Neuronal differentiation is accompanied by NSP-C expression.

Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto)chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.

A comparison of NSP-reticulons with conventional neuroendocrine markers in immunophenotyping of lung cancers.

Neuroendocrine-specific protein (NSP)-reticulons are endoplasmic reticulum-associated protein complexes, which have been identified as markers for neuroendocrine differentiation. In this study, the expression of two members of the family of NSP-reticulons, NSP-A and NSP-C, have been investigated in different types of lung cancer and compared with the expression patterns of five conventional neuroendocrine markers, the neural cell adhesion molecule (NCAM), synaptophysin, chromogranin A, Leu-7, and neurofilament proteins. NSP-A and NSP-C antibodies were reactive with most carcinoid tumour and small cell lung carcinoma (SCLC) cases, while atypical carcinoid tumours showed a variable expression. In the total group of neuroendocrine tumours, a high concordance of expression was found between NSP-A and NSP-C, while their expression correlated well with NCAM and synaptophysin positivity. Chromogranin A, Leu-7, and neurofilament proteins were shown to be expressed to a limited extent in these neuroendocrine tumours. In a selected group of non-SCLCs known to exhibit neuroendocrine features, NSP-A expression was detected at much higher frequency than NSP-C. In virtually all NSP-A positive cases, this expression was associated with one or more of the other neuroendocrine markers. NSP-A expression showed a stronger correlation with conventional neuroendocrine markers than NCAM. In detecting neuroendocrine differentiation in non-SCLC, NSP-A is more sensitive than synaptophysin, chromogranin A, Leu-7, and neurofilament proteins. It is concluded that NSP-reticulons are valuable markers in the diagnosis of neuroendocrine differentiation in non-SCLC and should be used in conjunction with NCAM.

An achaete-scute homologue essential for neuroendocrine differentiation in the lung.

In Drosophila and in vertebrates, the achaete-scute family of basic helix-loop-helix transcription factors plays a critical developmental role in neuronal commitment and differentiation. Relatively little is known, however, about the transcriptional control of neural features in cells outside a neuronal context. A minority of normal bronchial epithelial cells and many lung cancers, especially small-cell lung cancer, exhibit a neuroendocrine phenotype that may reflect a common precursor cell population. We show here that human achaete-scute homologue-1 (hASH1) is selectively expressed in normal fetal pulmonary neuroendocrine cells, as well as in the diverse range of lung cancers with neuroendocrine features. Strikingly, newborn mice bearing a disruption of the ASH1 gene have no detectable pulmonary neuroendocrine cells. Depletion of this transcription factor from lung cancer cells by antisense oligonucleotides results in a significant decrease in the expression of neuroendrocrine markers. Thus, a homologue of Drosophila neural fate determination genes seems to be necessary for progression of lung epithelial cells through a neuroendocrine differentiation pathway that is a feature of small-cell lung cancer, the most lethal form of human lung cancer.

Genomic organization of the human NSP gene, prototype of a novel gene family encoding reticulons.

Recently, cDNA cloning and expression of three mRNA variants of the human NSP gene were described. This neuroendocrine-specific gene encodes three NSP protein isoforms with unique amino-terminal parts, but common carboxy-terminal parts. The proteins, with yet unknown function, are associated with the endoplasmic reticulum and therefore are named NSP reticulons. Potentially, these proteins are neuroendocrine markers of a novel category in human lung cancer diagnosis. Here, the genomic organization of this gene was studied by analysis of genomic clones isolated from lambda phage and YAC libraries. The NSP exons were found to be dispersed over a genomic region of about 275 kb. The present elucidation of the genomic organization of the NSP gene explains the generation of NSP mRNA variants encoding NSP protein isoforms. Multiple promoters rather than alternative splicing of internal exons seem to be involved in this diversity. Furthermore, comparison of NSP genomic and cDNA sequences with databank nucleotide sequences resulted in the discovery of other human members of this novel family of reticulons encoding genes.

Neuroendocrine-specific protein C (NSP-C): subcellular localization and differential expression in relation to NSP-A.

