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Original Studies |
Departments of Endocrinology (A.C., E.M., S.M.W., R.C., A.d.L.), Pathology (X.M.-G.), and Nuclear Medicine (L.B.), Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona 08025, Spain
Address all correspondence and requests for reprints to: Eugenia Mato, Ph.D., Department of Endocrinology, Hospital de Sant Pau, Av.S.Antoni MaClaret n°167, Barcelona 08025, Spain. E-mail: emato{at}santpau.es
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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SRIF participates in the inhibition of hormonal secretion and hormonal regulation of cell proliferation and differentiation by specific membrane receptors (3), which were described in 1978 by Schonbrunn and Tashjian, and were cloned by Yamada et al. in 1992 and 1993 (4, 5). Five subtypes of SRIF receptors (SSTRs) have been characterized. Chromosomal localizations of the human SSTR genes have been located, dispersed on different chromosomes; and their pharmacological properties, tissue distribution, and signal transduction systems were shown to overlap but to differ one from one other (6, 7).
The role of SSTR gene expression and postreceptor events, in mediating tumor growth, remains to be elucidated. It is also unknown whether SSTR subtype messenger RNA (mRNA) expression may reflect underlying mechanisms of tumorigenesis and/or hormone expression of the tumor (8). Furthermore, SSTR2 has proved to be predominantly expressed in some neuroendocrine tumors (9), and it is characteristic for somatotroph pituitary adenomas (10). All these results tend to postulate that SSTR, and especially the subtype SSTR2, mediates the therapeutic effects of SRIF analogues (11).
In this study, we have assessed the expression of SRIF and SSTR subtypes in a series of MTC.
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Subjects and Methods |
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Fourteen patients with MTC (8 males and 6 females) were included in this study. Their ages ranged from 18–73 yr (mean 55). Eleven patients had sporadic tumors (negative genetic screening for the RET protooncogene germline mutation), whereas the remaining 3 belonged to a family with multiple endocrine neoplasia type 2A, in which a germline mutation in codon 634 of exon 11 of the RET protooncogene was identified. After resection of the primary tumor, in vivo SRIF scintigraphy was performed in 10 patients, but only in 5 of them was there a final evidence of tumor recurrence.
The study was approved by the institutional ethical committee, and informed consent was obtained from all patients.
Tissue samples
Tissue samples, obtained from 14 patients at surgery, were divided into several fragments; one of them was immediately frozen in liquid nitrogen and stored at -80 C; the remaining were fixed in buffered formaline and embedded in paraffin. The diagnosis of MTC was based on the pathological features of the tumors and was confirmed in all cases by positive immunoreactivity for CT and CEA. Tissue samples from rat brain were obtained as a source for control RNA for different SSTR subtypes, whereas DNA was obtained from the blood bank.
RNA and DNA isolation
Total RNA was extracted, following the guanidinium isothyocianate method (12). The RNA pellet was resuspended in water-diethylpirocarbonate solution and was treated with ribonuclease-free deoxyribonuclease I (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37 C and then at 95 C for 15 min. RNA concentration was estimated by ultraviolet spectrophotometry at 260 nm and 280 nm, and the integrity of mRNA was controlled by analyzing ribosomal RNA content on formaldehide/agarose gel and ethidium bromide staining. The RNA were stored at -80 C until used.
DNA was extracted by proteinase K (10 mg/mL; Boehringer Mannheim) and chloroform/isopropanolol-phenol (25:4:1), following the standard method previously described (13).
RT-PCR
One ug of total RNA was reversely transcribed in a buffer
solution containing 25 nmol/L MgCl2, 100 mmol/L Tris (pH 8.3), 500
mmol/L KCl, RNAguard (39 U/mL; Pharmacia, Uppsala, Sweden), M-MLV-RT
(200 U/mL; BRL, Gibco, Uxbridge, UK), 10 mmol/L deoxynucleotide
triphosphate(s), and random hexamer priming [d(N6)5'PO4; Pharmacia].
Incubations of 30 min at 42 C, 5 min at 94 C, and 5 min at 5 C were
carried out in a total vol of 20 µL. Complementary DNA (cDNA) was
stored at -80 C until used. SSTR PCR primers were selected as
previously described by Miller et al. in 1995 (10). PCR
conditions were: SSTR1, 3, 4, and 5 at 94 C for 1 min, at 60 C for 30
sec, and at 72 C for 75 sec; SSTR2 at 94 C for 1 min, at 57 C for 30
sec, and at 72 C for 75 sec. Human glyceraldehyde-3-phosphatase
dehydrogenase (GADPH) was amplified as a positive control from all
cDNA. PCR conditions for GADPH were: 94 C, 1 min; 62 C, 30 sec; and 72
C, 90 sec. SRIF was amplified using the primers described by Mato
et al. in 1994 (14), and the PCR conditions were: 92 C, 30
sec; 60 C, 30 sec; 72 C, 1 min. PCR reactions were carried out in a
total vol of 30 µL containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH
9.0), 3.5 mmol/L MgCl2, 40 µmol/L deoxynucleotide triphosphates, 2.5
U Taq polymerase (Ecotaq, Barcelona, Spain), for 40 cycles
in a thermocycler (Hybaid, Omnigene, Ashford, Kent, UK) (Table 1
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Immunohistochemical techniques
Immunostaining for SRIF was performed in paraffin-embedded sections of tumors, following the avidin-biotin-peroxidase method. A polyclonal antibody against SRIF was used (Dako Corp, Copenhagen, Denmark) at a final working dilution of 1:800 in PBS. Polyclonal antibodies against CT (Concepta, Barcelona, Spain) and CEA (Dako Corp) were also used at a final working dilution of 1:20 and 1:400, respectively. The secondary biotinized antibody was obtained from Vector Lab (BA-100, Lot 60226, Burlingame, CA) and used at a final working dilution of 1:400. ABC complex was prepared at 1:100 dilution. Diaminobenzidine was used as a chromogen. The sections were contrasted with Harris’ hematoxylin to magnify the localization of the immunolabeled antigen.
