Low Protein Z levels in patients with plasma cell neoplasms are inversely correlated with IL-6 levels.

Patients with multiple myeloma (MM) have an increased thrombotic risk, but pathogenesis remains uncertain. Low levels of Protein Z (PZ), a vitamin K-dependent plasma protein, are associated with venous as well as arterial thrombosis. The purpose of this study was to analyze PZ levels in patients with plasma cell neoplasms. PATIENTS AND METHODS: The study consisted of 64 plasma cells neoplasm patients and 42 healthy individuals. Clinical investigations included measurement of plasma PZ and IL-6 levels. RESULTS: PZ levels in patients with plasma cell neoplasms were significantly lower compared to healthy controls in the entire cohort (1392±659 vs.2010±603ng/mL, P<0.01), as well as in specific disease subgroups; symptomatic MM (1428±652ng/mL, p<0.01), smoldering MM (1437±883ng/mL, p=0.045) and monoclonal gammopathy of undetermined significance (MGUS) (1247±593ng/mL, p=0.01). PZ was negatively correlated with IL-6 levels in MM patients (r=-0.7, P<0.01). There was no significant difference in PZ levels between patients with or without thrombotic event. CONCLUSION: Plasma cell neoplasm patients have low levels of PZ. This is presumably related to the increased IL-6 production by the bone marrow microenvironment, and could have a potential role in the increased thrombotic tendency in those patients.

1,25-Dihydroxyvitamin D3 increases the growth-promoting activity of autocrine epidermal growth factor receptor ligands in keratinocytes.

Topical treatment of normal skin with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or its synthetic analogs results in enhanced keratinocyte proliferation. Autocrine growth factors belonging to the epidermal growth factor (EGF) family play a major role in controlling keratinocyte proliferation. 1,25-(OH)2D3 enhanced the autonomous proliferation of HaCaT human keratinocytes in the absence of exogenous growth factors. Autonomous and 1,25-(OH)2D3-stimulated proliferations were inhibited by a specific inhibitor of EGF receptor (EGFR) tyrosine kinase, an EGFR-neutralizing antibody, heparin, the heparin antagonist hexadimethrine, and the proteoglycan sulfation inhibitor chlorate. These results indicate the involvement of proteoglycan-dependent EGFR ligands. The initial events in EGFR (i.e. ErbB1) mitogenic signal transduction are dimer formation with another ErbB protein and tyrosine cross-phosphorylation. By immunoprecipitation followed by Western blotting we showed that ErbB1/ErbB3 heterodimers are the major mitogenic signaling entity in 1,25-(OH)2D3-stimulated cells. 1,25-(OH)2D3 did not affect the levels of the proteoglycan-dependent EGFR ligands amphiregulin and heparin-binding EGF nor the synthesis of proteoglycans, as assessed by 35S labeling and ion exchange chromatography. 1,25-(OH)2D3 caused a marked increase in the cellular contents of ErbB1, ErbB2, and ErbB3 proteins. The increase in ErbB proteins that mediates signal transduction by EGFR ligands can account for the stimulatory effect of 1,25-(OH)2D3 on autonomous keratinocyte proliferation.

1,25-Dihydroxyvitamin D3 increases the cellular content of the calcium-activated neutral protease mu-calpain in renal cell carcinoma.

mu-Calpain is a calcium-dependent neutral thiol protease activated by micromolar concentrations of calcium. mu-Calpain is implicated in various cellular functions regulated by calcium including exocytosis, cell fusion, apoptosis and control of cell proliferation. We studied the effect of 1,25-(OH)2D3 on mu-calpain levels in the human renal cell carcinoma line SK-RC-29 using monoclonal antibodies to the 80 kDa subunit of mu-calpain. Exposure of low density cultures (15000 cells/cm2) to 1,25-(OH)2D3 (100nM) for 48 hours resulted in 1.5-3 fold increase of mu-calpain cell content. The effect was not observed in higher density cultures (40000 cells/cm2). mu-Calpain content of high density cultures was higher than that of low density cultures and similar to that in low density cultures treated by 1,25-(OH)2D3. The cellular content of two other calcium binding proteins, annexin II and annexin VI was not affected by the hormone. 1,25-(OH)2D3 did not affect cell number or viability therefore its effect on mu-calpain is not secondary to changes in cell density. The effect of 1,25-(OH)2D3 was dose-dependent apparent already at 1nM and was not observed with 24,25-(OH)2D3. Increase in mu-calpain content may underlie some of the actions of 1,25-(OH)2D3 on classical and non classical target cells.

