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X marks the spot in lung cancer
MOLECULAR MEDICINE TODAY, APRIL 2000 (VOL. 6)
ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1a (HIF-1a). As a result, VEGF expression is upregulated (Fig. 1). Simons elaborates ‘Until now, scientists thought that there were two major stimulants of angiogenesis: inflammation and hypoxia. But there has never been a link between these two mechanisms. Here, an inflammatory response secretes a peptide that increases the amount of HIF-1a, so really what we thought were separate processes have become one.’ PR-39 also increases concentrations of receptors for fibroblast growth factors (FGF), another family of angiogenesis inducers. As the agent seems to induce multiple pathways, PR-39 is expected to be more potent in inducing angiogenesis in patients with heart failure than other factors tested so far. The peptide significantly increased vascularity along the infarct border zone in a mouse infarct model; PR-39 had been delivered for seven days via an intraperitoneally implanted infusion pump. Kenneth Chien at the University of California (San Diego, CA, USA) advocates that these results should be viewed with a mixture of both interest and caution. He points out that in current experimental models, healthy animals are injured to mimic a myocardial infarct. ‘This might not completely reflect the situation in older, atherosclerotic patients with stiffer blood vessels who have already shown that they are refractory to their own endogenous signals to develop collaterals. We clearly need to understand more about why they are refractory.’ ‘Blocking angiogenesis in the treatment of cancer is, in the long run, probably going to be easier to accomplish than promoting angiogenesis in patients with coronary artery disease’, says Chien. ‘Inhibition of a complex biological process, such as angiogenesis, is usually easier than activation, particularly if therapy is based on a single agent. We may need to go back and focus on some of the hard, fundamental work to determine whether multiple factors will ultimately be required to drive the formation of durable muscular arteries, as opposed to microcapillaries, in the atherosclerotic heart. Nevertheless, the findings are encouraging and provide further evidence that this is an area worthy of further pursuit.’ 1 Kamphaus, G.D. et al. (2000) Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J. Biol. Chem. 275, 1209–1210 2 Li, J. et al. (2000) PR39, a peptide regulator of angiogenesis. Nat. Med. 6, 49–55
Martina Habeck Freelance science writer
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X marks the spot in lung cancer Despite a number of recent advances, lung cancer remains one of the deadliest, least treatable and commonest neoplasms. In 1998, an estimated 171 500 US citizens and 184 200 Europeans developed lung cancer. However, a number of previous studies suggested that, irrespective of the level of tobacco-smoke exposure, women are 20–70% more likely than men to develop all major types of lung cancer. Now, two new studies suggest that genes on the X chromosome might partly explain this excess risk. A study by Sharon Shriver and colleagues (University of Pittsburgh Cancer Institute, PA, USA) suggests that part of the explanation could lie in women’s more frequent expression of the gastrin-releasing peptide receptor (GRPR), which might modulate proliferation of bronchial cells to gastrin-releasing peptide and chronic tobacco use1. The gene encoding GRPR is located on the X chromosome. In women, one copy of the X chromosome per cell is usually inactivated, but, as previous studies show, the GRPR gene escapes X-chromosome inactivation. The team examined GRPR expression in mainstream bronchus and peripheral biopsy specimens from 38 women and 40 men, including 58 lung cancer patients. They established primary cultures of bronchial epithelial cells and lung fibroblasts and determined GRPR mRNA expression. In some cases, they also used a polymorphism in exon 2 of the GRPR gene to detect allelespecific expression. When they stratified results according to tobacco exposure, they found that the GRPR gene was expressed more frequently in nonsmoking women than in non-smoking men: 55% of non-smoking women expressed GRPR mRNA in their bronchial epithelium, but no non-smoking men did. Moreover, GRPR gene expression was activated earlier among women smokers than in men: 75% of women with less than 25 pack-years exposure (a pack-year is one pack of 20 cigarettes smoked daily for a year) were GRPR positive compared with only 20% of men. However, gender-related differences disappeared among people who smoked more than this. The group also has preliminary information suggesting that activation of nicotinic acetylcholine receptors by nicotine might modulate GRPR gene expression: GRPR mRNA expression increased about
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
