**BIOTECH AND PHARMA NEWS**

[Preciouslife1]

Apoptosis: Activating the executioner

Sarah Seton-Rogers
Because all cells can undergo apoptosis, it has been difficult to find a target that can be exploited to induce death in cancerous, but not normal, cells. Paul Hergenrother and colleagues have now identified a small molecule that can induce apoptosis in various cancer cell lines and mouse tumours, but that seems to be relatively non-toxic to normal cells.
Many therapies being developed to trigger apoptosis are designed to activate early or intermediate steps in the apoptotic cascade, and therefore cancers with mutations that are downstream of these steps might be resistant. The authors reasoned that it would be ideal to activate a protein that is far downstream in the cascade, so they focused on the conversion of procaspase 3 to caspase 3. Caspase 3 is an 'executioner' caspase that catalyses the hydrolysis of many proteins. It is kept in its inactive procaspase 3 form in cells until needed, and, paradoxically, many tumour cells have high procaspase 3 expression whereas normal cells do not.

The authors screened a library of 20,500 small molecules for their ability to convert procaspase 3 to caspase 3 in vitro, and found one compound, procaspase-activating compound 1 (PAC1), that has a high specific dose-dependent activity. PAC1 induced apoptosis in various cancer cell lines, and there was a correlation between the levels of procaspase 3 expressed in the cells and PAC1-induced apoptosis. In vitro, PAC1 also effectively killed cancerous cells in resected colon tumours, but adjacent normal cells were resistant. To further examine the effect of PAC1 on normal cells, the authors also looked at several non-cancerous cell lines, and found that these cells had low procaspase 3 levels and were not affected by PAC1 treatment.
Will PAC1 be effective against tumours in vivo? The authors tested PAC1 in mouse xenograft models of renal and lung cancer. In the renal model they implanted a slow-release pellet of PAC1, and in the lung model they gave PAC1 orally. Both methods of delivering PAC1 slowed tumour growth in a dose-dependent manner, and PAC1 seemed to be well tolerated by the animals. Although the tumours did not regress, the authors propose that this might be because the doses of PAC1 used led to relatively low serum concentrations of the drug. Finally, they injected lung cancer cells into the tail vein of mice and gave PAC1 orally on days 1–4 and 7–11. After 28 days, the mice treated with PAC1 had a lower tumour burden than the control mice.
Hergenrother and colleagues have shown that PAC1 has the potential to treat cancers that have increased levels of procaspase 3, but a systematic analysis of procaspase 3 concentrations in different human cancer types is needed to determine which cancers will be most amenable to treatment with PAC1. Also, PAC1 might need to be modified to increase its oral availability and potency in humans. So, PAC1 might not be the magic bullet that cancer researchers are searching for, but it could be one step closer to it.

References and links

ORIGINAL RESEARCH PAPER

1. Putt, K. S. et al. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nature Chem. Biol. 27 August 2006 (doi:10.1038/nchembio814)

[Preciouslife1]

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis proteinAbstractActivation of effector caspases is a final step during apoptosis. Single-cell imaging studies have demonstrated that this process may occur as a rapid, all-or-none response, triggering a complete substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome-dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase-3-dependent feedback signalling and the X-linked-inhibitor-of-apoptosis-protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single-cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all-or-none response, with concentrations as high as 0.15 M enabling, but concentrations >0.30 M significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo.Keywords: apoptosis, caspases, single-cell imaging, systems biology, XIAP

[Preciouslife1]

