Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to obtain spectra of peptide-metal ion complexes formed by a zinc finger peptide of the transcription factor IIIA (Cys2-His2) type and zinc and cobalt ions, as well as peptide-enzyme complexes. Peptides and proteins analyzed by mass spectrometry are generally dissolved in 0.1% aqueous TFA (pH < 2.0). At this pH, peptides and proteins denature. We therefore reasoned that MALDI mass spectrometry might be able to detect noncovalently bound compounds if conditions were used to prepare the samples that allowed macromolecular assemblies to retain tertiary structure. Samples were dissolved in 1 M ammonium bicarbonate, and a saturated matrix solution was prepared using ethanolammonium bicarbonate (1:1) solution or ethanol-ammonium citrate (1:1) solution. All preparations of zinc finger peptides were done in a glovebox under nitrogen to prevent oxidation of the metal binding cysteine residues. Using this approach, we have been able to demonstrate that MALDI mass spectrometry can be used to study both noncovalent metal binding complexes and noncovalent peptide-enzyme complexes.

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G 1 to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Aspaldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E 276-395 (where cyclin E 276-395is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E 1-275 , cyclin E 276-395 deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E 276-395 , but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.The cyclins and their catalytic subunits, the cyclin-dependent kinases (CDKs), control cell cycle progression by regulating events that drive the transitions between cell cycle phases (13,14). The activity of these CDKs is regulated positively by cyclins, their associated catalytic partners, and negatively by binding of CDK inhibitors (CKIs). Activation of cyclin/CDK complexes results in a cascade of protein phosphorylations that ultimately induces cell cycle progression. Cyclins were first identified in clam and sea urchin embryos, where they were observed to accumulate during interphase and to be degraded during mitosis (16). The human G 1 cyclins, the D-and E-type cyclins, were identified functionally by screening of human cDNA libraries for sequences that could complement G 1 cyclin mutations in Saccharomyces cerevisiae (31,35,65). The cyclin E mRNA levels show a periodic pattern of expression, being synthesized during the G 1 phase of the cell cycle, with levels increasing sharply in late G 1 , followed by accumulation of cyclin E protein and then down regulation in S phase (1...

A workshop entitled “Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate” (held in Rockville, MD, September 19, 2016) was organized by the Radiation Research Program and Radiation Oncology Branch of the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), to identify critical research areas and directions that will advance the understanding of radiation-induced fibrosis (RIF) and accelerate the development of strategies to mitigate or treat it. Experts in radiation biology, radiation oncology and related fields met to identify and prioritize the key areas for future research and clinical translation. The consensus was that several known and newly identified targets can prevent or mitigate RIF in pre-clinical models. Further, basic and translational research and focused clinical trials are needed to identify optimal agents and strategies for therapeutic use. It was felt that optimally designed preclinical models are needed to better study biomarkers that predict for development of RIF, as well as to understand when effective therapies need to be initiated in relationship to manifestation of injury. Integrating appropriate endpoints and defining efficacy in clinical trials testing treatment of RIF were felt to be critical to demonstrating efficacy. The objective of this meeting report is to (a) highlight the significance of RIF in a global context, (b) summarize recent advances in our understanding of mechanisms of RIF, (c) discuss opportunities for pharmacological mitigation, intervention and modulation of specific molecular pathways, (d) consider the design of optimal clinical trials for mitigation and treatment and (e) outline key regulatory nonprescriptive frameworks for approval.

BACKGROUND.In a population of patients with brain metastases from melanoma,

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