of the
denatured labeled cDNA probe. After hybridization,
637
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
themembraneswerewashed twice in 2!SSC and 0.1%
SDS at room temperature for 10 min, followed by two
washes in 0.1! SSC and 0.1% SDS at 42 8C to remove
most of the background. Finally, the membranes were
exposed to KodakX-ray films (Kodak XAR-5; Eastman
Kodak) at K70 8C for 4–7 days or to phosphor-imager
(Fujifilm BAS-5000, Fujifilm IaI, Tokyo, Japan) for
4–24 h. The intensities of specific bandswere quantified
using the Tina software (Raytest, Stranbenhardt,
Germany) which came as an integrated program with
the phosphor-imager (Fujifilm BAS-500; El-Hefnawy
& Huhtaniemi 1998, El-Hefnawy et al. 2000, Manna
et al. 2001, Strassburg et al. 2002, Moran et al. 2006),
and related to those of the 28S rRNA in the gel stained
with ethidium bromide. The molecular sizes of the
mRNA species were estimated by comparison with the
mobility of the 18S and 28S rRNAs.
Hormone measurements
Serum levels of LH were measured by immunofluoro-
metric assay for rat (Delfia; Wallac, Turku, Finland) as
described previously (Haavisto et al. 1993). Cortico-
sterone levelsweremeasured fromdiethyl ether extracts
of the sera by RIA using a kit for rats and mice (MP
Biomedicals, Orangeburg, NY, USA) and progesterone
was measured by Delfia Progesterone Kit (Wallac). The
approximate assay sensitivities for LH, progesterone,
and corticosterone are 0.75 pg/tube, 50 fmol/tube, and
30 fmol/tube respectively. The intra- and inter-assay
coefficients of variations for these assays were below
10–15% respectively.
Immunoblotting analysis for GATA-4 and LHR
Western blotting was used to determine the concen-
tration changes in GATA-4 and LHR due to the
different treatments (Hecate or Hecate-CGb conju-
gate). Tissue samples were subjected to homogeni-
zation and protein concentration was measured using
the bicinchoninic acid (BCA) protein assay kit (Pierce,
Rockford, IL, USA). Electrophoresis was carried out
through a 12.5% SDS-PAGE gel (1 mg protein/well).
Dual Color Precision Plus Protein Standards (Bio-Rad)
were used as standards. After electrophoresis, proteins
were electrotransferred to Hybond-P PVDF Membrane
(Amersham Biosciences) using Trans-Blot SD cell
(Bio-Rad) by 20 V for 60 min. After blocking the
membrane with TBS containing 2% fat-free milk
powder C0.05 M Tween, the incubation with the
primary antibody was performed at C4 8C overnight.
Anti-GATA-4 (goat polyclonal antibody, dilution
1:100; C-20, sc-1237; Santa Cruz Biotechnology,
Santa Cruz, CA, USA) or anti-LHR (rabbit polyclonal
638
antibody, dilution 1:4000; Acris Antibodies, Hidden-
hausen, Germany) were used separately. As secondary
antibody, either bovine anti-goat IgG-HRP (sc-2350,
Santa Cruz Biotechnology) or ECL Anti-rabbit IgG,
HRP-linked whole AB (Amersham Biosciences) was
used in dilution 1:10 000. Signals were visualized
using ECL Plus Western blotting detection reagents
(Amersham Pharmacia Biotech) and finally exposed to
Fuji X-ray film (Super RX, Fuji Photo Film Ltd,
Bedford, UK). The intensities of specific bands were
quantified using the Tina Software (Raytest).
Laser pressure catapulting (LPC)
LPC was performed by Laser MicrobeamMicrodissec-
tion (PALM Microlaser Technologies, Bernried,
Germany) using a protocol described previously
(Westphal et al. 2002). Briefly, for laser microdissec-
tion, cryosections (5 mm) were prepared from three
independent specimens. Subsequent dehydration was
achieved using graded alcohol and xylene treatment as
follows: 95% ethanol for 30 s (twice), 100% ethanol
for 30 s (twice), and xylene for 5 min (twice). The
slides were dried under laminary flow for 10 min and
then stained with hematoxylin using RNase-free
conditions and kept on dry ice until LPC. LPC was
performed using a Zeiss inverted microscope PALM
Laser Micro-Beam System u.v. laser at 337 nm, linked
to a PC with the required software programs. The
tumor and non-tumor areas to be dissected were
selected by means of high-precision microcuts (the
specimens may range from as little as 1 to 1000 mm in
diameter). LPC dissection was performed using a few
shots each of 100 mm in diameter from both tumor and
non-tumor areas and from each slide. After catapulting
similar amounts of tumor and non-tumor area, the
material was removed from the caps for further
analysis. Total RNA was extracted from tumor tissues
using RNeasy kit (Qiagen).
RT-PCR and Southern hybridization
One microgram of total RNA was reverse transcribed
using 20 IU of avian myeloma virus reverse tran-
scriptase (RT), 50 IU RNase inhibitor, 25 pmol random
primers, and 10 mmol of each dNTP (all from
Promega), in 25 ml final volume for 1 h at room
temperature. For amplification of LHR and GATA-4
cDNA, specific primers were used as described
previously (Morrisey et al. 1997, Kero et al. 2000).
PCR was performed in a 25 ml final volume, 4 ml RTsolutions were mixed with 2 IU of the DyNAzyme II
DNA polymerase (Finnzymes Oy, Espoo, Finland),
10 pmol of each primer, and 10 mmol of each dNTP
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Endocrine-Related Cancer (2008) 15 635–648
(Zhang et al. 1994). The RT and PCRs were carried out
sequentially in the same assay tube. First, the RT
reaction was carried out (50 8C for 10 min), followed
by a denaturation period of 3 min at 97 8C. Thereafter,
a PCR with 40 cycles (96 8C for 1.5 min, 57 8C for
1.5 min, and 72 8C for 3 min, with a final extension
period of 10 min at 72 8C) was performed. The
sense primer for LHR corresponded to nucleotides
176–195 (5 0-CTCTCACCTATCTCCCTGTC-3 0) and
the antisense primer to nucleotides 878–858
(5 0-TCTTTCTTCGGCAAATTCCTG-3 0) of the
mouse 700 bp fragment of the LHR cDNA. The
sense primer for GATA-4 corresponded to sense
nucleotides 1527–1550 (5 0-AAACGGAAGCCCAA-
GAACCTGAAT-3 0) and the antisense primer to
1935–1953 (5 0-GGCCCCCACGTCCCAAGTC-3 0)
(expected size: 427 bp) of mouse GATA-4 cDNA
(Morrisey et al. 1997). As a control for RNA quality, a
395 bp fragment of the L19 ribosomal protein gene was
co-amplified with each sample (sense primer, 5 0-
GAAATCGCCAATGCCAACTC-3 0; antisense pri-
mer, 5 0-TCTTAGACCTGCGAGCCTCA-3 0). Kidney
RNA was used as a negative control. After RT-PCR,
20 ml aliquots of the reaction mixtures were loaded on
1% agarose gel containing ethidium bromide (0.4 mg/
l), to identify the amplified DNA fragments. Southern
hybridization was used, according to standard tech-
niques, to confirm the specificity of these PCR
products. Hybridization was carried out with the 5 0-
end labeled oligonucleotide 5 0-TGGAGAAGATGCA-
CAGTGGA-3 0, corresponding to nucleotides 641–660
of LHR cDNA and for GATA-4 with the 5 0-
AGTGGCACGTAGACGGGCGAGGAC-3 0, corre-
sponding to nucleotides 1676–1699. The membranes
were washed according to the manufacturer’s instruc-
tions and then exposed to Kodak X-ray film (XAR 5;
Eastman Kodak).
