No abstract

Hypoxia-inducible factor 1 (HIF-1) binds to cis-acting hypoxia-response elements within the erythropoietin, vascular endothelial growth factor, and other genes to activate transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix transcription factor composed of HIF-1␣ and HIF-1␤ subunits. Here, we demonstrate that HIF-1␣ contains two transactivation domains located between amino acids 531 and 826. When expressed as GAL4 fusion proteins, the transcriptional activity of these domains increased in response to hypoxia. Fusion protein levels were unaffected by changes in cellular O 2 tension. Two minimal transactivation domains were localized to amino acid residues 531-575 and 786 -826. The transcriptional activation domains were separated by amino acid sequences that inhibited transactivation. Deletion analysis demonstrated that the gradual removal of inhibitory domain sequences (amino acids 576 -785) was associated with progressively increased transcriptional activity of the fusion proteins, especially in cells cultured at 20% O 2 . Transcriptional activity of GAL4/HIF-1␣ fusion proteins was increased in cells exposed to 1% O 2 , cobalt chloride, or desferrioxamine, each of which also increased levels of endogenous HIF-1␣ protein but did not affect fusion protein levels. These results indicate that increased transcriptional activity mediated by HIF-1 in hypoxic cells results from both increased HIF-1␣ protein levels and increased activity of HIF-1␣ transactivation domains.Human cells require O 2 for essential metabolic processes, most notably oxidative phosphorylation. Hypoxia is a significant pathophysiologic component of many cardiovascular, hematologic, and pulmonary disorders (reviewed in Ref.

Apigenin is a nontoxic dietary flavonoid that has been shown to possess anti-tumor properties and therefore poses special interest for the development of a novel chemopreventive and/or chemotherapeutic agent for cancer. Ovarian cancer is one of the most common causes of cancer death among women. Here we demonstrate that apigenin inhibits expression of vascular endothelial growth factor (VEGF) in human ovarian cancer cells. VEGF plays an important role in tumor angiogenesis and growth. We found that apigenin inhibited VEGF expression at the transcriptional level through expression of hypoxia-inducible factor 1alpha (HIF-1alpha). Apigenin inhibited expression of HIF-1alpha and VEGF via the PI3K/AKT/p70S6K1 and HDM2/p53 pathways. Apigenin inhibited tube formation in vitro by endothelial cells. These findings reveal a novel role of apigenin in inhibiting HIF-1 and VEGF expression that is important for tumor angiogenesis and growth, identifying new signaling molecules that mediate this regulation.

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the musclespecific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation.The retroviral oncogene v-p3k codes for a homolog of the catalytic subunit p110␣ of phosphatidylinositol 3-kinase (PI 3-kinase); it induces oncogenic transformation of chicken embryo fibroblasts (CEF) in culture and causes hemangiosarcomas in young chicken (1). Overexpression of p3k in chicken embryo myoblasts (CEM) activates myotube formation and increases expression of muscle-specific proteins (2). PI 3-kinase is known to stimulate the differentiation of precursor cells of skeletal muscle (3,4). Interference with PI 3-kinase activity, either by the chemical inhibitor LY294002 or with a dominant negative mutant of p85, which is the regulatory subunit of PI 3-kinase, blocks myotube formation and the expression of muscle-specific proteins (2-4). These results strongly support the conclusion that PI 3-kinase activity is essential for skeletal myogenesis. An important role for PI 3-kinase has also been suggested for smooth muscle differentiation (5).Among the downstream targets of PI 3-kinase are phospholipase C (6-9), protein kinase C (10, 11), , and the serine-threonine kinase Akt͞protein kinase B. Akt was identified as the product of the oncogene v-akt in the lymphomagenic murine retrovirus, AKT8 (15). At the same time, Akt was named protein kinase B and RAC-PK (related to the A and C kinases) based on its homology with both protein kinase A and protein kinase C (16,17). Akt is an important component of survival signals from several growth factors (18)(19)(20)(21)(22)(23)(24)(25)(26). It can induce oncogenic transformation of CEF and, like p3k, it causes hemangiosarcomas in chickens (27). Here we show that constitutively active forms of Akt activate myogenic differentiation and that Akt is an essential participant in PI...