Eliot R. Spindel

Gastrin-Releasing Peptide (Mammalian Bombesin) Gene Expression in Health and Disease

In recent years much attention has been focused on gastrin-releasing peptide (GRP), the mammalian homologue of bombesin, both as a neuroregulatory hormone and as a tissue-specific growth factor in normal and neoplastic tissues. This paper will analyze the distribution and role of GRP in normal mammalian tissues and examine the potential involvement of GRP in diverse pathologic processes.

Mammalian bombesin-like peptides

Abstract Mammalian bombesin-like peptides share structural homology with the large family of amphibian bombesin-like peptides. At present there is evidence for two classes of mammalian bombesin-like peptides, the gastrin-releasing peptides (GRP) and the ranatensin-like peptides. GRPs are distributed throughout the mammalian central nervous system, peripheral nervous system, gastrointestinal (GI) tract and the lung where they act as neurotransmitters, autocrine hormones and growth factors. Less is known about the ranatensin-like peptides which to date have been detected only in the brain and spinal cord. The large number of amphibian bombesin-like peptides suggests that still more mammalian bombesin-like peptides remain to be characterized. The challenge will be to determine the key physiological roles of the entire family of these peptides.

Transient elevation of messenger RNA encoding gastrin-releasing peptide, a putative pulmonary growth factor in human fetal lung.

Gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian peptide bombesin, is present in pulmonary neuroendocrine cells and appears to be a growth factor for both normal and neoplastic pulmonary cells. Previously we have reported the cloning of the messenger RNAs (mRNAs) and gene that encode human GRP. We now report that GRP mRNAs are markedly elevated in human fetal lung during the canalicular phase of pulmonary development (from approximately 16 to 30 wk gestation). By RNA blot and in situ hybridization analyses, GRP mRNAs were first detectable in fetal lung at 9-10 wk, plateaued at levels 25-fold higher than in adult lungs from 16 to approximately 30 wk and then declined to near adult levels by 34 wk gestation. By contrast, GRP peptide levels remain elevated until several months after birth. Consistent with this, in situ hybridization and immunohistochemical studies showed that GRP mRNA and peptide consistently colocalized in early gestation lung but that in neonatal lung, many cells that contained GRP peptide no longer contained GRP mRNA. The transient expression of high levels of GRP mRNAs during an approximately 12-wk phase of fetal lung development suggests that the secretion of GRP or its COOH-terminal peptides from pulmonary neuroendocrine cells may play a role in normal lung development.

Gastrin-releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas

Gastrin-releasing peptide (GRP; mammalian bombesin) is present in the neuroendocrine cells of human fetal lung and in small cell lung carcinomas (SCLCs), where it may act as a growth factor. Considering the potential importance of GRP as a tumor marker, we have conducted a retrospective immunohistochemical analysis of 176 lung tumors for markers of GRP gene expression, as well as several other markers of neuroendocrine cell differentiation: chromogranin A, neuron-specific enolase, and calcitonin. The majority of carcinoids contained mature GRP, in contrast to only a minority of SCLCs and large cell lung carcinomas (LCLCs). However, a majority of SCLCs and LCLCs contained proGRP immunoreactivity. In situ hybridization did not add any information beyond what was obtained using proGRP antisera. In spite of sharing these neuroendocrine cell markers, SCLCs are associated with a graver prognosis than LCLCs. No prognostic significance was associated with immunostaining for GRP or several other markers of neuroendocrine cell differentiation.

Detection of human probombesin mRNA in neuroendocrine (small cell) carcinoma of the lung. In situ hybridization with cRNA probe.

The production of human bombesin (gastrin‐releasing peptide), a peptide with mitogenic action, is a recognized feature of neuroendocrine (small cell) carcinoma of the lung. However, immunostaining of bombesin is not always possible in these tumors, probably because of poor storage mechanisms or rapid release of hormone. Molecular biological analysis of the gene encoding human bombesin has revealed the DNA sequence of human pro‐bombesin. We have used in situ hybridization to study the expression of the human bombesin gene at the cellular level in small cell carcinoma of the lung. Probombesin cDNA was subcloned in pSP64 vector, linearized with Bam HI and transcribed in the presence of phosphorus 32 (32P)‐cytosine triphosphate (CTP) and SP6 polymerase. The cRNA probe was applied to tissue sections (from six cases of small cell carcinoma of the lung, freshly fixed in 4% paraformaldehyde), cell culture preparations (two different cell lines of small cell carcinoma), and cytologic specimens (smears of cells from three different cases of small cell carcinoma). Hybridization of probombesin mRNA was detected in tumor cells in all samples. Specificity of the signal was determined by control experiments, including the use of a probe which has a sequence identical to probombesin mRNA. Our results provide evidence for the expression of the bombesin gene in small cell carcinoma of the lung at a cellular level and show that probombesin mRNA is highly expressed in these tumors.

Conversion of methionine to homocysteine thiolactone in liver

Since hemocysteinemia is associated with arteriosclerosis, the conversion of methione to homocysteine thiolactone was studied in guinea pig liver in vivo. 60 min after intraperitoneal injection of [14C]methione, [14C]homocystein thiolactone was found to constitute 9.1% ± 0.2 of the lipid bound 14C and 20% ± 1.0 of the acid soluble 14C. This conversion is the first step of a new pathway by which the sulfur of methionine is transferred to phosphoadenosine phosphosulfate.

