Efficient expression of tum- antigen P91A by transfected subgenic fragments.

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. The gene encoding tum- antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.

cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.

Neuromedin B and gastrin-releasing peptide mRNAs are differentially distributed in the rat nervous system.

The bombesin-like peptides are a family of structurally related amidated peptide ligands that are known to have a variety of potent pharmacological actions on various cells, including neurons in the rat brain. Two mammalian representatives of the bombesin family of peptides have been identified, gastrin-releasing peptide (GRP) and neuromedin B (NMB). Previously, we cloned the rat preproGRP gene and determined the locations of neurons expressing this gene using in situ hybridization. In this study, we describe the structure and sequence of the rat preproNMB gene, and the first detailed cellular localization of preproNMB mRNA in rat brain using in situ hybridization. Nucleotide sequence analysis of cDNA and genomic clones reveals a 117 amino acid precursor whose overall structure is similar to that described for human preproNMB. Sequence similarity between the rat NMB and GRP genes is observed only over a limited 10 amino acid sequence encoding the carboxy termini of the GRP and NMB peptides, the region shown to be necessary and sufficient for high-affinity receptor binding. In situ hybridization studies performed with cRNA probes specific for NMB or GRP mRNA show that the distribution of cells expressing either mRNA in brain is very distinct. NMB mRNA is found most prominently in the olfactory bulb, dentate gyrus, and dorsal root ganglion. In contrast, the highest levels of GRP mRNA are observed in the forebrain (isocortex and hippocampal formation). This heterogeneity of mRNA distribution for these peptides suggests that these 2 structurally related peptides may have very distinct functions as neuropeptides in the rat nervous system.

Structural characterization of a brain-specific promoter region directing transcription of the rat prepro-gastrin-releasing peptide gene.

Expression of the mammalian prepro-gastrin-releasing peptide (preproGRP) gene has been shown to be restricted to neural and neuroendocrine cell types. In this paper, the structure and nucleotide sequence of the rat preproGRP gene coding regions and promoter is described and analyzed. The gene is divided into 3 exons, encoding a signal sequence, the 29 amino acid rat GRP, and a 92 amino acid extension peptide. While the overall prohormone structure is similar to that predicted from the sequence of the human gene, differences in transcription are apparent. Several forms of the rat preproGRP mRNA are found in brain: a 1.1 kb form which initiates in both brain and gut primarily from a TATAA-directed promoter, and less abundant forms of about 1.5 kb, whose initiation sites are heterogeneous, located 300-400 base pairs upstream of the 1.1 kb initiation site, and found only in spinal cord and a subset of brain nuclei expressing preproGRP mRNA. Comparison of the human and rat promoter region sequences identifies regions of high similarity upstream from both the 1.1 kb and 1.5 kb mRNA initiation sites, which may be important in the cell type-specific regulation of the preproGRP gene.

Distribution of two distinct messenger ribonucleic acids encoding gastrin-releasing peptide in rat brain.

There are two distinct mRNAs that encode the precursor to gastrin-releasing peptide (GRP) in rat brain. These two messages arise from separate transcription initiation sites located approximately 400 base pairs apart, which are presumably regulated by separate promoters. In the present study, we mapped the distribution of neurons containing GRP mRNAs by in situ hybridization using cRNA and synthetic DNA probes specific for the 1.5 kb GRP transcript and probes complementary to both the 1.5 kb and 1.1 kb transcripts. The distribution of neurons expressing GRP mRNA appears to be wider than that previously observed by immunohistochemical studies, suggesting an important functional role for this neuropeptide in a number of brain regions. We detected the 1.5 kb transcript only in cingulate cortex, Ammon's horn of the hippocampus and in subiculum. In contrast, the probe which hybridized to both GRP mRNAs labeled a broad range of brain areas, including those containing the 1.5 kb mRNA. These data suggest that the 1.5 kb mRNA encoding rat GRP is expressed only in specific parts of the limbic system, whereas the expression of the 1.1 kb GRP message is more widespread.

Gastrin-releasing peptide gene-associated peptides are expressed in normal human fetal lung and small cell lung cancer: a novel peptide family found in man.

