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Endocrinological Oncology |
Service de Biochimie Médicale (C.B.), Paris; INSERM U-401 (L.L., D.J.) and UPR 9055 (P.F.), Montpellier; Laboratoires Sandoz (A.R.), Rueil-Malmaison; and Centre Val d’Aurelle (P.R.), Montpellier, France
Address all correspondence and requests for reprints to: Dr. Dominique Joubert, INSERM U-401, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France.
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Together these data provide evidence for the existence of local production of SRIH and GHRH by human breast. Hypersecretion of GHRH by breast tumors indicates that this peptide could play a role in maintaining epithelial cell proliferation as is the case for other peptides produced locally.
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and -ß (2). SRIH is widely distributed throughout the
central nervous system and the gastrointestinal system. Its receptors
are present in various human cancers, among which are breast tumors
(3, 4, 5, 6). Its known antiproliferative effect on various systems has
opened the possibility of its use as a therapeutical tool in cancer
treatment (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). The antiproliferative effect of SRIH analogs has been demonstrated both in vitro and in vivo. In vitro, proliferation of human breast cancer cells (MCF-7 cells) is inhibited by the SRIH analog BIM 23014 (19). In vivo, continuous infusion of octreotide in nude mice bearing ZR-75-1 tumors induces a significant inhibition of tumor growth (20). In line with these data is the inhibitory effect of BIM 23014 on the growth of MCF-7 tumors in a nude mouse subrenal capsule assay (21). The antiproliferative effect of SRIH analogs is mediated through SRIH receptors that have been identified on these cells as well as in a fraction of human breast cancer biopsies.
As a neuroendocrine peptide, the physiological effects of SRIH on GH are intimately related to those of GHRH (22). However, whereas there are numerous studies on SRIH receptors and the biological effects of SRIH in various tissues other than the pituitary, very little attention has been devoted to GHRH in human cancers. The capacity of GHRH to stimulate cell proliferation has been demonstrated in pituitary somatotrophs (23, 24), and it is known that in human pituitary tumors, GHRH is released in higher amounts than from normal pituitaries, whereas maturation of the SRIH precursor molecule is impaired in these same tumors (25, 26). Therefore, the possibility that GHRH could play a role in the proliferation of cells other than sommatotrophs, as does SRIH, deserves attention, especially in the context of the potential use of SRIH analogs as therapeutic agents in the treatment of cancer.
The aim of the present study was to investigate the endogenous production of SRIH and GHRH by human normal and tumoral breast. We show that SRIH and GHRH are synthesized in and released in vitro from normal and tumoral breast samples. The release of both peptides is pulsatile when observed in perifusion, and tumoral samples hypersecrete GHRH compared to normal tissue. Finally, SRIH can stimulate GHRH release from normal breast tissue.
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Materials and Methods |
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RNAzol was obtained from Bioprobe (Montreuil-sous-Bois, France). The reverse transcriptase kit (superscript preamplification system) was obtained from Life Technologies (Cergy-Pontoise, France). Primers were synthesized by Eurogentec (Seraing, Belgium). The Expand polymerase was obtained from Boehringer Mannheim (Indianapolis, IN). The TA cloning kit (version 1.3) was purchased from Invitrogen (San Diego, CA), and the Sequenase (version 2.0) DNA sequencing kit was obtained from Amersham International (Aylesbury, UK).
Human breast tissue
Three groups of samples were used to perform 3 different sets of experiments: 1) 20 normal breast samples and 25 breast tumors were used for the study of pro-SRIH identification and GHRH messenger ribonucleic acid (mRNA) detection, GHRH HPLC characterization, and GHRH tissue content determination; 2) 3 normal samples and 4 tumors were used for perifusion studies; and 3) 3 normal samples and 6 tumors were used for static incubations. Normal samples were obtained from women who underwent surgery for mammary hypertrophy. The 36 tumors were collected in the operating room. They were histologically characterized as ductal breast tumors (grade 1, 2, or 3 in the classification of Scarff and Bloom); all were invasive, with or without lymph node metastasis. Fragments were randomly selected for the biochemical and in vitro studies from different parts of a specimen. Fat-like tissue was omitted. Fragments were frozen at -80 C; others were used immediately for perifusion or static incubation studies.
