| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Laboratoire d’Explorations Fonctionnelles Endocriniennes, INSERM U-515 (A.L., Y.L.B., C.G.), and Laboratoire d’Anatomie et Cytologie Pathologique (L.B.G.), Hôpital d’Enfants Armand Trousseau, 75012 Paris, France; Laboratoire de Biologie Hormonale, Hôpital Saint Louis (P.B.), 75010 Paris, France; and Service de Médecine Nucléaire (E.B., M.S.) and Laboratoire de Pharmacotoxicologie (G.V.), Institut Gustave Roussy, 96800 Villejuif, France
Address all correspondence and requests for reprints to: Dr. Christine Gicquel, Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau, 26 avenue Arnold Netter, 75012 Paris, France. E-mail: christine.gicquel{at}trs.ap-hop-paris.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4-androstenedione, 11-deoxycortisol, corticosterone, and
testosterone). Xenografts produced all three class of steroids, with
the preferential production of androgens of the 4
pathway. The H295R xenograft model is a good model of human adrenocortical carcinoma, as it mimics dysregulation of the IGF system usually found in these tumors. It also produces IGFBP-2 and steroids that can be used as tumor markers. This model may therefore be useful for evaluating therapeutic agents.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We recently showed that the insulin-like growth factor (IGF) system plays a major role in the malignant transformation of adrenocortical tumors. Indeed, most malignant tumors (90%) exhibit strong overexpression of the IGF-II gene and a maternal loss of heterozygosity of the imprinted 11p15 region (where the IGF-II gene maps) (4). Moreover, 11p15 loss of heterozygosity and the overexpression of the IGF-II gene in localized adrenocortical tumors are predictors of tumor relapse (our manuscript in preparation). The expression of IGF-binding proteins (IGFBPs) is also impaired, and malignant tumors contain large amounts of IGFBP-2, suggesting that IGFBP-2 is involved in IGF-II proliferative effects (5).
A human tumor cell line, H295, was recently established (6, 7). It was derived from an invasive, secreting primary adrenocortical carcinoma (ACC) (6). H295R cells retain the ability to produce all of the major steroids (mainly androgens). As in malignant adrenocortical tumors, cell proliferation is associated with an overexpression of both the IGF-II and IGFBP-2 genes (8). We have also shown that the proliferative effects of IGF-II on H295R cells are mediated by the type 1 IGF receptor (8).
Human tumor xenografts are well established tools for the preclinical screening of anticancer drugs, which is particularly interesting for rare tumors. The aim of this study was to establish and to characterize the first sc xenograft model of adrenal carcinoma for use in therapeutic trials. We found that the xenografts exhibited features typical of human ACC, dysregulation of the IGF system in particular. The xenografts also produced and secreted steroids and human IGFBP-2, and their plasma concentrations may be useful markers for the evaluation of anticancer drugs in mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animals and biological samples
Female nude mice (nu/nu) were bred at the INSERM U515
(Paris, France) animal house. They were fed on a standard diet and were
subjected to a regulated light cycle (12 h of light, 12-h of darkness).
Cell suspensions (100 µl) were bilaterally injected sc into the
flanks of 6-week-old mice (n = 20). For each passage, the tumor
was ground into small pieces in PBS, and the resulting suspension was
injected into the flanks of nude mice. Two perpendicular diameters of
the tumor were measured twice a week by the same investigator. The
volume of each tumor was calculated according to the following
equation: V (mm3) =
(d2 x D)/2, where d (mm) and D (mm) are the
smallest and largest perpendicular tumor diameters, respectively (9).
The take rate was defined as the number of tumors per number of
inoculation sites. Animals were decapitated, and blood was collected
into 4 mM EDTA. Plasma was isolated by
centrifugation, aliquoted, and stored at -20 C. Tumors were
immediately frozen in liquid nitrogen and stored at -80 C for RNA and
protein extraction or immersed in 10% formol for pathological
analysis. Experiments were carried out in accordance with the
guidelines of the Ministry of Agriculture (authorization 7574).
Pathological methods
Each tumor sample was weighed and fixed in formalin, and serial
sections were cut and stained with hematoxylin-eosin-safran.
IGF assays
The method used has been described in detail previously (10).
Plasma samples (25 µl) were incubated with 2 ml 0.01 N
HCl for 30 min at room temperature to dissociate IGFs from IGFBPs and
centrifuged on Centricon 30 (Amicon, Epernon, France) to separate IGFs
from IGFBPs. The ultrafiltrate containing IGFs was lyophilized,
dissolved in 0.1 M phosphate buffer (pH 7.4) and 1 mg/ml
BSA (BioMérieux, Paris, France), and incubated for 2–3 days in a
final volume of 400 µl with either 1) a specific polyclonal antihuman
IGF-I antibody that cross-reacts with murine IGF-I (gift from Dr. J.
