Identification of Hypoxically Inducible mRNAs in HeLa Cells Using Differential‐Display PCR

Abstract

Oxygen is an important regulator of gene expression in mammalian cells, though the extent of operation and the organization of the inducible mechanisms involved are still largely undetermined. To define better the response to hypoxia, we have used differential display PCR to identify genes whose expression is induced in HeLa cells exposed to 1 % oxygen. Among six genes whose induction by hypoxia was newly defined in this way, three were of known function, encoding the glucose transporter isoform 3 (Glut-3), adenylate kinase isoenzyme 3 (AK-3), and tissue factor, two were expressed sequence tags (ESTs), and one corresponded to a new sequence. One regulator of the transcriptional response to hypoxia has recently been identified as a heterodimeric DNA-binding complex termed hypoxia-inducible factor-1 (HIF-l), which is also inducible by the iron chelator, desferrioxamine. Of the six hypoxically regulated genes, at least four were also induced by exposure of the cells to desferrioxamine. To analyse further the mechanisms underlying induction of the genes identified in the differential display, inducible expression was compared in wild-type mouse hepatoma cells (Hepa-l), and mutant derivatives (c4) which fail to generate HIF-1, due to a functional defect in one component, HIF-1P. Two types of response were defined. For Glut-3 and AK-3, mutant (c4) cells showed almost complete loss of the inducible response to both hypoxia and desfemoxamine. In contrast, tissue factor mRNA was more inducible by both stimuli in c4 than wild-type cells. These studies demonstrate the critical importance of HIF-1P in newly recognized responses to hypoxia, and provide further evidence of the importance of this system of gene regulation in mammalian cells; they also demonstrate responses to both hypoxia and desfemoxamine which are independent of HIF-1P and which appear exaggerated in HIF-1P-deficient cells.

Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth

“…First, we studied expression of the glucose transporter GLUT3. We selected this gene for analysis because in cultured wt Hepa-1 cells it is expressed only at a low level in normoxia but shows a high degree of HIF-1␤͞ARNT-dependent induction by hypoxia (21). We reasoned that if the HIF-1 system of gene regulation were activated by hypoxia in the tumors there would be a large contrast in the expression of GLUT3 mRNA between wild-type and c4 xenografts.…”

“…These have provided an important tool for analysis of HIF-1, because one complementation group is functionally deficient in HIF-1␤͞ARNT (17) and is unable to form the HIF-1 heterodimer (18). Examination of oxygenregulated gene expression in these cells revealed reduced or absent induction by hypoxia of genes encoding glucose transporters, glycolytic enzymes, and vascular growth factors (18)(19)(20)(21)(22). This confirmed that HIF-1␤͞ARNT is critically involved in the induction of these genes in hypoxic cell culture, and suggested the utility of these mutant cells in defining further the consequences of HIF-1 activation.…”

The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF)

“…After siRNA treatment, cells were incubated under normoxic conditions or subjected to hypoxia to promote induction of HIF-responsive genes. When cells were treated with a control siRNA targeting GFP, two well characterized HIF target genes, Nip3 (23) and adenylate kinase 3 (AK3) (26), were both induced in response to hypoxia as assessed by Northern blot analysis (Fig. 1B).…”

Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist

“…In cells and tissues, hypoxia triggers a multifaceted adaptive response that is primarily driven by the heterodimeric hypoxiainducible factor 1 (HIF-1) (2). Under normal dioxygen levels, the ␣-subunit of HIF-1 is successively hydroxylated at proline residue 564 (3), ubiquitinated, and then degraded by the ubiquitinproteosome system.…”