Hyperactivation of the insulin-like growth factor receptor I signaling pathway is an essential event for cisplatin resistance of ovarian cancer cells.

Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas.

Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS.

An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.

Partial reduction and two-step modification of proteins for identification of disulfide bonds.

An experimental protocol was established to combine partial reduction, cyanylation, and a second modification step for the assignment of disulfide bonds in proteins that are resistant to proteolysis under native conditions. After proteolysis, disulfide bonds were assigned via MALDI mass spectrometry with subsequent semiautomatic interpretation using the program SearchXLinks, which enumerates all possible combinations of proteolytic fragments for all observed monoisotopic masses. The putative assignment of disulfide bonds was confirmed by ISD and PSD fragmentation of the corresponding protonated molecules.