Immunohistochemistry (IHC)
Paraformaldehyde-fixed paraffin sections (5 mm) of
mouse adrenal tumors were deparaffinized and rehy-
drated. For immunostaining, 3% H202 in water was
used to block endogenous peroxidases and all sections
were boiled by microwave treatment for 15 min in
10 mM citric acid (pH 6.0) for antigen retrieval. Two
washes were done after each step with 0.05 M Tris and
150 mM NaCl (TBS) with 0.1% Tween (TBS-T).
Serial sections of adrenal gland and testes as a positive
control were subjected to immunohistochemistry with
an anti-LHR antibody (dilution 1:1000, a rabbit
polyclonal antiserum directed against human peptide
sequence, KKLPSRETFVNLLEA; a gift of Dr A
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Fazleabbas) and/or commercial rabbit polyclonal
anti-GATA-4 IgG (dilution 1:800, Santa Cruz Bio-
technology). IHC for anti-steroidogenic factor (SF)-1
(goat polyclonal antibody, 1:400, Santa Cruz Bio-
technology) was also carried out. The slides were
incubated with primary antibody at 4 8C overnight, and
after incubation and washings with TBS–Tween buffer
the slides were incubated with the anti-rabbit (Vector
Laboratories, Inc., Burlingame, CA, USA) or anti-goat
(Santa Cruz Biotechnology) secondary antibody. The
avidin–biotin immunoperoxidase system was used to
visualize bound antibody (Vectastain Elite ABC Kit,
Vector Laboratories) with 3.3 0-diaminobenzidine
(Sigma) as a substrate. As a control for antibodies,
adjacent sections were incubated with either 1%
normal goat serum in PBS or rabbit IgG as a primary
antibody to differentiate unspecific staining from
specific staining.
Statistical analysis
Statistical analyses were carried out by ANOVA and/or
the non-parametrical Mann–Whitney Rank test using
SAS Enterprise Quide 3.0 program (SAS Institute Inc.,
Cary, NC, USA). Non-parametrical tests were used
when the variances were not equal. ANOVA was used
in other cases. P values !0.05 were regarded as
statistically significant. All the values are presented as
meanGS.E.M.
Results
Hecate-CGb conjugate treatment reduced
adrenal tumor volume
Hecate-CGb conjugate had an antineoplastic effect on
the adrenal tumors of inha/TagTGmice.Wehave earlier
characterized the ontogeny of adrenocortical tumorigen-
esis in these mice and observed tumors weighing 20- to
30-fold more than normal adrenals (290G69 mg) at
6–6.5 months of age following pre-pubertal gonadect-
omy (Rahman et al. 2004). Following a 1-month
treatment with Hecate-CGb conjugate, adrenal tumor
weight was reduced by an average of 59% in males
compared with Hecate treatment (364G147 vs 150G130 mg;Hecate versusHecate-CGb conjugate;P!0.01;
Fig. 1A). In females, the differencewas only 18% (292G156 vs 240G225 mg; Hecate versus Hecate-CGbconjugate; PO0.05; Fig. 1B). As the age-related
individual tumor progression rate was variable between
inha/Tag TGmice and has been observed already earlier
in inha/Tag andTagmice (Hanahan 1989,Kananen et al.
1995, 1996b, Rahman et al. 1998), we also analyzed the
639
Figure 1 Total adrenal weights of (A) male (nZ10) and(B) female (nZ10) inha/Tag transgenic (TG) and wild-type(WT) (WT male, nZ7 and female, nZ8) mice after 3 weeks ofHecate-CGb conjugate or Hecate treatment. Tumor burden,tumor weight/body weight in grams. The values aremeanGS.E.M. *P!0.01; Hecate-CGb conjugate versus Hecate.
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
tumor burden (tumor weight/body weight). This para-
meter also decreased after Hecate-CGb conjugate
treatment significantly in males in comparison with
Hecate treatment. In females, the treatment effect
remained non-significant (Fig. 1A and B). We did not
observe any statistically significant differences between
the total body weights of the mice between the treatment
groups (data not shown). No statistically significant
640
differences in tumor weights/burden between the sexes
could be observed in the present study, which was in line
with the earlier observations (Rahman et al. 2004). The
adrenal tumors metastasized in 10% (nZ10 for both
male and/or female inha/Tag mice) cases in the Hecate-
treated group to liver (proven by histopathological
analysis, data not shown), but never in Hecate-CGbconjugate-treated groups. According to earlier publi-
cations and observations, these tumorsmetastasized only
occasionally (around 10% cases) to liver, more
commonly in females thanmales (Kananen et al. 1996a).
LHR expression is higher in the adrenal tumors of
male mice
To find out why Hecate-CGb conjugate treatment was
less effective in female adrenal tumors, we quantified
LHR and GATA-4 mRNA expressions by northern
hybridization in male and female non-treated tumors
(Fig. 2). GATA-4 was included in the study due to its
close association with LHR expression and adreno-
cortical tumorigenesis (Kiiveri et al. 1999, Rahman et al.
2004).We alsomeasuredLHRandGATA-4 expressions
in the established Ca1 cell line derived from inha/Tagadrenocortical tumor (Kananen et al. 1996a, Rilianawati
et al. 1998) and from non-treated adrenocortical tumors.
A significantly higher level of LHR mRNA expression
was found in the male than in the female in adrenal non-
treated tumors (P!0.01; Fig. 2). No significant
differences inGATA-4mRNAlevels between the female
and male adrenocortical tumors could be observed.