Closset, Liege, Belgium) and [125I]hIGF-I (3000
cpm/tube) or 2) IGFBPs extracted from cerebrospinal fluid that have a
selective affinity for IGF-II (11) and
[125I]human IGF-II (3000 cpm/tube).
Steroid assays
Steroid secretion was assessed by determining the plasma
concentration of the steroid concerned. 17-Hydroxypregnenolone
(17OH-PREG), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone
(DHEA), 4-androstenedione (A),
11-deoxycortisol (11-DOF), corticosterone (B), and testosterone (T)
levels were determined by specific RIA methods after chromatographic
purification of plasma samples as previously described (12). The lower
limit of detection was 0.76 nmol/liter for 17OH-PREG, 0.84 nmol/liter
for 17OH-P, 1.08 nmol/liter for DHEA, 0.42 nmol/liter for
A, 0.51 nmol/liter for 11-DOF, 3.1 nmol/liter for B, and 0.19
nmol/liter for T (12). Cortisol (F) was determined directly using the
Gamma Coat [125I]F kit (DiaSoring, Stillwater,
MN) or was evaluated, with the same kit, after a chromatography step
(13). The cross-reactivity of the F assay with the main relevant
steroids was 6.3% for 11-DOF, 0.4% for B, and 1.2% for 17OH-P. The
lower limit of detection for F was 5.9 nmol/liter for the direct assay
(manufacturer’s data) and 7.0 nmol/liter after the purification
procedure (13).
Protein extraction
Frozen tissues (100 mg) were quickly homogenized on ice in 3 ml
ice-cold lysis buffer [50 mM HEPES (pH 7), 250
mM NaCl, 5 mM EDTA, 1 mM sodium
orthovanadate, 2 mM sodium pyrophosphate, and 0.1% Nonidet
P-40] containing protease inhibitors (1 mM dithiothreitol,
1 µg/ml aprotinin, 1 µg/ml leupeptin, and 50 µg/ml
phenylmethylsulfonylfluoride) using a Polytron homogenizer
(Brinkmann Instruments, Inc., Westbury, NY).
Homogenates were incubated for 1 h at 4 C and centrifuged at 15,000 x g for 30 min at 4 C. The supernatant was collected and frozen at -20 C. Aliquots of supernatant were collected for protein determination by the Bradford method (Bio-Rad protein assay, Bio-Rad Laboratories, Inc., Richmond, CA).
Western ligand and immunoblotting
Western ligand blotting and immunoblotting were performed as
previously described (14, 15). Briefly, plasma samples (3 µl) or
tumor proteins (200 µg) were denatured at 100 C for 2 min and
subjected to SDS-11% PAGE under nonreducing conditions. The proteins
were then electrotransferred onto nitrocellulose (BA 85,
Schleicher & Schuell, Inc., Dassel, Germany). The various
IGFBP species were detected 1) by incubation with a mixture of
[125I]IGF-I and
[125I]IGF-II (5 x
105 cpm each) at 4 C for 48 h, followed by
autoradiography (ligand blotting), and 2) by incubation with an
anti-bovine-IGFBP-2 polyclonal antibody that recognizes both human and
mouse IGFBP-2 (Upstate Biotechnology, Inc., Lake Placid,
NY). Blots were then incubated with an anti-IgG antibody coupled to
horseradish peroxidase (Biosource International,
Camarillo, CA), and complexes were detected by enhanced
chemiluminescence (Amersham Pharmacia Biotech, Uppsala,
Sweden).
Isolation and analysis of RNA
Total RNA was extracted from frozen tumors using the standard
CsCl/guanidine isothiocyanate method as previously described (16).
Total RNA (10 µg) was loaded onto a 1.0% agarose-formaldehyde gel,
subjected to electrophoresis, and transferred to a Hybond XL membrane
(Amersham Pharmacia Biotech). The RNA was covalently bound
to the membrane by baking at 80 C for 2 h. Northern blots were
probed with the human IGF-II complementary DNA (cDNA) (17).
Type 1 and type 2 IGF receptor expression was analyzed by RT-PCR. RNA samples (1 µg) were treated with deoxyribonuclease and reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), and the resulting cDNA was amplified by PCR. The sense and antisense primers for the type 1 IGF receptor were 5'-AAC CAC GAG GCT GAG AAG CT and 5'-CAG CAT AAT CAC CAA CCC TC, respectively (18). Oligonucleotide primers for the type 2 IGF receptor were designed using sequences from the 3'-untranslated region of the type 2 IGF receptor gene (19). The sense and antisense primers were 5'-TTG CCG GCT GGT GAA TTC AA and 5'-GTA TCA TGA GAA CCT GAA GAG, respectively. Deoxyribonuclease-treated RNA was subjected to PCR as a control for DNA contamination of the RNA samples. The amplification products were analyzed by electrophoresis in a 1.5% agarose gel.