A mouse monoclonal antibody RNL-4, as well as rabbit polyclonal antiserum POL-8 were raised against a synthetic peptide, encompassing the first twenty unique amino-terminal amino acid residues of NSP-C. The specificity of both immunoreagents was established in an ELISA assay using the synthetic peptide and by their immunoreactivity to NSP-C fusion proteins. Immunofluorescence analysis of COS-1 cells, transfected with NSP-C cDNA, showed staining of the endoplasmic reticulum with RNL-4 and POL-8. No cross-reactivity of these reagents with NSP-A or NSP-B was seen. Immunohistochemical studies in normal human tissues showed expression of NSP-C in tissues of neural and neuroendocrine origin, i.e. neurons of the central and peripheral nervous system, the neurohypophysis, adrenal medulla, adenohypophysis, pars intermedia, and in sporadic neuroedocrine cells of the lung. Expression of NSP-C was found in several small cell lung cancer (SCLC) cell lines, in non-SCLC cell lines with neuroendocrine features, but not in typical non-SCLC cell lines. Also, in a neuroblastoma cell line NSP-C expression was observed. Immunoblotting and immunoprecipitation studies with RNL-4 and POL-8 identified the 23 kDa NSP-C polypeptide in these cell lines. Immunofluorescence microscopy showed that also in these cell lines NSP-C is located at the endoplasmic reticulum, as shown before for NSP-A and NSP-B. In some of the cell lines coexpression of NSP-A and NSP-C was observed, while in others only one of the two could be detected. The differential expression of NSP-A and NSP-C in these cell lines is confirmed by immunoblotting and was also evident at the mRNA level. When NSP-A and NSP-C were coexpressed, the number of NSP-C-positive cells was always less than the number of NSP-A-positive cells. A partial colocalization of NSPs was observed in the endoplasmic reticulum. Cell fractionation studies revealed that both proteins are retained in the membranous fraction of the cell, from which they can be solubilized by Triton X-100. Immunoprecipitation analyses under native conditions indicate that NSP-C does not need to associate with NSP-A to form high molecular weight NSP-reticulons.

Subcellular localization and supramolecular organization of neuroendocrine-specific protein B (NSP-B) in small cell lung cancer.

We have recently isolated and characterized a novel gene that is expressed in a neuroendocrine-specific fashion and was therefore designated neuroendocrine-specific protein (NSP)-gene. The NSP-gene encodes three transcripts of different size, with unique 5'-sequences and completely overlapping 3'-sequences. The resulting proteins have an apparent molecular mass of 135 kDa as determined for NSP-A and 23 kDa as found for NSP-C. In the present study we focused on the biochemical characterization and subcellular localization of NSP-B, so far only found to be expressed in the neuroendocrine lung cancer cell line NCI-H82, and its relation to NSP-A. Transfection studies with the NSP-B transcript in COS-1 cells, followed by immunoprecipitation, resulted in a set of proteins ranging in molecular mass from 35 to 45 kDa, identical to NSP-Bs detected by immunoblotting in NCI-H82. In this cell line a major NSP-B triplet in the 43 to 45 kDa range and a 35 kDa NSP-B were consistently detected. Only the 45 kDa NSP-B was found to be phosphorylated. The observed pI values of the 43 to 45 kDa triplet ranged from 4.8 to 5.0, while the 35 kDa NSP-B has a more basic pI value of 5.7. Gel filtration studies show that NSP-A and NSP-B form supramolecular aggregates with a molecular mass of over 500 kDa, present to a minor extent in the phosphate buffered saline soluble cell fraction, but mainly occurring in the membranous pellet fraction from which they can be solubilized by Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)

NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum.

