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Max Planck Institute of Psychiatry, 80804 Munich, Germany [U. P., T. A., M. T., Y. G., G. K. S., D. S.]; UPR 9023 CNRS, Mécanismes Moléculaires des Communications Cellulaires, CCIPE, 34094 Montpellier Cedex 05, France [C. P., L. J.]; Institute of Pathology Marienkrankenhaus, 22087 Hamburg, Germany [W. S.]; and Neurosurgical Department, Hospital San Raffaele, 20132 Milan, Italy [M. L.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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protein, which are detected in a subset of ACROs
(2)
, and the loss of the tumor suppressor gene
p16INK4a in some NFPAs (3)
.
Recently, the new mouse gene Zac1 (4)
and its
human homologue ZAC (5)
were isolated by our
group from pituitary cDNA libraries. Zac1/ZAC reveals transactivation
and DNA binding activity compatible with a role as a transcription
factor (4
, 5)
. Zac1/ZAC expression in tumor cell lines
reduces proliferation rates, colony formation in soft agar, and tumor
formation in nude mice by regulating apoptosis and
G1 arrest (4
, 5)
. Abdollahi and
colleagues have identified a rat gene homologous to ZAC by
virtue of its loss of expression in rat ovary epithelial cells
undergoing spontaneous malignant transformation in vitro and
named it Lot1 (lost on
transformation; Ref. 6
). Expression of the
human homologue LOT1, which is identical to ZAC,
is decreased in human ovarian tumor cell lines (7)
.
Interestingly, expression of the Lot1 gene is down-regulated
by epidermal growth factor via the mitogen-activated protein kinase
pathway in rat ovarian epithelial cell culture, indicating a cross-talk
between mitogenic and antiproliferative signals (8)
. We
have recently shown that ablation of Zac1 expression by
antisense targeting in murine tumoral pituitary cell lines enhanced DNA
synthesis demonstrating a role for Zac1 in pituitary cell
proliferation (9)
. ZAC/LOT maps to chromosome
6q24-q25, a region known to harbor putative tumor suppressor genes
frequently involved in solid cancer tumors (10
, 11)
, and
it is expressed at high levels in rodent and human pituitaries
(4
, 5
, 9)
. Therefore, we analyzed ZAC gene
status and ZAC mRNA and protein expression in pituitary adenomas and
normal human pituitary to investigate its impact on neoplastic
pituitary transformation. |
Materials and Methods |
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Tumor allelotyping and DNA sequencing were performed as described previously (14) .
RT-PCR.
One µg of total RNA was reverse transcribed as described previously
(15)
. Two-µl reverse transcription products were
amplified with PCR SuperMix (Life Technologies, Inc., Karlsruhe,
Germany) in the presence of [-33P]dATP
(NEN, Cologne, Germany) using primer pairs located at the ZAC
(AJ006354) zinc finger region (primer 1, nt 214–240; primer 2,
nt 493–517) or at the COOH terminus (primer 3, nt 781–800; primer 4,
nt 1189–1208; Fig. 1A
). Primers for ß-actin
were as described previously (15)
. Conditions for ZAC and
ß-actin amplifications were 25 and 20 cycles, respectively, for 1 min
each at 94°C, 60°C, and 72°C. PCR was performed under exponential
conditions. To assess the kinetics of the PCR amplification reaction,
5-µl aliquots of the PCR products were collected every third cycle
starting from the sixteenth cycle onward. PCR products were quantified
by measuring the absorbance by digital analysis (Tina 4.0;
Raytest, Munich, Germany). Logs of absorbance versus the
number of cycles were plotted, and the cycle number corresponding to
the half-maximal concentration of PCR products was calculated. Negative
controls for RT-PCR were done without reverse transcriptase or
template. PCR products were electrophoresed in a 6% polyacrylamide
gel. Dried gels were exposed to X-ray film (Kodak, New Haven, CT) or to
a PhosphorImager. Absorbance for ZAC and ß-actin was quantified by
digital analysis. Relative ZAC expression levels were determined as the
AZAC:Aß-actin
ratio. PCR reactions were performed three times for each sample.