Human gastrin-releasing peptide gene is located on chromosome 18

Gastrin-releasing peptide (GRP), a bombesin-like peptide, increases plasma levels of gastrin, pancreatic polypeptide, glucagon, gastric inhibitory peptide, and insulin. GRP is produced in large quantities by small-cell lung cancer and acts as a growth factor for these cells. To determine if chromosomal changes in small-cell lung cancer are related to the expression of GRP, we chromosomally mapped the gene using human-mouse somatic cell hybrids. Twenty hybrids, characterized for human chromosomes, were analyzed by Southern filter hybridization of DNA digested with EcoRI. Human DNA cut with EcoRI yields a major band of 6.8 kb and a minor band of 11.3 kb. The 6.8 kb band segregated concordantly with chromosome 18 and the marker peptidase A. The chromosome 3 abnormalities seen in small-cell lung cancer do not correlate with the chromosomal location of GRP, suggesting that the elevated expression of this gene may be due to mechanisms other than chromosomal rearrangement.

Localisation of mRNA and co-expression and molecular forms of GRP gene products in endocrine cells of fetal human lung

SummaryThe presence of bombesin (gastrin-releasing peptide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP-and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using regionspecific antisera to pro-GRP. Additional antisera to chromogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining using antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro-GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreactive for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with immunocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-local-ised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP. Furthermore PGP 9.5 appears to be a useful marker for endocrine cells in the respiratory epithelium of human fetal lung.

Molecular biology of bombesin-like peptides. Comparison of cDNAs encoding human gastrin-releasing peptide, human neuromedin B, and amphibian ranatensin.

The large family of bombesin-like peptides is characterized by both sequence diversity and sequence homology. The techniques of molecular biology are particularly well suited to study the bombesin-like peptides, because the size of the family suggests that there will be a number of closely related genes and because the remarkable protein chemistry performed in isolating these peptidesJ4 provides an excellent starting point. Our laboratorys basic strategy has been to synthesize oligonucleotide probes based on the known amino acid sequence of a particular bombesin-like peptide and then to use these probes to screen appropriate cDNA libraries. Hybridizing cDNA clones are sequenced and the amino acid sequence of the propeptide deduced. This approach has a number of advantages. First, it allows the deduction of the amino acid sequence of bombesin-like peptides in multiple species; second, it allows deduction of the complete propeptide sequence; and third, it generates cDNA clones that can then be used as probes of gene expression. Two advantages of knowing the entire propeptide sequence are that the sequence conveys information as to how the mature bombesin-like peptide is processed from its precursor and that new peptide sequences, called cryptic peptides, are often found within the propeptide sequence. These cryptic peptides often have important biologic functions in their own right or can potentially be of diagnostic use. An example of this is the gastrin-releasing peptide (GRP) carboxy-terminal extension peptides (CTEPs), which may be potential tumor marker^.^ There are two factors critical to using oligonucleotides to screen cDNA libraries. First is the design of the oligonucleotide probe. The oligonucleotide probe is made complementary to a predicted mRNA sequence which encodes an amino acid sequence of interest. The amino acid sequence is chosen because it is known to be part of the peptide to be cloned, or because it is an amino acid sequence that is conserved across multiple species, or-for the best reason of all-because it is the only sequence available. The resulting probes are mixed sequences because of the degeneracy of the genetic code. In order to keep the mix as

Localization of Bombesin‐like Peptides in Tumors

The localization of bombesin gene products in neuroendocrine tumors was achieved by a number of techniques used in combination. These included immunocytochemistry, radioimmunoassay, and chromatographic procedures using a variety of region-specific antibodies recognizing separate portions of probombesin. In situ hybridization using cRNA probes was employed to analyze bombesin gene expression at a cellular level. A novel procedure using a divalent form of bombesin and gold-labeled monoclonal antibodies for the localization of bombesin binding sites at the ultrastructural level was employed in this study. Antibodies to neuron-specific enolase and electron microscopy were employed for the determination of neuroendocrine differentiation. Surgical samples of pulmonary (n = 250) and nonpulmonary (n = 28) small cell carcinomas, 49 carcinoids, and 62 atypical lung carcinoids were investigated and compared with 169 control tumors, including lymphomas, adenocarcinomas, squamous cell carcinomas, and non-small-cell undifferentiated tumors. Cell lines cultured from pulmonary small cell carcinoma and smear preparations of pleural effusions from patients with small cell carcinoma of the lung were also investigated. Strong immunostaining for neuron-specific enolase was noted in all neuroendocrine tumors investigated, and no immunoreactivity was noted in control cases. Electron-dense neurosecretory granules were abundant in carcinoid tumors, scattered in small cell carcinoids, and absent in control cases. Immunostaining for bombesin was particularly strong in benign carcinoids, whereas the more malignant neuroendocrine tumors (e.g., small cell carcinomas) stained best with antibodies to the carboxyl-terminal flanking portion of human probombesin (proGRP). These findings were further validated by radioimmunoassay and chromatography of tissue extracts. Specific binding sites for bombesin were demonstrated on the surface of small cell carcinoma cells maintained in culture. In situ hybridization demonstrated mRNA for preprobombesin in all small cell carcinomas investigated, including surgical samples, cytological preparations, and cell lines. Hybridization reactions varied in intensity, with some cells in autoradiograms almost masked by silver grains and others showing much lighter deposits.