The novel NSP gene was previously shown to encode, among a variety of neuroendocrine cell types, two 3'-overlapping transcripts, a 3.4 kb one for NSP-A (776 amino acids) and a 1.8 kb one for NSP-C (208 amino acids). The deduced proteins, which were predicted to possess distinct amino-terminal regions, appeared to exhibit some architectural resemblance to known neuroendocrine proteins. In this paper the biochemical characterization and subcellular localization of the two proteins is addressed. In vitro translation of NSP-A and -C RNA produced proteins of about 135 and 23 kDa, respectively. Proteins of similar molecular mass were also detected in immunoprecipitation and western blot analyses of neural and endocrine cells using specific anti-NSP-A or -C antisera; some heterogeneity of NSP-A was observed. NSP-A, but not NSP-C, appeared to be highly phosphorylated and preferentially on serine residues. In immunocytochemical studies, we demonstrated that NSP-A and -C are associated with the endoplasmic reticulum; NSP-A was found to co-localize with SERCA2b, a membrane-associated Ca(2+)-ATPase of the endoplasmic reticulum. In Purkinje cells, we found NSP-immunostaining in the perikaryon, the extensive dendritic tree and the axon, also suggesting association with the smooth endoplasmic reticulum. Biochemical studies of NSP-A provided evidence that NSP-A is strongly associated with microsomal membranes and analysis of deletion mutants of NSP-A revealed that the hydrophobic carboxy-terminal portion of the protein, which is also present in NSP-C, is critical for membrane binding. Through database searches, finally, we found two different NSP-related sequences, one in a sequenced region of human chromosome 19, and the second in a human, pancreatic islet-derived partial cDNA, suggesting that the NSP gene is the prototype of a larger gene family. The results of our studies seem to indicate that the NSP-encoded proteins are novel, membrane-anchored components of the endoplasmic reticulum for which we propose the name reticulons.

NSP-encoded reticulons are neuroendocrine markers of a novel category in human lung cancer diagnosis.

The NSP gene was recently shown to constitute the prototype of a novel gene family, to be selectively transcribed in neural and endocrine cells, and to encode three overlapping proteins, NSP-A, NSP-B, and NSP-C. These proteins were collectively designated reticulons, because they were found to be anchored to membranes of the endoplasmic reticulum through their common carboxy-terminal regions. The goal of the present study was to determine whether the reticulons might be used as markers for neuroendocrine differentiation in human lung tumors. Therefore, the tissue distribution of the NSP-A protein was studied and expression in human lung tumors was evaluated. Immunohistochemical analysis of normal tissues with monoclonal antibodies specifically recognizing the NSP-A protein indicated that NSP-A exhibits a distinct neuroendocrine distribution pattern since it was found to be expressed in a variety of cells with an established neuroendocrine phenotype but not in cells lacking such features. Results with specimens of a wide variety of primary human tumors provided further support for this claim. Immunohistochemical analysis of primary lung carcinomas revealed that NSP-A was readily detectable in small cell lung carcinoma (SCLCs) (8 of 12) and carcinoid tumors of the lung (3 of 3) but not in nonneuroendocrine non-SCLCs (0 of 10). In 13 of 27 non-SCLCs expressing the neural cell adhesion molecule and/or neurofilament proteins, however, NSP-A was found to be expressed. Northern blot analysis of human lung carcinoma cell lines revealed expression of NSP-A- and/or NSP-C-encoding mRNAs in all 18 SCLC cell lines that were studied, except one; however, no expression of these mRNAs could be detected in any of the 11 non-SCLC cell lines tested. The NSP transcript encoding NSP-B was found only in SCLC cell line NCI-H82. In conclusion, the results of our studies suggest that, in lung tumor cells, expression of NSP-A and most likely also NSP-C is restricted to cells with a neuroendocrine phenotype.

Molecular analysis of expression in rat brain of NSP-A, a novel neuroendocrine-specific protein of the endoplasmic reticulum.

Previous studies have established that the novel neuroendocrine-specific NSP gene encodes three carboxy-terminally overlapping proteins, NSP-A, NSP-B and NSP-C which are anchored to membranes of the endoplasmic reticulum. Here, we report results of studies in which expression of NSP-A in rat brain was investigated. Immunization of mice with a bacterial hybrid protein containing almost all NSP-A sequences led to the isolation of five monoclonal anti-NSP-A antibodies. The corresponding epitopes were found to be mapping to two regions unique to NSP-A. In Western blot analysis of rat cerebrum and cerebellum using these antibodies, proteins of about 145 kDa were detected. An immunohistochemical study of rat brain revealed the presence of NSP-A in many brain regions, particularly in cerebellar Purkinje cells, in neurons of the superior colliculus and of the pyriform and enthorhinal cortex, in fibers of the basal ganglia and several hippocampal regions including CA3 (stratum lucidum) and the dentate gyrus, in the induseum griseum and in the subcommissural organ, suggesting a role of NSP-A in many areas of the brain.

