Interaction of HIF-1α and Notch3 Is Required for the Expression of Carbonic Anhydrase 9 in Breast Carcinoma Cells.

Expression of carbonic anhydrase 9 (CA9) is associated with poor prognosis and increased tumor aggressiveness and does not always correlate with HIF-1α expression. Presently, we analyzed the regulation of CA9 expression during hypoxia by HIF-1α, Notch3, and the von Hippel-Lindau (VHL) in breast carcinoma cells. Both HIF-1α and Notch3 were absolutely required for the expression of CA9 mRNA, protein, and reporter. Reciprocal co-immunoprecipitation of HIF-1α, Notch3 intracellular domain (NICD3), and pVHL demonstrated their association. The presence of common consensus prolyl hydroxylation and pVHL binding motifs (L(XY)LAP);LLPLAP(2191) suggested an oxygen-dependent regulation for NICD3. However, unlike the HIF-1α protein, NICD3 protein levels were not modulated with hypoxia or hypoxia-mimetic agents. Surprisingly, mutations of the common prolyl hydroxylation and pVHL binding domain lead to the loss of CA9 mRNA, protein, and reporter activity. Chromatin immunoprecipitation assay demonstrated the association of NICD3, HIF-1α, and pVHL at the CA9 promoter. Further, the NICD3 mutant defective in prolyl hydroxylation and subsequent pVHL binding caused a reduction in cell proliferation of breast carcinoma cells. We show here for the first time that the interaction of HIF-1α with NICD3 is important for the regulation of CA9 expression. These findings suggest that although CA9 is a hypoxia-responsive gene, its expression is modulated by the interaction of HIF-1α, Notch3, and VHL proteins. Targeting the expression of CA9 by targeting upstream regulators could be useful in cancer/stem cell therapy.

A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.

Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.