Endocrine consequences of the treatments
As sham-treated group (castrated TG mice treated with
vehicle) shows identical hormonal values with the
Hecate-treated group, which has been demonstrated
earlier for testicular and ovarian tumors (Bodek et al.
2005b), ovarian xenograft model (Gawronska et al.
2002), and also rat mammary gland tumor model
(Zaleska et al. 2004), in this study the sham-treated
group was not taken into account in order to concentrate
on the differences between the Hecate and Hecate-CGbconjugate groups. In the WT groups, gonadectomy-
induced elevated levels of LH were clear due to the lack
of negative feedback from the gonads. After 1 month of
treatment with Hecate-CGb conjugate, no statistically
significant differences could be seen in Hecate and
Hecate-CGb conjugate treatment groups in the LH,
corticosterone, or progesterone levels in either sex
(Fig. 3), although progesterone levels were twofold
lower, but not statistically significant in Hecate-CGbconjugate-treated males in comparison with Hecate
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Figure 2 Northern hybridization analysis of (A) LHR mRNA and(B) western blot analysis of the protein expression in inha/TagTG adrenal tumors of male and female mice and from tumor-derived Ca1 cells. Here, in three independent experiments,each time two different tumors for both sexes were used (nZ6for both males and females). RNA was extracted from one-halfof the tumor and protein from the other half of the tumor.(A) Each lane contains 20 mg total RNA. The migration of the28S and 18S rRNAs is shown on the left side of the LHR panel.The sizes of the different LHR mRNA splice variants (in kb) arepresented on the right. Two lanes for each type of sample aredepicted. The upper panel shows, on the top, the ethidiumbromide (EtBr) staining of the 28S rRNA for RNA loadingcontrol. The middle and lower panels show northern hybrid-ization for GATA-4 and LHR mRNA respectively. The bottompanel shows densitometric quantification of the longest (7.0 kb)LHR mRNA splice variant (filled bars) and GATA-4 (open bars)in arbitrary densitometric units (the mean of Ca1 tumor cells forGATA-4 or LHR was designated as 100%) corrected forintensity of the 28S rRNA band. Each bar represents themeanGS.E.M. of three independent experiments, each time twodifferent tumors for both sexes and from two different cellextracts. Densitometric quantification of the six different bandsis represented by one bar on the densitometric analysis. Eachband shows a different tumor/mouse. **P!0.01, *P!0.05(P!0.01 and P!0.05; male versus female for LHR, maleand/or female versus Ca1 cells for LHR). Ca1, adrenaltumor cell line derived from a transgenic mice expressing(inha)/Simian Virus 40 T-antigen (Tag) adrenal tumor; tu, tumor.
Endocrine-Related Cancer (2008) 15 635–648
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group (in females nodifferences inhormonal values seen,
data not shown), due to the high individual variations
between the serum progesterone values (Fig. 3). Signi-
ficant change in LH levels was seen between gonadecto-
mized WT mice and the TG mice for both Hecate and
Hecate-CGb conjugate-treated groups (P!0.05; Fig. 3).
Serum progesterone levels were three- to four-fold
higher in the TG mice than in WT mice, which might
explain the lower level of LH in adrenal tumor-bearing
TG mice (P!0.05; Fig. 3). This finding is in agreement
with the earlier findings in adrenal tumors and adrenal
tumor-derived Ca1 cells (Kananen et al. 1996a)
indicating their steroidogenic activity, e.g., progesterone
secretion.
Hecate-CGb conjugate selectively destroyed the
LHR possessing adrenal tumor cells
To assess whether the Hecate-CGb conjugate treatment
can eradicate specifically the LHR-expressing adreno-
cortical tumor cells, wemeasured the LHR and GATA-4
protein expression levels in Hecate-CGb conjugate-
treated adrenals in comparison with the Hecate-treated
and non-treated adrenocortical tumor LHR protein
levels. Western blotting analysis revealed that there
was a concomitant significant decrease in LHR and
GATA-4 in the Hecate-CGb conjugate-treated adrenal
tumors (P!0.01 Hecate-CGb conjugate versus non-
treated or Hecate-treated adrenal tumors; Fig. 4),
indicating selective destruction of LHR and GATA-4
positive adrenocortical tumor cells. Higher GATA-4
expression compared with LHR after Hecate-CGbconjugate might reflect the previously described
phenomenon that these adrenocortical cells might
express GATA-4 without LHR, but not vice versa and
their expression patternwas higher/broader (Kiiveri et al.
1999, 2002). The LHR or GATA-4 protein expression in
WT control mouse adrenals could not be detected.
Histopathological analysis demonstrating anti-
tumoral effects of Hecate-CGb conjugate
treatment
Histopathological analysis revealed that Hecate-CGbconjugate treatment of TG adrenocortical tumor mice
(B) The upper panel shows GATA-4, middle panel LHR, andlower panel b-actin protein expressions in western blottinganalysis. Each lane represents three independent experiments,each time three different tumors for both sexes and fromthree different cell extracts. Each band shows a differenttumor/mouse. Ca1, adrenal tumor cell line derived from atransgenic mice expressing (inha)/Simian Virus 40 T-antigen(Tag) adrenal tumor; tu, tumor; on the right side, the molecularweights of the proteins in kilodaltons are indicated.
641
Figure 4 Western blot analysis for LHR and GATA-4. Proteinextracts from male inha/Tag TG mice treated with Hecate orHecate-CGb conjugate or from non-treated tumor sampleswere analyzed by western blotting. The upper panel showsLHR, GATA-4, and b-actin expressions and the lower paneltheir densitometric analysis. The representative band datashown are from one of the four different adrenal tumors/mouseper group/independent experiments (nZ4). Each bar indensitometric quantification shows the quantification of fourseparate independent tumor samples/experiments. Each barrepresents the meanGS.E.M. of four independent experiments,each time different tumor sample. Hecate-CGb conjugatedownregulated significantly both LHR and GATA-4 expressionscompared with non-treated control adrenal tumor or Hecate-treated tumor (**P!0.001). No detectable LHR or GATA-4 wasdetected in WT C57Bl mouse adrenals. On the right side, themolecular weights of the proteins in kilodaltons are indicated.
Figure 3 Serum (A) LH, (B) progesterone, and (C) corticoster-one concentrations in transgenic inha/Tag male mice aftera 3-week treatment with Hecate-CGb conjugate or Hecate.WT, wild-type or TG, inha/Tag mice treated with Hecate-CGbconjugate or Hecate. The values are meanGS.E.M.; nZ5,*P!0.05; Hecate-CGb conjugate-treated TG mice versusHecate or Hecate-CGb conjugate-treated WT mice.
