Rapamycin-sensitive pathway regulates mitochondrial membrane potential, autophagy, and survival in irradiated MCF-7 cells.

Radiation-induced inhibition of rapamycin-sensitive pathway and its effect on the cellular response to radiation were studied in the human breast cancer cell line MCF-7. Both radiation and rapamycin shared molecular targets and induced similar physiologic responses. Each of these treatments increased immunostaining of mammalian target of rapamycin (mTOR) in the nucleus, and radiation led to decreased phosphorylation of its autophosphorylation site Ser2481. In addition to dephosphorylation of established mTOR downstream effectors 4E-binding protein 1 and p70 ribosomal S6 kinase, both treatments decreased the level of eukaryotic initiation factor 4G. Experiments with the potentiometric dye, JC-1, revealed an oligomycin-dependent increase in mitochondrial membrane potential following radiation or rapamycin treatment, suggesting that both lead to reversal of F0F1ATPase activity. Both radiation and rapamycin induced sequestration of cytoplasmic material in autophagic vacuoles. In both cases, appearance of autophagic vacuoles involved the participation of microtubule-associated protein 1 light chain 3 (LC3). Transient cotransfection of green fluorescent protein-LC3 with either wild-type or dominant-negative mTOR further showed that inactivation of mTOR pathway is sufficient to induce autophagy in these cells. Finally, administration of rapamycin in combination with radiation led to enhanced mitochondria hyperpolarization, p53 phosphorylation, and increased cell death. Taken together, these experiments show that radiation-induced inhibition of rapamycin-sensitive pathway in MCF-7 cells causes changes in mitochondria metabolism, development of autophagy, and an overall decrease in cell survival.

The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment.

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.