From the Cover: Autophagy promotes MHC class II presentation of peptides from intracellular source proteins -- Data Supplement - HTML Page - 01190SuppResults.htslp -- Proceedings of the National Academy of Sciences

Supporting Results
Microarray Analysis of Starved Cells.
To obtain a detailed impression of human genes involved in autophagy, a comparative gene expression analysis by using oligonucleotide microarrays was performed. Genes that were reproducibly up- or down-regulated at 6 and 24 h were assigned to functional categories based on the Gene Ontology (go) classification system (1). Categories showing a significantly enhanced proportion of regulated genes compared with the overall proportion of regulated genes were filtered out and are shown in Tables 5 and 6. Several characteristics of autophagy as a process to ensure cell survival in a nutritionally deprived environment are reflected in these categories, exhibiting a distinct transcriptional signature of starved cells.

The formation of autophagic vacuoles is illustrated by the go terms "small GTPase mediated signal transduction" and "ARF guanyl-nucleotide exchange factor activity," which comprise up-regulated genes involved in the control of vesicular transport and membrane trafficking, especially in the endosomal/lysosomal pathway, as, for example, Rab proteins (2). In contrast, genes regulating rather exocytotic transport processes appear decreased under the term "Golgi stack." In response to amino acid starvation, cells seem to up-regulate in particular genes involved in amino acid transport. This finding has been described in yeast (3). Amino acid-dependent metabolic and proliferative activities can be expected to be reduced to a minimum under these conditions. This result is reflected in the down-regulation of various genes implicated in protein and nucleic acid synthesis. Gene categories such as DNA replication and repair, ribosome biogenesis and assembly, or rRNA, tRNA, and mRNA transcription and processing among the down-regulated biological functions demonstrate this phenomenon. Consequently, genes mediating cell cycle arrest are up-regulated. Enduring starvation may ultimately lead to cell death. Autophagic phenotypes have been associated with programmed cell death other than apoptosis, suggesting a particular autophagic death pathway independent of caspase activation (4–6). We observed an up-regulation of genes related to apoptosis during starvation, most of which are classified in the context of apoptosis inhibition, thus supporting the model of autophagic death independent of apoptosis. Interestingly, ceramide has been described as an important mediator of autophagy and autophagic cell death (7, 8) and the go terms "sphingoid metabolism" and "ceramide metabolism" appeared among the up-regulated categories in our experiment.

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