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). Oligodeoxynucleotides
were 3' end-labeled with [-33P]dATP
(NEN, Cologne, Germany) by terminal transferase (Roche,
Mannheim, Germany). Sections were exposed to PhosphorImager, dipped in
Ilford K5 photoemulsion, and developed after 28 days or exposed to
ßmax hyperfilms (Amersham-Pharmacia) for 14 days. As a negative
control, a 100-fold excess of nonlabeled oligodeoxynucleotide was added
to the radioactive probe.
Generation of ZAC Antibody and Western Blotting.
A cDNA fragment encoding part of the ZAC COOH-terminal region (Fig. 1A)
(residues 226–443) was subcloned into pGEX-5X-3
(Amersham-Pharmacia). The fusion protein was purified by SDS-PAGE and
electroelution. Rabbits were immunized twice with 40 µg of the fusion
protein at 2-week intervals, and antisera were collected weekly. The
specificity of the antibody (1:10,000) was tested by Western blotting
of mock- and ZAC-transfected SaOs-2 osteosarcoma cells as described
previously (4
, 9)
. The antibody was preabsorbed by
incubating 1 µl of unpurified serum with glutathione-Sepharose beads
loaded with an equivalent amount of GST or GST-ZAC
ZF in 1 ml of TBS
Tween/5% nonfat dried milk for 8 h at 4°C. After sedimentation
of the beads, the supernatant was diluted 10-fold in TBS Tween/5%
nonfat dried milk to reach the final antiserum dilution as described
above.
Immunohistochemistry and Quantification of ZAC Immunoreactivity.
Primary antibodies and dilutions were as follows: polyclonal rabbit
antiserum anti-ZAC (1:800), monoclonal mouse antibody antihuman ß-FSH
(1:500), ß-LH (1:500), ß-thyreotrophin-stimulating hormone (1:800),
prolactin (1:400), -subunit (1:500; all from Immunotech, Hamburg,
Germany), adrenocorticotrophin hormone (1:100; Dako, Hamburg, Germany),
and two different antibodies against human growth hormone [1:100
(Sigma, Deisenhofen, Germany) and 1:800 (a gift from Dr. C. J.
Strasburger; University of Munich, Munich, Germany). Cell proliferation
was determined with Mib-1 monoclonal mouse antibody (1:200; Dako). Mono
and double immunohistochemistry was performed as reported previously
(9)
. For negative controls, the primary antibody was
omitted, or, in the case of ZAC staining, sections were incubated with
preimmune serum. ZAC immunoreactivity was quantified by two independent
investigators unaware of the tumor diagnosis. Staining was scored as
absent (0), weak (1)
, moderate (2)
, and strong (3)
, and the percentage
of cells in each category was determined. Intensity of immunoreactivity
was calculated: 0 x the percentage of unstained
cells + 1 x the percentage of weakly stained
cells + 2 x the percentage of moderately
stained cells + 3 x the percentage of
strongly stained cells. Each value was divided by 300 (a hypothetical
maximum for 100% of the cells being strongly stained), providing final
values between 0 (no immunoreactivity) and 1 (maximum
immunoreactivity).
EGFR.
ZAC and EGFR mRNA levels were compared in an additional 16 pituitary
adenomas (5 ACROs, 2 PROLs, 1 TSH, 3 GONAs, and 5 NULLs). ZAC was
amplified using 30 cycles; products were separated in 1.2% agarose gel
and visualized by ethidium bromide staining. EGFR wild type and variant
vIII (EGFRvIII) were examined by nested PCR using NP1/2 (NP1, nt
167–180; NP2, nt 1339–1362) as the outer primer pair and NP6/7 (NP6,
nt 245–265; NP7, nt 1227–1247) or JS3/4 (JS3, nt 519–540; JS4, nt
887–910) as the inner primer pair (16)
. NP6/7 amplifies a
1002-bp fragment in case of EGFR wild type or a 201-bp fragment
remaining from the deletion of the coding region of exon 2 through exon
7, characteristic of EGFRvIII. JS3/4 revealed a 391-bp band in all
samples expressing EGFR or EGFRvIII. Human anterior pituitary,
anaplastic meningioma, a plasmid containing an EGFRvIII fragment (201
bp) recognized by NP6/7, and a plasmid containing the 391 bp of the
EGFR recognized by JS3/4 served as positive controls.