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
induced a definite reduction of the adrenocortical tumor
mass where residual tumor tissue could be observed in a
reduced area of the zona glomerulosa in hematoxylin–
eosin-stained sections (Fig. 5B). This finding in the
representative sample was reproducible as the same
phenomenon was observed in all stained samples of the
same treatment groups (nZ5 from each group). The
adrenal tumors metastasized in 10% (nZ10 for both
males and/or female inha/Tag mice) cases in the
Hecate-treated group to liver (proven by histopatholo-
gical analysis, data not shown), but never in Hecate-
CGb conjugate-treated groups. According to earlier
publications and observations, these tumors metasta-
sized only occasionally (around 10% cases) to liver,
more commonly in females than males (Kananen
et al. 1996a).
642
LHR and GATA-4 expressions are colocalized in
the tumors
We next demonstrated that LHR and GATA-4 are
co-localized in the same adrenocortical tumor region.
The data obtained by LPC of the tumorous and non-
tumorous tissues from the same slice demonstrated the
co-localization of GATA-4 and LHR mRNA messages
in adrenal tumor tissue and their absence in normal
adrenal cortex (Fig. 6). The finding in the representa-
tive sample was reproducible as the same phenomenon
was observed in all other three samples.
We then investigated whether the expression
patterns of these proteins were affected by the Hecate
or Hecate-CGb conjugate treatment. GATA-4
expression was found to directly localize to the
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Figure 5 Hematoxylin–eosin staining of adrenal tumors aftertreatments with (A and C) Hecate or (B and D) Hecate-CGbconjugate. (A and B, 20! magnification, C and D, 10!magnification). The picture from (C) Hecate-treated tissue istaken from the contralateral side of the big tumor to show theexpression of the tumorous tissue in the whole adrenal gland.
Figure 6 RT-PCR/Southern hybridization of the PALM micro-dissected samples. (A and B) Hematoxylin-stained specimensof cryosections of the tumorous adrenal tissue. (A) The tumor
Endocrine-Related Cancer (2008) 15 635–648
H–E-stained tumor area that was in line with our
previous findings (Rahman et al. 2004; Fig. 7A and C).
In Hecate-CGb conjugate-treated sections, no LHR
staining could be detected even in the same H–E-
stained, marginalized, and reduced tumorous area
(Fig. 7A and D). We also found SF-1 expression in
the same tumor areas where GATA-4 expression could
be observed (Fig. 7B and C).
sections (dark-stained cells) and (B) non-tumor tissues (light-stained area in the middle) were dissected out and catapulted.(C) The catapulted tumor (WT, wild-type; adr tum, adrenaltumor) and (D) normal non-tumorous tissues in the cap of anEppendorf tube (magnification 63!; adr non-tu, non-tumorusadrenal tissue).Discussion
The lack of a suitable animal model for adrenocortical
tumorigenesis has been a major obstacle for unraveling
the molecular mechanisms involved in their patho-
genesis, as well as for improving their diagnostic and
therapeutic strategies. Thus, establishment of suitable
animal models is of extremely high importance in order
to study human adrenal tumorigenesis. Chronically
elevated LH, ectopic/upregulated LHR expression,
hyperplasia–adenoma–adenocarcinoma sequence tumor
formation, slow tumor growth, late discernible tumor
incidence (analogous to human postmenopausal/andro-
pausal age), low metastases frequency (5–10%): these
characteristics make the inha/Tag TG murine model a
good tumor relevance model for human ACCs studies.
Our inha/Tag TG mouse is at the moment one of the
rare and unique murine models in which adrenocortical
tumorigenesis and its molecular mechanisms or any
treatment strategies can be investigated. Although the
physiologic significances of extragonadal LHR
expression in reproductive function is questioned
(Pakarainen et al. 2005, 2007), some recent studies
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(Rao Ch et al. 2004, Alevizaki et al. 2006, Vuorenoja
et al. 2007) suggest that the adrenal cortex expresses low
levels of LHR (Pabon et al. 1996) whose function
becomes significantwhenLH levels are elevated, as after
gonadectomyorpostmenopausally. In light of the present
study, we could postulate that the upregulated LHR
expression in adrenal tumors is functionally relevant and
can be used to target the tumors by the cytotoxic Hecate-
CGb conjugate. This model serves as an example
demonstrating how ectopically expressed receptors in
tumors can be used as therapeutic target.
We used in the present study an established inha/TagTG mouse model for post-gonadectomy adrenocortical
tumorigenesis, where the pathophysiological and
endocrine responses induced by tumorigenesis are
well characterized. We found that Hecate-CGb con-
jugate treatment reduced the total adrenocortical tumor
weights without detectable side effects, e.g., adrenal
643
Figure 7 Hematoxylin–eosin staining (A) and immunohisto-chemistry for (B) SF-1, (C) GATA-4 and (D) LHR in Hecate-CGbconjugate-treated adrenal tumor sections.
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
insufficiency. Curiously, the treatment was, for
unknown reason, ineffective in female with adrenocor-
tical tumors. The adrenal response to elevated LH in
inha/Tag mice was probably more sensitive in females
andwe believe it was alsomore aggressive, which could
explainwhy immortalization of Ca1 cells from a female
inha/Tag founder mouse tumor was possible (Kananen
et al. 1996a, Rilianawati et al. 1998). It has been shown
that the castrated male mice developed adrenocortical
tumors more slowly than gonadectomized females
along with the ectopic LHR expression (Bielinska
et al. 2003). Moreover, the elevated LH levels induced
abundant ectopic LHR expression and acted as a tumor
promoter selectively only in the intact female double-
positive TG mice, where bLHb-CTP mice producing
constitutively elevated levels of LH (Risma et al. 1995)
were crossbred with inha/Tag TG mice (Mikola et al.
2003). This never happened in the male double-positive
mice (Mikola et al. 2003). In humans, ACCs are also
more common in postmenopausal women than in aging
males (Schulick & Brennan 1999a,b). Therefore, it
could be an established phenomenon that female
adrenals respond more readily to high LH and
tumorigenesis might be more aggressive in them,
which might imply that the suppression of LH action
needed to block tumorigenesis in females should be
more complete/stronger, whereas male tumorigenesis
can be blocked with less-effective suppression. It is
likely that this mechanism occurred in our Hecate-CGbconjugate-treated inha/Tag mice where, with the
applied dose of the drug, we achieved much a better
response in the males than in the females. Probably, this
phenomenon was additionally helped by the higher
concentration of LHR in the tumor mass in the male
644
tumors attracting larger amounts of Hecate-CGbconjugate, exerting consequently a more severe lytic
effect. The reason for the higher LHR expression in
male adrenal tumors remains unknown. The putative
difference in tumor cell membrane charges between the
male and females could also be one of the potential
mechanistic reasons behind this differential treatment
effects, which needs to be further evaluated in future
studies.
