With improved resolution, tissue-specific molecular markers and precise timing, University of Oregon biologist James A. Weston and colleagues have possibly overturned a long-standing assumption about the origin of embryonic cells that give rise to connective and skeletal tissues that form the base of the skull and facial structures in back-boned creatures from fish to humans.

Weston and co-authors from the Max Planck Institute of Immunology in Germany and the French National Scientific Research Centre at the Curie Institute document their potentially textbook-changing case in an article appearing online this week (May 19-23) ahead of regular publication in the Proceedings of the National Academy of Sciences.

The cells in question, they argue, do not come from a portion of embryonic neural epithelium called the neural crest, as widely believed, but rather from a distinct thin layer of epidermal epithelial cells next to it. “Our results,” Weston said, “could lead to a better understanding of the etiology of craniofacial defects, as well as the evolution of the head that distinguishes vertebrates from other creatures.”

The neural crest was first identified by classical embryologists in the late 19th and early 20th centuries and has been one of the most studied embryonic tissues. Conventional wisdom says that the neural crest gives rise to skeletal and connective tissue of the head and face, as well as a wide diversity of other stem cells that migrate to many places in the vertebrate embryo, where they spawn the cells that create the peripheral nervous system, and pigment cells in skin and hair (or scales and feathers).

The new study is part of research done over 25 years in Weston’s quest to understand early development of the neural crest and explore alternative explanations for sometimes differing findings involving its assumed cell lineages. Weston noted that mutations in mice that adversely affected development of the peripheral nervous system or pigmentation did not affect craniofacial structures, whereas mutations that caused abnormal development of skeletal and connective tissue of the head and face did not alter neural crest-derived pigment or peripheral nervous system cells.

This paradox, he said, led him to wonder if different genetic programs were required to function in distinct embryonic precursors of these tissues. “In our new paper,” he said, “we finally were able to re-examine some of the underlying assumptions that have led to the conventional wisdom about the source of the embryonic cell lineages that give rise to the skeleton and connective tissue of the head and face.”

In the mouse embryo at eight days gestation, Weston and collaborators used high-resolution imaging and immunostaining techniques to identify and track the dispersal of cells known to jump start connective and skeletal tissue development. They were able to see clearly that these cells came from the non-neural layer of cells rather than from the neural crest. The same distinction also exists in chicken embryos during the first few days of gestation, Weston noted. “Looking at the right time is very important,” he said.

Weston argues that this non-neural epithelium is indeed distinct from the neural crest, because its cells contain characteristically different molecules. He and colleagues dispute suggestions that this non-neural structure is simply a sub-domain of the neural crest. “These cells emerge at a different time in development and disperse in the embryo before neural crest cells begin to migrate,” Weston said.

“New technologies let us see cell types more clearly than ever before,” said Weston, a member of the UO’s Institute of Neuroscience. “We previously had discovered that a molecule that marks cell surfaces in the non-neural epithelium reveals a very sharp boundary between this non-neural epithelium and the neural tissue connected to the neural crest. In this study, we took a closer look.”

They located a population of cells in the non-neural epithelium that express other molecules that “do not appear to originate from the neural crest,” said Weston, who retired in 2001 but continued to teach in the College of Arts and Sciences until 2006. He still collaborates in some research with colleagues at the UO and at various labs around the world.

“I think our results have two important messages,” he said. “First, it is important to identify and validate — rather than ignore — assumptions; and second, because we identified an alternative embryonic cell lineage as the source of the head and facial structures, we can now more effectively analyze and understand the molecular-genetic mechanisms that regulate the normal and abnormal development of these structures.”

A study released today reveals a cellular mechanism involved in alcohol dependence. The study, in the May 28 issue of The Journal of Neuroscience, shows that gabapentin, a drug used to treat chronic pain and epilepsy, reduces alcohol intake in alcohol-dependent rats by normalizing chemical communication between neurons, which is altered by chronic alcohol abuse.