Tissue extraction and Sephadex G-50 filtration
Normal and tumoral breast samples (50–100 mg) were boiled for 5 min in 2 mol/L acetic acid, homogenized (1:10, wt/vol), and kept frozen overnight. After thawing, homogenates were centrifuged at 2000 x g for 20 min. Proteins were assayed in pellets (containing >95% of the total proteins) by the method of Bradford (27). Aliquots of supernatants (100 µL) were lyophilized and then assayed for GHRH contents. The remaining supernatants were loaded onto a Sephadex G-50 column (90 x 2 cm) and eluted at 4 C with 5.2 mol/L acetic acid. Synthetic SRIH-14 and SRIH-28 were also applied to determine their elution volumes. Fractions (1.7 mL) were collected at 10-min intervals, lyophilized, and assayed for immunoreactive (IR-) SRIH by RIA.
In vitro experiments
Perifusion was conducted as previously described (25). Fragments (1 mm3) were perifused in an isolated chamber at 37 C containing 0.4 g Bio-Gel P2 (Bio-Rad, Richmond, CA). The perifusion medium contained 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.18 mmol/L KH2PO4, 2.5 mmol/L CaCl2, 25 mmol/L NaHCO3, 1.18 mmol/L MgSO4, and 14 mmol/L glucose supplemented with 5 g/L BSA and a mixture of amino acids. The medium was constantly gassed with 5% CO2-95% O2. Fractions (1 mL) were collected every 2 min, and immediately frozen at -20 C until hormone assay.
For static incubation, between 50–100 mg tissue were cut into small fragments (1 mm3) and incubated at 37 C in Krebs-Ringer perifusion medium (1.5 mL) with or without 10-8 mol/L GHRH or 10-7 mol/L SRIH; these two concentrations are known to induce maximal effects on GH secretion. Sample sizes were too small to allow dose-dependent studies. After a 4-h incubation, medium and fragments were separated by centrifugation and frozen separately until used. Results are expressed as femtomoles per mg protein extracted from the incubated tissues.
Hormone assays
IR-SRIH materials (extracted Pro-SRIH and released SRIH) were assayed by RIA, using, as previously described (26), a polyclonal antibody (Amersham) raised against SRIH-14 that reacted equally with SRIH-14 and SRIH-28. This antibody recognizes the SRIH precursor as the SRIH-14 sequence in the C-terminal part of the molecule. As SRIH-14 is used as the standard in the assay, pro-SRIH quantification is expressed equivalent to SRIH-14.
The assay procedure was as follows. Standard or sample (0.1 mL), 0.1 mL diluted antiserum (final dilution, 1:25,000), and 0.1 mL [125I]SRIH (3,500 cpm/tube; Amersham) were incubated at 4 C for 24 h. The buffer used for dilutions was 50 mmol/L sodium phosphate buffer, pH 7.2, containing 0.3% BSA and 10 mmol/L ethylenediamine tetraacetate. Separation of free from bound SRIH was achieved using dextran-coated charcoal. The sensitivity of the assay was 1.22 fmol/tube, and the intra- and interassay coefficients of variation were 5% and 9%, respectively.
IR-GHRH was assayed by RIA. The GHRH-(1–40) antibody was a generous gift from M. C. Thonon (Laboratory of Dr. Vaudry, URA CNRS 650, Faculté des Sciences, Université de Rouen, Mont Saint-Aignan, France).
The assay procedure was as follows. Standard or sample (0.1 mL), 0.1 mL diluted antiserum (the same as that used for in vitro experiments; final dilution, 1:60,000), and 0.1 mL [125I]human (h) GHRH-(1–44) (4,500 cpm/tube) were incubated at 4 C for 3 days. Separation was achieved using dextran-coated charcoal. All dilutions were performed with potassium phosphate buffer, pH 7.4, containing 0.1% Triton X-100 and 0.2% sodium azide. The sensitivity of the assay was 0.8 fmol/tube, and the intra- and interassay coefficients of variation were 8% and 14%, respectively. The GHRH antibody reacted equally with human (h) GHRH-(1–44), hGHRH-(1–40), and hGHRH-(1–31). The specificity of the assay is attested by the lack of cross-reactivity of the antibody with other peptides (25).
For static incubations, assays were performed on 100 µL medium in triplicate.
GHRH, pro-SRIH, and SRIH characterization by high performance liquid chromatography (HPLC)
For HPLC characterization of GHRH present in breast tissue, lyophilized extracts corresponding to 250 µL tissue homogenate were reconstituted in 0.5 mL 10 mmol/L trifluoroacetic acid and centrifuged at 10,000 x g for 15 min at 4 C. Aliquots of supernatant (100 µL) were applied to a reverse phase Ultrapore RPMC column (250 x 4.6 mm). For HPLC characterization of GHRH present in perifusion medium, 100 µL medium were directly applied to the RPMC column. Extracts or medium were eluted isocratically with 27% acetonitrile and 10 mmol/L trifluoroacetic acid (10 min), followed by linear acetonitrile gradients of 1%/15 min (60 min) and 1%/min (10 min) at a flow rate of 1 mL/min. Fractions were collected at 1-min intervals, lyophilized, and assayed for GHRH by RIA. Standard [synthetic GHRH-(1–44)] was diluted in 10 mmol/L trifluoroacetic acid perifusion medium.