As we did not find significant changes in the serum
endocrine profiles of the Hecate- or Hecate-CGbconjugate-treated mice, the treatment outcome could
not be monitored by serum hormone measurements.
The progesterone levels are decreased in Hecate-CGbconjugatemale in comparisonwithHecate group but the
high individual variations made them non-significant.
LH levels were higher in gonadectomized WT than in
TGmales, which indicated increased endocrine activity
of the tumors. The hormone with increased production
by the tumors exerting a negative feedback action of LH
secretion is probably progesterone. However, the
adrenal tumors can also produce sex steroids (androgens
or estrogens; Leinonen et al. 1991, Kananen et al.
1996a, Lacroix et al. 1999, Bielinska et al. 2005). No
effect of the treatment could be seen on corticosterone
levels between Hecate and Hecate-CGb conjugate
treatment groups, which indicates that the Hecate-
CGb conjugate treatment does not destroy normal
adrenal tissue or induce adrenocortical hypofunction.
This additionally supports the principle that this peptide
attacks only the negatively charged tumor cells and
spares the healthy ones.
We have shown earlier a precise co-initiation of
GATA-4 along with the LHR expression during
adrenocortical tumorigenesis ontogeny (Rahman et al.
2004). GATA-4 has been proposed to be a potential
marker for adrenocortical tumorigenesis (Kiiveri et al.
1999), along with LHR (Rahman et al. 2004). Further
proof is needed to determine whether GATA-4works as
a specific co-activator for LHR (or even vice versa) in
adrenocortical tumorigenesis. Thus, we wanted to see
whether GATA-4 expression would also diminish due
to the Hecate-CGb conjugate treatment, which could
lead us to further mechanistic studies in the future. As
there are some difficulties with the availability of good
LHR antibodies, GATA-4 immunohistochemistry
could also additionally serve as a marker for treatment
efficacy. We found that both LHR and GATA-4 protein
expressions decreased significantly after Hecate-
CGb conjugate treatment compared with Hecate. By
immunohistochemistry, we could detect GATA-4 in
residual tumor area indicating that some tumor
steroidogenic cells are still left, as GATA-4 is expressed
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Endocrine-Related Cancer (2008) 15 635–648
only in tumor adrenocortical steroidogenic cells
(Kiiveri et al. 1999, 2002, Peterson et al. 2004), but
LHR staining was no longer detectable in the Hecate-
CGb conjugate-treated samples. These results indicated
that Hecate-CGb conjugate killed specifically the tumor
cells expressing LHR. This finding is well in line with
the adrenal tumor weight results. Even after the Hecate-
CGb conjugate treatment, the adrenal has not returned
to totally normal size. However, GATA-4 and SF-1
were colocalized in the same tumor area. SF-1 is known
to be another factor activating adrenocortical steroido-
genesis and it is shown to transactivate both GATA-4
and LHR activities (Tremblay & Viger 1999). The
expression pattern of SF-1 may implicate its role as a
co-activator of GATA-4 and LHR in adrenocortical
tumorigenesis of the inha/Tag TG mice.
Human adrenal LH/hCG responsive tumors have been
well described (Leinonen et al. 1991, Lacroix et al.
1999). Higher basal levels of corticosteroids in post-
menopausal women are also associated with higher
circulating LH and LHR expressions in adrenals (Pabon
et al. 1996, Alevizaki et al. 2006). A recent publication
confers that ectopic LHR may induce adrenal hyper-
plasia- and Cushing’s syndrome-like symptoms as well
as hCG-mediated steroidogenic activity (Mazzuco et al.
2006). Ectopic expression of LHR in the adrenal gland
has been shown to be associated with not only
adrenocorticotropin-independent Cushing’s syndrome
but also benign aldosterone-producing adenomas
(Saner-Amigh et al. 2006). There are also reports about
malignant androgen-producing LH-dependent adrenal
tumors (de Lange et al. 1980, Leinonen et al. 1991).
Hence, LH/hCG-dependent mechanisms of adrenal
pathogenesis do exist and they may be clinically
important in circumstances with elevated LH levels,
e.g., in connectionwith polycystic ovarian syndrome and
after menopause in women.
Taken together, the present data provide novel
evidence that targeted destruction of adrenocortical
tumors expressing ectopically LHR by the Hecate-CGbconjugate is possible. In addition to the currently usedTG
mouse model, our findings prove the more general
principle that receptors expressed ectopically in malig-
nant cells can be exploited in targeted cytotoxic therapies
without affecting the normal healthy cells.
Acknowledgements
Thisworkwas supported by grants fromFinnishCultural
Foundation at Varsinais-Suomi, the Finnish Cancer
Society, the Moikoinen Cancer Research Foundation,
the Academy of Finland, and Turku University Hospital.
We thank Dr A Fazleabbas for the kind donation of the
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LHR antibody and Dr Sonia Bourguiba for fruitful
discussions. The authors also thank Ms Johanna
Lahtinen, Ms Tarja Laiho and Ms Heli Niittymaki for
their technical assistance and the personnel of Turku
University Animal Facility for taking good care of the
mice. This project was supported by grants from the
Finnish Cultural Foundation at Varsinais-Suomi (S V),
the Moikoinen Cancer Research Foundation (N R), the
Academy of Finland (I H, J T, N R), and the Turku
University Hospital (J T). We declare that there is no
conflict of interest that would prejudice thismanuscript’s
impartiality.
References
Ahlman H, Khorram-Manesh A, Jansson S, Wangberg B,
Nilsson O, Jacobsson CE & Lindstedt S 2001 Cytotoxic
treatment of adrenocortical carcinoma. World Journal of
Surgery 25 927–933.
Alevizaki M, Saltiki K, Mantzou E, Anastasiou E &
Huhtaniemi I 2006 The adrenal gland may be a target of
LH action in postmenopausal women. European
Journal of Endocrinology 154 875–881.
Arceci RJ, King AA, Simon MC, Orkin SH & Wilson DB
1993 Mouse GATA-4: a retinoic acid-inducible GATA-
binding transcription factor expressed in endodermally
derived tissues and heart.Molecular and Cellular Biology
13 2235–2246.
Barbosa AS, Giacaglia LR, Martin RM, Mendonca BB & Lin
CJ 2004 Assessment of the role of transcript for GATA-4
as a marker of unfavorable outcome in human adreno-
cortical neoplasms. BMC Endocrine Disorder 4 3.
