Guillem JG, Ã'Brian CA, Fitzer CJ, Forde KA, LoGerfo P, Treat M. & Weinstein IB 1987 Altered levels of protein kinase C and. Ca-dependent protein kinases in ...
Analysis of a protein kinase pituitary tumours U F
C-\g=a\mutation in human
Schiemann, R Assert, D Moskopp, R Gellner, Gullotta, W Domschke and A Pfeiffer
K
Hengst,
'Department of Medicine B, 2Department of Neurosurgery and 'Institute of Neuropathology of the Münster University Hospital, and department of Medicine of the Bergmannsheil Hospital, Ruhr-University, 44789 Bochum, Germany (Requests for offprints should be addressed 48129 Münster, Germany)
to U
Schiemann, Department of Medicine
of the Münster University
48129 Münster,
Germany
Hospital, Albert-Schweitzer-Strasse 33,
Abstract
generally accepted that protein kinase C-\g=a\(PKC-\g=a\) is important enzyme in the cellular regulation of growth and differentiation by phosphorylating proteins. Recent It is
an
studies have described a point mutation of PKC-\g=a\ (position 908 of the genetic sequence, codon GAC becoming GGC) in invasive human pituitary tumours which leads to an exchange of amino acids in the protein. We investigated 11 human pituitary tumours to evaluate the data obtained previously. cDNA was subcloned and up to ten individual clones were sequenced from each tumour, resulting in 85 clones analyzed in total. All of the
Introduction
Pituitary tumours comprise nearly 10% of all intracranial neoplasms (Gerdes et al. 1984) and consist of proliferating adenohypophysial endocrine cells of varying types. Most are cytologically benign and grow slowly with compression of the surrounding structures. They are able to extend superiorly into the sella diaphragm and cause compression of the optic chiasm. Inferiorly they penetrate the sella floor and may gain access to the sphenoid sinus and to the nasal cavity, and tumours showing lateral extension permeate
the cavernous sinus, cranial nerves and vessels. A number of descriptions of so called 'invasive' adenomas have appeared in the literature (Martins et al. 1965, Scheithauer et al. 1986, Selman et al. 1986, Buchfelder et al. 1995). Invasion means durai infiltration or perforation of the endosteum (Buchfelder et al. 1995). The rate of surgically recognized invasive adenomas is about 25% and corre¬ sponds to the volume of the tumour (Buchfelder et al.
1995). Using computerized tomography or magnetic reso¬ imaging, neuroradiologists categorize tumours by
nance
size, invasiveness and degree of suprasellar extension.
Macroadenomas (>10mm diameter) are more likely to show durai invasion than microadenomas (Scheithauer et al. 1986). Microscopic durai invasion has been identified in 66% of microadenomas and in about 90% of
adenomas showed a normal wild-type sequence of PKC-\g=a\ DNA. Even if the tumour was 'invasive' (infiltration of the dura mater) no mutation at position 908 of the sequence was found. Moreover, using Western blot analyses we did not observe any differences in PKC-\g=a\ protein expression in invasive as compared with non\x=req-\ invasive pituitary adenomas. Until now we have been unable to confirm the data of other investigators, suggesting that mutated PKC-\g=a\ is an inconsistent feature of
pituitary
invasive pituitary tumours. Journal of Endocrinology (1997) 153, 131\p=n-\137
macroadenomas (Selman et al. 1986). The incidence of malignant alteration of the adenohypophysis is very low. Thirty-six cases of pituitary gland carcinoma exhibiting craniospinal or extracranial métastases have been reported in the literature since 1900 (Hoffmann & Duffner 1985, Hashimoto et al. 1986). Up to now the etiology of pituitary tumours has still not been clarified. At first, altered hypothalamic stimulation was thought to induce adeno¬ matous proliferation of the pituitaries. In the meantime, molecular biological and genetic analyses of these tumours have pointed out that changes of signal transducing pathways in the tumour itself are able to cause cell proliferation. Landis et al. (1989) revealed that about 40% of GH-secreting tumours