1,25-Dihydroxyvitamin D3 potentiates the cytotoxic effect of TNF on human breast cancer cells.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the cytotoxic action of TNF on MCF-7 human breast cancer cells and on adult bovine aortic endothelial cells. 1,25(OH)2D3 increased the effect of TNF on MCF-7 cells but not on endothelial cells over a wide TNF concentration range. At a suboptimal concentration (1 ng/ml) the potentiation was twofold. The effect of 1,25(OH)2D3 was specific, dose-dependent and apparent at a physiological concentration (0.1 nM) of the hormone. The potentiating effect of 1,25(OH)2D3 on TNF action was abolished by cycloheximide indicating that their interaction requires protein synthesis. Addition of 1,25(OH)2D3 13 h after TNF in a 28-h assay was sufficient to induce its full potentiating effect indicating that the hormone modulates a late event in the cytokine's action. These data suggest that some of the in vivo antitumor effects of 1,25(OH)2D3 may be due to an increase in the anticancer activity of the immune system.

Niche-modulated and niche-modulating genes in bone marrow cells.

Bone marrow (BM) cells depend on their niche for growth and survival. However, the genes modulated by niche stimuli have not been discriminated yet. For this purpose, we investigated BM aspirations from patients with various hematological malignancies. Each aspirate was fractionated, and the various samples were fixed at different time points and analyzed by microarray. Identification of niche-modulated genes relied on sustained change in expression following loss of niche regulation. Compared with the reference ('authentic') samples, which were fixed immediately following aspiration, the BM samples fixed after longer stay out-of-niche acquired numerous changes in gene-expression profile (GEP). The overall genes modulated included a common subset of functionally diverse genes displaying prompt and sustained 'switch' in expression irrespective of the tumor type. Interestingly, the 'switch' in GEP was reversible and turned 'off-and-on' again in culture conditions, resuming cell-cell-matrix contact versus respread into suspension, respectively. Moreover, the resuming of contact prolonged the survival of tumor cells out-of-niche, and the regression of the 'contactless switch' was followed by induction of a new set of genes, this time mainly encoding extracellular proteins including angiogenic factors and extracellular matrix proteins. Our data set, being unique in authentic expression design, uncovered niche-modulated and niche-modulating genes capable of controlling homing, expansion and angiogenesis.

Upregulation of the calcium-dependent protease, calpain, during keratinocyte differentiation.

Calpain is a ubiquitous neutral calcium-activated thiol protease that is implicated in various cellular functions including exocytosis, cell fusion, apoptosis and proliferation. The calpain system is composed of the enzymes mu-calpain and m-calpain and their endogenous inhibitor, calpastatin. We employed the spontaneously immortalized human HaCaT keratinocytes, which retain their ability to differentiate in vitro and in vivo, to study the modulation of the calpain system during keratinocyte differentiation. The cellular levels of keratinocyte differentiation markers and of the components of the calpain system were monitored by immunoblotting. Three established differentiation stimuli: increase in cell density as a function of time in culture, elevation of extracellular calcium concentration and exposure to 1,25-dihydroxyvitamin D3 enhanced the expression of the three keratinocyte differentiation markers keratin 10, involucrin and transglutaminase. The differentiation of HaCaT cells was accompanied by elevation of the components of the calpain system, although the pattern of increase varied according to the specific differentiation stimulus. A higher increase in calpains as compared with the increase in calpastatin suggests an increase in net calpain activity during differentiation. Such an increase may play a part in the differentiation process itself and/or in the regulation of key events in differentiating keratinocyte metabolism.