PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Background: The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some functions are regulated via intracellular signaling cascades, others by involvement of the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, understanding of their functions and the exact nature of these interactions remains incomplete. Methods: IGF-I was PEGylated at its lysine sites - K27, K65 and K68. Binding of PEG-IGF-I to the IGFBPs was analyzed using BIAcore and its ability to activate the IGF-IR was assessed using IGF-IR phosphorylation assay. Furthermore, functional consequences of PEGylating the lysine residues of IGF-I was investigated using cell viability and cell migration assays. In addition, particular downstream signaling pathways regularly implicated in these mechanisms were also dissected using phospho-AKT and phospho-ERK1/2 assays. Results: In this study, IGF-I specifically PEGylated at lysine 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) were employed. Receptor phosphorylation was only reduced by 2-fold with PEG-K65 and PEG-K68 over all the time points tested, and as observed in two cell types, 3T3 fibroblasts and MCF-7 breast cancer cells. PEGylation at K27 resulted in a much larger effect, with more than 10-fold lower activation for 3T3 fibroblasts and a ~3 fold reduced IGF-IR activation for MCF-7 breast cancer cells over 15 minutes. In addition, all PEG-IGF-I variants demonstrated a ten-fold reduction in the association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants completely lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes as compared to IGF-I; in contrast, cell viability was fully preserved. Further investigations into the downstream signaling pathways revealed that the PI3-K/AKT pathway was preferentially affected upon treatment with the PEG-IGF-I variants compared to the MAPK/ERK pathway. Conclusion: PEGylation of IGF-I has an impact on cell migration but not cell viability. General significance: PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on its interaction with its receptor as well as key extracellular proteins such as VN and IGFBPs.

Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73, by Spearman's ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the myelin basic protein (MBP) and glutathione S-transferase (GST) M1, nerve growth factors, such as nerve injury-induced protein 1 (NINJ1), X-ray and excision DNA repair factors (XRCC9 and ERCC5) and X-linked genes such as the ribosomal protein, RPS4X. Primers were then designed for seven array-selected genes, including transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), GSTP1, crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1) and tubulin β-5 (TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (r=0.75, Pvalue<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.

Polymorphic variants of NFKB1 and its inhibitory protein NFKBIA, and their involvement in sporadic breast cancer

Nuclear factor kappa-beta (NF-kappaB) is a transcription factor responsible for modulating the expression of many genes involved in cell proliferation, differentiation, apoptosis and metastasis. NF-kappaB interacts with IkappaB inhibitory proteins to regulate gene expression. This study investigated common variants within the genes coding for NF-kappaB and IkappaB, NFKB1 and NFKBIA, for involvement in sporadic breast cancer. Genotypes were determined in a population of breast cancer affected individuals and age-matched controls. Results do not support an involvement of the tested NFKB1 and NFKBIA polymorphisms in susceptibility to sporadic breast cancer, in the tested Caucasian population.