harbour point mutations within the Gsa-subunit gene. These so called gsp mutations lead to defective adenylate cyclase activity with consequent elevated intracellular cyclic AMP levels which result in increased growth hormone (GH) secretion. Ras mutations play no role in pituitary adenomas (Karga et al. 1992). Alvaro et al. (1993) revealed that invasive human pituitary tumours express a point-mutated protein kinase C(PKC- ) which is known as an important intracellular enzyme for proliferation and differentiation. Our interest was to confirm this genetic alteration by investigating 11 human pituitary tumours and discuss whether this PKC- mutation is really involved in the tumorigenesis
pituitary adenomas or not. Moreover, the same mutation was recently described as occurring in thyroid adenomas (Prévostel et al. 1995) which would suggest that it may be more widespread and of general significance. of
Materials and Methods Patients
pituitary tumours (ten non-secreting prolactin (PRL)-secreting tumour) from patients (seven women, four men) who were oper¬ ated by trans-sphenoidal adenomectomy in the Depart¬ ment of Neurosurgery of the Münster University Hospital. Our study was approved by the local ethical committee according to the guidelines issued by the Royal College of Physicians of London in September 1984, and amended in November 1984 and August 1986. Consent was obtained from each patient after a full explanation of the purpose and nature of all procedures used. The patients were 34—81 years (average 57 years). Tumour biopsies (60— 140 mg) were collected in the operating room, directly frozen in liquid nitrogen and stored at 20 °C until assay.
We
investigated
tumours,
one
11
GH and
—
Materials extraction The Ultraturrax T25 for tissue homogenization was obtained from Janke and Kunkel (Staufen i.Br., Germany). The RNA denaturating solutions (guanidinium-thiocyanate, lauryl-sarcosine, Nacitrate) came from Merck (Darmstadt, Germany) and Sigma (Deisenhofen, Germany). For measurement of the RNA concentration of the samples we used photometric analysis with the Gene Quant RNA/DNA Calculator from Pharmacia (Uppsala, Sweden).
RNA
RT-PCR The materials employed were obtained as follows: RNAsin from Promega (Madison, WI, USA), reverse transcriptase (RT) from Gibco BRL Life Technologies, Inc. (Gaithersburg, MD, USA), Taq DNA polymerase from Perkin Elmer (Vaterstetten, Germany), deoxynucleotides from Serva (Heidelberg, Germany), random primers from Boehringer Mannheim (Germany) and PCR primers from Biometra (Göttingen,
Germany).
Subcloning procedure and restriction analysis Escherichia coli (DH-5a), caseine, yeast, pAMP 1 vector DNA and Uracil DNA glycosylase were purchased from Gibco BRL Life Technologies, Inc. and lysozyme and restriction enzymes (EcoRI, Hindlll) were from Boehringer Mannheim. Sequence analysis RNAse was from Boehringer Mannheim, primers were obtained from Biometra and
dideoxynucleotides were purchased (Weiterstadt, Germany).
from
Applied
Bio-
systems
Western blot
analysis
All chemicals
were
from Merck except for
purchased
aprotinin (Bayer, Wuppertal, Germany), phenylmethylsulphonyl fluoride (Boehringer Mannheim) and Tween 20, 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium (NBT) (Sigma). Polyclonal PKC- antibodies (catalogue no. 539601) and synthetic peptide of PKC- were obtained from Calbiochem (La Jolla, CA, USA). Laser densitometer and Quantimage came from Molecular Dynamics (Sunnyvale, CA, USA).
Morphological studies We used Hardy's to characterize tumours.
classification (Comtois
al.
et
the size and invasiveness of the
1991) pituitary
neuropathologist examined a standardized series of biopsies of the mucosa of the sphenoid sinus, the basal dura mater and the pituitary tumours by light microscopy (haematoxylin/eosin and van Gieson staining). It was proved that the samples from which cDNA was subcloned were histologically homogenous. The tumours were classified as 'invasive' and 'non-invasive' adenomas by examining whether cellular infiltration of the dura mater The
was
found.