Mammalian gastrin-releasing peptide (GRP) is found in cells of neuroendocrine and neural origin, and GRP mediates a variety of physiological and trophic responses when it binds to high affinity cell surface receptors on effector cells. Analysis of cDNA clones derived from prepro-GRP mRNAs predict the concurrent expression of a unique series of peptide hormones, the GRP gene-associated peptides (GGAPs). Alternative RNA splicing of the primary GRP gene transcript results in mRNAs that could encode 3 distinct forms of GGAPs. Using specific antisera directed against synthetic peptides representing portions of the predicted GGAPs, we found multiple GGAP forms in the endocrine cells of human fetal lung and in human small cell lung cancer cells (SCLC). Within a single pulmonary endocrine cell, at least 2 of these 3 predicted forms could be demonstrated using immunohistochemical techniques. In addition, concordant expression of GRP and GGAPs was found in 10 SCLC cell lines and 3 human SCLC tumors. These findings establish GGAPs as a novel peptide family in man and warrant further investigation into their potential role in normal and malignant growth.

Transcriptional activation and DNase I hypersensitive sites are associated with selective expression of the gastrin-releasing peptide gene.

The gastrin-releasing peptide (GRP) is a neuropeptide hormone and growth factor produced normally by neural and neuroendocrine cells, as well as by human small-cell lung cancer (SCLC) tumors and derived cell lines. This study compares the structure of the human prepro-GRP gene in four SCLC cell lines that express variable levels of steady-state GRP mRNA. The regulation of GRP gene expression appears to be at the level of primary transcription based on nuclear run on studies. In the two SCLC cell lines expressing GRP we find a single transcription start site for GRP mRNA, and near this site we find four DNase I hypersensitive sites. These hypersensitive sites are absent in the two cell lines that do not express GRP. The presence of DNase hypersensitive sites in the promoter region of the GRP gene is the structural feature that best correlates with transcriptional activation. These four DNase hypersensitive sites are candidates for cis acting regulatory regions, which may be important in determining the level of transcription of the human prepro GRP gene.

Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor.

The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands.

The rat prepro gastrin releasing peptide gene is transcribed from two initiation sites in the brain.

The gastrin-releasing peptide (GRP) is a gastrointestinal hormone, a neuropeptide, and a growth factor. To study systematically GRP gene expression in embryonic and in adult mammals, we cloned the rat prepro-GRP gene from brain cDNA libraries. Analysis of these cDNA clones along with organ-specific mRNA studies show that there are prepro-GRP transcripts initiating in the brain, but not in the duodenum, from a novel promoter. The latter is located at least four hundred base pairs upstream from a second promoter that is active in both duodenum and in brain. In contrast to human prepro-GRP, there is no alternative processing of the 3'-region of rat brain and duodenum prepro-GRP transcripts; hence, these mRNAs encode a single 147 amino acid prepro-GRP hormone that is homologous to the third isoform of the human prepro-GRP hormone.

Swiss 3T3 mouse embryo fibroblasts transfected with a human prepro-GRP gene synthesize and secrete pro-GRP rather than GRP.

A prepro-gastrin-releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a approximately 15-kd pro-GRP hormone but not fully processed 2.8-kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro-GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the transfected cells.

Monoclonal antibodies for the in vitro detection of small cell lung cancer metastases in human bone marrow.

Three rat monoclonal antibodies were selected for the immunodetection of small cell lung cancer metastases in bone marrow and other hematologic samples. By membrane immunofluorescence, they define three distinct surface antigens here termed lung cancer-associated antigens or LCAs. The latter are widely expressed on small cell lung cancer and non-small cell lung cancer cells/cell lines, but not detectable on a variety of normal and transformed bone marrow, blood and lymphoid cells. Anti-LCA1 (IgM) is similar to the many anti-lacto-N-fucopentaose III IgM antibodies rasied against human tumors. In contrast, anti-LCA2 (IgG2b) and anti-LCA3 (IgG2a) define surface proteins of 29, 32, 41 and 98 kilodaltons, respectively, that have not been reported earlier. These three reagents have immunodiagnostic potential, since in combination they label all 49 lung cancer cell lines tested. Their ability to detect lung cancer metastases in patient's bone marrow samples is documented in an accompanying paper.

Immunodetection of small cell lung cancer metastases in bone marrow using three monoclonal antibodies.