Tumorigenesis: Rare and informative

Nicola McCarthy
An increased risk of cancer development is found in the rare syndrome xeroderma pigmentosum (XP), whereas the development of segmental progeria (aspects of accelerated ageing) is observed in Cockayne syndrome (CS) and trichothiodystrophy (TTD). Patients with these distinct syndromes all have defects in the nucleotide excision DNA repair pathway. Remarkably, in some instances mutations in the same gene can give rise to XP, CS or TTD. In exceptionally rare cases, patients present with both XP and CS or TTD symptoms, known as XPCS or XPTTD. Mouse models of XP, CS and TTD have provided useful insights into these diseases, so to investigate how patients can develop XPCS, Jan Hoeijmakers and colleagues used a mutation found in an XPCS patient — a single point mutation (G602D) in the XPD gene, the product of which forms part of the TFIIH DNA repair and transcription complex — to produce an XPCS mouse that has both an increased risk of tumour development and segmental progeria.
As in patients with XPCS, the expression level of the TFIIH complex was reduced by 50% in cells of the XPCS mouse compared with wild-type cells. As XP and CS patients are hypersensitive to UV-induced DNA damage, the authors examined the UV sensitivity of XPCS mice. UV hypersensitivity was seen after 1 week in XPCS mice exposed to an environmentally relevant dose of UV for 4 days. CS patients do not have an increased risk of skin tumour development despite hypersensitivity to UV radiation, but XP and XPCS patients do. To test for a predisposition to skin cancer, the XPCS mice were exposed to a daily low dose of UV, and the incidence of tumour development was compared with another cancer-prone XP mouse model — XPA mice that have no nucleotide excision repair function. After 17 weeks, all ten XPCS mice had skin or eye tumours compared with none of the XPA mice, indicating that the XPCS mutation produces a profound increase in the risk of UV-mediated tumour development, consistent with the development of a skin tumour in the original XPCS patient at 2.5 years of age.
Why are XPCS mice, which retain some nucleotide excision repair, more cancer prone than XPA mice that have no nucleotide excision repair function? The removal of a UV-induced DNA adduct involves an incision (cut) and patch of the DNA and removal of the lesion. Through a number of experiments the authors found that although cells from both XPA and XPCS mice lack the capacity to remove UV-induced DNA lesions, XPCS cells seem to have uncoupled cut and patch mechanisms that result in persistent DNA strand breaks owing to the lesion not being patched. The authors propose that it is this characteristic that further increases the risk of tumour development.

So, although syndromes such as TTD and CS are associated with accelerated ageing and no increase in cancer risk, the XPCS mouse model shows that these two processes are not always antagonistic and can coexist. This is crucially dependent on the specific mutation that has occurred and the processes that are consequently affected.

References and links

ORIGINAL RESEARCH PAPER

1. Andressoo, J. -O. et al. An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria. Cancer Cell 10, 121–132 (2006)

[Preciouslife1]

Angiogenesis: Moving downstream

Sarah Seton-Rogers
Vascular endothelial growth factor (VEGF) increases tumour angiogenesis, but now Laura Benjamin and colleagues have looked downstream of VEGF and shown that activation of the Akt pathway — one of the pathways activated in response to VEGF — can recapitulate the effects of VEGF on tumour blood vessels.
To investigate the role of endothelial-cell-autonomous Akt activation in blood vessels in an otherwise normal microenvironment, the authors used transgenic mice that expressed a tetracycline-repressible myristoylated AKT1 (myrAKT1) that is constitutively active specifically in endothelial cells. They observed distinct differences in the vasculature of mice with endothelial expression of myrAKT1 compared with those without myrAKT1 induction. The surface area of the skin of mice with activated myrAKT1 was covered by twice as many blood vessels, and these vessels had larger diameters and were more tortuous. Furthermore, the mice had increased blood volume, and there was a trend towards increased permeability of the myrAKT1 vessels. These abnormal blood vessels that mimic tumour vasculature but are formed in the absence of a tumour are called 'pathological blood vessels'.
Is the continued activation of AKT1 required to maintain these pathological blood vessels? The authors activated myrAKT1 expression in mice by withdrawing tetracycline and then restored tetracycline to suppress myrAKT1 expression. They found that the effects of myrAKT1 on blood vessels were completely reversed when myrAKT1 expression was suppressed, indicating that the vessels were dependent on sustained AKT1 signalling.
One downstream effector of Akt is mammalian target of rapamycin (mTOR), and the mTOR inhibitor rapamycin is currently being investigated as an anticancer agent. Treating mice with rapamycin at the same time as tetracycline was withdrawn to induce myrAKT1 expression blocked the formation of pathological blood vessels. The inhibition of mTOR by rapamycin not only blocks signalling downstream of mTOR, but also reduces Akt activation. The authors attribute the observed effects in endothelial cells to this inhibition of the Akt pathway. They also injected tumour cells that expressed VEGF into nude mice, and showed that rapamycin treatment significantly reduced tumour growth, endothelial Akt activation and the permeability of tumour blood vessels.

The work by Benjamin and colleagues highlights the importance of the Akt–mTOR pathway in angiogenesis. Furthermore, it indicates that the inhibition of this pathway, for example by rapamycin, is a valid therapeutic strategy for tumours with increased angiogenesis. The ability of rapamycin to inhibit pathological angiogenesis and vascular leak might also facilitate the more effective delivery of chemotherapeutic agents to tumours. As VEGF-inhibitors are already being used in patients with various cancers, it will be interesting to see whether the inhibition of the Akt–mTOR pathway replicates or amplifies the effects of VEGF inhibition.