Tissue The
biopsies
pieces,
of the
pituitary tumours
were cut
and blood and connective tissues
were
into small removed.
RNA extraction, RT-PCR and sequence
analysis Pituitary tumours were homogenized in a denaturating solution consisting of 4 m guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0-5% sarcosyl and 0-1 m 2-mercaptoethanol by using the Ultraturrax T25. RNA from the pituitary tumours was extracted according to the method of Chomczynski & Sacchi (1987). This procedure was followed by the production of cDNA with reverse transcription (Kawasaki 1990) in a reaction containing 1 µg RNA. Fifty percent of the cDNA volume was amplified in a PCR using 2 U DNA polymerase from Thermus aquaticus (Taq) and 30 pmol of each primer. The following primers were used: 5'-CCAAACGGG CTTTCAGAT-3' (5'-pnmer; nucleotides 589-606)
and 5'-TTCACTCGGTCAAGGTTGTT-3' (3'-primer; nucleotides 993—1012). We performed the amplification in 28 cycles of 40 s at 94 °C, 1 min at 64 °C and 1 min and 30 s at 72 °C. The amplified DNA was subcloned in the pAMP 1 vector according to the instructions provided, using DH-5a as competent cells. To examine the
subcloning
reactions
we
performed
restriction
analysis
Table 1 Age of patient, invasiveness human pituitary tumours
(Hardy's classification),
hormonal
activity and sequenced clones of the
Hardy
Age (years)
classificatlon
ACTH
TSH
GH
PRL
LH
(pg/ml)
(µ /ml)
(ng/ml)
(ng/ml)
(U/l)
81
II 111
95 18 I !
1-14
0-2
0-8 0-01 0-02 1-31 008 2*14 1-28 0-84 1-81 0-93
0-5 0*9
FSH
(U/l)
Sequenced
clones
Pituitary tumour 1 2
r>!
3 4 5 6 7 8 9 10
53 63 73 65 56 70 34 35 53
I 1
16 II
22
IV
24 29
II III III IV
18 5 25 17
1 U EcoRI and 1 U Hindlll for each subclone. were incubated with 10 µg RNAse for 30 min at 37 °C and extracted by a phenol/
using
Afterwards, all subclones chloroform
procedure. Finally
we
performed
analysis employing the method of Sanger et al.
a
sequence
(1977) using
dideoxynucleotides. Amplification was accomplished in 30 cycles of 25 s at 95 °C and 4 min and 15 s at 60 °C. For each PCR fragment up to ten subclones were sequenced. Western blot
Pituitary
analysis
tumours
lOniM EGTA, 2
homogenized in 50 mM Tris, EDTA, pH 7-4 with 0-1% (v/v)
0-4 Oil 5-3
»2 I 04 48
9 72 2 26 21 215
1-5 4-5 6-7 3-2 2-8 9-0 1-2 6-2 1-8 4-2 81
0-9 5-7 1-2 (1-8
8-4
14-3 M
3-1 5-4 3-2 4-7
6 10
7 8 6 9 7 9
For evaluation of the specificity of PKC- signals, 4 µg immunization peptide of PKC-a antibodies was carried out. Visualization of PKC- signals was achieved with protein A conjugated to alkaline phophatase and BCIP/NBT. Prestained molecular weight markers were run in parallel with protein probes. Intensities of PKC- expression were determined by laser densitometry integrating the PKC signal subtracted from background signal using Quantimage software. For statistical analysis the unpaired f-test was used. ture.
competition with
were
mM
2-mercaptoethanol, 0-3 niM aprotinin, 1 mM phenylmethylsulphonyl fluoride and 100 µ leupeptine by using the Ultraturrax T25. Afterwards, samples were sonified for 3 x 10 s at 100 W and centrifuged at 100 000 g for 45 min. The supernatant was collected as cytosolic fraction and the sediment was again sonified in homogenization buffer containing 1% (v/v) nonidet P-40. After incubation for 30 min on ice and centrifugation for 15 min at 15 000 £ the supernatant
0-2 0-3 0-2 0-3 0-2
35 54
was
collected
as
membrane fraction.
