LONDON ¿ The discovery that a protein that is mutated in a rare, inherited form of cancer plays a key role in regulating the cellular response to shortage of oxygen has brought scientists closer to understanding how cells detect oxygen levels.

The finding will have implications for researchers studying how to stimulate angiogenesis ¿ the growth of new blood vessels ¿ in tissues that have been deprived of oxygen, such as heart muscle following a myocardial infarction. It also provides insights into the metabolism of cancer cells, which behave as though they lack oxygen, even when they do not.

Peter Ratcliffe, professor of renal medicine at the Institute of Molecular Medicine at the John Radcliffe Hospital in Oxford, U.K., told BioWorld International, ¿The Holy Grail in our field is to understand the cell¿s oxygen-sensing mechanisms, and this finding suggests that we might be close to that. It also puts metabolism back onto the center stage of molecular oncology. It makes us think that the metabolic abnormalities of cancer cells are probably more central to tumorigenesis than people have previously believed.¿

Ratcliffe, together with colleagues at the Wellcome Trust Centre for Human Genetics in Oxford, and from the University of Birmingham, reports the study in a paper in the May 20 issue of Nature titled, ¿The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.¿

Starting with the observation that many mammalian genes with important functions are regulated by oxygen levels, Ratcliffe¿s group embarked on finding out how cells sensed oxygen. They and others identified sections in the control sequences of these genes, which they called hypoxia response elements, and showed that these bound a group of transcription factors, called the hypoxia inducible factors (HIFs). These transcription factors regulate many different genes, including, for example, that encoding vascular endothelial growth factor (VEGF), which stimulates angiogenesis.

Ratcliffe said: ¿We noticed that many of the genes that were upregulated by hypoxia in normal cells were often upregulated constitutively in cancer cells. In other words, the oncogenic process has in some way led to an obligatory hypoxia pattern, even when the cancer cells have normal oxygen levels. This led us to believe that this system might be important in cancer and that oncogenic mutations might activate this system.¿

Connection Drawn With VHL Disease

At the same time, other researchers were studying a rare inherited cancer called von Hippel-Lindau (VHL) disease. This affects one in 20,000 people, causing highly vascularized tumors of the eye, brain, and spine, as well as tumors of the adrenal glands and kidneys. People with the disease inherit one copy of the VHL gene, and tumors develop when a mutation occurs in their second copy of the gene. In addition, about 70 percent of sporadic kidney cancers are caused by mutations in the VHL gene.

Studies of tumor cells from people with VHL disease showed they also had abnormal upregulation of the messenger RNAs that are normally switched on by hypoxia ¿ and that normal levels of these molecules could be restored by transfecting into the cells a copy of the wild-type (normal) VHL gene.

¿This stimulated our interest in VHL, because it meant there was something about VHL that was connected with hypoxic gene regulation,¿ Ratcliffe said.

In the Nature paper, Ratcliffe and his colleagues show that the VHL protein is needed for the destruction of HIFs in cells with normal oxygen levels. He told BioWorld International: ¿So if oxygen levels are good, the HIFs are consumed and stay at vanishingly low levels; when hypoxia occurs, HIFs accumulate because the rate of breakdown is reduced. When VHL is defective, however, HIFs remain at a high level ¿ the system is constitutively switched on.¿ The paper shows that cells lacking VHL fail to break down HIFs even when their oxygen levels are normal.

Researchers Explore Iron Dependency

Other experiments carried out by the team demonstrated that the interaction between VHL and HIF is iron-dependent. ¿One possible explanation,¿ Ratcliffe said, ¿is that there is an iron protein in the VHL-HIF complex. It has already been proposed that it is an iron protein that senses oxygen and enables cells to respond in this way. So this may provide a clue to the location of the oxygen-sensing system. It might be that the sensor is local to HIF, rather than acting by a long signal cascade.¿

One hypothesis the team is working on is that the iron protein interacts with a partially reduced oxygen species, for example hydrogen peroxide. This is known as a Fenton reaction ¿ the oxidation of a molecule by a combination of iron and hydrogen peroxide.

¿The Fenton reaction has become a paradigm for theories about how free radicals affect molecules in biological systems,¿ Ratcliffe said. ¿In this case, we are proposing that the iron somehow causes the oxidation of the protein, and this is the signal for VHL to mark it for destruction.¿

In a News and Views article in the same issue of Nature, William G. Kaelin Jr., of the Howard Hughes Medical Institute and department of adult oncology at the Dana-Farber Cancer Institute and departments of medicine, Brigham and Women¿s Hospital and Harvard Medical School in Boston, pointed out that some features of VHL suggest it is analogous to part of a multiprotein complex found in yeast called SCF. He wrote: ¿These complexes target other proteins in the cell for degradation via a process called ubiquitination. The target is bound by a protein called ubiquitin, which labels it for destruction in the cavity of a protein-digesting complex called the proteosome.¿ VHL might regulate protein turnover in the same way, he suggested.

Ratcliffe added: ¿We think the VHL functions as part of an SCF-type complex to regulate the destruction of HIF, most probably by regulating the linkage to the polypeptide, ubiquitin. The HIF/VHL interaction gives specificity to the tagging with ubiquitin. Once it is linked to ubiquitin, it has had it and joins a general system for destruction by the proteosome.¿

Ratcliffe¿s agenda in the near future includes projects to determine the nature of the complex between HIF and VHL; to find out if iron is present and, if so, where; and to find out how HIF is modified by oxygen.