RNA treatment kills mice

Using RNA interference to shut down harmful genes can have fatal flaw.

The research shows that mice can die when injected with high doses of a form of RNA. The RNA molecules, which were folded into structures called short hairpin RNAs (shRNAs), were able to overwhelm a cell's normal RNA-processing mechanisms, with fatal consequences.

Researchers say the work emphasizes the need to proceed carefully towards human trials of RNA interference, a scheme by which RNA is used to shut down harmful genes. Such trials are already underway for a condition known as macular degeneration and a childhood infection called respiratory syncytial virus. And a slew of new trials are planned to treat conditions such as HIV, hepatitis and even bird flu.

Safety concerns about RNA interference have been raised before. Scientists have already reported that some forms of the technique can shut down unintended genes, or trigger a cellular defence mechanism known as the interferon response2. That could lead to harmful side effects.

Source:
Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.
Nature 441, 537-541 (25 May 2006)

[email protected]

Bird flu vaccine

Biologists looking for an efficient way to halt the spread of avian flu have hit upon a promising twofer. By inserting an avian flu gene into a vaccine that protects against another bird malady, researchers have developed a vaccine that could combat both. If successful, the vaccine may offer an alternative to the mass slaughterings that have cost the world's poultry industry millions in lost sales.

Most commercial chicken farms vaccinate animals against the highly contagious Newcastle virus, which can decrease egg production or kill domestic poultry and some wild birds. Research teams in Germany and the United States, working independently, wanted to know if the Newcastle disease vaccine (NDV) could be altered to deliver protection against avian influenza. There currently is no vaccine for avian flu, which is caused by a highly contagious virus that is often fatal to birds and can be passed to humans.

A group led by molecular biologist Angela Römer-Oberdörfer of the Friedrich-Loeffler Institute in Riems, Germany, added a gene from the bird flu virus to a commercially available Newcastle vaccine. The gene, called H5, is one of 16 subtypes of hemagglutinin, a protein that binds the avian influenza virus to the cells it infects. When the researchers exposed chickens to lethal doses of the avian influenza virus and the Newcastle virus, birds inoculated with the recombinant vaccine produced antibodies against both viruses, offering protection against both diseases.

Microbiologist Peter Palese from Mount Sinai Medical School in New York City led the second team, which added the avian flu H7, another hemagglutinin subtype, to a weakened strain of the Newcastle vaccine. When exposed to an H7 strain of avian influenza, 90% of the vaccinated chickens were protected, and all were immune to the Newcastle virus.

Source:

Man-Seong Park, John Steel, Adolfo García-Sastre, David Swayne, and Peter Palese.Engineered viral vaccine constructs with dual specificity: Avian influenza and Newcastle disease. PNAS | May 23, 2006 | vol. 103 | no. 21 | 8203-8208

Kelli Whitlock Burton
ScienceNOW Daily News
22 May 2006

Naphthalene suppress apoptosis

A study of the nematode worm Caenorhabditis elegans suggests that chemicals in mothballs shut down the natural process by which cells commit suicide, allowing cancer cells to divide and conquer.

The finding is serendipitous. When a neighboring lab became infested by mites, biochemist Ding Xue of the University of Colorado in Boulder tried to protect his C. elegans by putting mothballs in their containers. But the balls may have done more harm than good. Some of the worms' cells that were programmed to apoptose--or kill themselves--kept living indefinitely, a problem that has been linked to cancer in humans.

To figure out what was going on, Xue and colleagues isolated a chemical commonly used in mothballs called naphthalene. When worms were grown on top of an oil film containing the chemical, about a fifth of them had at least one cell survive that should have killed itself. And when worms with a defective apoptosis enzyme known as a caspase encountered naphthalene at levels similar to those seen with human mothball use, the animals had seven more cells survive in their pharynx than normal worms did. Unexposed mutant worms had on average only 1.5 extra cells, indicating that naphthalene exacerbates the problem.

The caspase itself appears to be napthalene's target. Incubating the worm caspase or a related human caspase directly with naphthalene nearly wiped out the enzyme's activity, the team reports online 14 May in Nature Chemical Biology. The lack of a working caspase would throw a wrench into the entire apoptotic pathway, Xue says, because it would prevent necessary downstream signals from firing and inducing cell death. He notes that many proteins in the cell death pathway are conserved between C. elegans and humans, so it's likely that naphthalene has a similar effect in people and could promote tumor growth with regular exposure.

