The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.

We report a method for Selective Depletion of abundant RNA (SDRNA) species from Human total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, here demonstrating removal of ribosomal and mitochondrial transcripts from clinical FFPE tissue RNA archived up to 20 years. Importantly, SDRNA removes 98% of targeted RNAs while preserving relative abundance profiles of non-targeted RNAs, enabling routine whole transcriptome analysis of clinically valuable archival tissue specimens by Next-Generation Sequencing.

BackgroundMolecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues.Methodology/Principal FindingsThe method we describe is based on the widely used TaqMan® real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess.Conclusions/SignificanceASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan® protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability.

Summary The rice resistance gene Xa21 confers resistance against the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). The molecular genetic mechanism controlling the integration of the Xa21‐mediated disease resistance response with the developmental program in rice is under study in this model system. Reproducible means of infecting plants at certain developmental stages were designed based on the timing of full expansion of the leaf. Xa21‐resistance progressively increases from the susceptible juvenile leaf 2 stage through later stages, with 100% resistance at the adult leaf 9/10 stage. We found that Xa21 expression is independent of plant developmental stage, infection with Xoo, or wounding. Expression of the Xa21 gene transcript is not correlated with expression of Xa21 disease resistance indicating that the developmental regulation of Xa21‐resistance is either controlled post‐transcriptionally or by other factors.

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.