Immunohistochemistry for EGFR wild type was performed in all 65
pituitary adenomas examined for ZAC and in the 16 additional tumors.
Specificity of mouse monoclonal antibody against the intracellular
domain of the human EGFR (amino acids 985–996; Sigma) was confirmed
after preabsorption with the immunogen (Bachem, Heidelberg, Germany).
Five glioblastomas and five meningiomas were used as positive controls
for EGFR.
Statistics.
Only the tumor groups with sample sizes of 
7 were considered (ACROs,
CUSHs, PROLs, GONAs, and NULLs). The means of the final values for the
ZAC immunoreactivity within these groups were compared by a one-way
ANOVA, followed by pairwise comparisons using Scheffé’s
post hoc test in the case of significant group effects. To
approach normality and homogeneity in the data, the final values for
the ZAC immunoreactivity were first transformed with the "arcsin"
transformation and then used in the ANOVA. Associations between final
values for ZAC immunoreactivity and proliferation index or grade of
invasiveness for each tumor were investigated by using the Spearman
correlation coefficient. < 0.05 was accepted as a
nominal level of significance. It was reduced (adjusted according to
Bonferroni procedure) for all post hoc tests to keep the
type I error 
0.05.
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Results |
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and were sequenced
as reported previously (14)
. No mutation was found,
suggesting that the coding region of ZAC is not frequently mutated.
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and localized in endocrine cells (Fig. 2c)
.
Only faint signals were found in the posterior pituitary (Fig. 2, a and b)
. These findings were confirmed by
immunohistochemistry in parallel sections. The specificity of the
anti-ZAC antibody had been tested by immunoblotting (Fig. 1B)
. In normal anterior pituitaries, ZAC immunoreactivity
was very intense in the nuclei but weak in the cytoplasm (Fig. 2, e and f)
. These findings were in agreement with
the nuclear localization of recombinant ZAC as described previously
(5)
. Double immunohistochemistry revealed that all types
of hormone-producing cells were positive for ZAC, indicating that its
expression is not restricted to a single cell type (examples of
colocalization are shown in Fig. 2, g and h
).
RT-PCR analysis and quantification of 25 pituitary adenomas
versus normal human anterior pituitary (Fig. 3B)
showed a highly variable
expression of the ZAC gene, which was highest in anterior
normal pituitary; slightly reduced in ACROs, PROLs, TSHs, and CUSHs;
and significantly reduced or absent in NFPAs. In the latter group, loss
of ZAC mRNA expression was more frequent in NULLs than in GONAs. The
same results were obtained for different primer pairs. In addition, no
signals or only weak signals were observed in the vast majority of
NFPAs by in situ hybridization in a larger number of
pituitary adenomas (65 cases). The eight different
oligodeoxynucleotides revealed no variation in the hybridization signal
in each tumor tested. Again, NULL was the subclass with the most
frequent loss of ZAC mRNA (Fig. 3D)
. As shown in Fig. 3D
and quantified in Fig. 3C
, different subtypes
of pituitary tumors showed significant differences in ZAC
immunoreactivity (ANOVA, F = 4.58; sig. of F < 0.0001). In agreement with our findings at the mRNA level, ZAC protein
staining was faint or absent in NFPAs and significantly less intense in
both GONAs and NULLs compared with the other subclasses of tumors
(Scheffé’s post hoc tests, P < 0.05). ZAC immunoreactivity was heterogeneously distributed in
a number of NFPAs. Using double immunohistochemistry, we investigated
whether areas immunopositive for ZAC were restricted to areas
immunopositive for hormones. ZAC staining was independent of the
hormonal status of the cells and was present or absent in
gonadotrophin-producing cells and gonadotrophin-negative areas/cells
(data not shown). No significant correlation between ZAC
immunoreactivity and the proliferation index or the invasiveness of the
adenomas was found (data not shown).