References and links

ORIGINAL RESEARCH PAPER

1. Phung, T. L. et al. Pathological angiogenesis is induced by sustained Akt signalling and inhibited by rapamycin. Cancer Cell 10, 159–170 (2006)

[Preciouslife1]

Ageing: Longevity mutations inhibit tumours

Patrick Goymer
The link between cancer and ageing is well established — the older animals of many species are also more likely to develop tumours. Cynthia Kenyon and colleagues now extend the link further by showing that mutations that extend lifespan in Caenorhabditis elegans also inhibit tumour growth.

To examine the links between ageing and cancer, the authors studied the interactions between mutations that affect the two processes. Mutations in the C. elegans insulin-receptor gene daf-2 extend lifespan by more than twofold. By contrast, mutations in the gld-1 tumour-suppressor gene cause germ cells to re-enter mitosis, overproliferate and give rise to tumours that kill the animal. When the authors combined the two mutations, however, the lifespan was indistinguishable from that of the daf-2 single mutant — the tumorigenic effect of gld-1 was completely abolished.
How might the lack of insulin signalling prevent tumour development? The authors found that cell division decreased and apoptosis increased in the germ lines of the daf-2; gld-1 double mutant compared with the gld-1 single mutant. Interestingly, daf-2 mutations affected only germline mitosis in the tumour, and not in the normal germ lines of otherwise wild-type individuals. Also, they found that p53 was required for increased apoptosis and that introducing a p53 mutation into the double mutant reduced lifespan. This is in contrast to wild-type animals, in which p53 mutations reduce apoptosis but do not affect lifespan.
Are these effects specific to mutations in the insulin-signalling pathway, or do they occur with other lifespan-increasing mutations? Mutations in genes that affect food uptake and mitochondrial pathways also extend lifespan. Worms with such mutations, unlike daf-2 mutants, were not completely unaffected by the gld-1 mutation; however, the daf-2; gld-1 double mutants lived longer than gld-1 animals. These mutations did not affect apoptosis, but they did still reduce tumour incidence. They might therefore have more indirect downstream effects.
A connection between insulin signalling and tumour growth has also been shown in mammals, and it will be interesting to carry out *************************alent experiments in mice. These results also suggest that, in contrast to p53, many mutations will have opposing effects on ageing and cancer.

References and links

ORIGINAL RESEARCH PAPER

1. Pinkston, J. M. et al. Mutations that increase the life span of C. elegans inhibit tumor growth. Science 313, 971–975 (2006)

[Preciouslife1]

Trial Watch: Chemoprevention with COX2 inhibitors?

Colorectal cancer often develops from adenomatous polyps (adenomas), so finding agents that can prevent or regress colorectal adenomas has been a goal of researchers. Cyclooxygenase 2 (COX2) is overexpressed in human colorectal adenomas and tumours, but not in normal colorectal tissue, indicating that COX2 inhibitors might prevent colorectal cancer.
The primary results of two large trials that investigated the use of the COX2 inhibitor celecoxib for the prevention of colorectal adenomas were reported recently. These trials were both stopped in late 2004 following reports that celecoxib and other COX2 inhibitors could increase the risk of cardiovascular events. Although the safety data from these trials were published rapidly, it was not possible to assess the risk–benefit ratio of celecoxib for chemoprevention until now.
The placebo-controlled Adenoma Prevention with Celecoxib (APC) and Prevention of Colorectal Sporadic Adenomatous Polyps (PreSAP) trials were similarly designed, and randomized 2,035 and 1,561 patients, respectively. Only patients who had had an adenoma removed during a previous colonoscopy were enrolled, and both trials were planned to have 3 years of active treatment with colonoscopies after 1 and 3 years. Different dosing regimens were used; patients in the APC trial received 200 mg or 400 mg of celecoxib twice a day or placebo, and patients in the PreSAP trial received 400 mg of celecoxib once a day or placebo.
Both trials found that celecoxib was significantly more effective than placebo for preventing adenomas over 3 years of treatment. In the APC trial, the cumulative incidence of adenomas was 60.7% for patients who received placebo, and 43.2% and 37.5% for patients who received 200 mg or 400 mg of celecoxib twice a day, respectively. In the PreSAP trial, the cumulative incidence of adenomas was 49.3% in the placebo group and 33.6% in the celecoxib group. Both trials were too small to assess whether celecoxib decreased colorectal cancer rates, but reduction in adenoma is considered an excellent surrogate endpoint for colorectal cancer.
However, both trials also found that celecoxib was associated with an increased risk of cardiovascular events compared with placebo. A hypothetical risk–benefit analysis in an accompanying editorial suggested that celecoxib could have an advantage over no treatment for preventing colorectal cancer. However, because cardiovascular events are much more common than colorectal cancer, the increase in cardiovascular events with celecoxib clearly outweighed any possible decrease in colorectal cancer.
So, although celecoxib can prevent colorectal adenomas, it cannot be recommended for this indication owing to an increased risk of cardiovascular events.

