ygen-sensitive HIF-1 subunit, which is degraded through the ubiquitin–proteasome pathway upon recognition by the von Hippel-Lindau protein as part of the E3 ubiquitin ligase. The VHL protein binds to HIF-1 and its paralog HIF-2 by recognizing two highly conserved, hydroxylated proline residues for polyubitylation. Hypoxia inhibits prolyl hydroxylation, thereby preventing HIF-1 degradation. Subsequently, stabilized HIF-1 and HIF-2 undergo nuclear translocation, dimerization with ARNT, and recruitment of the transcription coactivators p300/CBP, resulting in transcriptional activation of a series of genes for angiogenesis, metabolism, and survival. Whereas HIF-1 is ubiquitously expressed, HIF-2 expression seems restricted to certain tissues in development and physiology. The abundance of HIF-1 as well as HIF-2 is frequently detected in the vast majority of human cancers. Although these transcription factors were initially thought to share overlapping functions in tumor progression, each seems to possess unique and sometimes opposing activities through specific target gene activation and differential interactions with other proteins. Specifically, their opposing activities have been shown in the regulation of cell cycle and DNA repair: Whereas HIF-1 inhibits cell-cycle progression PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19776382 and DNA repair by antagonizing c-Myc activities, HIF-2 does the reverse by enhancing c-Myc activities. Furthermore, the roles of HIF-1 and HIF-2 in cancer seem context dependent. Whereas HIF-2 acts as a tumor suppressor in glioma, non-small cell lung cancer, and hepatocellular carcinoma, it drives tumorigenesis and growth of VHL-deficient renal clear-cell carcinoma. In keeping with this, EPAS1 polymorphisms have been identified as one of the two susceptibility loci in renal cell carcinoma. In addition, somatic, gainof-function mutations in HIF-2 have been linked to the development of paraganglioma and somatostatinoma in patients. Likewise, HIF-1 has been implicated as a tumor suppressor especially in kidney cancer, even though substantial evidence in the literature support a critical role of HIF-1 in progression and metastasis. The tumor-suppressing activity of HIF-1 is strongly indicated by the genetic evidence that focal, homozygous deletions of HIF1A gene are found in many VHL-deficient renal clear-cell carcinoma cell lines and the functional evidence that HIF-1 inhibits cell proliferation and tumor growth. All these studies suggest complex roles 
for HIF-1 and HIF-2 in cancer. As a step towards understanding the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19778700 complexity of cancer biology, we employed intermittent induction of HIF-1 and HIF-2 in various cancer cell types and investigated their differential effects on malignant progression in immunodeficient mice. LOXO-101 Materials and Methods Plasmid construction and viral production An oxygen-resistant HIF-1, HIF1, with P402A and P564A substitutions, was cloned into pLenti6.3/TO/V5-DEST through homologous recombination reactions. Similarly, HIF-2 with P405A and P531A substitutions and a 3xFLAG at the amino terminus was cloned into the same vector. To produce lentiviruses, 293FT cells derived from a human embryonic kidney cell line were transfected with a lentiviral vector and Virapower packaging mix using Lipofectamine 2000. Lentiviral supernatant was harvested 3 days after transfection and filtered through a 0.45-m sterile syringe filter. The filtered virus was aliquoted and stored at -80C. Viral titers were determined according to the manufacturer’s instruction.