Denaturing gel electrophoresis was performed with 20 µg cytosolic or membrane protein on 8% polyacrylamide gels according to Laemmli (1970). Protein concen¬ tration was determined by a modification (Stoscheck 1990) of the Bradford assay (1976). Proteins were blotted by alkaline semidry transfer (Towbin et al. 1979, Burnette 1981) to polyvinylidene fluoride (PVDF) membranes. Polyclonal antibodies were generated against the C terminus (amino acids 662-672) of rabbit PKC- which
is identical to the human sequence. After blocking the PVDF membrane for 1 h, PKC- antibodies were incubated in a final dilution of 1/170 in TBS (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0-05% (v/v) Tween 20 with 1% (w/v) non-fat dry milk) for 2 h at room tempera-
Results
pituitary adenomas were characterized as hormonal non-secreting tumours when the clinical features of the patients and laboratory measurements were considered (Table 1). Only tumour 11 showed an increased serum PRL level (215 ng/ml) which could be considered as significant hyperprolactinaemia. The GH level in the same tumour was slightly elevated (5-3 ng/ml). Radiologically none of the pituitary tumours was characterized as enclosed microadenoma (10mm, Hardy II), four tumours as locally invasive (Hardy III) and two tumours as diffusely invasive (Hardy IV). The pituitary tumours of Hardy classes III and IV bore characteristics of histological invasiveness. The collagen layers of the basal dura mater were dissociated and diffusely infiltrated by epithelial cells of the adeno¬ hypophysial tissue (Fig. la). In contrast to this, the nonTen
invasive tumours showed a clear demarcation between adenomatous tissue and dura mater (Fig. 1/;). The PKC- DNA fragments, amplified by RT-PCR,
had a length of 423 base pairs. The sequencing of the PKC- DNA fragments (position 589-1012) extracted
1 (a) Invasive pituitary adenoma: dura mater sheets dissociated by infiltrating tumour cells: Cieson stain, (b) Non-invasive pituitary adenoma: no infiltration of the dura mater is evident: Gieson stain.
Figure van van
from 11
pituitary adenomas revealed no point mutation at
908 of the cDNA. Invasive and non-invasive tumours showed the same genetic sequence of PKC-a DNA which is identical to wild-type PKC- . The results were verified by performing sequence analysis with the upstream primer (5' direction; Fig. 2d) as well as with the downstream primer (3' direction; Fig. 2b). For high
position
sensitivity of PKC mutations we analyzed single colonies of clonai DNAs. Sequencing of at least eight independent colonies of six patients with invasive pituitary adenomas largely excluded the possibility that mutated PKC-a transcripts were not detected. Moreover, no mutations observed within 200 nucleotides upstream and 90 nucleotides downstream of position 908 of PKC-a. were
Discussion
(a) CCATTCCGGAAGGGGACQAGGAAGGA 290 2»
A
ACÁ TG
300
The PKC- , one of 12 PKC isoenzymes, has been characterized as an important enzyme for cell proliferation and differentiation (Nishizuka 1992). It is activated by phospholipids and diacylglycerol (second messenger) and changes proliferative cellular activity by phosphorylating proteins. Several publications have described altered PKC activities in different human tumours, for example, decreased activity in colon tumours (Guillem et al. 1987, Kopp et al. 1991) and elevated activity in breast tumours (O'Brian et al. 1989) and pituitary adenomas (Alvaro et al. 1992, 1993). It was suggested that changes in PKC activity are involved in the tumorigenesis of these different human tissues. Alvaro et al. (1993) tried to explain the increased expression and activity of PKC- in human pituitaries compared with normal rat pituitaries with a genetic alteration of the enzyme. These authors revealed the presence of a point mutation at position 908 of the cDNA (V3 region of the protein), codon GAC becoming GGC, which leads to a substitution of a negatively charged