The central amygdala, a part of the brain involved in emotions such as stress and fear, is important in regulating alcohol consumption. Most central amygdala neurons communicate via a chemical signal known as GABA, which is an inhibitory neurotransmitter. Alcohol dependence has been associated with the strengthening of inhibitory synapses in this brain region.

Gabapentin (known commercially as Neurontin) is structurally similar to GABA and increases GABA neurotransmission. In alcoholics, gabapentin has been shown to effectively treat alcohol withdrawal and reduce alcohol consumption and cravings following detoxification. However, how gabapentin acts in the brain to combat alcohol dependence has been unclear.

The study’s authors, led by Marisa Roberto, PhD, at the The Scripps Research Institute, made rats dependent on alcohol by chronically exposing them to ethanol in an aerosol or in their food. They then tested how much alcohol the rats voluntarily drank and examined neural signaling in the central amygdala.

The study authors found that gabapentin reduced alcohol intake in rats chronically exposed to alcohol, but not in rats that were chronically unexposed. Gabapentin reduced alcohol intake in alcohol-dependent rats whether it was given systemically or infused directly into the central amygdala, supporting the importance of the central amygdala in alcohol dependence.

“What I find to be important about this paper is that gabapentin’s effect on alcohol consumption is only seen in alcohol-dependent rats,” said Julie Blendy, PhD, at the University of Pennsylvania, an expert unaffiliated with the study. “For me, this speaks volumes to the addiction field, in that therapeutic targets for addiction—which have been few and far between—may be missed when examined in animal studies that use only minor exposures of alcohol,” said Blendy.

Gabapentin corrected the cellular effects of chronic alcohol exposure. Both gabapentin and alcohol increase GABA neurotransmission in the central amygdala of non-alcohol-dependent rats, but in alcohol-dependent rats, gabapentin reduced it, suggesting that altered GABA neurotransmission is key to alcohol dependence.

In the study, gabapentin and chronic alcohol exposure both affected GABA B (GABAB) receptors. The authors believe that alcohol abuse alters the function of these receptors, and gabapentin may be able to counteract alcohol dependence by regulating their function.

“This study provides important mechanistic insights,” said Robert Messing, MD, at the Ernest Gallo Clinic and Research Center at the University of California at San Francisco, an expert also uninvolved with the study. “Because gabapentin is well tolerated, this paper provides a strong rationale for large clinical trials testing whether gabapentin is an effective treatment for alcoholism in both detoxified and actively drinking alcoholics,” Messing said.

A novel type of drug can shrink primary breast cancer tumors significantly in just 6 weeks Research provides leads to a new target in cancer treatment -the cancer stem cell.

(Berlin, Germany) A drug that targets the cell surface receptors that play an important role in many types of cancer can bring about significant tumour regression in breast cancer after only six weeks of use, a scientist told the 6th European Breast Cancer Conference (EBCC-6) today (Thursday 17 April).  Dr. Angel Rodriguez, from the Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, USA, said that the work demonstrated for the first time that the tyrosine kinase inhibitor lapatinib could decrease tumour-causing breast cancer stem cells in the primary breast cancers of women receiving neoadjuvant treatment (treatment given before the primary surgery for the disease).

Dr. Rodriguez and colleagues studied 45 patients with locally advanced breast cancer in which the gene HER-2 was over-expressed.  The patients received lapatinib for six weeks, followed by a combination of weekly trastuzumab and three-weekly docetaxel, given over 12 weeks, before primary surgery.  Biopsies were performed at the time of diagnosis and also after six weeks of lapatinib and cells from the tumours were obtained and analyzed.

“We saw significant tumour regression after six weeks of single agent lapatinib,” said Dr. Rodriguez.  “Bi-dimensional tumour measurements showed a median decrease of minus 60.8%. We had previously showed that tumour-causing breast cancer stem cells were resistant to conventional preoperative chemotherapy; indeed, residual cancers that were exposed to such chemotherapy showed an increase in tumour-causing cells and enhanced tumour initiation by the formation of mammospheres, small tumours that form when tumour-causing cells are cultured in a test tube, which reflect the capacity of the cells to self-renew.  So we were excited to see that the results with lapatinib were different.”

