Human Chondrosarcoma Cells Acquire an Epithelial-Like Gene Expression Pattern via an Epigenetic Switch: Evidence for Mesenchymal-Epithelial Transition during Sarcomagenesis

Chondrocytes are mesenchymally derived cells that reportedly acquire some epithelial characteristics; however, whether this is a progression through a mesenchymal to epithelial transition (MET) during chondrosarcoma development is still a matter of investigation. We observed that chondrosarcoma cells acquired the expression of four epithelial markers, E-cadherin,desmocollin 3, maspin, and 14-3-3σ, all of which are governed epigenetically through cytosine methylation. Indeed, loss of cytosine methylation was tightly associated with acquired expression of both maspin and 14-3-3σ in chondrosarcomas. In contrast, chondrocyte cells were negative for maspin and 14-3-3σ and displayed nearly complete DNA methylation. Robust activation of these genes was also observed in chondrocyte cells following 5-aza-dC treatment. We also examined the transcription factor snail which has been reported to be an important mediator of epithelial to mesenchymal transitions (EMTs). In chondrosarcoma cells snail is downregulated suggesting a role for loss of snail expression in lineage maintenance. Taken together, these results document an epigenetic switch associated with an MET-like phenomenon that accompanies chondrosarcoma progression

Human Adipocyte Conditioned Medium Promotes In Vitro Fibroblast Conversion to Myofibroblasts

Abstract Adipocytes and adipose tissue derived cells have been investigated for their potential to contribute to the wound healing process. However, the details of how these cells interact with other essential cell types, such as myofibroblasts/fibroblasts, remain unclear. Using a novel in-vitro 3D human adipocyte/pre-adipocyte spheroid model, we investigated whether adipocytes and their precursors (pre-adipocytes) secrete factors that affect human dermal fibroblast behavior. We found that both adipocyte and pre-adipocyte conditioned medium induced the migration of fibroblasts, but only adipocyte conditioned medium induced fibroblast differentiation into a highly contractile, collagen producing myofibroblast phenotype. Furthermore, adipocyte mediated myofibroblast induction occurred through a TGF-β independent mechanism. Our findings contribute to a better understanding on the involvement of adipose tissue in wound healing, and may help to uncover and develop fat-related wound healing treatments

RABL6A Regulates Schwann Cell Senescence in an RB1-Dependent Manner

Schwann cells are normally quiescent, myelinating glia cells of the peripheral nervous system. Their aberrant proliferation and transformation underlie the development of benign tumors (neurofibromas) as well as deadly malignant peripheral nerve sheath tumors (MPNSTs). We discovered a new driver of MPNSTs, an oncogenic GTPase named RABL6A, that functions in part by inhibiting the RB1 tumor suppressor. RB1 is a key mediator of cellular senescence, a permanent withdrawal from the cell cycle that protects against cell immortalization and transformation. Based on the RABL6A-RB1 link in MPNSTs, we explored the hypothesis that RABL6A promotes Schwann cell proliferation and abrogates their senescence by inhibiting RB1. Using sequentially passaged normal human Schwann cells (NHSCs), we found that the induction of replicative senescence was associated with reduced expression of endogenous RABL6A. Silencing RABL6A in low passage NHSCs caused premature stress-induced senescence, which was largely rescued by co-depletion of RB1. Consistent with those findings, Rabl6-deficient MEFs displayed impaired proliferation and accelerated senescence compared to wildtype MEFs. These results demonstrate that RABL6A is required for maintenance of proper Schwann cell proliferation and imply that aberrantly high RABL6A expression may facilitate malignant transformation

Expression of adipocyte markers in differentiated cells.

<p>NPADs and DPADs (white bars) were differentiated into NDADs and DDADs (black bars), respectively. The expression of adipocyte markers was examined by quantitative reverse transcriptase PCR (q-RT-PCR). (A) Peroxisome proliferator-activated receptor gamma (PPARγ); (B) CCAAT/enhancer binding protein-alpha (C/EBPα); (C) lipoprotein lipase (LPL); (D) fatty acid binding protein 4 (FABP-4); (E) adipsin; (F) adiponectin; and (G) leptin were all induced to high levels upon differentiation. (H) A marker of pre-adipocytes termed C-C motif chemokine 2 (CCL2) was expressed in the undifferentiated DPAD and NPAD cells but reduced upon differentiation. Values were normalized to GAPDH and made relative to undifferentiated NPADs and represent 3 replicates. All transcript level changes in differentiated cells as compared to undifferentiated cells were statistically significant (*represents p<0.05; **represents p<0.01, Students t-test). For all genes except leptin, differences between DDADs and NDADs were significant (#p<0.05, Students t-test).</p

Staphylococcal <i>α</i>-toxin was cytotoxic for DDADs.

<p>DDAD cells were treated with various concentrations of α-toxin for 24 h. The Cell Titer 96® Aqueous One Solution Proliferation Assay and propidium iodine were used to assess the cellular toxicity and damage. (a) At 5 ug/ml and 10 ug/ml of α-toxin significant cell death occurred, while no effect was observed at lower concentrations (below 1 ug/ml). (b) Consistent with the cytotoxic effect, significant amounts of PI were incorporated into DDADs at 5 ug/ml and 10 ug/ml of α-toxin, but not at lower concentrations (*represents p<0.05, Students t-test).</p

α-toxin failed to induce IL-6 and IL-8 production in DDADs.

<p>DDADs were treated with low concentrations of α-toxin for 24 h. The culture supernates were collected for IL-6 (a) and IL-8 (b) quantification by ELISA.</p

Genes that were analyzed by q-RT-PCR with respective primers.

<p>Genes that were analyzed by q-RT-PCR with respective primers.</p

TSST-1 and SEB induced IL-6 and Il-8 production in DDADs and NDADs.

<p>Differentiated adipocytes were treated with 50/ml and 100 ug/ml of TSST-1 or SEB for 24 h. The culture supernates were collected from DDADs for IL-6 (a) and IL-8 (b) and NDADs IL-6 (c) and IL-8 (d) for quantification by ELISA (*represents p<0.05; **p<0.01, Students t-test). Fold changes in IL-6 (e) and IL-8 (f) production were calculated in DDADs and NDADs after TSST-1 and SEB exposure. Values represent fold changes relative to PBS-treated controls.</p

Endotoxin (LPS) induced IL-6 and Il-8 production in both DDADs and NDADs.

<p>After differentiation, DDADs and NDADs were treated with endotoxin (5 ng/ml) for 24 h. The culture supernates were collected for IL-6 and IL-8 quantification by ELISA. For both IL-6 and IL-8, relative differences between DDADs and NDADS were significant (**represents p<0.01, Students t-test).</p

TSST-1 and endotoxin (LPS) cooperatively induce IL-6 and IL-8 production in DDADs.

<p>DDADs were treated with TSST-1 (50 ug/ml), endotoxin (5 ng/ml), or TSST-1 (50 ug/ml)+endotoxin (5 ng/ml) for 24 h. The culture supernates were collected for IL-6 and IL-8 quantification by ELISA. TSST-1+ endotoxin induced more IL-6 (a) and IL-8 (b) production in DDADs than PBS, TSST-1, or endotoxin alone (*represents p<0.05; **p<0.01, Students t-test).</p