Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. Although it usually promotes tumor cell survival under hypoxia, it has also been implied to trigger apoptosis. Although the impact of hypoxia has been extensively studied in many adult solid tumors, its role in most childhood tumors, for example, in rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), has not yet been addressed. Here, we report that hypoxia protects A204 RMS and A673 ES cells against anticancer drug-or tumor necrosis factor-related apoptosis-inducing ligandinduced apoptosis and that Hif-1a plays a key role in conferring apoptosis resistance under hypoxia. Although a functional HIF-1 pathway and proapoptotic proteins such as p53 and Bcl-2/E1B 19 kDa interacting protein 3 were activated under hypoxia in both A204 RMS and A673 ES cells, these cells remained refractory to apoptosis. Concomitant analysis of antiapoptotic proteins revealed that hypoxia induced expression of Bcl-2 and inhibitor of apoptosis proteins (IAP)-2 as well as proteins associated with anaerobic metabolism such as the glucose transporter protein GLUT-1 and the glycolytic enzyme Aldolase A. Specific downregulation of Hif-1a by RNA interference significantly enhanced apoptosis under hypoxia by preventing the hypoxia-mediated increase in GLUT-1 expression without altering expression levels of the antiapoptotic proteins Bcl-2 or cIAP-2. Moreover, glucose deprivation-induced apoptosis of A204 RMS and A673 ES cells was inhibited under hypoxic conditions in a Hif-1a-dependent manner. As GLUT-1 was induced via Hif-1a under hypoxia in A204 RMS and A673 ES, these findings suggest that the Hif-1a-mediated increase in glucose uptake plays an important role in conferring apoptosis resistance. Thus, hypoxia-inducible genes may represent novel targets for therapeutic intervention in some pediatric tumors, which warrants further investigation.

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To explore mechanisms controlling sonic hedgehog (Shh) expression in human cancers, we investigated regulation of Shh by the transcription factor NF-kappaB. We identify putative NF-kappaB binding sites in the human Shh promoter region that specifically bind NF-kappaB complexes. Further, NF-kappaB activation by tumor necrosis factor alpha (TNF-alpha) or p65 overexpression stimulates Shh promoter activity and p65 binds to Shh promoter in vivo. NF-kappaB-mediated transcriptional activation of Shh is mapped to a minimal NF-kappaB consensus site at position +139 of Shh promoter. NF-kappaB activation results in increased Shh mRNA and protein expression in vitro and, notably, also in vivo in a genetic mouse model of inducible NF-kappaB activity. Specific NF-kappaB inhibition by inhibitory NF-kappaBalpha (Ikappa-Balpha) superrepressor or p65 knockdown inhibits NF-kappaB-induced Shh promoter activation and Shh expression. NF-kappaB-mediated Shh expression promotes proliferation and confers resistance to TRAIL-induced apoptosis. Silencing of Shh prevents NF-kappaB-stimulated proliferation, while the addition of Shh rescues the proliferation defect imposed by NF-kappaB inhibition. Notably, NF-kappaB-stimulated tumor growth is significantly impaired by Shh knockdown in an in vivo model of pancreatic cancer. By demonstrating that NF-kappaB regulates Shh expression, which contributes to NF-kappaB-mediated proliferation and apoptosis resistance in vitro and in vivo, our findings have important implications to target aberrant Shh expression in human cancers.

Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.