Bielinska M, Genova E, Boime I, Parviainen H, Kiiveri S,
Leppaluoto J, Rahman N, Heikinheimo M & Wilson DB
2005 Gonadotropin-induced adrenocortical neoplasia in
NU/J nude mice. Endocrinology 146 3975–3984.
Bielinska M, Parviainen H, Porter-Tinge SB, Kiiveri S,
Genova E, Rahman N, Huhtaniemi IT, Muglia LJ,
Heikinheimo M & Wilson DB 2003 Mouse strain
susceptibility to gonadectomy-induced adrenocortical
tumor formation correlates with the expression of GATA-
4 and luteinizing hormone receptor. Endocrinology 144
4123–4133.
Bodek G, Rahman NA, Zaleska M, Soliymani R, Lankinen H,
Hansel W, Huhtaniemi I & Ziecik AJ 2003 A novel
approach of targeted ablation ofmammary carcinoma cells
through luteinizing hormone receptors using Hecate-CGbconjugate. Breast Cancer Research and Treatment 79
1–10.
Bodek G, Kowalczyk A, Waclawik A, Huhtaniemi I & Ziecik
AJ 2005a Targeted ablation of prostate carcinoma cells
through LH receptor using Hecate-CGb conjugate:
functional characteristic and molecular mechanism of cell
death pathway. Experimental Biology and Medicine 230
421–428.
645
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
Bodek G, Vierre S, Rivero-Muller A, Huhtaniemi I, Ziecik
AJ & Rahman NA 2005b A novel targeted therapy of
Leydig and granulosa cell tumors through the luteinizing
hormone receptor using a hecate-chorionic gonadotropin
beta conjugate in transgenic mice. Neoplasia 7 497–508.
Bornstein SR, Stratakis CA & Chrousos GP 1999 Adreno-
cortical tumors: recent advances in basic concepts and
clinical management. Annals of Internal Medicine 130
759–771.
Bourdeau I, D’Amour P, Hamet P, Boutin JM & Lacroix A
2001 Aberrant membrane hormone receptors in inciden-
tally discovered bilateral macronodular adrenal hyper-
plasia with subclinical Cushing’s syndrome. Journal of
Clinical Endocrinology and Metabolism 86 5534–5540.
Cattanach BM, Iddon CA, Charlton HM, Chiappa SA & Fink
G 1977 Gonadotrophin-releasing hormone deficiency in a
mutant mouse with hypogonadism. Nature 269 338–340.
Chomczynski P & Sacchi N 1987 Single-step method of
RNA isolation by acid guanidinium thiocyanate- phenol-
chloroform extraction. Analytical Biochemistry 162
156–159.
El-Hefnawy T & Huhtaniemi I 1998 Progesterone can
participate in down-regulation of the luteinizing hormone
receptor gene expression and function in cultured murine
Leydig cells. Molecular and Cellular Endocrinology 137
127–138.
El-Hefnawy T, Manna PR, Luconi M, Baldi E, Slotte JP &
Huhtaniemi I 2000 Progesterone action in a murine
Leydig tumor cell line (mLTC-1), possibly through a
nonclassical receptor type. Endocrinology 141 247–255.
Feelders RA, Lamberts SW, Hofland LJ, van Koetsveld PM,
Verhoef-Post M, Themmen AP, de Jong FH, Bonjer HJ,
Clark AJ, van der Lely AJ et al. 2003 Luteinizing
hormone (LH)-responsive Cushing’s syndrome: the
demonstration of LH receptor messenger ribonucleic acid
in hyperplastic adrenal cells, which respond to chorionic
gonadotropin and serotonin agonists in vitro. Journal of
Clinical Endocrinology and Metabolism 88 230–237.
Gawronska B, Leuschner C, Enright FM & Hansel W 2002
Effects of a lytic peptide conjugated to beta HCG on
ovarian cancer: studies in vitro and in vivo. Gynecologic
Oncology 85 45–52.
Givens JR, Andersen RN, Wiser WL, Donelson AJ &
Coleman SA 1975 A testosterone-secreting, gonado-
tropin-responsive pure thecoma and polycystic ovarian
disease. Journal of Clinical Endocrinology and
Metabolism 41 845–853.
Goodarzi MO, Dawson DW, Li X, Lei Z, Shintaku P, Rao CV
& Van Herle AJ 2003 Virilization in bilateral macro-
nodular adrenal hyperplasia controlled by luteinizing
hormone. Journal of Clinical Endocrinology and
Metabolism 88 73–77.
Haavisto AM, Pettersson K, Bergendahl M, Perheentupa A,
Roser JF & Huhtaniemi I 1993 A supersensitive
immunofluorometric assay for rat luteinizing hormone.
Endocrinology 132 1687–1691.
646
Hanahan D 1989 Transgenic mice as probes into complex
systems. Science 246 1265–1275.
Jaffe RB, Seron-Ferre M, Crickard K, Koritnik D, Mitchell
BF & Huhtaniemi IT 1981 Regulation and function of the
primate fetal adrenal gland and gonad. Recent Progress in
Hormone Research 37 41–103.
Kananen K, Markkula M, Rainio E, Su JG, Hsueh AJ &
Huhtaniemi IT 1995 Gonadal tumorigenesis in transgenic
mice bearing the mouse inhibin alpha-subunit promoter/-
simian virus T-antigen fusion gene: characterization of
ovarian tumors and establishment of gonadotropin-
responsive granulosa cell lines.Molecular Endocrinology
9 616–627.
Kananen K, Markkula M, Mikola M, Rainio EM, McNeilly A
& Huhtaniemi I 1996a Gonadectomy permits adrenocor-
tical tumorigenesis in mice transgenic for the mouse
inhibin alpha-subunit promoter/simian virus 40 T-antigen
fusion gene: evidence for negative autoregulation of the
inhibin alpha- subunit gene. Molecular Endocrinology 10
1667–1677.
KananenK,MarkkulaM, El-Hefnawy T, Zhang FP, Paukku T,
Su JG, Hsueh AJ & Huhtaniemi I 1996b Themouse inhibin
alpha-subunit promoter directs SV40 T-antigen to Leydig
cells in transgenic mice.Molecular and Cellular
Endocrinology 119 135–146.
Kananen K, Rilianawati, Paukku T, Markkula M, Rainio
EM & Huhtanemi I 1997 Suppression of gonadotropins
inhibits gonadal tumorigenesis in mice transgenic
for the mouse inhibin alpha-subunit promoter/simian
virus 40 T-antigen fusion gene. Endocrinology 138
3521–3531.
