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RESEARCH BRIEFS
Gene Therapy Inefficiency ProbedBecause congestive heart failure remains a pervasive problem in the ever increasing elderly population, the potential rewards from correcting genetic defects of the aging heart are enormous. Gene therapy may be an answer; however, older hearts are not as easily infected with transfer viruses as younger hearts.
The coxsackievirus and adenovirus receptor (CAR) binds to an adenovirus, then integrins pull it through the membrane. Finally, the virus releases therapeutic DNA. (Image courtesy of Roger Hajjar) Research published in the March 4 Circulation by a team from Massachusetts General Hospital led by Roger Hajjar, HMS associate professor of medicine, may reveal part of the answer. The researchers found that the decreased infection efficiency is caused, in part, by decreased production of integrin subreceptors. Integrins internalize one commonly used vector, adenovirus, once the virus binds to the coxsackievirus and adenovirus receptor (CAR). In this study of rats at either 6 or 26 months of age, the scientists discovered that though expression of CAR increased in older rats, production of many integrins decreased. According to Hajjar, the reduction in integrin production may be part of an attempt by the aging body to compensate for a fading immune system. "This could be a way of becoming more resistant to viruses," he said. To be certain that the decrease in integrins was responsible for the decreased infection efficiency, the scientists blocked the integrin binding site by adding monoclonal antibodies to beta integrin or arginine-glycine-aspartate peptides that mimic the adenovirus binding domain. Infectivity decreased by half in both cases. When the scientists added laminin, however, they got a surprise. Laminin is a component of basal membranes and anchors cells by binding to integrins; the expectation was that it, too, should block infection. Instead, infection increased. This unexpected finding may provide part of the solution to the adenoviral uptake dilemma. "Laminin can increase integrin activity, so you may have to mix the adenovirus with products like laminin to enhance the gene transfer," said Hajjar. "As we design gene therapy trials, we have to incorporate this knowledge into how we treat elderly patients." --Jennifer Frazer
Mechanism Revealed for Acquired Resistance to "Smart" DrugWhen it was first approved to treat chronic myelogenous leukemia (CML) two years ago, imatinib (Gleevec) was one of the first of a new generation of "smart" cancer drugs designed to target cancer-causing molecules within certain cells, leaving normal cells uninjured. But cancer cells in some patients mutate further and become resistant to the drug's effect. Now, using a new cell-screening technique, researchers have identified 112 mutations that can confer drug resistance. They also discovered new mechanisms of acquired drug resistance in the protein kinase made by the mutated CML gene BCR-ABL. The scientists used recombinant DNA methods to randomly mutate BCR-ABL to mimic potential variations that might be found in CML patients. The mutated genes were then transferred into millions of mouse blood cells and exposed to imatinib. Though most succumbed, some of the cells with specific variations thrived. "We looked for those cells that continued to grow, extracted them, and sequenced their genes," said lead author Mohammad Azam, a postdoctoral researcher at the Whitehead Institute at MIT. They found 15 mutations that other researchers had linked to imatinib resistance in CML patients--plus 97 more. The mutations may be used for clinical prognosis of drug dosage and effectiveness, to assemble a more effective drug "cocktail," and as a tool in developing drugs that anticipate and block acquired resistance, said senior author George Daley, HMS assistant professor of medicine at the Whitehead and Massachusetts General Hospital. The findings are reported in the March 21 Cell with related structural analysis of the ABL protein. "Kinases have become fashionable targets for the pharmaceutical industry, not the least because of the clinical success of imatinib in treating CML," wrote Howard Hughes investigator Stephen Harrison, HMS professor of biological chemistry and molecular pharmacology, in an accompanying review. "Drug developers should look well beyond the catalytic sites of their enzymes." --Carol Cruzan Morton
Death Proteins Play New RoleThe drama of programmed cell death plays out some of its most compelling scenes at the mitochondria. It is by inserting themselves into the mitochondrial membrane, thereby releasing cytochrome c into the cytosol, that the death-dealing proteins BAX and BAK help bring about the cell's demise. It now appears that the deadly duo may also control apoptosis by regulating calcium levels at the endoplasmic reticulum. "Within the intrinsic cell death pathway, the endoplasmic reticulum is also a key site of regulation. And the mediator there, the currency, looks like it is calcium," said Stanley Korsmeyer, the Sidney Farber professor of pathology at the Dana-Farber Cancer Institute. The findings, reported in the March 7 Science, arose as the result of a puzzling discovery. Luca Scorrano and Scott Oakes, postdoctoral fellows in pathology at HMS and DFCI, found that cells lacking BAX and BAK were resistant to agents known to kill cells by flooding them with calcium. For example, the chemical thapsigargin blocks calcium reuptake pumps located in the endoplasmic reticulum, resulting in toxic levels of calcium. Scorrano and Oakes knew that the endoplasmic reticulum harbors 10 to 15 percent of a cell's BAX and BAK and that the pair controls steady-state calcium levels in that organelle. To see if the endoplasmic reticulum's calcium stores might be regulating apoptosis, the researchers restored normal calcium levels in the mutant cells by introducing functioning calcium reuptake pumps. They found they could reactivate apoptosis, but only in response to certain signals, such as those from oxidative stress and lipid second messengers. To reinitiate cell death in response to other signals, the researchers had to selectively introduce BAK to the mitochondria. A third set of signals required corrections at both the endoplasmic reticulum and mitochondria. Strategies for fighting cancer and degenerative diseases, which aim to kill cells in the first case and keep them alive in the second, could make use of these new findings. "One might attempt to control calcium at the level of the endoplasmic reticulum," said Korsmeyer. "That would potentially give another avenue to try to regulate the death response." --Misia Landau |