References and links

ORIGINAL RESEARCH PAPERS

1. Arber, N. et al. Celecoxib for the prevention of colorectal adenomatous polyps. N. Engl. J. Med. 355, 885–895 (2006)

[Preciouslife1]

Vaccines: Everything in moderation

Ezzie Hutchinson

Tumour vaccines aim to elicit potent immunity against a tumour, but one obstacle that they must overcome is the poor reactivity of T cells for tumour-associated antigens (TAAs). Mimotopes are mimics of tumour epitopes that can be used in vaccines to increase the number and function of TAA-specific T cells, but tumour regression in patients often does not correlate with the magnitude of the T-cell responses that are caused. Jill Slansky and colleagues have now shown that it is not the peptides that bind with highest affinity to the T-cell receptors (TCRs) that stimulate the most effective anti-tumour response, but those that bind with moderate affinity.
The authors identified six mimotopes from a combinatorial peptide library that, when complexed with the major histocompatibility complex molecule H-2Ld, increased affinities for a representative TCR that recognizes the AH1 T-cell epitope from a mouse colon tumour. All six mimotopes stimulated the production of interferon- (IFN), which is a potent activator of macrophages and is crucial for an effective response against solid tumours, from a T-cell clone in cell culture. They also all showed a correlation between their binding affinity to the TCR and their functional stimulation of the T cells.
So, does this correlation between binding affinity and functional stimulation hold in vivo? Mice were first vaccinated with mimotopes and then challenged with colon tumour cells. The AH1 peptide provided no protection from tumour growth, as expected. Unexpectedly, however, the intermediate-affinity mimotopes were most effective — a significant number of mice remained tumour free for more than 60 days compared with mice vaccinated with high-affinity mimotopes. When the high-affinity peptides were examined further, Slansky and colleagues found that although numbers of AH1-specific T cells with the expected expression of activation markers were increased in the mice, these T cells had defects in effector function. Tumour-infiltrating T cells in the mice that were vaccinated with high-affinity mimotopes produced significantly less IFN than those in mice that were vaccinated with the intermediate-affinity mimotopes. Interestingly, this was specific to stimulation with the AH1 mimotopes, as treatment of the mice with other T-cell stimuli induced higher IFN production.
These data show that it is the quality and not the quantity of the T-cell response that determines the effectiveness of the immune response to these tumour vaccines in vivo. This should be taken into account when designing peptide vaccines for anticancer therapy in the future.

References and links

ORIGINAL RESEARCH PAPER

1. McMahan, R. H. et al. Relating TCR-peptide-MHC affinity to immunogenecity for the design of tumour vaccines. J. Clin. Invest. 116, 2543–2551 (2006)

[Preciouslife1]