12-fold when cultured lung fibroblasts were exposed to nicotine levels equivalent to those in smokers’ blood, and the researchers located high-affinity, saturable nicotinebinding sites on lung fibroblasts and bronchial epithelial cells, which also bind nicotinic antagonists. The authors conclude that molecular markers, such as GRPR expression, might identify individuals – especially women – at high-risk of developing lung cancer. However, GRPR isn’t the only factor that recent studies link to increased lung cancer susceptibility. Another report2 links a new addition to the epidermal growth factor (EGF) repeat superfamily to lung and other cancers. Proteins encoded by this gene family contain a 30–40-residue EGF repeat domain composed of conserved cysteine and glycine residues. They are widely expressed, and some family members – including EGF itself, CD97 (a recently identified human leukocyte antigen involved in lymphocyte activation) and the Notch family – modulate development. Notch proteins also seem to play a role in a diverse range of diseases, including stroke, dementia, leukaemia and breast cancer. Researchers at Hyseq (Sunnyvale, CA, USA) have now identified a new member of the EGF-repeat superfamily, known as EGFL6, using a high-throughput genetic screen. Paradoxically, EGFL6 also maps to the human X-chromosome. The encoded repeat sequence of EGFL6 more closely resembles the repeat sequence of Notch and CD97 than EGF, and Hyseq researchers predict that it might encode a secreted protein, as the gene contains a putative signal-peptide-encoding sequence. Apart from EGF-like repeats, the predicted protein sequence also contains two N-linked glycosylation sites, an integrin-association motif and a tyrosine phosphorylation site. The Hyseq study indicates that only specific tumour and fetal tissues express EGFL6. ‘Importantly, the gene appears to be uniquely expressed in certain rapidly dividing tissues,’ explains John Ford, Hyseq’s Senior Director of Functional Genomics. ‘Characterizing the gene product should help to determine if EGFL6 plays a role in cellular proliferation,’ he adds. Despite the prima facie evidence, the paper doesn’t causally link cancer and EGFL6, ‘The data associate the expression of
139
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w
s
EGFL6 with certain tumours. Therefore, EGFL6 and its encoded protein could represent valuable screening markers. More research should help to determine if the gene product plays a functional role in lung cancers,’ Ford comments. He guardedly adds that the association with the X chromosome is also ‘interesting’ given the gender distribution of certain cancers. Once again, however, he stresses that more work is
MOLECULAR MEDICINE TODAY, APRIL 2000 (VOL. 6)
needed: ‘This is still preliminary research. Cancer is a complicated disease on so many levels; there may never be a single answer. But studies such as these help us understand the regulatory processes underlying specific cancers – which should help us improve diagnosis and treatment. 01 Shriver, S.P. et al. (2000) Sex-specific expression of gastrin-releasing peptide
How HIV hijacks host cells Using gene chips, Gary Geiss (University of Washington, Seattle, WA, USA) and colleagues have discovered that at least 20 Tcell genes were differentially expressed soon after infection with HIV-1 (Ref. 1) (Box 1). This is the first time that gene-chip technology has been used to identify hostcell changes in gene expression brought about by HIV infection, but soon it is likely to be used to elucidate the effects of other viruses on host-cell gene expression. ‘Eventually it may be possible to identify genes and/or pathways that are regulated by many different viral infections; this analysis is only the first step in that direction,’ says Geiss. The precise role of the differentially expressed genes is not yet known, but several are involved in T-cell signalling, sub-cellular trafficking and the regulation of transcription. For example, mRNAs for the T-cell receptor a-chain and CD8-a are involved in antigen recognition via interactions with major
histocompatibility molecules, which might explain the presence of HIV-1-infected CD81 cells or the increase in CD81 cells observed in HIV infected people. Genes possibly involved in cellular trafficking and transport are translocase of the outer mitochondrial membrane (TOM34) and prothymosin-a. TOM34 could be involved in mitochondrial transport and other interactions, and the downregulation of prothymosin-a might contribute to the export of unspliced viral RNAs. Finally, receptor-interacting protein 140 (RIP140) could influence lipid metabolism via the regulation of receptor transcription, and might be linked with the dorsocervical fat-pad enlargement – ‘buffalo hump’ – that occurs in some HIV infected people. ‘One problem with analysing the expression of thousands of genes is the ability to make sense of the data’, says Geiss. He goes on: ‘I chose to speculate on how a few may be relevant to HIV, but it is possible,
Box 1. Some of the differentially expressed genes in CD41 T cells after HIV-1 infection Upregulated genes T-cell receptor a-chain c-region CD8-a precursor – potential role in T-cell interactions Human neurogranin Receptor-interacting protein 140 (RIP140) – potential role in regulation of transcription Transfer valyl-tRNA synthetase Translocase of the outer mitochondrial membrane (TOM34) – potential role in mitochondrial transport
