ABSTRACT
BACKGROUND: Compared with the standard qPCR, nanoliter-scale qPCR can use smaller quantities of RNA and increase throughput. The TaqMan™ OpenArray® NT Cycler System was assessed for use with degraded RNA from formalin-fixed paraffin-embedded (FFPE) tumors. RESULTS: Expression of candidate prognostic genes was quantified using the OpenArray platform and matching fresh frozen and FFPE patient renal cell carcinomas. Reverse transcription, with gene-specific reverse transcription and preamplification, optimized the percentage of detectable transcripts. When using high quality RNA from fresh frozen tumors, the OpenArray platform identified 30 prognostic genes. However, when using RNA from FFPE tumors, only 13 prognostic genes were identified, but this increased to 33 with addition of preamplification. CONCLUSION: The OpenArray platform can be optimized to quantify gene expressions from FFPE tumors.
Subject(s)
Carcinoma, Renal Cell/genetics , Gene Expression Profiling/methods , Kidney Neoplasms/genetics , Microfluidics/methods , Real-Time Polymerase Chain Reaction/methods , Carcinoma, Renal Cell/pathology , Formaldehyde , Humans , Kidney Neoplasms/pathology , Paraffin EmbeddingABSTRACT
Tamoxifen significantly reduces tumor recurrence in certain patients with early-stage estrogen receptor-positive breast cancer, but markers predictive of treatment failure have not been identified. Here, we generated gene expression profiles of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen monotherapy. An expression signature predictive of disease-free survival was reduced to a two-gene ratio, HOXB13 versus IL17BR, which outperformed existing biomarkers. Ectopic expression of HOXB13 in MCF10A breast epithelial cells enhances motility and invasion in vitro, and its expression is increased in both preinvasive and invasive primary breast cancer. The HOXB13:IL17BR expression ratio may be useful for identifying patients appropriate for alternative therapeutic regimens in early-stage breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Homeodomain Proteins/genetics , Interleukin-17/genetics , Tamoxifen/therapeutic use , Aged , Aged, 80 and over , Biomarkers, Tumor , Cell Line, Tumor , Cell Movement , Female , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/biosynthesis , Humans , In Situ Hybridization , Interleukin-17/biosynthesis , Logistic Models , Middle Aged , Neoplasm Invasiveness , Prognosis , ROC Curve , Reverse Transcriptase Polymerase Chain Reaction , Treatment OutcomeABSTRACT
Although distinct pathological stages of breast cancer have been described, the molecular differences among these stages are largely unknown. Here, through the combined use of laser capture microdissection and DNA microarrays, we have generated in situ gene expression profiles of the premalignant, preinvasive, and invasive stages of human breast cancer. Our data reveal extensive similarities at the transcriptome level among the distinct stages of progression and suggest that gene expression alterations conferring the potential for invasive growth are already present in the preinvasive stages. In contrast to tumor stage, different tumor grades are associated with distinct gene expression signatures. Furthermore, a subset of genes associated with high tumor grade is quantitatively correlated with the transition from preinvasive to invasive growth.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Gene Expression Profiling , Disease Progression , Enzymes/genetics , Epithelial Cells/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Image Processing, Computer-Assisted , In Situ Hybridization , Neoplasm Proteins/genetics , Neoplasm Staging , Polymerase Chain ReactionABSTRACT
We investigated the effect of nitric oxide (NO) donors on the activities of annexin II tetramer (AIIt), a member of the Ca2+- dependent phospholipid-binding protein family. Incubation of purified AIIt with S-nitrosoglutathione (GSNO) led to the inhibition of AIIt-mediated liposome aggregation. This effect was dose-dependent with an IC50 of approximately 100 micro m. Sodium nitroprusside, another NO donor also inhibited AIIt-mediated liposome aggregation, whereas reduced glutathione, nitrate, or nitrite had no effects. GSNO also inhibited AIIt-mediated membrane fusion, but not the binding of AIIt to the membrane. GSNO only has a modest effect on liposome aggregation mediated by annexins I, III or IV. The binding of AIIt to the membrane protected the reactive sites of GSNO on AIIt. GSNO did not inhibit AIIt-mediated liposome aggregation in the presence of dithiothreitol. Taken together, our results suggest that GSNO inactivates AIIt possibly via S-nitrosylation and/or the formation of disulfide bonds.