Mechanisms of disease: Perils of ageing

Asher Mullard

A molecular mechanism has now been proposed that explains why Alzheimer's disease (AD), which typically strikes in 50–80-year olds, is linked with ageing. Reporting in Science, researchers showed that the transcription factors HSF-1 and DAF-16, which are regulated by a central ageing pathway, have opposing disaggregation and aggregation activities that function together to prevent AD.
AD, like other late-onset neurodegenerative diseases, is correlated with toxic aberrant protein aggregation — specifically, amyloid precursor protein breaks down into A1–42 peptides, which aggregate. But why aggregate-mediated toxicity is linked with age has remained unclear.
Cohen, Bieschke and colleagues investigated whether increasing the lifespan (or slowing the ageing) of Caenorhabditis elegans would delay the onset of aggregation. If so, then the late onset of AD could be due to a detoxifying activity that becomes compromised with ageing. If not, a stochastic time-related build-up of toxic aggregates to a threshold could explain the late onset of AD.
To distinguish between these possibilities, researchers disrupted the insulin-signalling pathway, which is central in the regulation of ageing in worms, flies and mammals. In C. elegans, a sole insulin receptor, DAF-2, transduces a signal that reduces the expression of genes that are regulated by the transcription factors DAF-16 and HSF-1, resulting in a shortened lifespan. By knocking down daf-2 in C. elegans that expressed human A1–42, researchers showed that worms with longer lifespans had reduced toxic aggregation — late onset is therefore due to a compromised detoxifying activity rather than a stochastic accumulation. Double knockdown of daf-2 with either daf-16 or hsf-1, however, reversed this effect.
So, how do DAF-16 and HSF-1 inhibit the toxicity of protein aggregation? Examining the amounts of high-molecular-weight A1–42 aggregates and small A1–42 aggregates, Cohen and colleagues made several interesting findings. First, HSF1 regulates the disaggregation of A1–42 aggregates, but DAF-16 does not. By contrast, DAF-16 mediates the formation of high-molecular-weight A1–42 aggregates, but these aggregates do not correlate with toxicity. Last, small A1–42 aggregates correlate with toxicity.
Together, these results are indicative of a mechanism that links ageing with late-onset AD. As aggregates develop, HSF-1 activity mediates their disaggregation. DAF-16 activity supports an alternative pathway (which perhaps functions as a back-up pathway) that mediates the formation of low-toxicity, high-molecular-weight aggregates from high-toxicity small aggregates. Because both detoxification pathways are mediated by the ageing-related insulin-signalling pathway, both can become compromised with ageing, leading to aggregate build-up.

A molecular mechanism has now been proposed that explains why Alzheimer's disease ... is linked with ageing.

Interestingly, as the insulin-signalling pathway is also associated with the formation of other toxic aggregates, such as those responsible for Huntington's disease, further research into this pathway could yield therapeutic targets for the general prevention of late-onset aggregation-linked neurodegenerative diseases.

References and links

ORIGINAL RESEARCH PAPER

1. Cohen, E. et al. Opposing activities against age onset proteotoxicity. Science 10 August 2006 (doi:10.1126/science.1124646)

[Preciouslife1]

Ageing: Balancing self-renewal and ageing

Ekat Kritikou

Mammalian ageing has been associated with reduced regenerative capacity. Three new studies now provide a potential link between the tumour suppressor p16INK4a, the replicative capacity of stem cells and regenerative capacity in ageing tissues.
Expression of p16INK4a increases with age, and increased p16INK4a expression has been associated with cellular senescence and has been postulated to contribute to ageing. However, whether increased p16INK4a expression functionally contributes to ageing by causing a decline in stem-cell function in vivo remained unclear.
Krishnamurthy et al. assessed the impact of p16INK4a deficiency and overexpression in pancreatic-islet proliferation. The authors induced p16INK4a expression (to a level similar to that observed in old mice) in young transgenic mice that harboured an extra copy of the p16INK4a gene. These transgenic animals had reduced islet proliferation compared with controls, which prompted the authors to propose that increased levels of p16INK4a in old animals inhibit islet proliferation. Further analysis showed that the loss of p16INK4a did not alter islet proliferation in young mice, but did rescue the age-induced decrease in proliferation.
Because the tumour-prone nature of p16INK4a-deficient animals limited the functional studies that could be carried out, Krishnamurthy et al. used a model of islet regeneration. Prolonged survival after a single, diabetes-causing treatment with a -cell toxin requires the regeneration of significant numbers of functional -cells. Animals that were deficient in p16INK4a exhibited enhanced survival. The greatest differences in survival were observed in the oldest mice, which indicates that p16INK4a mediates a decline in the replicative capacity of islets that is associated with ageing.
Molofsky et al. observed increased p16INK4a expression with age in neural progenitors, which prompted them to examine the percentage of progenitors, proliferation and neurogenesis in the forebrain lateral ventricle subventricular zone (SVZ) in young and old p16INK4a-deficient mice. The percentage of stem cells, their potential to self-renew and the overall proliferation rate all decline with age in the SVZ. Loss of p16INK4a rescued the age-related decline in cells that can form stem-cell colonies in culture, and partially rescued the overall decline in SVZ proliferation and neurogenesis. The authors proposed that p16INK4a expression could be developmentally programmed to increase with age in order to counter the increasing incidence of cancer in the ageing nervous system. Alternatively, increased p16INK4a expression might reflect the induction of senescence in the ageing cells in response to damage that accumulates with age.
In a third report, Janzen et al. showed that p16INK4a expression increased with age in haematopoietic stem cells (HSCs). Studies of differentiation and homing capacity in p16INK4a-deficient mice indicated that the age-associated increase in p16INK4a expression restricts the number of HSCs. Further analysis of cell proliferation in young and old wild-type and p16INK4a-knockout mice showed no differences in young animals. However, old p16INK4a-deficient mice had higher numbers of stem-cells and increased stem-cell function, which was associated with increased proliferation.
What is the mechanism of p16INK4a function? Based on the analysis of markers that have been associated with stem-cell self-renewal, such as Bmi1 and Hes1, Janzen et al. proposed the following model: in aged animals, the increase in p16INK4a expression is associated with a reduced repopulating capacity and decreased expression of Hes1, whereas p16INK4a deletion is accompanied by an improved repopulating capacity of stem cells and increased expression of Hes1.
Taken together, these results from diverse tissues indicate that p16INK4a contributes to mammalian ageing by limiting the self-renewal of regenerative cells — at least in the bone marrow, endocrine pancreas and brain. Whether the balance between self-renewal and ageing is also controlled by p16INK4a in other tissues remains to be investigated.