Dr. Rodriguez’s results suggest that specific signalling inhibitors of the pathways responsible for stem cell self-renewal could provide a possible therapy for eliminating tumour-causing cells in order to achieve the long-term eradication of cancer.

Cancer stem cells help maintain the malignant tissue in the tumour by regenerating the tumour after attack from chemotherapy drugs.  “This indicates that the stem cells themselves should be the specific target of chemotherapy drugs,” said Dr: Rodriguez.  “Rather than the broad brush approach, in which cells are killed indiscriminately, targeting the stem cells may be more effective and also prevent some of the unpleasant side effects associated with conventional chemotherapy treatment.”

Scientists believe that cancer stem cells come into being through damage to their own DNA, which affects the regulation of their self-renewal.  Other cells divide into two ‘daughter’ cells, but a stem cell can divide into a new stem cell and a ‘progenitor’ cell.  The progenitor cell loses the power of self-renewal, but can still change into the cell type of the tissue served by the stem cell.  The stem cell population then continues to renew itself as it generates new cells for the tissue.  “This means that, unlike other cells, the stem cell has lost control over its own population size,” said Dr. Rodriguez.

Lapatinib has few side effects, and those that exist are minimal, including diarrhoea and acne.  But it is expensive.  “In the US it costs between $2000 and $3000 a month,” he said.

“This is an exciting finding, and we will be starting further studies on stem cells in order to confirm it.  We will also look into its applicability in testing novel agents targeting tumour-initiating cells.  This finding should also apply to other types of cancers and research of tumour-initiating stem cells in other cancers is ongoing,” said Dr. Rodriguez.

“International studies are currently underway looking at the effect of lapatinib in lung, colon, head and neck, gastric, oesophageal, and bladder cancer and lymphoma, among others,” he said.

Note:  Lapatinib has not yet been licensed for use in the EU, although it has been approved in Switzerland and received a positive opinion regarding a conditional marketing authorisation from the European Medicines Agency in December.  This conditional authorisation refers to its use in patients with advanced or metastatic breast cancer with HER-2 over-expression in the tumours.

Catalogue no: 204, Thursday 18 April, 17.15 hrs CEST (Hall 1)

Researchers have uncovered details about how dietary restriction slows down aging. A team of University of Washington scientists have uncovered details about the mechanisms through which dietary restriction slows the aging process.  Working in yeast cells, the researchers have linked ribosomes, the protein-making factories in living cells, and Gcn4, a specialized protein that aids in the expression of genetic information, to the pathways related to dietary response and aging.  The study, which was led by UW faculty members Brian Kennedy and Matt Kaeberlein, appears in the April 18 issue of the journal Cell.

Previous research has shown that the lifespan-extending properties of dietary restriction are mediated in part by reduced signaling through TOR, an enzyme involved in many vital operations in a cell.  When an organism has less TOR signaling in response to dietary restriction, one side effect is that the organism also decreases the rate at which it makes new proteins, a process called translation.

In this project, the UW researchers studied many different strains of yeast cells that had lower protein production.  They found that mutations to the ribosome, the cell’s protein factory, sometimes led to increased life span.  Ribosomes are made up of two parts -the large and small subunits -and the researchers tried to isolate the life-span-related mutation to one of those parts.

“What we noticed right away was that the long-lived strains always had mutations in the large ribosomal subunit and never in the small subunit,” said the study’s lead author, Kristan Steffen, a graduate student in the UW Department of Biochemistry.

The researchers also tested a drug called diazaborine, which specifically interferes with synthesis of the ribosomes’ large subunits, but not small subunits, and found that treating cells with the drug made them live about 50 percent longer than untreated cells.  Using a series of genetic tests, the scientists then showed that depletion of the ribosomes’ large subunits was likely to be increasing life span by a mechanism related to dietary restriction -the TOR signaling pathway.

