Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo

Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.

Preferences for chemotherapy in patients with advanced non-small cell lung cancer: descriptive study based on scripted interviews

Objective: To determine how patients with lung cancer value the trade off between the survival benefit of chemotherapy and its toxicities.

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Inherited susceptibility to uterine leiomyomas and renal cell cancer

Herein we report the clinical, histopathological, and molecular features of a cancer syndrome with predisposition to uterine leiomyomas and papillary renal cell carcinoma. The studied kindred included 11 family members with uterine leiomyomas and two with uterine leiomyosarcoma. Seven individuals had a history of cutaneous nodules, two of which were confirmed to be cutaneous leiomyomatosis. The four kidney cancer cases occurred in young (33- to 48-year-old) females and displayed a unique natural history. All these kidney cancers displayed a distinct papillary histology and presented as unilateral solitary lesions that had metastasized at the time of diagnosis. Genetic-marker analysis mapped the predisposition gene to chromosome 1q. Losses of the normal chromosome 1q were observed in tumors that had occurred in the kindred, including a uterine leiomyoma. Moreover, the observed histological features were used as a tool to diagnose a second kindred displaying the phenotype. We have shown that predisposition to uterine leiomyomas and papillary renal cell cancer can be inherited dominantly through the hereditary leiomyomatosis and renal cell cancer (HLRCC) gene. The HLRCC gene maps to chromosome 1q and is likely to be a tumor suppressor. Clinical, histopathological, and molecular tools are now available for accurate detection and diagnosis of this cancer syndrome.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity

A covalent conjugate (NR-LU-10/SA) was prepared between streptavidin (SA) and NR-LU-10, a mAb that binds an antigen expressed on the surface of most human carcinomas. NR-LU-10/SA was injected into nude mice bearing human tumor xenografts. Injection of biotinylated galactosyl-human serum albumin reduced the circulating levels of conjugate by 95%. Subsequent administration of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin achieved peak uptake at the tumor within 2 hr while >80% of the radioactivity was eliminated in the urine. A single dose of 600–800 μCi of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin produced cures in 10/10 mice with established (>200 mm3) s.c. human small cell lung or colon cancer xenografts and 8/10 cures in mice with human breast cancer xenografts without significant toxicity.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

Mammalian Scratch: A neural-specific Snail family transcriptional repressor

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix–loop–helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Poly (alpha 2,8-deaminoneuraminic acid) is expressed in lung on a single 150-kDa glycoprotein and is an oncodevelopmental antigen.

Homopolymers of alpha 2,8-linked N-acetylneuraminic acid [poly(alpha 2,8-Neu5Ac)] of the neural cell adhesion molecule NCAM have been shown to be temporally expressed during lung development and represent a marker for small cell lung carcinoma. We report the presence of a further polysialic acid in lung that consists of oligo/polymers of alpha 2,8-linked deaminoneuraminic acid residues [poly (alpha 2,8-KDN)], as detected with a monoclonal antibody in conjunction with a specific sialidase. Although the various cell types forming the bronchi, alveolar septs, and blood vessels were positive for poly (alpha 2,8-KDN) by immunohistochemistry, this polysialic acid was found on a single 150-kDa glycoprotein by immunoblot analysis. The poly(alpha 2,8-KDN)-bearing glycoprotein was not related to an NCAM protein based on immunochemical criteria. The expression of the poly (alpha 2,8-KDN) was developmentally regulated as evidenced by its gradual disappearance in the rat lung parenchyma commencing 1 week after birth. In adult lung the blood vessel endothelia and the smooth muscle fibers of both blood vessels and bronchi were positive but not the bronchial and alveolar epithelium. The poly (alpha 2,8-KDN)-bearing 150-kDa glycoprotein became reexpressed in various histological types of lung carcinomas and cell lines derived from them and represents a new oncodevelopmental antigen in lung.

Species-specific metastasis of human tumor cells in the severe combined immunodeficiency mouse engrafted with human tissue.

We have attempted to model human metastatic disease by implanting human target organs into the immunodeficient C.B-17 scid/scid (severe combined immunodeficiency; SCID) mouse, creating SCID-hu mice. Preferential metastasis to implants of human fetal lung and human fetal bone marrow occurred after i.v. injection of human small cell lung cancer (SCLC) cells into SCID-hu mice; the homologous mouse organs were spared. Clinically more aggressive variant SCLC cells metastasized more efficiently to human fetal lung implants than did cells from classic SCLC. Metastasis of variant SCLC to human fetal bone marrow was enhanced in SCID-hu mice exposed to gamma-irradiation or to interleukin 1 alpha. These data indicate that the SCID-hu mice may provide a model in which to study species- and tissue-specific steps of the human metastatic process.

Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53

The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21waf1 promoter and to stimulate p53RE-dependent transcription but not basal transcription in vitro. Its DNA-binding activity was inhibited by the wild type but not mutant p53RE-containing DNA oligomers. Also, this p53RE-binding activity was found in human p53 null Saos-2 osteosarcoma and H1299 small cell lung carcinoma cells. Interestingly, this activity exhibited a p53RE sequence preference that was distinct from the p53 protein. The activity is neither p53 nor p73, because anti-p53 or anti-73 antibodies were unable to detect this purified protein nor were the antibodies able to alter the p53-like activity, the p53RE-protein complex. These results demonstrate that, besides p73, an additional p53-like protein exists in cells, which is named NBP for non-p53, p53RE binding protein.