Kero J, Poutanen M, Zhang FP, Rahman N, McNicol AM,
Nilson JH, Keri RA & Huhtaniemi IT 2000 Elevated
luteinizing hormone induces expression of its receptor
and promotes steroidogenesis in the adrenal cortex.
Journal of Clinical Investigation 105 633–641.
Ketola I, Rahman N, Toppari J, Bielinska M, Porter-Tinge
SB, Tapanainen JS, Huhtaniemi IT, Wilson DB &
Heikinheimo M 1999 Expression and regulation of
transcription factors GATA-4 and GATA-6 in developing
mouse testis. Endocrinology 140 1470–1480.
Kiiveri S, Siltanen S, Rahman N, Bielinska M, Lehto VP,
Huhtaniemi IT, Muglia LJ, Wilson DB & Heikinheimo M
1999 Reciprocal changes in the expression of transcrip-
tion factors GATA-4 and GATA-6 accompany adreno-
cortical tumorigenesis in mice and humans. Molecular
Medicine 5 490–501.
Kiiveri S, Liu J, Westerholm-Ormio M, Narita N, Wilson
DB, Voutilainen R & Heikinheimo M 2002 Tran-
scription factors GATA-4 and GATA-6 during mouse
and human adrenocortical development. Endocrine
Research 28 647–650.
Lacroix A, Hamet P & Boutin JM 1999 Leuprolide acetate
therapy in luteinizing hormone–dependent Cushing’s
syndrome. New England Journal of Medicine 341
1577–1581.
www.endocrinology-journals.org
Endocrine-Related Cancer (2008) 15 635–648
de LangeWE, Pratt JJ & Doorenbos H 1980 A gonadotrophin
responsive testosterone producing adrenocortical ade-
noma and high gonadotrophin levels in an elderly woman.
Clinical Endocrinology 12 21–28.
LaPolt PS, Oikawa M, Jia XC, Dargan C & Hsueh AJ 1990
Gonadotropin-induced up- and down-regulation of rat
ovarian LH receptor message levels during follicular
growth, ovulation and luteinization. Endocrinology 126
3277–3279.
Larson BA, Vanderlaan WP, Judd HL & McCullough DL
1976 A testosterone-producing adrenal cortical adenoma
in an elderly woman. Journal of Clinical Endocrinology
and Metabolism 42 882–887.
Leinonen P, Ranta T, Siegberg R, Pelkonen R, Heikkila P &
Kahri A 1991 Testosterone-secreting virilizing adrenal
adenoma with human chorionic gonadotrophin receptors
and 21-hydroxylase deficiency. Clinical Endocrinology
34 31–35.
Leuschner C & Hansel W 2004 Membrane disrupting lytic
peptides for cancer treatments. Current Pharmaceutical
Design 10 2299–2310.
Leuschner C, Enright FM, Melrose PA & Hansel W 2001
Targeted destruction of androgen-sensitive and -insensi-
tive prostate cancer cells and xenografts through
luteinizing hormone receptors. Prostate 46 116–125.
Leuschner C, Enright FM, Gawronska B & Hansel W 2003a
Membrane disrupting lytic peptide conjugates destroy
hormone dependent and independent breast cancer cells
in vitro and in vivo. Breast Cancer Research and
Treatment 78 17–27.
Leuschner C, Enright FM, Gawronska-Kozak B & Hansel W
2003b Human prostate cancer cells and xenografts are
targeted and destroyed through luteinizing hormone
releasing hormone receptors. Prostate 56 239–249.
Manna PR, El-Hefnawy T, Kero J & Huhtaniemi IT 2001
Biphasic action of prolactin in the regulation of murine
Leydig tumor cell functions. Endocrinology 142 308–318.
Mazzuco TL, Chabre O, Feige JJ & Thomas M 2006
Aberrant expression of human luteinizing hormone
receptor by adrenocortical cells is sufficient to provoke
both hyperplasia and Cushing’s syndrome features.
Journal of Clinical Endocrinology and Metabolism 91
196–203.
Mijnhout GS, Danner SA, van de Goot FR & van Dam EW
2004 Macronodular adrenocortical hyperplasia in a
postmenopausal woman. Netherlands Journal of
Medicine 62 454–455.
Mikola M, Kero J, Nilson JH, Keri RA, Poutanen M &
Huhtaniemi I 2003 High levels of luteinizing hormone
analog stimulate gonadal and adrenal tumorigenesis in
mice transgenic for the mouse inhibin-alpha-subunit
promoter/Simian virus 40 T-antigen fusion gene.
Oncogene 22 3269–3278.
Miyamura N, Taguchi T, Murata Y, Taketa K, Iwashita S,
Matsumoto K, Nishikawa T, Toyonaga T, Sakakida M &
Araki E 2002 Inherited adrenocorticotropin-independent
macronodular adrenal hyperplasia with abnormal cortisol
www.endocrinology-journals.org
secretion by vasopressin and catecholamines: detection of
the aberrant hormone receptors on adrenal gland.
Endocrine 19 319–326.
Moran DS, Eli-Berchoer L, Heled Y, Mendel L, Schocina M
& Horowitz M 2006 Heat intolerance: does gene
transcription contribute? Journal of Applied Physiology
100 1370–1376.
Morrisey EE, Ip HS, Tang Z & Parmacek MS 1997 GATA-4
activates transcription via two novel domains that are
conserved within the GATA-4/5/6 subfamily. Journal of
Biological Chemistry 272 8515–8524.
Pabon JE, Li X, Lei ZM, Sanfilippo JS, Yussman MA & Rao
CV 1996 Novel presence of luteinizing hormone/chor-
ionic gonadotropin receptors in human adrenal glands.
Journal of Clinical Endocrinology and Metabolism 81
2397–2400.
Pakarainen T, Zhang FP, Poutanen M & Huhtaniemi I 2005
Fertility in luteinizing hormone receptor-knockout mice
after wild-type ovary transplantation demonstrates
redundancy of extragonadal luteinizing hormone action.
Journal of Clinical Investigation 115 1862–1868.
Pakarainen T, Ahtiainen P, Zhang FP, Rulli S, Poutanen M &
Huhtaniemi I 2007 Extragonadal LH/hCG action-not yet
time to rewrite textbooks. Molecular and Cellular
Endocrinology 269 9–16.
Peterson RA II, Kiupel M, Bielinska M, Kiiveri S,
Heikinheimo M, Capen CC & Wilson DB 2004
Transcription factor GATA-4 is a marker of anaplasia in
adrenocortical neoplasms of the domestic ferret (Mustela
putorius furo). Veterinary Pathology 41 446–449.
Rahman NA, Kananen Rilianawati K, Paukku T, Mikola M,
Markkula M, Hamalainen T & Huhtaniemi IT 1998
Transgenic mouse models for gonadal tumorigenesis.