“We knew that dietary restriction decreased TOR signaling, and that decreased TOR signaling reduced translation or protein production, but this was the first direct evidence that all three were acting in the same genetic pathway,” said Kennedy, an associate professor of biochemistry.

“The big question then became what’s happening in these translation-deficient cells to slow aging,” added Kaeberlein, an assistant professor of pathology.  “That’s when Vivian MacKay, a co-author on the study, had the idea to look at Gcn4.”

Gcn4 is a specialized protein called a transcription factor, which helps transfer genetic information during cell growth.  The protein is activated when a cell is starving for amino acids.  What made Gcn4 interesting to the UW team was its unique mode of regulation.

“When ribosomes aren’t working at 100 percent capacity, most proteins are made less efficiently, but Gcn4 is different,” explained Dr. MacKay, a research professor of biochemistry.  “Sometimes, you actually get more Gcn4 produced even when everything else is going down.  That’s precisely what we found in the longer-lived yeast strains with mutations in the large subunit of the ribosome.”

To make the link between Gcn4 and longevity, the scientists then asked whether preventing the increase of Gcn4 would block life span extension.  In every case, cells lacking Gcn4 did not respond as strongly as Gcn4-positive cells.

“The increased production of Gcn4 in long-lived yeast strains, combined with the requirement of Gcn4 for full life-span extension, makes a compelling case for Gcn4 as an important downstream factor in this longevity pathway,” Kaeberlein said.

Although scientists don’t yet know whether Gcn4 plays a similar role in organisms other than yeast, Kennedy points out that worms, flies, mice and humans all have Gcn4-like proteins that appear to be regulated in a similar way.

“The role of TOR and translation in aging is known to be conserved across many different species, so it’s plausible that this function of Gcn4 is conserved as well,” Kennedy said.  Future research will be aimed at testing this hypothesis.

“Clearly TOR signaling is one component, and perhaps the major component, of the beneficial health effects associated with dietary restriction,” said Kaeberlein.  “The difficulty with TOR as a therapeutic target, however, is the potential for negative side effects.  As we learn more of the mechanistic details behind how TOR regulates aging, we will hopefully be able to identify even better targets for treating age-associated diseases in people.”

A team of researchers at Yale School of Medicine have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer’s recurrence and its resistance to chemotherapy.

“These results bring us closer to more effective and targeted treatment for epithelial ovarian cancer, one of the most lethal forms of cancer,” said Gil Mor, M.D., associate professor in the Department of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine.

Mor presented his findings recently at the annual meeting of the American Association for Cancer Research (AACR) Meeting in San Diego, California.

Cancerous tumors are made up of cells that are both cancerous and non-cancerous.  Within cancerous cells, there is a further subclass referred to as cancer stem cells, which can replicate indefinitely.

“Present chemotherapy modalities eliminate the bulk of the tumor cells, but cannot eliminate a core of these cancer stem cells that have a high capacity for renewal,” said Mor, who is also a member of the Yale Cancer Center.  “Identification of these cells, as we have done here, is the first step in the development of therapeutic modalities.”

Mor and colleagues isolated cells from 80 human samples of either peritoneal fluid or solid tumors.  The cancer stem cells that were identified were positive for traditional cancer stem cell markers including CD44 and MyD88.  These cells also showed a high capacity for repair and self-renewal.

The isolated cells formed tumors 100 percent of the time.  Within those tumors, 10 percent of the cells were positive for cancer stem cell marker CD44, while 90 percent were CD44 negative.

Mor and his team were able to isolate and clone the ovarian cancer stem cells.  They found that these cells were highly resistant to conventional chemotherapy while the non-cancer stem cells responded to treatment.  “Isolating and cloning these cells will lead to development of new treatments to target and eliminate the cancer stem cells and hopefully prevent recurrence,” said Mor.