Serum YB-1 (Y-box binding protein 1) as a biomarker of bone disease progression in patients with breast cancer and bone metastases.

YB-1 (Y-box binding protein 1) is a multifunctional cold-shock protein that has been implicated in all hallmarks of cancer. Elevated YB-1 protein level was associated with poor prognosis in several types of cancers, including breast cancer (BC), where it is a marker of decreased overall survival (OS) and distant metastasis-free survival across all subtypes. YB-1 is also secreted by different cell types and may act as an extracellular mitogen; however the pathological implications of the secreted form of YB-1 (sYB-1) are unknown. Our purpose was to retrospectively evaluate the association between YB-1 measured by ELISA in serum and disease characteristics and outcomes in patients with BC and bone metastases (BM). In our cohort, sYB-1 was detected in the serum of 22 (50%) patients, and was associated with the presence of extra-bone metastases (p=0.044). Positive sYB-1 was also associated with faster bone disease progression (HR 3.1, 95% CI 1.09-8.95, P=0.033), but no significant differences were observed concerning OS, and time to development of skeletal-related events. Moreover, patients with positive sYB-1 also had higher levels of IL-6, a known osteoclastogenic inducer. Therefore, detection of sYB-1 in patients with BC and BM may indicate a higher tumor burden, in bone and extra-bone locations, and is a biomarker of faster bone disease progression.

Cell-specific regulation of acetylcholinesterase expression under inflammatory conditions.

Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were surprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.

Unilateral tumid lupus erythematosus.

Few cases of inflammatory blaschkolinear dermatoses have been described. We report a case of blaschkolinear cutaneous tumid lupus erythematous and discuss the potential association between cutaneous genetic mosaicism, Blaschko lines and lupus susceptibility.

Positive tetracycline control of expression of p15INK4B from an Epstein-Barr autonomous plasmid in a human melanoma cell line.

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control ('TET ON' system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

Use of a recombinant parvovirus to facilitate screening for human melanoma cell clones expressing tetracycline-responsive transactivators.

The tetracycline regulatory (TET) system provides a useful means of controlling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100-200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.