Determination of the in vivo degradation mechanism of PEGDA hydrogels.

Poly(ethylene glycol) (PEG) hydrogels are one of the most extensively utilized biomaterials systems due to their established biocompatibility and highly tunable properties. It is widely acknowledged that traditional acrylate-derivatized PEG (PEGDA) hydrogels are susceptible to slow degradation in vivo and are therefore unsuitable for long-term implantable applications. However, there is speculation whether the observed degradation is due to hydrolysis of endgroup acrylate esters or oxidation of the ether backbone, both of which are possible in the foreign body response to implanted devices. PEG diacrylamide (PEGDAA) is a polyether-based hydrogel system with similar properties to PEGDA but with amide linkages in place of the acrylate esters. This provides a hydrolytically-stable control that can be used to isolate the relative contributions of hydrolysis and oxidation to the in vivo degradation of PEGDA. Here we show that PEGDAA hydrogels remained stable over 12 weeks of subcutaneous implantation in a rat model while PEGDA hydrogels underwent significant degradation as indicated by both increased swelling ratio and decreased modulus. As PEGDA and PEGDAA have similar susceptibility to oxidation, these results demonstrate for the first time that the primary in vivo degradation mechanism of PEGDA is hydrolysis of the endgroup acrylate esters. Additionally, the maintenance of PEGDAA hydrogel properties in vivo indicates their suitability for long-term implants. These studies serve to elucidate key information about a widely used biomaterial system to allow for better implantable device design and to provide a biostable replacement option for PEGDA in applications that require long-term stability.

Drying and storage effects on poly(ethylene glycol) hydrogel mechanical properties and bioactivity.

Hydrogels based on poly(ethylene glycol) (PEG) are increasingly used in biomedical applications because of their ability to control cell-material interactions by tuning hydrogel physical and biological properties. Evaluation of stability after drying and storage are critical in creating an off-the-shelf biomaterial that functions in vivo according to original specifications. However, there has not been a study that systematically investigates the effects of different drying conditions on hydrogel compositional variables. In the first part of this study, PEG-diacrylate hydrogels underwent common processing procedures (vacuum-drying, lyophilizing, hydrating then vacuum-drying), and the effect of this processing on the mechanical properties and swelling ratios was measured. Significant changes in compressive modulus, tensile modulus, and swelling ratio only occurred for select processed hydrogels. No consistent trends were observed after processing for any of the formulations tested. The effect of storage conditions on cell adhesion and spreading on collagen- and streptococcal collagen-like protein (Scl2-2)-PEG-diacrylamide hydrogels was then evaluated to characterize bioactivity retention after storage. Dry storage conditions preserved bioactivity after 6 weeks of storage; whereas, storage in PBS significantly reduced bioactivity. This loss of bioactivity was attributed to ester hydrolysis of the protein linker, acrylate-PEG-N-hydroxysuccinimide. These studies demonstrate that these processing methods and dry storage conditions may be used to prepare bioactive PEG hydrogel scaffolds with recoverable functionality after storage.

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis.

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

c-Abl phosphorylation of ΔNp63α is critical for cell viability.

The p53 family member p63 has been shown to be critical for growth, proliferation and chemosensitivity. Here we demonstrate that the c-Abl tyrosine kinase phosphorylates the widely expressed ΔNp63α isoform and identify multiple sites by mass spectrometry in vitro and in vivo. Phopshorylation by c-Abl results in greater protein stability of both ectopically expressed and endogenous ΔNp63α. c-Abl phosphorylation of ΔNp63α induces its binding to Yes-associated protein (YAP) and silencing of YAP by siRNA reduces the c-Abl-induced increase of ΔNp63α levels. We further show that cisplatin induces c-Abl phosphorylation of ΔNp63α and its binding to YAP. Overexpression of ΔNp63α, but not the c-Abl phosphosites mutant, protects cells from cisplatin treatment. Finally, we demonstrate the rescue of p63 siRNA-mediated loss of viability with p63siRNA insensitive construct of ΔNp63α but not the phosphosites mutant. These results demonstrate that c-Abl phosphorylation of ΔNp63α regulates its protein stability, by inducing binding of YAP, and is critical for cell viability.

JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis.

Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP's function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP's ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP's function.

Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia.

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.