...p16INK4a contributes to mammalian ageing by limiting the self-renewal of regenerative cells...

Reflinks<H5 minmax_bound="true">ORIGINAL RESEARCH PAPERS </H5>

1. <LI id=B1 minmax_bound="true">Krishnamurthy, J. et al. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 6 Sep 2006 (doi:10.1038/nature05192)
   • Article
   <LI id=B2 minmax_bound="true">Molofsky, A.V. et al. Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature 6 Sep 2006 (doi:10.1038/nature05091)
   • Article
2. Janzen, V. et al. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 6 Sep 2006 (doi:10.1038/nature05159)
   • Article

[Preciouslife1]

Membrane trafficking: Kiss and patch up

James Pickett
Coupling of mucus secretion and membrane repair. Adapted from figure 7 in the highlighted paper.

The cell lining of the gastrointestinal tract is continually damaged by mechanical stresses and scratching by partially digested food as it traverses the gut. Potentially, this could have fatal consequences for the cells, but the digestive tract has evolved two defence mechanisms: the formation of membrane patches that plaster over holes in the membrane, and the secretion of a lubricating mucus that cushions the membrane against further abrasions. Miyake et al. have now shown that during cell injury, both of these processes are rapidly activated to protect the gastrointestinal tract.
Miyake et al. monitored mucus secretion using fluorescently labelled lectins that bind to the glycoproteins found in mucus granules. Wounding the mucosal cells, by repetitively drawing them up through a syringe, induced the release of lectins into the surrounding medium. The amount of lectin secretion increased with the amount of cell injury but was inhibited in the absence of calcium, which is known to be an important regulator of membrane-fusion events.
The authors developed a second method for assessing plasma membrane resealing after wounding. Cells were incubated with a lipophilic fluorescent dye that labels the plasma membrane. When cell membranes were punctured by a laser insult, the cytoplasm was quickly labelled by the dye in the absence of calcium. In the presence of calcium, no intracellular labelling was observed, which indicated that the plasma membrane was rapidly resealed following injury.
By combining these two techniques, Miyake et al. conclusively showed that membrane resealing occurs as a consequence of mucus secretion. After injury, cells that lacked cytosolic staining by the lipophilic dye were abundantly labelled on their membranes by fluorescent lectins, indicating that mucus secretion had occurred. Furthermore, cells that survived plasma-membrane disruption were depleted of intracellular mucus, which was instead secreted. Therefore, it seems that an increase in calcium promotes the secretion of mucus-laden vesicles. It is the 'spent' vesicle membranes themselves that subsequently remain on the plasma membrane and patch over the hole.

...membrane resealing occurs as a consequence of mucus secretion.

Importantly, when these experiments were repeated in segments of rat colon, the same conclusions were reached. This work has raised some key questions. For example, how is calcium involved in mucus secretion and membrane resealing? And, could these 'healing' processes be defective in pathological conditions of the gastrointestinal tract, as has been demonstrated to be the case for skeletal muscle?

References and links

ORIGINAL RESEARCH PAPER

1. Miyake, K. et al. Disruption-induced mucus secretion: repair and protection. PLoS Biol. 4, e276 (2006)

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News In Brief...you can click on blue links for more in depth details.