For pro-SRIH characterization, IR-SRIH fractions obtained after Sephadex G-50 filtration of breast samples or rat hypothalamus (our sample reference) were reconstituted in 2 mol/L acetic acid (50 µL), loaded on an Ultrapore C18 column (5 µm; 250 x 4.6 mm), and eluted first isocratically with 23% acetonitrile and 0.1% trifluoroacetic acid (40 min), followed by a linear acetonitrile gradient (3%/min for 25 min) at a constant flow rate (1 mL/min). For HPLC characterization of SRIH released during perifusion, 1 mL perifusion medium corresponding to pooled perifusion fractions containing an estimated SRIH concentration of 44 fmol/mL was isocratically eluted with 22% acetonitrile and 10 mmol/L trifluoroacetic acid (10 min) followed by linear acetonitrile gradients of 1%/7.5 min (30 min) and 1.5%/min (10 min) at a flow rate of 1 mL/min. Fractions were collected at 1-min intervals, lyophilized, and assayed for IR-SRIH by RIA. SRIH-28 and SRIH-14 were used as standards; they were diluted in either 2 mol/L acetic acid or perifusion medium.
PCR amplification of breast tissue complementary DNA (cDNA)
Total RNA was extracted from breast tissue (two tumors and two normal samples) homogenates by RNAzol using a method derived from that of Chomzynski (27a). Human pituitary was used as the positive control for GHRH gene expression (28).
First strand cDNA was produced in a reaction containing 20 mg total RNA, oligo(deoxythymidine) primer, and reverse transcriptase using the Superscript kit according to the manufacturer’s recommendations. cDNA was amplified by PCR. The PCR mixture contained 2.5 U DNA polymerase (Expand, Boehringer Mannheim) and 10 pmol of the following primers determined according to the human GHRH cDNA sequence (15): 5'-TATGCAGATGCATCTTCAC-3' (5'-primer) and 5'-GGAGTTCCTGCTGTGCTTCT-3' (3'-primer) in a total volume of 50 mL. PCR of 40 cycles was performed, consisting of denaturation (30 s at 94 C), annealing for 30 s at 55 C, and extension at 75 C for 1 min. To detect the presence of contamination, DNA was replaced by water. Amplification was accomplished in a Perkin-Elmer/Cetus Thermocycler (Norwalk, CT). After completion, amplified DNA was electrophoresed on a 2% agarose gel, and the size of the amplified DNA was compared to that of the appropriate molecular size marker by ethidium bromide staining. DNA at the expected size (224 bp) was then cryoeluted and ethanol precipitated.
Sequencing
The purified PCR products were subcloned using the TA Cloning PCRTM II vector kit (In Vitrogen, San Diego, CA). Sequencing was performed on the antiparallel strand using 5 pmol sequencing primer 5'-GGAGTTCCTGCTGTGCTTCT-3', chain-terminated dideoxynucleotides [35S]deoxy-ATP labeling, and the Sequenase polymerase according to the manufacturer’s instructions.
Statistical analysis
Results were analyzed using Student’s t test when comparing release or content between normal and tumoral samples. Paired Student’s t test was used when comparing release and content in the presence or absence of stimuli.
Pulsatile profiles of SRIH and GHRH release were first analyzed by a Fourrier analysis, which gives a spectrum of amplitude of the series to analyze. A dominant frequency was obtained (or not) from the raw data (29). The presence of peaks of secretion was then determined by the method of Santen and Bardin (30). A peak is detected when a value is higher than a threshold of 4 times the intraassay coefficient of variation and if it is followed by a value equal or lower than this. This method was applied after smoothing of the secretory profiles by the method of Savitsky-Golay (31). A window of five points was taken for this smoothing, avoiding a significant modification of amplitudes. These analysis were performed with ORIGIN (version 4.0, Microcal, Northampton, MA).
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Figure 1
shows the IR-SRIH elution profile of an
acetic acid extract of a normal sample after Sephadex G-50 filtration.
Two peaks were identified that were previously characterized as
containing authentic pro-SRIH (26). The IR material contained in these
peaks had apparent molecular masses of 17 (peak 1; elution volume, 66
mL) and 12 (peak 2; elution volume, 74 mL) kDa, respectively (26).