acid by an apolar glycin. Our results cannot confirm these previous data. We investigated 11 human pituitary tumours (ten non-secreting and one GH and PRL-secreting) and only the normal wild-type sequence of PKC- cDNA in up to ten subclones of each PCR fragment could be found. On the other hand, Alvaro et al. (1993) stated that all invasive adenomas seemed to be mutated, but that the number of their clones bearing normal PKC- cDNA was higher than that of clones bearing the mutated PKC- cDNA. However, since a relatively large number of individual clones were sequenced from each tumour it is unlikely that we failed to detect the mutation. It is conceivable that PKC-a mutations are an inconsistent feature of invasive pituitary tumours and that they are only expressed in a small subpopulation of tumour cells. It is also possible that racial differences, age or environmental factors, such as food or radiation, play an important role for genetic heterogeneity and may influence the frequency of mutations as described previously (Landis et al. 1989, Hosoi et al. 1993). Protein expression of PKC- varied widely between the different samples and did not reveal significant differences between invasive and non-invasive pituitary adenomas in contrast to the data by Alvaro et al. (1992, 1993). Invasive as well as non-invasive pituitary tumours showed PKC-a expression with high as well as low signal intensity so we suggest that interindividual differences of protein expres¬ sion should be considered. The majority of PKC- was membrane-associated, suggesting marked activation of the enzyme. Remarkably, the degree of membrane translocation was slightly higher in non-invasive pituitary adenomas than in invasive ones. Our data thus do not confirm the proposal that invasive adenomas express higher levels of PKC- . We did not investigate rat pituitary tissues as a reference for human adenomatous
aspartic
Figure
2 (a)
5'-sequencing of the
PKC-
fragment of an
invasive
pituitary adenoma showing codon GAC at positions 907-909. (fa) 3'-sequencing of the PKC- fragment of an invasive pituitary adenoma
positions
showing codon
CTG
(complementary codon
to
GAC)
at
907-909.
In order to evaluate protein expression of PKC-a, Western blot analyses of the same pituitaries investigated for PKC- mutations were carried out. Membrane and
cytosolic
fractions contained
80 kDa which
an
immunoreactive band
completely eliminated by preadsorption of the antibody with the peptide used for immunization indicating its specificity (Fig. 3a and b). Respectively, two samples (cytosolic and membrane frac¬ tion) of nine adenomas were analyzed on a single blot to allow relative comparisons (Fig. 3c). Intensities of PKC-a signals were determined by laser densitometry which showed a ratio of cytosolic to membrane enzyme of at
was
0-75 ± 0-13 in six invasive adenomas and of 0-49 ± 0-12 in three non-invasive adenomas. This difference was not significant, although the amount of PKC- in the mem¬ brane fraction was somewhat greater in the non-invasive adenomas. The cytosolic PKC- (in arbitrary units) was 2601 ±751 and 1906 ±638, and the membraneassociated PKC- was 3181 ±644 and 3450 ± 638 in invasive and non-invasive adenomas respectively which was not significantly different.
Figure 3 Expression of PKC- in invasive and non-invasive pituitary adenomas. Protein (20 µg) of cytosolic and membrane fraction was separated by SDS-PAGE and isotype-specific detection was performed with polyclonal PKC- antibodies, (a) Specific detection of PKC-a at 80 kDa In two pituitary adenomas in cytosolic (c) and membrane (m) fractions, (fa) as (a) with preincubation of PKC- antibodies with the peptide used for immunization, (c) Invasive adenomas were analyzed in lanes 1, 3, 5, 7, 8 and 9, non-invasive adenomas in lanes 2, 4 and 6.