Source:
Cancer Agent Is a Stinker
By Katherine Unger
ScienceNOW Daily News
15 May 2006

Kokel D, Li Y, Qin J, Xue D.
The nongenotoxic carcinogens naphthalene and para-dichlorobenzene suppress apoptosis in Caenorhabditis elegans
Nat Chem Biol. 2006 May 14

Software for identifying disease genes

Drawing on various databases, ENDEAVOUR gathers all the data about genes that are known to be connected with a disease or a biological process and integrates these data into a mathematical model. With the aid of this model, scientists study the similarities between the 'known genes' and genes whose biological function is not yet known. ENDEAVOUR then indicates whether these genes might possibly underlie a certain disorder.

ENDEAVOUR has been fine-tuned and tested in the laboratory. The researchers took the data for a number of known genes from the mathematical model and then entered the genes as 'unknown' into ENDEAVOUR. For the majority of the syndromes tested (such as Alzheimer's disease, leukemia, colon cancer, and Parkinson's disease), ENDEAVOUR found the underlying genes and thus proved its validity.

As an extra validation of the program, the researchers used ENDEAVOUR to look for new disease genes that underlie hereditary disorders. Among other things, they wanted to identify a new gene that can be correlated with DiGeorge syndrome − a genetic disorder that affects more than 1 in 4000 newborn children. The infants have deformed features and blood vessel abnormalities in the heart. ENDEAVOUR identified one gene as a possible disease gene: YPEL1.

To confirm this mathematical prediction biologically, the researchers used the zebra fish as model system to replicate the disease. They studied zebra fish that could not produce the zebra fish YPEL1 gene. The embryos of these fish showed several abnormalities that are comparable to the symptoms of DiGeorge syndrome. This study provided the ultimate proof that ENDEAVOUR is a very useful tool for identifying new disease genes.

ENDEAVOUR can accelerate research into a number of disorders by providing the tools for rapidly identifying genes that play a role in the disorders.

ENDEAVOUR can be used via: www.esat.kuleuven.ac.be/endeavour or www.bits.vib.be/endeavour.

Growth hormone, insulin, and longevity

A number of studies have shown that restricting calories increases the lifespan of animals, but the biological basis for this has remained elusive. A new report hints that growth hormone, as well as insulin, are key factors in the life-extending effects of calorie restriction.

"The implication ... for pharmaceutical development would be that the signaling pathways of growth hormone and insulin may be logical targets for development of anti-aging medicine," Dr. Andrezej Bartke from Southern Illinois University in Springfield told Reuters Health.

"Although it would be irresponsible to recommend that healthy people start using anti-diabetic drugs," said Bartke, "it is reasonable to suggest that treatment(s) causing an improvement in insulin sensitivity combined with modest reduction in insulin release would reduce risk of age-related disease and likely also delay aging."

Bartke's team tested whether growth hormone and insulin are tied to the life-extending effects of calorie restriction in a series of experiments with normal mice and mutant mice deficient in growth hormone.

The mutant mice do not express the receptor for growth hormone (and are therefore growth hormone resistant), have profoundly suppressed insulin levels, and are known to live longer and age more slowly than normal mice, the researchers note in Proceedings of the
National Academy of Sciences.

As expected, the team observed that restricting food increases longevity in normal healthy mice. Reduced feeding increased lifespan by about 19 percent in normal male mice and by about 28 percent in normal female mice.

However, in sharp contrast to its effects in normal mice, calorie restriction failed to increase lifespan in mutant mice lacking growth hormone receptor. "The present findings show that growth hormone resistant mice fail to respond normally to calorie restriction, a very effective life-extending intervention," Bartke said.

"The key implication of this study is that growth hormone receptor and thus presumably the normal, physiological actions of growth hormone are important in regulation of aging and life span," Bartke said.

The team also found that calorie restriction for 12 months improves insulin sensitivity in normal mice but fails to further enhance the "remarkable insulin sensitivity" in growth hormone knockout mice.

This finding, Bartke said, "supports our hypothesis that increased sensitivity to the actions of insulin is a very important and perhaps the key mechanism of delayed aging and prolonged longevity in growth hormone deficient and growth hormone resistant mice."

SOURCE: PNAS, May 16, 2006.

Cancer Stem Cells

Close on the heels of the discovery that cancer has its own rejuvenating stem cells, a University of Michigan research team has found a way to distinguish these bad-actors from the normal stem cells that they so closely resemble, and to kill the cancer stem cells without harming the normal stem cells in the same tissue.

The progression of some cancers, including leukemia, appears to be driven by cancer stem cells - rare cancer cells that have a greater ability to proliferate than other cancer cells and which are therefore the most malignant. To have any hope of curing cancer it is necessary to develop therapies that kill these cancer stem cells. Yet these cells frequently have properties that are similar to normal stem cells in the same tissue, making it difficult to kill cancer stem cells without damaging the normal stem cells.