Detection of bone marrow metastases by indirect immunofluorescence methods was investigated using three monoclonal antibodies (MoAbs) raised against small cell lung cancer (SCLC). These antibodies, designated anti-LCA1, -LCA2 and -LCA3, recognize three different antigens on the surface of SCLC cells. Eighty-four bone marrow samples from 74 different patients were studied. Whereas tumor cells were found in 32 (38%) by MoAb staining, only 10 (12%) were positively identified using conventional morphological methods. Nine out of the morphologically positive specimens showed reactivity with at least two monoclonal antibodies. Among the 32 samples proven positive by immunofluorescence, an important antigenic variability was noted. Anti-LCA1 recognized tumor cells in 62%, anti-LCA2 and anti-LCA3 in 53%. Due to the recognition of bone marrow involvement by fluorescence methods in 26% of the 34 patients classified as limited disease, a new subgroup of limited disease patients was defined whose prognosis remains undetermined. Our results confirm the utility of immunodetection in the diagnosis of SCLC bone marrow metastases and emphasize the advantage of using a panel of MoAbs with different antigenic specificities. Further study is needed to determine the prognostic significance of bone marrow involvement established by immunodetection.

Detection of small cell lung cancer bone marrow metastases by immunofluorescence.

It is well known that small cell lung cancer (SCLC) has a high propensity to metastasize to the bone marrow and that such involvement has a prognostic significance. A more accurate detection of these bone marrow metastases is thus mandatory. In this study, we analysed the results of the detection of these metastases using an indirect immunofluorescence test. For this purpose, 3 anti-SCLC rat monoclonal antibodies (MoAbs) specific for 3 different antigens (LCA1, LCA2, LCA3) have been utilized to examine 59 bone marrow samples from patients at time of diagnosis and 20 samples from chemotherapy treated patients. Eight patients had bone marrow clearly involved by morphological analysis. They all had fluorescent cells recognized at immunodetection with at least 2 MoAbs positive for 7 out of the 8 samples. Fifteen samples, negative by morphological analysis, were proven positive by immunofluorescence. In 12 cases, involvement was detected only by 1 MoAb (6 anti-LCA1, 2 anti-LCA2, 4 anti-LCA3). A correlation was found between the number of samples proven positive by morphological analysis and the number of positive MoAbs for these samples (p less than 0.005). Among the bone marrow samples provided by the 32 limited disease patients, LCA positive cells were detected in 9 (28%) compared to 14 out of the 27 (52%) samples from extensive disease patients (p less than 0.05). We concluded that the indirect immunofluorescence with a panel of MoAbs increases the rate of detection of bone marrow SCLC metastases.

Expression of biologically active and antigenically authentic parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein by a recombinant baculovirus.

The hemagglutinin-neuraminidase (HN) gene of human type 3 parainfluenza virus has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. HN protein produced in insect cells by the recombinant baculovirus appeared to be glycosylated, was transported to the cell surface, and was biologically active. All of the HN epitopes previously mapped functionally to a region(s) involved in neuraminidase and/or hemagglutination activities were conformationally unaltered on the recombinant protein. The HN produced in this system also induced a protective immune response in immunized cotton rats. From these studies we conclude that the HN expressed in insect cells represents a source of authentic HN glycoprotein suitable for structural analysis and immunization.

Human gastrin-releasing peptide gene maps to chromosome band 18q21.

A complementary DNA clone encoding human pre-pro gastrin-releasing peptide, a 27-amino acid neuropeptide and putative growth factor, was used to determine the chromosomal location of this gene. Southern blot hybridization to genomic DNA isolated from a panel of human-rodent somatic cell hybrids unambiguously maps this gene to human chromosome 18. In situ chromosomal hybridization confirms the hybrid data and further localized the gene to chromosome band 18q21. Karyotypic abnormalities in tumors and inherited disease states which involve chromosome band 18q21 may now be studied for correlated changes in the structure and expression of the human GRP gene.

Expression of the gastrin-releasing peptide gene in human small cell lung cancer. Evidence for alternative processing resulting in three distinct mRNAs.

cDNA clones to human prepro-gastrin-releasing peptide (prepro-GRP) mRNA detect synthesis of prepro-GRP-related transcripts in 4 of 7 small cell lung cancer (SCLC) cell lines and 1 of 2 metastatic SCLC tumors examined. A correlation is noted between prepro-GRP gene expression and the occurrence of bombesin-related immunoreactivity in SCLC cell lines. Examination of the structure of prepro-GRP transcripts found in SCLC reveals three types of prepro-GRP mRNA which differ in the structure of a putative GRP-associated peptide in the pro-GRP precursor. The subcellular distribution of prepro-GRP-related RNAs and structure of SCLC-derived prepro-GRP cDNA clones suggest that all three types of transcript could function as mRNAs, although there are differences in the prevalence of the different RNA types in different cellular compartments. Comparison of the sequence of cDNA clones with the sequence of a genomic prepro-GRP clone reveals that the three forms of prepro-GRP mRNA arise from a single primary transcript which undergoes alternative processing from two splice donor sites to two splice acceptor sites. The predicted amino acid sequence of the translated products of the three mRNAs are quite distinct, leading to predicted pro-GRP molecules of differing structure.