SRIH-(1–14) and SRIH-(1–28) were not detected (elution volume, 118
mL). When a fraction of these peaks was analyzed by HPLC (Fig. 2), IR-SRIH was eluted at the same retention time as
pro-SRIH extracted from rat hypothalamus.
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GHRH is synthesized by human breast tissue
As shown in Fig. 3, reverse transcription-PCR
amplification indicated that GHRH mRNAs are present in human normal and
tumoral breast tissue. Sequencing demonstrated the complete identity of
the sequence of the amplified DNA fragment with that of GHRH cDNA (data
not shown). Figure 4 shows that IR-GHRH present in the
tissue eluted from the HPLC column with the same retention time as that
of authentic GHRH-(1–44).
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In vitro release of SRIH and GHRH in perifusion
As shown in Table 1, when fragments of either
normal samples or tumors were perifused, IR-SRIH and IR-GHRH could be
measured by RIA. IR-SRIH was released in detectable amounts in only two
of the four tumoral samples analyzed. When it was released, there were
no differences from the amounts released by normal samples. GHRH was
released from all tumoral samples. Release was higher than that from
normal samples in three of the four cases studied. HPLC
characterization (Figs. 4
and 5) showed identity of
IR-GHRH with GHRH-(1–44) and of IR-SRIH with SRIH-(1–28) for normal
as well as tumoral samples.
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, top panel), whereas
GHRH release decreased rapidly during the first minutes of perifusion
and then reached a value that was maintained for the rest of the
experiment (Fig. 6b, top panel).
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, the release of both peptides was pulsatile, as
determined by Fourrier analysis (middle panel) and the peak
detection analysis of Santen and Bardin (lower panel).
Results are summarized in Table 1. For SRIH release from normal tissue
(n = 3), a unique frequency was identified; one peak occurred
every 41.5 ± 0.7 min (mean ± SEM of Fourrier
and Santen and Bardin determinations). For GHRH release, a major
harmonic was detected by Fourrier analysis (frequency, 42.1 ± 0.3
min), and a minor one was found using the Santen and Bardin method
(frequency, 23.9 ± 0.5 min). SRIH release from tumoral tissue was
detected in only two of the four cases studied. It was pulsatile in one
case. In this case, pulse frequency was 40.8 min (mean value of
Fourrier and Santen and Bardin determinations; see Table 1). GHRH
release was detected for all tumoral samples, but pulsatility was
present in only one case; frequency was not different from that
estimated for normal samples. In vitro release and regulation in static incubation
The release of SRIH and GHRH was also observed in static incubation.
GHRH basal release (Table 2 and Fig. 7)
was significantly different (P < 0.05): 29.6 ±
4.6 fmol/mg proteins for normal samples (n = 3) vs.
382 ± 92 fmol/mg proteins for tumors (n = 6). SRIH basal
release was, by contrast, not different between normal and tumoral
tissue (Table 2). Values ranged from 5.8–8.6 fmol/mg proteins for
normal samples and from 0–61.6 fmol/mg proteins for tumors.
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and Fig. 7; 56.7 ± 8.9 vs. 29.6 ± 4.6
fmol/mg proteins; P < 0.05). The presence of SRIH did
not affect GHRH release from tumors.
When incubation was performed in the presence of 10-8
mol/L GHRH, SRIH release was not affected (Table 2).
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The presence of IR-SRIH in breast cancer specimens has been inconsistently shown in the literature (32, 33), and no information were available regarding its presence in normal breast. Three human breast cancer cell lines, ZR-75-1, MDA-MB-436, and MCF-7, have been described to contain SRIH-like immunoreactivity (34). Demonstration of endogenous peptide synthesis is proved here for SRIH by identification of pro-SRIH. Among the two IR-SRIH forms detected in breast tissue, only the 12-kDa form has been described in the hypothalamus (35) or pancreas (36). The 17-kDa form (elution volume, 66 mL) has been described in normal and tumoral human pituitaries (26). It contains the tetradecapeptide sequence and the dibasic site for its release from the precursor. The molecular significance of this increase in apparent size has not been established. It should be noted that in the case of provasopressin, for example, high molecular forms were also reported to be produced, especially under stress conditions and in tumoral cells (37, 38). SRIH-14 or SRIH-28 could not be detected in tissue extracts, which could mean that the mature peptide is not stored in the mammary tissue, as we observed in pituitary tissue by contrast to hypothalamus (26).
Quantification of pro-SRIH after Sephadex G-50 filtration in normal and tumoral breast tissue showed a wide range of concentrations in the two tissues. The range is wider for the tumors, but there are no statistical differences between normal and tumoral breast.