"This study proves that it is possible to identify differences in the mechanisms that maintain normal stem cells and cancer stem cells, and to therapeutically exploit these differences to kill the cancer stem cells without harming normal stem cells in the same tissue," said Sean J. Morrison PhD, lead author on the study and the Director of U-M's Center for Stem Cell Biology, LSI research associate professor and associate professor of internal medicine in the Medical School. Morrison is also a Howard Hughes Medical Institute investigator.

Three years ago, Morrison was part of a U-M team that made the breakthrough discovery that breast cancer has its own stem cells. These cells appear to be the primary drivers in tumor growth and possibly metastasis and the discovery pointed to a new direction in cancer therapy: Rather than trying to eliminate every last tumor cell, cancer therapies might be more specifically targeted at just the stem cells in cancer.

But telling the bad guys from the good guys - normal adult stem cells that the body relies on to replace cells damaged by injury, disease and old age - is a big problem, because they have so many features in common, Morrison explained.

In a new paper appearing as the advance online publication in Nature for April 5, 2006 (to be published in print in the near future), Morrison's team has found a way to tell stem cell friends from foes, as well as a drug that can make them behave differently.

"In many types of tumors, the gene Pten is missing or turned off," Morrison said. "Because Pten regulates cell proliferation and survival, we studied its role in the maintenance of normal blood-forming stem cells."

Omer Yilmaz, a graduate student in the Morrison laboratory, deleted the Pten gene from adult blood-forming stem cells in mice and found that the loss of Pten led to leukemia, marked by the generation of leukemic stem cells. Transplantation of even a single leukemic stem cell from these Pten -deficient mice into a second mouse caused leukemia, demonstrating the powerful cancer-causing ability of these cells.

In addition to pumping up leukemic stem cell numbers, Pten deletion also caused normal blood-forming stem cells to start dividing, but over time the normal stem cells became depleted in the absence of Pten. Additional experiments by the team established that every blood-forming stem cell needs Pten to maintain itself, in contrast to leukemic stem cells that thrive without Pten.

This finding of a key difference between normal stem cells and cancer stem cells suggested that drugs that target the metabolic pathway in which Pten acts should have opposite effects on normal blood-forming stem cells and leukemic stem cells. To test this, the team treated the mice with rapamycin, a drug that reduces the activity of this metabolic pathway. The drug is used to prevent tissue rejection in transplant patients, and is currently being tested in clinical trials for activity against a variety of cancers.

They found that rapamycin inhibited the creation and maintenance of leukemic stem cells. And even better, it "rescued" the function of normal blood-forming stem cells in these mice, that otherwise crashed without Pten.

Mice that were put on rapamycin immediately after Pten deletion failed to develop leukemia. Mice that already had leukemia were kept alive longer by the drug.

These results suggest that by better understanding the mechanisms that regulate the maintenance of normal stem cells it will be possible to develop new anti-cancer therapies that are more effective and less toxic. This is particularly important issue for leukemia patients that often cannot be cured with current therapies, and for whom existing therapies sometimes have fatal side-effects.

"The ability to strategically target and kill leukemia-initiating stem cells will undoubtedly have a significant impact on our capacity to treat these often fatal diseases more effectively," said Riccardo Valdez, M.D. an assistant professor of pathology at U-M who participated in the study. "At the same time, it would minimize potentially life-threatening side effects caused by conventional drugs."

Morrison is quick to point out that these findings are limited to mice so far, and cannot be immediately extrapolated to human patients. However, the study raises the possibility that rapamycin could be effective in depleting leukemic stem cells for at least certain patients.

Clinical trials will be required to test this in human patients.

Source:
Life Science Institute - University of Michigan
Science Daily

Gene Associated With Heart Rhythm

Using a new genomic strategy that has the power to survey the entire human genome and identify genes with common variants that contribute to complex diseases, researchers at Johns Hopkins, together with scientists from Munich, Germany, and the Framingham Heart Study, U.S.A., have identified a gene that may predispose some people to abnormal heart rhythms that lead to sudden cardiac death, a condition affecting more than 300 thousand Americans each year.

The gene called NOS1AP, not previously flagged by or suspected from more traditional gene-hunting approaches, appears to influence significantly one particular risk factor - the so-called QT interval length - for sudden cardiac death. The work will be published online at Nature Genetics on April 30.

"In addition to finding a genetic variant that could be of clinical value for sudden cardiac death, this study also demonstrates how valuable large-scale genomics studies can be in detecting novel biological targets," says the study's senior author, Aravinda Chakravarti, Ph.D., director of the McKusick-Nathans Institute for Genetic Medicine at Hopkins. "This study, conducted during the early days of a new technology, would have been impossible without the pioneering support of the D.W. Reynolds Foundation in their generous support of our clinical program in sudden cardiac death here at Hopkins."

