Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor–β1‐mediated regulatory abnormalities including up‐regulation of C‐Myc and MTA1

Abstract

Objective To examine whether and to what extent the intracellular trafficking features of HLA–B*2705, which is associated with the development of spondylarthritis (SpA), differ from those of HLA–B*2709 and HLA–B*0702, which are not associated with SpA. Methods HeLa cells were transfected with complementary DNA encoding for HLA–B proteins fused to Renilla luciferase or yellow fluorescent protein. The subcellular distribution of properly folded and unfolded/misfolded HLA–B proteins was examined by flow cytometry and confocal microscopy of cells labeled with ME1 and HC‐10 antibodies, respectively. HLA–B/HLA–B interactions were monitored in endoplasmic reticulum (ER)– and plasma membrane–enriched subcellular fractions, by bioluminescence resonance energy transfer (BRET). Results All 3 HLA–B alleles displayed a similar distribution pattern (properly folded heavy chain at the cell surface, unfolded/misfolded proteins only in the cytoplasm). By means of BRET, we provided evidence that both HLA–B*2705 and HLA–B*2709 formed more oligomers in the ER and the plasma membrane than did HLA–B*0702. The propensity of HLA–B*2705 to form oligomers in the ER was partly attributable to residue Cys67 of the molecule. For all 3 alleles, increased expression of HLA–B proteins was associated with intracytoplasmic accumulation of unfolded/misfolded proteins and intracellular vesicles, probably corresponding to expanded ER–Golgi intermediate compartments, in which these proteins accumulated together with the stress sensor BiP. Conclusion Our results suggest that the difference in disease susceptibility conferred by HLA–B*2705 and HLA–B*2709 cannot be explained by their different propensity to form dimers or misfolded proteins, thus presumably implicating other, still unknown factors.

The genomic landscape of small intestine neuroendocrine tumors

“…Moreover, preclinical data from studies in cell lines suggest that the TGF-β pathway might be a therapeutic target in small bowel NENs (22,23). The SI-NET cell line KRJ was induced to proliferate by TGF-β1 (24), increasing c-myc expression, downregulating p21, and cross-activating ERK1/2 in KRJ SI-NET cells, but not in normal small intestinal enterochromaffin cells. Furthermore, SMAD alterations might adversely affect responses to fluorouracil-based therapy,…”

Increased Expression of 14-3-3β Promotes Tumor Progression and Predicts Extrahepatic Metastasis and Worse Survival in Hepatocellular Carcinoma

“…37,38 In brief, 1,500 cells were seeded onto the flat bottom of 96-well plates in 200 L serum-free medium. After cells attached, the medium was replaced with fresh medium containing 10% FBS for incubation at the indicated times (0, 24, 48, 74 hours).…”

Inhibition of proliferation of small intestinal and bronchopulmonary neuroendocrine cell lines by using peptide analogs targeting receptors

“…KRJ-I cells were kept in Ham F12 medium (Gibco, Gaithersburg, Md) containing 10% fetal bovine serum (FBS) (SigmaAldrich, St. Louis, Mo), penicillin 100 U/mL, and streptomycin (100 mg/mL). 21,29,30 For NCI-H720, a 1:1 solution of Ham F12 and Dulbecco minimal essential medium (DMEM) supplemented with final concentrations of FBS (5%), insulin (0.005 mg/mL), transferrin (0.01 mg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), b-estradiol (10 nM), HEPES medium (10 mM), and L-glutamine (2 mM) was used. 20 The adhesive growing NCI-H727 cells were kept at 378C in RPMI 1640 medium containing final concentrations of FBS (10%), L-glutamine (2 mM), sodium pyruvate (1 mM), and glucose (2.5 g/L).…”

“…29,30 Lanes 1 and 2 contained negative (medium only) and positive (cell suspension) controls. Drugs were diluted in PBS and applied at final concentrations from 1 nM to 100 nM (n 5 6 wells for each concentration/plate).…”