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Results |
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; Fig. 1). A correlation between SSTR expression
and the results of the in vivo scintigraphy was only
possible in the 5 patients with tumor recurrence. In all these
patients, octreotide scans were positive; and in all of them, SSTRs
were expressed (and the SSTR pattern was SSTR2, 3, and 5 in 2 cases;
SSTR2 and 5 in 1 case; SSTR2 in 1 case; and SSTR5 in 1 case).
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). SRIF expression by RT-PCR
was detected in all 14 cases; however, no correlation between SRIF
expression and immuno-SRIF detection was found (Table 3). No correlation between SRIF
expression and any clinicopathological features was found.
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Discussion |
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The introduction of SSTR scintigraphy for the localization of primary and metastatic SSTR-rich tumors allowed the identification of these receptors in a wide variety of tumors, including in MTC (8). Furthermore, this simple and effective technique has proved to be very useful in visualizing a large number of unsuspected metastases of these tumors (15). In an earlier series from our hospital, the use of this technique could detect clinically nonevident recurrences in 54.5% of MTC patients (16).
This is the first report in which mRNA expression of SSTR subtypes and the expression of SRIF have been studied in a series of human MTCs, in an attempt to support the existence of an autocrine/paracrine role for SRIF in MTC, as well as to provide additional evidences in favor of the use of SRIF analogues in the therapy of MTC patients. Interestingly, we identified the SSTR2 and SSTR5 as the most frequent subtype receptors in this tumor, whereas the SSTR4 was not detected in any case. The studies on the role of these two receptor subtypes indicate that they may mediate the antiproliferative effects of SRIF, through a protein tyrosine phosphatase or other different cellular mechanisms not yet identified (17). Our findings are in concordance with the recent reports by Miller et al. in 1995 (10), who, in human pituitary adenomas and normal tissue, found expression of multiple SSTR gene transcripts; SSTR5 was expressed in neoplastic and normal pituitary tissue, whereas SSTR1, although present, was expressed in a variable fashion in GH-secreting tumors, which were responsive to SRIF in vitro. Finally, the SSTR4 was not detected in normal or neoplastic human pituitary tissue.
In our series, only two cases with negative receptors were observed. This may reflect that more receptor subtypes probably still remain to be identified. In fact, receptors purified from rat brain and identified in AtT-20 cells have characteristics that differ from the five cloned SSTRs, and antibodies that recognize the purified brain SSTRs do not cross-react with the five cloned ones (18, 19); alternatively, the stability of these mRNAs may be low.
In humans, there is considerable controversy in the literature, regarding SRIF as a tumor marker. The reports concerning elevated plasma SRIF levels in MTC patients differ considerably. Elevated levels were found in up to 50% (20) or only 2 of 55 patients (2), and the authors speculated that this discrepancy might be caused either by intermittent SRIF release, a short plasma half-life, or low plasma levels resulting from nonsecretion of the peptide (21). Immunohistochemical studies have shown that SRIF can be found in 30–65% of primary MTCs; in addition, immunopositivity was linked to a longer survival rate (20, 22). However, the significance of these results is questionable, because the number of patients studied was very small. Moreover, in most cases, SRIF positivity is restricted to a few scattered cells, and metastatic tissue is less frequently positive than are primary tumors.
The simultaneous expression of SRIF and SSTR genes supports the hypothesis that SRIF might have an autocrine or paracrine action in normal and neoplastic C cells, suggesting that its binding may have an inhibitory influence on tumor metabolism and cell growth. In fact, an antiproliferative effect of SRIF analogues has been reported on the growth of experimental cell lines and explants (23, 24). The growth inhibitory activity of SRIF on tumor cells would support the use of SRIF analogues in the treatment of MTC patients (25). However, some studies showed no correlation between SSTR status and SRIF tumor levels. Therefore, although SRIF seems to be present in MTC and may act as a regulatory peptide on C cells, its function in these tumors remains unclear.
In conclusion, these studies may contribute to the further understanding of the correlation between SSTR mRNA expression and the functionality of these receptors, capable of mediating the effects of SRIF analogues for clinical application; they also contribute to the understanding of the pathogenetic mechanisms of medullary thyroid carcinoma.
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Footnotes |
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Received October 20, 1997.
Revised February 23, 1998.
Accepted April 7, 1998.
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References |
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