The release of SRIH-(1–28) and GHRH-(1–44) was observed in both perifusion and static incubation. Surprisingly, when observed in perifusion, the release of SRIH-(1–28) and GHRH-(1–44) was pulsatile, with differences between normal and tumoral samples. The release of IR-SRIH and GHRH from normal breast was always present. It showed a unique frequency for SRIH, with one peak occurring every 41.5 min, and a major and a minor harmonic for GHRH, with frequencies of, respectively, 41.5 and 23.9 min. Pulsatility was observed during the entire perifusion, although there was a tendency for a slight decrease in basal values over the 4 h of perifusion. Peak periods did not always occur simultaneously for the two peptides, indicating that the two peptides are probably not stored in the same secretory vesicle. However, if secretory profiles were always pulsatile from normal samples, this was not the case for the tumors. Indeed, SRIH release, observed in two of the four cases studied, was pulsatile in only one case, with a pulse frequency identical to that observed for normal samples. GHRH, by contrast to SRIH, was released from all tumoral samples, but similarly to SRIH, it was pulsatile in only one case. The tumors that did not secrete SRIH did secrete GHRH, indicating that the absence of SRIH release was not just a general lost of differentiation of breast tissue posterior to cell transformation. Also, in the sample for which GHRH pulsatility was not present, SRIH pulsatility was still present, showing that the regulations involved in the onset of pulsatility for the two peptides may be different. In this regard, it was surprising to observe a pulsatile release if one considers the experimental protocol used. Indeed, breast specimens were cut into small fragments, with several fragments in each perifusion chamber. Pulsatility could, therefore, be due to the fact that the cells have retained in vitro the intrinsic property to generate pulses at a rhythm predetermined in vivo. It could also signify the release of an unknown factor able to synchronize secretion from the different fragments. Nevertheless, that a peptide can be released in a pulsatile manner from a tumor indicates that the transformed cells can still be synchronized and are not always, at least under our in vitro conditions, functionally dissociated from one another. The functional significance of the pulsatile release of SRIH and GHRH, however, remains to be determined in both normal and tumoral tissues.
Quantification of the SRIH and GHRH released during perifusion showed that in three of four cases studied, GHRH release from tumors was higher than that from normal samples, whereas SRIH release was in the same range when it occurred. This is in agreement with the results obtained in static incubation. Indeed, there was a striking difference in the amounts of GHRH released from the tumors compared to those released from normal breast; tumors released 13 times more GHRH than did normal tissue (note that this higher release was inversely correlated to GHRH tissue contents, as expected). This is an interesting observation if one considers the known proliferative effect of GHRH. Transgenic mice with the hGHRH gene have pituitary hyperplasia, sometimes resulting in tumor formation (39), and pituitary tumors hypersecrete GHRH as do breast tumors (25). However, the first consequence of GHRH hypersecretion is not neoplastic transformation, but only hyperplasia. This means that GHRH overproduction might favor and entertain cell proliferation, but is certainly not an initiator of cell transformation. It should also be noted that the amount of GHRH released during the 4 h of incubation largely exceeds the initial GHRH contents, suggesting either a de novo synthesis or a maturation of the GHRH precursor during incubation. This is what occurs for SRIH. Indeed, breast tissue contains the SRIH precursor, but not SRIH-(1–28) or SRIH-(1–14).
GHRH release from normal breast tissue is regulated by SRIH, which, in contrast to its known inhibitory effects, elicits a stimulation of GHRH release. This regulation is not observed with tumoral tissue, probably due to the already high levels of GHRH secreted in vitro. That the effect of SRIH on GHRH release is direct or indirect can be discussed. A negatively coupled receptor cannot account for a stimulation, and the known SRIH receptors are negatively coupled to the transduction mechanisms (40). Therefore, the existence of another factor has to be hypothesized that would mediate the action of SRIH on GHRH. This factor should be an inhibitor of GHRH release, and its secretion should be inhibited by SRIH. The exact significance of GHRH release regulation by SRIH can be questioned, as there is no SRIH in the tissue. However, there are no arguments suggesting that the in vitro SRIH release cannot occur in vivo.
SRIH and its analogs are known to be antiproliferative factors in various cell types, including breast cancer cells (41, 42, 43), which has been at the origin of a potential use of the SRIH analogs in cancer. Our data, however, show that the major disorder observed in breast cancer does not concern SRIH but, rather, GHRH. One can speculate that as these two peptides have opposite physiological effects, SRIH analogs could profit by a blockade of GHRH action and, consequently, have their therapeutical benefits potentiated.
Received April 23, 1996.
Revised September 19, 1996.
Accepted November 7, 1996.
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