QT interval measures the period of time it takes the heart to recover from the ventricular beat - when the two bottom chambers of the heart pump. Corresponding to the "lub" part of the "lub-dub" pattern of the heartbeat, an individual's QT interval remains constant. This interval is partly dependent on one's genetic constitution and, moreover, genes also play a role in sudden cardiac death.

"There's a great deal of evidence out there that having a too long or too short QT interval is a risk factor for sudden cardiac death," says the study's co-first author, Dan Arking, Ph.D., an instructor in the McKusick-Nathans Institute. "This makes it appealing to study because it can be measured non-invasively with an EKG, and each person's QT interval, in the absence of a major cardiovascular event, is stable over time, making it a reliable measure."

Identifying those at high risk for sudden cardiac death before fatalities occur has been challenging, both at the clinical and at the genetic level, says the study's other first author, Arne Pfeufer, M.D., of the Institute of Human Genetics at the Technical University in Munich, Germany. Doctors estimate that in more than one third of all cases, sudden cardiac death is the first hint of heart disease. It is widely believed that many factors, genetic and environmental, contribute to irregular heartbeat and other conditions that may lead to sudden cardiac death.

Being able to identify predisposed individuals can save their lives by prescribing beta-blockers and other drugs that regulate heart rhythm, and even by implanting automatic defibrillators in those with the highest risk.

In an effort to identify risk factors with a genetic foundation, the researchers took the unconventional approach of starting from scratch and not looking at genes already known or suspected to be involved in heart rhythm.

"Studying individual genes is not going to open new areas of research," says Chakravarti. "Using a whole-genome approach allows us to find new targets that we never would have imagined."

So instead of focusing on so-called candidate genes with known functions that are highly suspect in heart beat rhythm, the team first focused on people who have extremely long or short QT intervals. The researchers used subjects from two population-based studies, about 1800 American adults of European ancestry from the Framingham Heart Study of Framingham, Mass., and about 6,700 German adults from the KORA-gen study of Augsburg, Germany.

The research team then searched for any specific DNA sequences that showed up more frequently in people who have longer or shorter QT intervals than in those with normal QT intervals. To do this, they examined the DNA sequences of both long and short QT people. The human genome contains 3 billion letters, known as nucleotides. Each person's genome differs from the next person's by as many as 10 million nucleotides. The researchers looked for single nucleotide variations - known as single nucleotide polymorphisms, or SNPs for short - that track with having a long or short QT interval.

Only one particular SNP correlated with QT interval. That SNP was found near the NOS1AP gene, which has been studied for its function in nerve cells and was not previously suspected to play a role in heart function. However, the research team found that the NOS1AP gene is turned on in the left ventricle of the human heart. And the "lub" part of the "lub-dub" heartbeat corresponds to ventricular contraction. So NOS1AP is active in the right place and time to play a role in QT interval.

Further studies revealed that approximately 60 percent of people of European descent may carry at least one copy of this SNP in the NOS1AP gene. According to the researchers, this particular SNP is responsible for up to 1.5 percent of the difference in QT interval, meaning that other genes, missed in this study, certainly contribute to QT length.

Now that researchers know that variants of the NOS1AP gene correlate with QT interval length, they hope to figure out exactly how the DNA sequence variations alter the function of the gene, and how changes in gene function affects heart rhythm.

The Hopkins researchers were funded chiefly by the D.W. Reynolds Clinical Cardiovascular Research Center, which focuses on sudden cardiac death. Additional support was provided by the Johns Hopkins University, the National Institutes of Health, a GlaxoSmithKline Competitive Grants Award Program for Young Investigators, an unrestricted grant from Pfizer Inc., and the German Federal Ministry of Education and Research in the context of the German National Genome Research Network (NGFN). The Framingham Heart Study is supported by the National Heart, Lung and Blood Institute of the National Institutes of Health and Boston University School of Medicine and the Cardiogenomics Program for Genomic Applications.

Authors on the paper are Arking, Wendy Post, Linda Kao, Morna Ikeda, Kristen West, Carl Kashuk, Eduardo Marbán, Peter Spooner, and Chakravarti, all of Johns Hopkins; Pfeufer, Mahmut Akyol, Siegfried Perz, Shapour Jalilzadeh, Thomas Illig, Christian Gieger, Erich Wichmann, and Thomas Meitinger of the GSF National Research Center of Environment and Health in Neuherberg, Germany; Christopher Newton-Cheh, Chao-Yu Guo, Martin Larson, and Christopher O'Donnell of the National Heart, Lung and Blood Institute's Framingham Heart Study; Joel Hirschhorn of Harvard Medical School, and Stefan Kaab of Ludwig-Maximilians University of Munich.

Source:
Science Daily