Statistical and mathematical analysis
Values are medians (minimum-maximum) unless otherwise stated.
The Mann-Whitney test was used to evaluate differences between groups.
P < 0.05 was considered significant. An exponential
function was fitted to the tumor growth curve, and doubling time was
calculated for r2 > 0.95.
Plasma sample volumes were small, so for statistical analysis, the first calibration point (loss-corrected) was retained for steroids with concentrations lower than the detection threshold (A, DHEA, 17OH-P, and 11-DOF).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Differences were also seen between
the tumors growing on the left and right sides of individual mice.
However, analysis of tumor growth curves for the second and third
passages showed that growth rate was stable through the serial
passages. Most xenografts had a doubling time of 12 days (range, 6–26;
Fig. 1B).
|
). Indeed, the xenografts contained
rather small, uniform cells with prominent nucleoli, a high mitotic
index, and foci of necrosis. No vascular invasion was observed. A thin
capsule surrounded the tumor, interrupted in a few places by small
bulging extracapsular nodules.
|
.
|
Unlike control mice, xenografted mice had high steroid levels.
17OH-PREG (the main precursor of androgens in H295R cells) was
metabolized to give both 17OH-P and DHEA. High
concentrations of A were derived from 17OH-P and DHEA, but
also from 11-DOF. Like NCI H295R cells, the xenografts produced large
amounts of 11-DOF, suggesting an alteration in the 11ß-hydroxylase
activity. There was a significant difference in F levels
(P = 0.006) between the control and xenografted mouse
groups (Fig. 3
). Indeed, more than half of the xenografted mice had F
levels above those in the control group (Fig. 3). Although there is
cross-reaction with other steroids, in the direct
[125I]F assay, this difference between controls
and xenografted animals is consistent with F production by xenografts.
Due to the plasma volumes required for F assay after chromatography
extraction, F assays could not be carried out for this series of
xenografted mice. Instead, we used another series of 10 xenografted
mice. We found that mice with F levels above 70 nmol/liter in the
direct assay had detectable cortisol levels after the purification
procedure (data not shown). Moreover, xenografts producing large
amounts of one steroid also produced large amounts of the other
steroids.
Expression of the components of the IGF system in mice
IGF-II expression was expected in xenografts because they were
derived from the NCI H295R ACC cell line. Northern blot analysis of RNA
extracts from xenografts showed that the species of IGF-II messenger
RNA (mRNA) produced (2.2, 4.8, and 6.0 kb) were the same as those in
NCI H295R cells, with the preferential expression of the 2.2 kb
P3-driven species (Fig. 4).
|
In Western ligand blot analysis of the proteins extracted from
xenografts and from xenograft-bearing mouse plasma, we detected a band
that migrated to the same position as the 34-kDa band of a control
human serum but was absent from control mouse plasma (Fig. 5A). This 34-kDa band was identified as
human IGFBP-2 by immunoblotting. On the immunoblot probed with the
anti-IGFBP-2 antibody, a 32-kDa band was also detected in plasma from
both xenografted and control mice. This band corresponds to mouse
IGFBP-2 (the IGFBP-2 antibody recognizes both mouse and human
IGFBP-2).
|
). By contrast, in
xenografts, an additional band migrating at 25 kDa was detected on
IGFBP-2 immunoblots. This band has also been detected in human ACC (5)
and probably corresponds to a proteolytic fragment (Fig. 5B).
Other IGFBPs were detected by Western ligand blotting in tumors, and in
control and xenograft-bearing mouse plasma, a 39- to 42-kDa protein
corresponding to IGFBP-3, a 32-kDa protein corresponding to IGFBP-1 and
IGFBP-2, and a 24-kDa protein corresponding to IGFBP-4 (Fig. 5A).
Finally, we showed that both IGF receptors (types 1 and 2) were
expressed similarly in xenografts and H295R cells (Fig. 6).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Until recently, no experimental model was available. Only a few human ACC cell lines have been established (6, 21), and no ACC xenograft model has previously been described. We recently characterized the IGF system of the human adrenal tumor H295R cell line and demonstrated the involvement of IGF-II in cell proliferation (8). This cell line is, therefore, a useful in vitro model for adrenal carcinoma. However, cell lines alone are insufficient for therapeutic evaluation, and the main limitation to the development of new treatment strategies for ACC has been the lack of an appropriate animal model.