Molecular and Cellular Endocrinology 145 167–174.
Rahman NA, Kiiveri S, Siltanen S, Levallet J, Kero J, Lensu
T, Wilson DB, Heikinheimo MT & Huhtaniemi IT 2001
Adrenocortical tumorigenesis in transgenic mice: the role
of luteinizing hormone receptor and transcription factors
GATA-4 and GATA-61. Reproductive Biology 1 5–9.
Rahman NA, Kiiveri S, Rivero-Muller A, Levallet J, Vierre S,
Kero J, Wilson DB, Heikinheimo M&Huhtaniemi I 2004
Adrenocortical tumorigenesis in transgenic mice expres-
sing the inhibin alpha-subunit promoter/simian virus
40 T-antigen transgene: relationship between ectopic
expression of luteinizing hormone receptor and transcri-
ption factor GATA-4. Molecular Endocrinology 18
2553–2569.
Rao ChV, Zhou XL & Lei ZM 2004 Functional luteinizing
hormone/chorionic gonadotropin receptors in human
adrenal cortical H295R cells. Biology of Reproduction 71
579–587.
Reincke M, Karl M, Travis WH, Mastorakos G, Allolio B,
Linehan HM & Chrousos GP 1994 p53 Mutations in
human adrenocortical neoplasms: immunohistochemical
and molecular studies. Journal of Clinical Endocrinology
and Metabolism 78 790–794.
647
S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor
Rilianawati, Kero J, Paukku T & Huhtaniemi I 2000 Long-
term testosterone treatment prevents gonadal and adrenal
tumorigenesis of mice transgenic for the mouse inhibin-
alpha subunit promoter/simian virus 40 T-antigen fusion
gene. Journal of Endocrinology 166 77–85.
Rilianawati, Paukku T, Kero J, Zhang FP, Rahman N,
Kananen K & Huhtaniemi I 1998 Direct luteinizing
hormone action triggers adrenocortical tumorigenesis in
castrated mice transgenic for the murine inhibin alpha-
subunit promoter/simian virus 40 T-antigen fusion gene.
Molecular Endocrinology 12 801–809.
Risma KA, Clay CM, Nett TM, Wagner T, Yun J & Nilson
JH 1995 Targeted overexpression of luteinizing hormone
in transgenic mice leads to infertility, polycystic ovaries,
and ovarian tumors. PNAS 92 1322–1326.
Rivero-Muller A, Vuorenoja S, Tuominen M, Waclawik A,
Brokken LJ, Ziecik AJ, Huhtaniemi I & Rahman NA 2007
Use of hecate-chorionic gonadotropin beta conjugate in
therapy of lutenizing hormone receptor expressing
gonadal somatic cell tumors. Molecular and Cellular
Endocrinology 269 17–25.
Saner-Amigh K, Mayhew BA, Mantero F, Schiavi F, White
PC, Rao CV & Rainey WE 2006 Elevated expression of
luteinizing hormone receptor in aldosterone-producing
adenomas. Journal of Clinical Endocrinology and
Metabolism 91 1136–1142.
Schulick RD & Brennan MF 1999a Long-term survival after
complete resection and repeat resection in patients with
adrenocortical carcinoma. Annual Surgery and Oncology
6 719–726.
Schulick RD & Brennan MF 1999b Adrenocortical carci-
noma. World Journal of Urology 17 26–34.
Sheeler LR 1994 Cushing’s syndrome and pregnancy.
Endocrinology and Metabolism Clinics of North America
23 619–627.
Smith HC, Posen S, Clifton-Bligh P & Casey J 1978
A testosterone-secreting adrenal cortical adenoma.
Australia andNewZealand Journal ofMedicine 8 171–175.
Strassburg CP, Strassburg A, Kneip S, Barut A, Tukey RH,
Rodeck B & Manns MP 2002 Developmental aspects of
human hepatic drug glucuronidation in young children
and adults. Gut 50 259–265.
648
Takahashi H, Yoshizaki K, Kato H, Masuda T, Matsuka G,
Mimura T, Inui Y, Takeuchi S, Adachi H &Matsumoto K
1978 A gonadotrophin-responsive virilizing adrenal
tumour identified as a mixed ganglioneuroma and adreno-
cortical adenoma. Acta Endocrinologica 89 701–709.
Tremblay JJ & Viger RS 1999 Transcription factor GATA-4
enhances Mullerian inhibiting substance gene transcrip-
tion through a direct interaction with the nuclear receptor
SF-1. Molecular Endocrinology 13 1388–1401.
Utsugi T, Schroit AJ, Connor J, Bucana CD & Fidler IJ 1991
Elevated expression of phosphatidylserine in the outer
membrane leaflet of human tumor cells and recognition
by activated human blood monocytes. Cancer Research
51 3062–3066.
Vuorenoja S, Rivero-Muller A, Kiiveri S, Bielinska M,
HeikinheimoM,WilsonDB,Huhtaniemi IT&RahmanNA
2007 Adrenocortical tumorigenesis, luteinizing hormone
receptor and transcription factors GATA-4 and GATA-6.
Molecular and Cellular Endocrinology 269 38–45.
Werk EE Jr, Sholiton LE & Kalejs L 1973 Testosterone-
secreting adrenal adenoma under gonadotropin control.
New England Journal of Medicine 289 767–770.
Westphal G, Burgemeister R, Friedemann G, Wellmann A,
Wernert N, Wollscheid V, Becker B, Vogt T, Knuchel R,
Stolz W et al. 2002 Noncontact laser catapulting: a basic
procedure for functional genomics and proteomics.
Methods in Enzymology 356 80–99.
Wy LA, Carlson HE, Kane P, Li X, Lei ZM & Rao CV 2002
Pregnancy-associated Cushing’s syndrome secondary to a
luteinizing hormone/human chorionic gonadotropin
receptor-positive adrenal carcinoma. Gynecological
Endocrinology 16 413–417.
Zaleska M, Waclawik A, Bodek G, Zezula-Szpyra A, Li X,
Janowski T, Hansel WH, Rahman NA & Ziecik AJ 2004
Growth repression in diethylstilbestrol/dimethylbenz[a]-
anthracene-induced rat mammary gland tumor using
Hecate-CGbeta conjugate. Experimental Biology and
Medicine 229 335–344.
Zhang FP, Hamalainen T, Kaipia A, Pakarinen P &
Huhtaniemi I 1994 Ontogeny of luteinizing hormone
receptor gene expression in the rat testis. Endocrinology
134 2206–2213.
www.endocrinology-journals.org