140
Downregulated genes Adenylosuccinate lyase Prothymosin-a – potential role in cellular trafficking Phosphoglycerate kinase Glucose phosphate kinase p18 protein mRNA/stathmin Signal reduction particle 72 Placental protein 5 Type IVA protein tyrosine phosphatase Tyrosine kinase
receptor: relationship to smoking history and risk of lung cancer. J. Natl. Cancer Inst. 92, 24–33 02 Yeung, G. et al. (1999) Cloning of a novel epidermal growth factor repeat containing gene EGFL6: expressed in tumor and fetal tissues. Genomics 62, 304–307
Mark Greener Freelance science writer
even likely, that not all of these genes will be biologically relevant to HIV infection. Some of the genes differentially regulated, such as phosphoglycerate kinase, are involved in cellular metabolism and may be an indication of the growth or metabolic state of the cell’. Other changes in gene expression are likely to occur both before and after three days, but the study was limited because infected cells deteriorate rapidly after this time. In addition, other changes are likely to occur in genes not tested in this study. Geiss, Michael Katz and Roger Bumgarner (University of Washington) constructed gene chips using 1506 target human cDNAs; ‘We now have over 15 000 genes and are currently doing similar studies with our new gene set. We are very interested in doing time courses of infection, particularly at earlier time points to identify genes regulated at early times during infection,’ comments Geiss. Although these results might not shed light directly on the mode of action of HIV, they could provide clues as to the effects of HIV and other viruses on host cells, such as the initiation of the host cell’s defence or apoptotic pathways. ‘Once genes and pathways have been identified it will be possible to look at them in more detail and design experiments to test their functions. For example genes that are downregulated can be artificially overexpressed while the outcome of infection is monitored. Conversely, genes that are upregulated can be knocked out or inhibited (potentially by drugs) to see what effect that may have on infection’, says Geiss. In the future, the genes and pathways identified by this research might offer therapeutic targets. However, Geiss says, ‘We cannot conclude that at this time, but ask me in a few more years.’ 01 Geiss, G.K. et al. (2000) Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology 266, 8–16
Sharon Dorrell Freelance science writer
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1a (HIF-1a). As a result, VEGF expression is upregulated (Fig. 1). Simons elaborates ‘Until now, scientists thought that there were two major stimulants of angiogenesis: inflammation and hypoxia. But there has never been a link between these two mechanisms. Here, an inflammatory response secretes a peptide that increases the amount of HIF-1a, so really what we thought were separate processes have become one.’ PR-39 also increases concentrations of receptors for fibroblast growth factors (FGF), another family of angiogenesis inducers. As the agent seems to induce multiple pathways, PR-39 is expected to be more potent in inducing angiogenesis in patients with heart failure than other factors tested so far. The peptide significantly increased vascularity along the infarct border zone in a mouse infarct model; PR-39 had been delivered for seven days via an intraperitoneally implanted infusion pump. Kenneth Chien at the University of California (San Diego, CA, USA) advocates that these results should be viewed with a mixture of both interest and caution. He points out that in current experimental models, healthy animals are injured to mimic a myocardial infarct. ‘This might not completely reflect the situation in older, atherosclerotic patients with stiffer blood vessels who have already shown that they are refractory to their own endogenous signals to develop collaterals. We clearly need to understand more about why they are refractory.’ ‘Blocking angiogenesis in the treatment of cancer is, in the long run, probably going to be easier to accomplish than promoting angiogenesis in patients with coronary artery disease’, says Chien. ‘Inhibition of a complex biological process, such as angiogenesis, is usually easier than activation, particularly if therapy is based on a single agent. We may need to go back and focus on some of the hard, fundamental work to determine whether multiple factors will ultimately be required to drive the formation of durable muscular arteries, as opposed to microcapillaries, in the atherosclerotic heart. Nevertheless, the findings are encouraging and provide further evidence that this is an area worthy of further pursuit.’ 1 Kamphaus, G.D. et al. (2000) Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J. Biol. Chem. 275, 1209–1210 2 Li, J. et al. (2000) PR39, a peptide regulator of angiogenesis. Nat. Med. 6, 49–55
Martina Habeck Freelance science writer
N
e
w
s