Molecular genetic analysis reveals chromosomal deletion, gene amplification, and autocrine growth factor production in the pathogenesis of human lung cancer.

These studies of lung cancer suggest that a number of molecular mechanisms may be important in the pathogenesis of lung cancer, especially SCLC. An inherited predisposition to develop SCLC may correlate with a nonfunctional, recessive allele for a gene (McKusick #18228, McKusick 1986) that maps to chromosome region 3p(14-23). Individuals at risk would be heterozygous for this allele in their germ line, carrying one copy of a normal functional gene and one mutant, recessive allele. Exposure to carcinogens, in particular cigarette smoke, can produce somatic genetic changes such as chromosomal deletion or gene mutation in the functional allele of this gene, unmasking the nonfunctional allele. Loss of this normal gene may alter the regulation of cell growth, perhaps by allowing the deregulated expression of proto-oncogenes of the myc family, or autocrine growth factors such as GRP and/or its receptor. Alternatively, loss of this gene may result in the cell returning to a less differentiated developmental state where growth regulation is less stringent. Persons with this mutant gene should be at increased risk to develop SCLC, and further RFLP analysis of the 3p region in SCLC may allow identification of specific haplotypes with increased risk of developing lung cancer. If this notion is correct, one might expect to find an increased frequency of second tumors in lung cancer patients and the presence of similar chromosomal deletions in second tumors arising in SCLC patients. In this regard, cured lung cancer patients, including those with SCLC, have a tenfold increased risk of developing a second lung cancer (Fontana 1977; Cortese et al. 1983; Johnson et al. 1986b). In fact, a chromosome 3p deletion along with other chromosomal abnormalities was identified in acute erythroleukemia cells arising in a long-term survivor of SCLC (Bradley et al. 1982), implicating this same region in the pathogenesis of both tumors. Other predictions include the correction of at least a portion of the defect by introducing a normal chromosome 3 into SCLC cells. While c-myc is expressed in many fetal and adult tissues, high-level expression of N- and L-myc is very restricted as to tissue and stage in the developing mouse, with N-myc expressed in the fetal but not adult lung, whereas the lung was the only adult tissue where L-myc expression was detected (Zimmerman et al. 1986). Could these patterns provide a clue to the differential expression of c-, N-, and L-myc found in different lung cancers (Nau et al. 1986)?(ABSTRACT TRUNCATED AT 400 WORDS)

Rat AL2, AL3, AL4 and AL5 monoclonal antibodies bind to the common acute lymphoblastic leukaemia antigen (CALLA gp 100).

Four distinct rat monoclonal antibodies against the common ALL antigen (CALLA, gp 100) were obtained in a single fusion. Rat AL2, AL3, AL4, AL5 and the previously reported mouse J5 monoclonal antibodies identified the same subsets of leukaemic cells. AL2 and AL3 reacted weakly with terminal transferase-positive cells in normal bone marrow and foetal liver, as well as with a minority of mature granulocytes in blood. Immunoprecipitation experiments and competitive binding assays demonstrated that the four rat antibodies and J5 bound to the same glycoprotein of approximately 100,000 mol. wt. This set of rat monoclonal antibodies directed against CALLA has not only a diagnostic usefulness but may also be of therapeutic value.

Rat hybridoma technology: culturing of rat myeloma cell line IR983F prior to cell fusion.

The nonsecreting IR983F rat myeloma cell line grew exponentially with a mean generation time of 16 h when reset daily at 5 X 10(5) cells/ml in culture medium with horse serum. High yields of rat-rat hybrids were systematically obtained when fusing exponentially growing IR983F cells with syngeneic immune spleen cells. The yields of hybrids secreting specific antibodies varied greatly according to the immunisation procedure. In the best conditions, 22 hybrids secreting anti-human leukocyte antibodies were recovered per 10(7) plated cells.