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.
EGFR wild-type mRNA was detected in 7 (4 ACROs, 1 PROL, 1 GONA, and 1
NULL) of 16 tumors by RT-PCR. Authenticity of amplification products
was verified by probing with EGFR cDNA fragments (data not shown).
EGFRvIII transcripts were not detected in any of the 16 tumors examined
(Fig. 4A)
. EGFR protein was highly expressed in 10 of 81
samples, whereas in 22 cases, EGFR immunoreactivity was detectable only
in a few scattered endocrine cells (data not shown). The
immunohistological examination of all pituitary tumors revealed no
correlation between the absence of ZAC and the presence of EGFR. In
most NFPAs, in which ZAC expression was reduced or lost, none or only a
few EGFR-positive endocrine cells were present. In the few tumors
showing strong EGFR immunoreactivity, double immunolabeling for EGFR
and ZAC confirmed that ZAC expression was independent of EGFR status
(Fig. 4B)
.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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In conclusion, we observed a strong reduction or loss of ZAC expression in NFPAs in comparison with clinically active pituitary adenomas indicative of different molecular pathways operating in the pathogenesis of pituitary adenomas. Therefore, ZAC may be a useful marker in the molecular diagnosis of this subgroup of pituitary adenomas. Furthermore, the absence of ZAC expression in NULLs, the most undifferentiated type of pituitary tumor, suggests a possible role of ZAC not only in growth regulation but also in the differentiation of the pituitary. Elucidation of ZAC-dependent molecular pathways in NFPAs, which represent 25% of all pituitary neoplasias, may lead to new therapeutical approaches for these tumors, whose growth cannot be pharmacologically limited at present.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Deutsche Forschungsgemeinschaft
(DFG) (Grants Pa 647/1-1 and, in part, Sp 386/3-1), the Centre
Nationale de la Recherche Scientifique, the Ligue Nationale contre le
Cancer, and the Association pour la Recherche contre le Cancer. 
2 U. P. and T. A. contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Max Planck Institute of Psychiatry, Kraepelinstrasse 10,
80804 Munich, Germany.
4 G. K. S. and D. S. are joint senior
authors.
5 The abbreviations used are: PROL, prolactinoma;
ACRO, acromegalic-associated tumor; NFPA, nonfunctioning pituitary
adenoma; CUSH, Cushing’s adenoma; TSH, thyreotrophinoma; GONA,
gonadotrophinoma; NULL, null-cell adenoma; RT-PCR, reverse
transcription-PCR; FSH, follicle-stimulating hormone; LH, luteinizing
hormone; LOH, loss of heterozygosity; EGFR, epidermal growth factor
receptor; nt, nucleotide(s); GST, glutathione
S-transferase.
Received 1/27/00. Accepted 10/31/00.
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represents PCR
reaction without template. The graph shows quantitative analysis of the
AZAC:Aß-actin
ratio in the same samples, with the mean of the ZAC:ß-actin ratios
(0.642 ± 0.03) set as 100% of seven normal anterior
pituitaries. The values of pituitary adenomas are presented as a
percentage of normal pituitaries. Similar results were obtained from
two other independent experiments. C, the intensity of
ZAC immunoreactivity was calculated in 65 pituitary adenomas as
described in "Materials and Methods." The means observed in GONAs
and NULLs were significantly lower than those observed in the other
groups (Scheffé’s post hoc test;
P < 0.05). Values are reported as the
mean ± SE, and asterisks (*) indicate
statistically significant differences. D, expression of
ZAC mRNA and protein in five different types of pituitary adenomas
(#3, #6, #10, #15, and #23). Top
row, PhosphorImager pictures showing ZAC mRNA expression.
Middle row, histoautoradiographs of the same sections.
Bottom row, ZAC protein expression in adjacent sections
of the same adenomas. ZAC mRNA and protein expression is intense in
ACROs, PROLs, and CUSHs; dramatically decreased in GONAs; and lost in
NULLs. Cells were counterstained with toluidine blue. The
oligodeoxynucleotide used for this experiment was 614–659.