tissue because it is not known if even normal human pituitary tissue is comparable with it. To normalize greater interindividual differences in PKC- expression it would be necessary to examine normal human pituitary tissue from the same patients which could not be taken because of ethical reasons. Consequently we did not find indi¬ cations of a correlation of PKC- expression, degree of PKC-a activation and PKC- mutation. Nevertheless there is a widespread agreement to suggest that dominant mutations, particularly gsp mutations are involved in the pathogenesis of pituitary tumours (Vallar
al. 1987, Landis et al. 1989, Lyons et al. 1990). Whether defective adenylate cyclase activity with consequently increased GH secretion leads directly to cellular prolifer¬ ation of the pituitary gland in patients with acromegaly is not quite clear. Adams et al. (1995) compared the clinical and biochemical characteristics of GH-secreting tumours harbouring gsp mutations (gsp-positive) with the characteristics of gsp-negative tumours and found no significant difference in tumour size and mitotic potential examined by DNA polymerase activity and Ki-67 et a
staining.
questionable whether hormonal activity of the is correlated with cellular proliferation. tumours pituitary On the one hand, invasive adenomas show a significantly It is also
expression of Ki-67 antigen, a marker for proliferative cellular activity (Gerdes et al. 1984, Knosp et al. 1989) in the immunoperoxidase staining without increased
correlation with the hormonal content. On the other hand, Scheithauer et al. (1986) indicated that pituitary tumours of varying hormonal type, regardless of whether they are clinically functional or not, show differing frequencies of invasion (adrenocorticotrophin cell adenoma 62%, prolactinomas 52%, GH-secreting adenomas 30%). Up to now there has been no evidence that invasiveness of pituitary adenomas is caused directly by oncogene activity like point-mutated PKC- . It is possible that invasive and non-invasive pituitary adenomas represent different tumour groups with regard to their biological behaviour. It is, however, probable that the term 'invasive' does not express a degree of proliferative activity, but is a description of tumour stage supposing that each pituitary adenoma is non-invasive at the beginning and is able to become invasive in the course of progression which is then frequently associated with clinical symptoms. References Adams EF, Buchfelder M & Fahlbusch R 1995 Molecular biological aspects of acromegaly. Acta Neurochimrgica 133 225-226. Alvaro V, Touraine Ph, Raisman Vozari R, Bai-Grenier F, Birman & Joubert (Bression) D 1992 Protein kinase C activity and expression in normal and adenomatous human pituitaries. International Journal of Cancer 50 724—730. Alvaro V, Levy L, Dubray C, Roche A, Peillon F, Quérat & Joubert D 1993 Invasive human pituitary tumours express a point mutated -protein kinase C.Journal of Clinical Endocrinology and Metabolism 77 1125-1129. Bradford MM 1976 A rapid and sensitive technique for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72 248—254. Buchfelder M, Fahlbusch R, Adams EF, Roth M & Thierauf 1995 Invasive pituitary adenomas: frequency and proliferation. Acta Neurochimrgica 133 223-224. Burnette WN 1981 Western blotting: Electrophoretic transfer of proteins from SDS-polyacrylamide gels to unmodified nitrocellulose and radiographie detection with antibody and radioiodinated protein A. Analytical Biochemistry 112 195-203. Chomczynski & Sacchi 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate—phenol—chloroform extraction. Analytical Biochemistry 162 156—159. & Comtois R, Beauregard H, Somma M, Serri O, Aris-Jilwan Hardy J 1991 The clinical and endocrine out-come to transsphenoidal microsurgery of nonsecreting pituitary adenomas. Cancer 68 860-866. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U & Stein H 1984 Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. Journal of Immunology 133 1710—1715. Guillem JG, CA, Fitzer CJ, Forde KA, LoGerfo P, Treat M & Weinstein IB 1987 Altered levels of protein kinase C and Ca-dependent protein kinases in human colon carcinomas. Cancer Research 47 2036-2039.
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a
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Jameson JL
&
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Received 25 June 1996 Revised manuscript received 28 October 1996 Accepted 13 November 1996