Drug Discovery
Three-hybrid for mammalian cells
As an extension of the yeast two-hybrid systems for detecting protein-protein interactions, yeast three-hybrid systems are used to screen for proteins that interact with a small molecule or vice versa. For drug discovery, however, it would be advantageous to perform these types of screens in mammalian cells. Caligiuri et al. now describe MASPIT, or mammalian small molecule–protein interaction trap, a mammalian three-hybrid system.

Caligiuri, M. et al. Chem. Biol. 13, 711–722 (2006).

Chemical Biology
A new tag-probe system for labeling proteins
Short peptide tag–fluorescent small molecule pairs such as the tetracysteine motif–FlAsH dye are indispensable for cell-biology applications. Ojida et al. report that an oligo-aspartate tag and a fluorescently labeled multinuclear zinc(II) complex can serve as a new system for labeling cell-surface proteins. Notably, the properties of the probe can be easily tuned to suit the application.

Ojida, A. et al. J. Am. Chem. Soc. 128, 10452–10459 (2006).

Cell Biology
Multiplex measurements of neuronal signaling
Signal propagation through individual neurons is often studied using electrophysiology techniques. Patolsky et al. have now designed a cell culture–compatible field-effect transistor array of silicon nanowire for the multiplex arraying of neurons. This array facilitates highly sensitive, spatially resolved detection of neuronal signaling, with potential applications in drug discovery and testing.

Patolsky, F. et al. Science 313, 1100–1104 (2006).

[Preciouslife1]

Proteomics
Measuring the half-lives of proteins
Using a TAP-tagged yeast library and western blotting after inhibition of protein synthesis, Belle et al. have measured the half-lives of more than 3750 proteins in the yeast proteome. This was the final piece of the puzzle that has now allowed them to construct a quantitative model of protein ********************bolism, using previous large-scale measurements of mRNA levels, translation rates and protein abundances.

Belle, A. et al. Proc. Natl. Acad. Sci. USA 103, 13004–13009 (2006).

Imaging and visualization
Quadruplex molecular beacons
The hairpin-shaped molecular beacons are becoming increasingly important tools for highly sensitive and specific DNA and RNA detection. When closed, the fluorophore on one end of the molecular beacon is quenched by the quencher at the other end. When bound to a target sequence, however, the hairpin opens and fluorescence is restored. Bourdoncle et al. now demonstrate that molecular beacons employing a G-quadruplex motif can be constructed, which may give the user more control over their thermodynamic and kinetic properties.

Bourdoncle, A. et al. J. Am. Chem. Soc. 128, 11094–11105 (2006).

[Preciouslife1]

Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes

Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein derivative FlAsH. This labeling allowed us to image both intracytoplasmic and intranuclear HIV-1 complexes in three dimensions over time (4D) in human cells and enabled us to analyze HIV-1 kinetics by automated 4D quantitative particle tracking. In the cytoplasm, HIV-1 complexes underwent directed movements toward the nuclear compartment, kinetically characteristic of both microtubule- and actin-dependent transport. The complexes then adopted smaller movements in a very confined volume once associated with the nuclear membrane and more diffuse movements once inside the nucleus. This work contributes new insight into the various movements of HIV-1 complexes within infected cells and provides a useful tool for the study of virus-host cell interactions during infection.

Nature Methods - 3, 817 - 824 (2006)
Published online: 21 September 2006; | doi:10.1038/nmeth928
Nathalie Arhel1, 6, Auguste Genovesio2, 4, 6, Kyeong-Ae Kim5, Sarah Miko1, Emmanuelle Perret3, Jean-Christophe Olivo-Marin2, Spencer Shorte3 & Pierre Charneau1

[Preciouslife1]

Neural precursor cells possess multiple p53-dependent apoptotic pathways



Neural precursor cells (NPCs) are markedly sensitive to apoptotic insults. p53-dependent transcriptional activation of proapoptotic genes has been hypothesized to regulate NPC death in response to DNA damage. Recent studies of non-NPCs have also indicated that p53 may directly interact with Bcl-2 molecules and thereby regulate death independently of transcription. The contribution of transcription-independent p53 activation in NPC death has not been characterized. In this study, we found that apoptosis caused by chemotherapeutic agents in NPCs required p53 expression and new macromolecular synthesis. In contrast, NPC death induced by staurosporine, a broad kinase inhibitor, is regulated by p53 in the absence of macromolecular synthesis. The apoptosis effector molecules Bax and Bak, Apaf-1, and caspase-9 were shown to be downstream of p53 in both pathways. These findings indicate that p53 is in a unique position to regulate at least two distinct signaling portals that activate the intrinsic apoptotic death pathway in NPCs.
Keywords:

caspase, neural stem cells, genotoxic injury, Bax, transcription

Abbreviations:

AraC, cytosine arabinoside; BAF, BOC-aspartyl(Ome)-fluoromethyl ketone; CHX, cycloheximide; NPC, neural precursor cell; STS, staurosporine

Edited by V De Laurenzi
R S Akhtar1,2, Y Geng1, B J Klocke1 and K A Roth1,2

1. <LI id=aff1 minmax_bound="true">1Department of Pathology, Division of Neuropathology, University of Alabama at Birmingham, SC 961, 1530 3rd Avenue South, Birmingham, AL 35294-0017, USA
2. 2Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA

Correspondence: KA Roth, Department of Pathology, Division of Neuropathology, University of Alabama at Birmingham, SC 961, 1530 3rd Avenue South, Birmingham, AL 35294-0017, USA. Tel: +205-934-5802; Fax: +205-934-6700; E-mail: kroth@path.uab.edu
Received 23 August 2005; Revised 22 November 2005; Accepted 21 December 2005; Published online 3 March 2006.

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News and Analysis


Nature Reviews Drug Discovery 5, 715-716 (September 2006) | doi:10.1038/nrd2134
From the analyst's couch: Hepatitis C virus therapies

Richard E. T. Smith1
The hepatitis C virus (HCV) is believed to have infected approximately 170 million people worldwide (Fig. 1). Often referred to as the silent killer, patients can remain asymptomatic for decades before developing liver cirrhosis and/or hepatocellular carcinoma. Approximately 40–50% of liver transplants in the US are the result of HCV infection. Six major HCV genotypes (HCV1–6) have been identified, which vary in their geographic distribution and response to treatments.
Figure 1 | Worldwide prevalence of hepatitis C in millions.

Although Europe and the Americas represent the major drug markets for HCV treatments, they comprise only a fraction of the worldwide patient population. Source: Ref. 2.

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Standard of care is lacking

HCV patients currently receive a combination of pegylated interferon-alfa (PEG–IFN) — either 2a (Roche's Pegasys) or 2b (Schering-Plough's PEG-Intron) — and ribavirin (Rebetol (Schering-Plough), Copegus (Roche) and various generics). However, because of the once-a-week injections, many patients are unable to withstand the 24-week (for HCV2/3) or 48-week (for HCV1) course of treatment. Coupled with this, only around 50% of patients with the predominate genotype in the US and Europe, HCV1, achieve a sustained virological response (SVR) or 'cure' (Fig. 2). New approaches that improve response rates are therefore desperately needed.
Figure 2 | Sustained virological response rates with treatments for hepatitis C.

DA, direct antiviral; IFN, interferon; PEG, pegylated interferon; RBV, ribavirin.

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Polymerase inhibitors

As with HIV drug development, the first direct antivirals to be developed for HCV were the nucleoside analogues that inhibit the non-structural protein 5B (NS5B) polymerase, an essential enzyme required for viral replication. Ribavirin is a weak inhibitor, but is very synergistic when combined with interferon. The problem of ribavirin-induced anaemia led to the development of a prodrug (Viramidine; Valeant) that seeks to circumvent this issue, as the prodrug is activated in the liver; however, efficacy results have been disappointing so far.
Newer nucleoside analogues are now in development, the most advanced being valopicitabine (NM283; Idenix/Novartis), which is currently in Phase II combination trials with PEG–IFN. Early results in both treatment-naive and treatment non-responder HCV1 patients proved encouraging; however, the higher incidence of gastrointestinal side effects in the high-dose arms led to the lowering of the valopicitabine dose in both trials. Phase III trials are expected to start in the first half of 2007, following the completion of ribavirin drug–drug interaction studies and further studies to investigate different doses and dosing schedules. Another nucleoside analogue in development is Roche's R1626. Interim results from a Phase I trial indicate that R1626 is effective and well tolerated, and Phase II combination trials with PEG–IFN and ribavirin are anticipated to start in the third quarter of 2006.
Non-nucleoside analogue inhibitors (NNI) that also target the HCV polymerase are in earlier stages of development. ViroPharma/Wyeth's HCV-796 is the most advanced member of this drug class. Interim Phase I data indicate that the drug is effective and well tolerated. The early development of resistance is not uncommon with this class, as is evident with NNIs for HIV, but like HIV these drugs will probably have to be used in combination with other therapies.