X marks the spot in lung cancer Despite a number of recent advances, lung cancer remains one of the deadliest, least treatable and commonest neoplasms. In 1998, an estimated 171 500 US citizens and 184 200 Europeans developed lung cancer. However, a number of previous studies suggested that, irrespective of the level of tobacco-smoke exposure, women are 20–70% more likely than men to develop all major types of lung cancer. Now, two new studies suggest that genes on the X chromosome might partly explain this excess risk. A study by Sharon Shriver and colleagues (University of Pittsburgh Cancer Institute, PA, USA) suggests that part of the explanation could lie in women’s more frequent expression of the gastrin-releasing peptide receptor (GRPR), which might modulate proliferation of bronchial cells to gastrin-releasing peptide and chronic tobacco use1. The gene encoding GRPR is located on the X chromosome. In women, one copy of the X chromosome per cell is usually inactivated, but, as previous studies show, the GRPR gene escapes X-chromosome inactivation. The team examined GRPR expression in mainstream bronchus and peripheral biopsy specimens from 38 women and 40 men, including 58 lung cancer patients. They established primary cultures of bronchial epithelial cells and lung fibroblasts and determined GRPR mRNA expression. In some cases, they also used a polymorphism in exon 2 of the GRPR gene to detect allelespecific expression. When they stratified results according to tobacco exposure, they found that the GRPR gene was expressed more frequently in nonsmoking women than in non-smoking men: 55% of non-smoking women expressed GRPR mRNA in their bronchial epithelium, but no non-smoking men did. Moreover, GRPR gene expression was activated earlier among women smokers than in men: 75% of women with less than 25 pack-years exposure (a pack-year is one pack of 20 cigarettes smoked daily for a year) were GRPR positive compared with only 20% of men. However, gender-related differences disappeared among people who smoked more than this. The group also has preliminary information suggesting that activation of nicotinic acetylcholine receptors by nicotine might modulate GRPR gene expression: GRPR mRNA expression increased about
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
12-fold when cultured lung fibroblasts were exposed to nicotine levels equivalent to those in smokers’ blood, and the researchers located high-affinity, saturable nicotinebinding sites on lung fibroblasts and bronchial epithelial cells, which also bind nicotinic antagonists. The authors conclude that molecular markers, such as GRPR expression, might identify individuals – especially women – at high-risk of developing lung cancer. However, GRPR isn’t the only factor that recent studies link to increased lung cancer susceptibility. Another report2 links a new addition to the epidermal growth factor (EGF) repeat superfamily to lung and other cancers. Proteins encoded by this gene family contain a 30–40-residue EGF repeat domain composed of conserved cysteine and glycine residues. They are widely expressed, and some family members – including EGF itself, CD97 (a recently identified human leukocyte antigen involved in lymphocyte activation) and the Notch family – modulate development. Notch proteins also seem to play a role in a diverse range of diseases, including stroke, dementia, leukaemia and breast cancer. Researchers at Hyseq (Sunnyvale, CA, USA) have now identified a new member of the EGF-repeat superfamily, known as EGFL6, using a high-throughput genetic screen. Paradoxically, EGFL6 also maps to the human X-chromosome. The encoded repeat sequence of EGFL6 more closely resembles the repeat sequence of Notch and CD97 than EGF, and Hyseq researchers predict that it might encode a secreted protein, as the gene contains a putative signal-peptide-encoding sequence. Apart from EGF-like repeats, the predicted protein sequence also contains two N-linked glycosylation sites, an integrin-association motif and a tyrosine phosphorylation site. The Hyseq study indicates that only specific tumour and fetal tissues express EGFL6. ‘Importantly, the gene appears to be uniquely expressed in certain rapidly dividing tissues,’ explains John Ford, Hyseq’s Senior Director of Functional Genomics. ‘Characterizing the gene product should help to determine if EGFL6 plays a role in cellular proliferation,’ he adds. Despite the prima facie evidence, the paper doesn’t causally link cancer and EGFL6, ‘The data associate the expression of
139
N
e
w
s
EGFL6 with certain tumours. Therefore, EGFL6 and its encoded protein could represent valuable screening markers. More research should help to determine if the gene product plays a functional role in lung cancers,’ Ford comments. He guardedly adds that the association with the X chromosome is also ‘interesting’ given the gender distribution of certain cancers. Once again, however, he stresses that more work is
MOLECULAR MEDICINE TODAY, APRIL 2000 (VOL. 6)