Regional mapping of the human NSP gene to chromosome region 14q21-->q22 by fluorescence in situ hybridization analysis.

Genetic sequences of the novel NSP gene, which encodes neuroendocrine-specific proteins, were isolated from cDNA libraries constructed with mRNA isolated from human lung carcinoma cells. Hybridization analysis of a panel of human x mouse cell hybrids with an 0.8-kb NSP cDNA probe indicated that the human NSP gene is probably located on chromosome 14. Fluorescence in situ hybridization analysis of metaphase chromosomes using overlapping genomic clones of NSP as a probe localized the NSP gene to chromosome region 14q21-->q22.

Cluster-10 lung-cancer antibodies recognize NSPs, novel neuro-endocrine proteins associated with membranes of the endoplasmic reticulum.

We have identified a novel gene (the NSP gene) encoding 3 transcripts and coding for 3 neuroendocrine-specific proteins (NSPs), by screening a cDNA expression library of the small-cell lung-cancer (SCLC) cell line NCI-H82 with the cluster-10 lung-cancer antibodies RNL2 and RNL3. The 3 transcripts code for NSPs with apparent molecular weights of 135 kDa (NSP-A), 43 to 45 and 35 kDa (NSP-B) and 23 kDa (NSP-C). NSP-A and NSP-B are recognized by antibodies RNL2 and RNL3, while second-generation antibodies, specifically recognizing NSP-A and NSP-C, have been produced after immunization with a hybrid protein obtained after bacterial expression of the largest NSP-transcript or with a synthetic peptide specific for NSP-C. The NSPs exhibit a highly restricted distribution pattern and are found mainly in neural and neuro-endocrine cell types, and in neuro-endocrine tumours. Of the different types of lung tumours, mainly SCLC and carcinoids were positive in immunocytochemical assays using the anti-NSP antibodies, while non-SCLC were in general negative. The subcellular distribution of the NSPs was studied in human SCLC cell lines. They do not co-localize with components typical of neuro-endocrine granules, such as synaptophysin and chromogranin. The use of NSP antibodies in the immunofluorescence technique applied to cultured SCLC cells, made it obvious that these proteins localize in the endoplasmic reticulum. Cell fractionation procedures, monitored by immunoblotting assays, indicated an association of the NSPs with the microsomal fraction, from which they could be solubilized with Triton X-100. Gel filtration studies with this solubilized fraction revealed that NSPs form supramolecular aggregates with a molecular weight of more then 500 kDa.

Cloning and expression of alternative transcripts of a novel neuroendocrine-specific gene and identification of its 135-kDa translational product.

Monoclonal antibodies RNL-2 and RNL-3 were previously shown to react with four 35-45-kDa proteins, expressed only in small cell lung carcinoma NCI-H82 cells, but to stain a subset of neuroendocrine tissues and neoplasms (Broers, J. L. V., Mijnheere, E. P., Klein Rot, M., Schaart, G., Sijlmans, A., Boerman, O. C., and Ramaekers, F. C. S. (1991) Cancer 67, 619-633). We used RNL-2 and RNL-3 to isolate cDNA sequences that code for proteins containing the two corresponding epitopes and utilized such cDNAs to develop second generation antibodies. Using these antibodies, we identified a novel 135-kDa protein. The corresponding cDNAs were found to belong to a previously unknown gene with a neuroendocrine-specific expression pattern, tentatively designated NSP gene. NSP transcription appeared to result in mRNAs of 3.4 and 1.8 kilobases (kb). In the NCI-H82 cells only, an apparently aberrant transcript of 2.3 kb was found. cDNAs containing coding sequences of the 3.4-, 2.3-, and 1.8-kb transcripts were isolated, and nucleotide sequence analysis revealed extensive sequence overlap and open reading frames for proteins of 776, 356, and 208 amino acids, respectively. The three deduced proteins all have a common carboxyl-terminal part with two large hydrophobic regions. Transfection of the complete coding sequences of the 3.4-kb transcript resulted in the production of a protein with an apparent molecular mass of 135 kDa. This protein is predicted to be highly negatively charged (calculated pI of 4.35), to be rich in proline and serine, and to contain multiple potential phosphorylation sites.