Targeting MAGE-C1/CT7 expression increases cell sensitivity to the proteasome inhibitor bortezomib in multiple myeloma cell lines

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26-27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70-80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.     

Human StarD5, a cytosolic StAR-related lipid binding protein

Recently identified StarD5 belongs to the StarD4 subfamily, a subfamily of steroidogenic acute regulatory related lipid transfer (START) domain proteins that includes StarD4 and StarD6, proteins whose functions remain unknown. The objective of this study was to confirm StarD5's protein localization and sterol binding capabilities as measures to pursue function. Using rabbit polyclonal antibody against newly purified human histidine-tagged/StarD5 protein, StarD5 was detected in human liver. In parallel studies, increased expression of StarD5 in primary hepatocytes led to a marked increase in microsomal free cholesterol. Cell fractionation studies demonstrated StarD5 protein in liver cytosolic fractions only, suggesting StarD5 as a directional cytosolic sterol carrier. Supportive in vitro binding assays demonstrated a concentration-dependent binding of cholesterol by StarD5 similar to that of the cholesterol binding START domain protein StarD1. In contrast to selective cholesterol binding by StarD1, StarD5 bound the potent regulatory oxysterol, 25-hydroxycholesterol, in a concentration-dependent manner. StarD5 binding appeared selective for cholesterol and 25-hydroxycholesterol, as no binding was observed for other tested sterols. The ability of StarD5 to bind not only cholesterol but also 25-hydroxycholesterol, a potent inflammatory mediator and regulatory oxysterol, raises basic fundamental questions about StarD5's role in the maintenance of cellular cholesterol homeostasis.     

Organometallic nickel(II) complexes with dithiophosphate, dithiophosphonate and monothiophosphonate ligands

The hydroxo complex [NBu₄]₂[Ni₂(C₆F₅)₄(μ-OH)₂] reacts with ammonium O,O^′-dialkyldithiophosphates, O-alkyl-p-methoxyphenyldithiophosphonate acids and ammonium O-alkylferrocenyldithiophosphonates in dichloromethane under mild conditions to give, respectively, [NBu₄][Ni(C₆F₅)₂{S(S)P(OR)₂}] (R=Me (1), Et (2), ⁱPr (3)) and [NBu₄][Ni(C₆F₅)₂{S(S)P(OR)Ar}] (Ar=p-MeOC₆H₄, R=Me (4), Et (5), ⁱPr (6); Ar=ferrocenyl; R=Me (7), Et (8), ⁱPr (9)). The monothiophosphonate nickel complexes [NBu₄][Ni(C₆F₅)₂{S(S)P(OR)(ferrocenyl)}] (R=Et (10), ⁱPr (11)) are obtained by reaction of the hydroxo complex with O-alkylferrocenyldithiophosphonate acids. Analytical (C, H, N, S), conductivity, and spectroscopic (IR, ¹H, ¹⁹F and ³¹P NMR, and FAB-MS) data were used for structural assignments. A single-crystal X-ray diffraction study of [NBu₄][Ni(C₆F₅)₂{S(S)P(OMe)(p-MeOC₆H₄)}] (4) and [NBu₄][Ni(C₆F₅)₂{S(O)P(OEt)(ferrocenyl)}] (10) shows that in both cases the coordination around the nickel atom es essentially square planar with NiC₂S₂ and NiC₂SO central cores, respectively.     

Extracellular calcium acts as a "third messenger" to regulate enzyme and alkaline secretion

It is generally assumed that the functional consequences of stimulation with Ca2+ -mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ "sensor" raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a "third messenger" via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+.     

Non-smoking coke oven workers show an occupational PAH exposure-related increase in urinary mutagens

We examined the urinary mutagenicity in the YG1024 Salmonella typhimurium strain in the presence of S9 mix, of 31 male non-smoking coke oven workers and an equal number of controls matched for gender and dietary habits. Occupational PAH exposure to the workers was assessed by means of the individual urinary post-shift excretion of 1-pyrenol (mean +/- S.D.: 5.41 +/- 6.06 micromole/mol creatinine). Eleven urinary extracts of workers (35.5%) were clearly mutagenic (with at least a doubling of the number of spontaneous revertants), against only two samples in the control group (6.5%) (chi2-test; chi2 = 7.883; P < 0.01). Moreover, the mean mutagenic activity level corrected for dilution/concentration of the urine was about three times higher in coke oven workers than in matched controls (mean +/- S.D. (range) 495 +/- 407 (89.7-1603) versus 186 +/- 113 (14.2-524) net revertants/mmol creatinine; Mann-Whitney U-test, z = 3.86, P < 0.001). Simple linear regression analysis showed that the coke workers' urinary mutagenic activity is associated with the PAH occupation-related urinary excretion of 1-pyrenol (r = 0.41, P = 0.0215). This study definitely demonstrates an occupation-related exposure of coke oven workers' bladder epithelium to mutagenic PAH metabolites. This factor, mainly in the case of high exposure studied here, may account for a higher bladder cancer risk in coke oven workers.     

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.     

Identification of a novel sulfonated oxysterol, 5-cholesten-3beta,25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria

This study reports the discovery of a novel sulfonated oxysterol found at high levels in the mitochondria and nuclei of primary rat hepatocytes after overexpression of the gene encoding steroidogenic acute regulatory protein (StarD1). Forty-eight hours after infection of primary rat hepatocytes with recombinant adenovirus encoding StarD1, rates of bile acid synthesis increased by 4-fold. Concurrently, [(14)C]cholesterol metabolites (oxysterols) were increased dramatically in both the mitochondria and nuclei of StarD1-overexpressing cells, but not in culture medium. A water-soluble [(14)C]oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Enzymatic digestion, HPLC, and tandem mass spectrometry analysis identified the water-soluble oxysterol as 5-cholesten-3beta,25-diol 3-sulfonate. Further experiments detected this cholesterol metabolite in the nuclei of normal human liver tissues. Based upon these observations, we hypothesized a new pathway by which cholesterol is metabolized in the mitochondrion.