Characterization of the functional and growth properties of long-term cell cultures established from a human somatostatinoma.

In somatostatinoma, a rare malignant somatostatin (SST)-secreting neoplasia, tumour regression is rarely observed, implying the need for novel antiproliferative strategies. Here, we characterized a long-term culture (SST-secreting cancer (SS-C cells)) established from a human somatostatinoma. High concentrations of SST and chromogranin A were released by SS-C cells and SST release was stimulated by depolarizing stimuli and inhibited by the SST analogue, octreotide. SS-C cells expressed mRNA for SST receptor (SSTR) subtypes 1, 2 and 4, being also able to bind native SST. Moreover, SS-C cells were positively stained with an antibody to SSTR2. SS-C cells also expressed interferon-gamma (IFN-gamma) receptor mRNA and measurable telomerase activity. Our findings indicate that in vitro exposure of SS-C cells to native SST-28, to octreotide, to IFN-gamma, or to 3'-azido-3'deoxythymidine (AZT), a telomerase inhibitor, results in inhibition of SS-C cell proliferation. Concomitant with growth inhibition, apoptosis was detected in SST-, octreotide-, IFN-gamma- or AZT-treated SS-C cell cultures. Taken together our results characterized native SST, SST analogues, IFN-gamma and a telomerase inhibitor as growth-inhibiting and proapoptotic stimuli in cultured human somatostatinoma cells. Based on these findings, the potential of SST analogues, IFN-gamma and AZT, alone or in combination, should be further explored in the medical treatment of somatostatinoma.

Estrogen metabolism in human colorectal cancer cells.

Epidemiological and "in vitro" studies support a direct role of estrogens in the pathogenesis and/or progression of colorectal cancer (CRC). Recent observations suggest a local synthesis of 17beta-estradiol (E(2)). In the present study, the CRC estrogen receptor beta (ERbeta) positive HCT8, HCT116, DLD-1 and LoVo cell lines were evaluated for expression of functional 17beta-hydroxysteroid dehydrogenase (17betaHSD) types 1, 2, 3, and 4. RT-PCR analysis revealed that while 17betaHSD1 and 17betaHSD4 were expressed in all the four cell lines, 17betaHSD2 and 17betaHSD3 were expressed in a cell-specific manner. The interconversion of tritiated estrone (E(1)) or E(2) evaluated by thin layer chromatography of conditioned media revealed that in HCT8, HCT116, and DLD-1 cells both reductive and oxidative activities were present, the latter showing K(m) values (approximately 10 microM) 40-fold higher than the former (approximately 250 nM). On the contrary, in LoVo cells, estrogens were almost (approximately 90%) completely metabolized to hydrophile compounds. Charcoal-dextrane (DC) stripped fetal calf serum (FCS) (10%), E(2) (10nM), Vitamin D(3) (100nM) and the combined E(2) and Vitamin D(3) treatment were evaluated for modulation of 17betaHSD isoenzymes gene expression and activity. Gene expression and activity of 17betaHSD reductive and oxidative isoenzymes were respectively inhibited and enhanced by Vitamin D(3) in HCT8 and LoVo cells. Surprisingly, DC-FCS induced a marked increase of estrogen metabolism toward hydrophile metabolites in all four cell lines. In conclusion, our results clearly show that metabolism of estrogens by 17betaHSD isoenzymes is functional and modulated by external stimuli in continuous neoplastic colonic epithelial cell lines.

Ribozyme-mediated compensatory induction of menin-oncosuppressor function in primary fibroblasts from MEN1 patients.

Multiple endocrine neoplasia type 1 (MEN1) syndrome is characterized by the occurrence of tumors of parathyroids, neuroendocrine cells of the gastro-enteropancreatic tract and anterior pituitary. MEN1 gene encodes menin-oncosuppressor protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors. We have analyzed the MEN1 mRNA and menin expression in fibroblasts from normal skin biopsies and from MEN1 patients (two with a frameshift 738del4 (exon 3) mutation, introducing a premature stop codon, and an individual with an R460X (exon 10) nonsense mutation). The expression of full-length menin protein did not differ between MEN1 and normal fibroblasts. Wild-type alleles mRNAs were expressed in MEN1 patients, whereas mutant alleles were partially degraded by nonsense-mediated mRNA decay pathway, suggesting a mechanism of compensation for allelic loss by the up-regulation of wild-type menin expression at a post-transcriptional level. Small-interfering RNA silencing of the wild-type mRNA allele abolished menin compensation, whereas the ribozyme silencing of the MEN1-mutated mRNA allele resulted in strongly enhanced wild-type menin expression. Gel-retardation analysis showed that in vitro-specific RNA-protein complexes bound to MEN1 mRNA. These findings contribute to the understanding of tumorigenesis in MEN1, offering the basis for the development of RNA-based therapies in MEN1 gene mutation carriers.

A comparison of methods for the analysis of low abundance proteins in desmoid tumor cells.

The desmoids are a group of rare clinically diverse, deep-seated fibrous neoplasms. The exact etiology is unknown, but several factors are considered to be positively correlated with their development and growth, i.e., genetic and hormonal factors and trauma. These tumors may be sporadic or associated with a genetic disease such as familial adenomatous polyposis (FAP). Devoid of metastatic potential, they tend to form large, infiltrative masses which, if not completely excised, recur repeatedly. Although surgery is widely accepted as the first-line treatment for extra-abdominal and abdominal wall desmoids, a proportion of cases are successfully palliated with either estrogen antagonists (tamoxifen, toremifene, and raloxifene) or nonsteroidal anti-inflammatory drugs. We describe and compare four methods for evaluating the expression of estrogen receptors alpha/beta and COX-1 and COX-2 in desmoid tumor-derived cells and tissues: immunocytochemistry, immunohistochemistry, RT-PCR, and two-color Western blot detection with the Odyssey infrared imaging system. Through this comparative analysis, Western blot with Odyssey was recognized as the best method to analyze the expression particularly of low expressed proteins in desmoid-derived cells. The use of a specific and reliable assessment method becomes fundamental in the evaluation of the presence and modulation of proteins which are important but weakly expressed in these rare tumors.