In this study we used the H295R cell line, which has abnormalities of the IGF system similar to those of human ACC and characteristics typical of tumorigenic cells (immortality, loss of contact inhibition, growth factor-independent proliferation, and anchorage-independent growth) (6, 7, 8) to establish and characterize an in vivo murine xenograft model.
We successfully transplanted H295R cells into nude mice with a take rate of almost 100%. The latency period for tumor growth was 6 weeks at the first passage and remained constant for sequential passages. Tumorigenicity has to date been maintained for approximately 24 months (five passages). The xenografted tumors reproduced the features of human ACC and exhibited histological features similar to those of the original tumor (6). No metastases were observed in xenografted mice.
NCI H295R xenografts appear to be a useful ACC model for two major reasons: xenografts reproduce the dysregulation of the IGF system found in malignant human tumors and in the H295R cell line. Indeed, most malignant adrenocortical tumors strongly overexpress the IGF-II gene, and efficient translation results in the production of large amounts of IGF-II protein, mostly in precursor forms (5). We showed that IGF-II is involved in adrenocortical tumor cell proliferation by demonstrating that cell proliferation is reduced by blocking antibodies directed against the IGF-II peptide or the type 1 IGF receptor (8). IGF-II effects are restricted to tumors via an auto/paracrine pathway, and systemic plasma levels of IGF-II from patients with ACC are in the normal range (22). In this study plasma levels of IGF-II from xenograft-bearing mice are also in the same range as those in control mice.
The high level of IGF-II production by malignant tumors should also be seen in the light of chemoresistance. A recent study suggested that the antiapoptotic function of IGF-II may result in a decrease in sensitivity to various chemotherapeutic agents (23). It also demonstrated that sensitivity to chemotherapeutic agents is highly enhanced if IGF-II is blocked. Various antimitotic agents (mostly cisplatin based) have been tested, but partial and transient tumor responses have been observed in only a small percentage of patients (24, 25, 26, 27, 28, 29). Clearly, trials with other drug combinations are required, but the rarity of adrenal carcinoma considerably limits the evaluation of chemotherapy. Thus, H295R xenografts provide a much-needed model for testing adjuvant therapies, particularly therapies directed against growth factors.
Another key advantage of this xenograft model is the production by xenografts and secretion into mouse plasma of IGFBP-2 and steroids that could be used as tumor markers. The xenografts produced human IGFBP-2. IGFBPs locally modulate the actions of IGF (30). We previously showed the specific overexpression of IGFBP-2 in human ACC (5) and the exclusive expression of IGFBP-2 in the H295R cell line (8). Several studies have suggested that IGFBP-2 expression may be associated with malignancy. Both stimulatory and inhibitory effects of IGFBP-2 on IGF activity have been reported. Among stimulatory effects, IGFBP-2 proteolysis, by decreasing IGF-II affinity, may increase IGF-II bioavailability and enhance its proliferative and/or its antiapoptotic effects. Although IGFBP-2 proteolysis did not occur in H295R cells (8), it exists in vivo, both in human ACC (5) and in xenografts, suggesting that IGFBP-2 proteolysis requires other cell components. A recent report (31) suggests that IGFBP-2 enhances cell proliferation in adrenal tumors by an IGF-independent mechanism. Human IGFBP-2 was secreted into mouse plasma. We have previously shown that IGFBP-2 is secreted in human ACC and that IGFBP-2 levels are correlated with tumor spread (Boulle, N., E. Baudin, C. Gicquel, A. Logié, J. Bertherat, A. Penformis, X. Bertagna, J. Luton, M. Schlumberger, and Y. Le Bouc, manuscript submitted).
In conclusion, this mouse model, involving the xenografting of H295R cells, should make it possible to study the involvement of the IGF system in chemoresistance and to evaluate new therapeutic agents. Angiostatic therapy seems to be of potential interest, as recent data have suggested that the angiogenic switch is activated during the early premalignant stages of adrenocortical tumorigenesis (32).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received March 29, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. HOEFLICH, M. M. WEBER, T. FISCH, S. NEDBAL, C. FOTTNER, M. W. ELMLINGER, R. WANKE, and E. WOLF Insulin-like growth factor binding protein 2 (IGFBP-2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF-I excess on adrenocortical cells in vivo FASEB J, November 1, 2002; 16(13): 1721 - 1731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hoeflich, R. Reisinger, H. Lahm, W. Kiess, W. F. Blum, H. J. Kolb, M. M. Weber, and E. Wolf Insulin-like Growth Factor-binding Protein 2 in Tumorigenesis: Protector or Promoter? Cancer Res., December 1, 2001; 61(24): 8601 - 8610. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