needed: ‘This is still preliminary research. Cancer is a complicated disease on so many levels; there may never be a single answer. But studies such as these help us understand the regulatory processes underlying specific cancers – which should help us improve diagnosis and treatment. 01 Shriver, S.P. et al. (2000) Sex-specific expression of gastrin-releasing peptide
How HIV hijacks host cells Using gene chips, Gary Geiss (University of Washington, Seattle, WA, USA) and colleagues have discovered that at least 20 Tcell genes were differentially expressed soon after infection with HIV-1 (Ref. 1) (Box 1). This is the first time that gene-chip technology has been used to identify hostcell changes in gene expression brought about by HIV infection, but soon it is likely to be used to elucidate the effects of other viruses on host-cell gene expression. ‘Eventually it may be possible to identify genes and/or pathways that are regulated by many different viral infections; this analysis is only the first step in that direction,’ says Geiss. The precise role of the differentially expressed genes is not yet known, but several are involved in T-cell signalling, sub-cellular trafficking and the regulation of transcription. For example, mRNAs for the T-cell receptor a-chain and CD8-a are involved in antigen recognition via interactions with major
histocompatibility molecules, which might explain the presence of HIV-1-infected CD81 cells or the increase in CD81 cells observed in HIV infected people. Genes possibly involved in cellular trafficking and transport are translocase of the outer mitochondrial membrane (TOM34) and prothymosin-a. TOM34 could be involved in mitochondrial transport and other interactions, and the downregulation of prothymosin-a might contribute to the export of unspliced viral RNAs. Finally, receptor-interacting protein 140 (RIP140) could influence lipid metabolism via the regulation of receptor transcription, and might be linked with the dorsocervical fat-pad enlargement – ‘buffalo hump’ – that occurs in some HIV infected people. ‘One problem with analysing the expression of thousands of genes is the ability to make sense of the data’, says Geiss. He goes on: ‘I chose to speculate on how a few may be relevant to HIV, but it is possible,
Box 1. Some of the differentially expressed genes in CD41 T cells after HIV-1 infection Upregulated genes T-cell receptor a-chain c-region CD8-a precursor – potential role in T-cell interactions Human neurogranin Receptor-interacting protein 140 (RIP140) – potential role in regulation of transcription Transfer valyl-tRNA synthetase Translocase of the outer mitochondrial membrane (TOM34) – potential role in mitochondrial transport
140
Downregulated genes Adenylosuccinate lyase Prothymosin-a – potential role in cellular trafficking Phosphoglycerate kinase Glucose phosphate kinase p18 protein mRNA/stathmin Signal reduction particle 72 Placental protein 5 Type IVA protein tyrosine phosphatase Tyrosine kinase
receptor: relationship to smoking history and risk of lung cancer. J. Natl. Cancer Inst. 92, 24–33 02 Yeung, G. et al. (1999) Cloning of a novel epidermal growth factor repeat containing gene EGFL6: expressed in tumor and fetal tissues. Genomics 62, 304–307
Mark Greener Freelance science writer
even likely, that not all of these genes will be biologically relevant to HIV infection. Some of the genes differentially regulated, such as phosphoglycerate kinase, are involved in cellular metabolism and may be an indication of the growth or metabolic state of the cell’. Other changes in gene expression are likely to occur both before and after three days, but the study was limited because infected cells deteriorate rapidly after this time. In addition, other changes are likely to occur in genes not tested in this study. Geiss, Michael Katz and Roger Bumgarner (University of Washington) constructed gene chips using 1506 target human cDNAs; ‘We now have over 15 000 genes and are currently doing similar studies with our new gene set. We are very interested in doing time courses of infection, particularly at earlier time points to identify genes regulated at early times during infection,’ comments Geiss. Although these results might not shed light directly on the mode of action of HIV, they could provide clues as to the effects of HIV and other viruses on host cells, such as the initiation of the host cell’s defence or apoptotic pathways. ‘Once genes and pathways have been identified it will be possible to look at them in more detail and design experiments to test their functions. For example genes that are downregulated can be artificially overexpressed while the outcome of infection is monitored. Conversely, genes that are upregulated can be knocked out or inhibited (potentially by drugs) to see what effect that may have on infection’, says Geiss. In the future, the genes and pathways identified by this research might offer therapeutic targets. However, Geiss says, ‘We cannot conclude that at this time, but ask me in a few more years.’ 01 Geiss, G.K. et al. (2000) Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology 266, 8–16
Sharon Dorrell Freelance science writer
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.