Podcasts about bmi1

    This week we have some encouraging findings on Alzheimer’s as researchers hunt for a cure, there were two research studies released from the University of Toronto: one involving aneurysms, and the other discussing ‘invisible’ pain, as well as, a recent discovery that could change the face of opioid manufacturing. Keep listening to hear all the details!  +++++  The population of the world is steadily living longer, increasing the likelihood of one in three developing a form of Alzheimer’s in their lifetime. Much work has been done to combat this debilitating disease, but as of yet, there is no cure. After a decade of work a team led by Hôpital Maisonneuve-Rosemont researcher and Université de Montréal associate professor Dr. Gilbert Bernier has shed promising light on the origin of the most common and prevalent form of Alzheimer’s hoping to someday help mitigate or even reverse the progress of the disease.  There are genetic variables when it comes to being diagnosed with this disease, but age is the principal risk factor. Many researchers are trying to better understand the genetic and pathophysiological risks, but few studies have focused on the origin of Alzheimer’s and its relationship with the aging of the brain.  Working from the idea that the most prevalent form of Alzheimer’s disease is not genetic but instead epigenetic, Dr. Bernier and his team carried out an extensive scientific investigation to better understand the role of a specific gene, BMI1, in the onset and development of the disease.  In a study published in 2009, researchers found that, in mice models, the mutation of the BMI1 gene triggered an accelerated and pathological aging of the brain and eyes. Based on this finding, Dr. Bernier’s team deduced that if the BMI1 gene stopped functioning in a human, it would also cause accelerated aging of the brain and the development of conditions related to Alzheimer’s disease.  By comparing the brains of deceased Alzheimer’s patients (taken from samples in the Douglas Bell Canada Brain Bank) with those of deceased non-Alzheimer’s patients of the same age, the team observed a marked decrease of the BMI1 gene only in patients who died of the disease. To verify that this decrease was not simply a consequence of the disease, the researchers repeated the process with patients who died of early onset Alzheimer’s disease, which is a genetic and much rarer form of the that strikes before the age of 50 and sometimes even before 40. The researchers discovered that there was no change in BMI1 gene expression in these cases.  In a third step, the team examined the brains of individuals who died from other aging-related dementias, and once again observed no change in BMI1 gene expression. Finally, using a complex method, the researchers recreated in the laboratory, neurons found in Alzheimer’s disease patients and healthy individuals. Once again, BMI1 gene expression only decreased in neurons of Alzheimer’s-disease patients.  The team concluded that the loss of BMI1 gene expression in the brains and neurons of patients with the common form of Alzheimer’s was not a consequence of the disease and could, therefore, be the cause.  This led to the researchers wanting to test their hypothesis that the loss of BMI1 plays a direct role in the development of Alzheimer’s disease. To do so, they created healthy human neurons in the lab. Once the neurons reached maturity, they deactivated the BMI1 gene using a genetic method. The results were remarkable. All the neuropathological markers of Alzheimer’s were reproduced in the lab, concluding to the researchers that the loss of BMI1 function in human neurons was enough to activate the disease.  Encouraged by their unexpected findings, the researchers also ran molecular studies to understand how the loss of BMI1 triggers the disease. These studies revealed that the loss of BMI1 causes an increase in production of beta-amyloid and tau proteins and a decrease in the neurons’ natural capacity to eliminate toxic proteins.  The researchers believe that the restoration of BMI1 gene expression in the neurons of Alzheimer’s disease patients in the preliminary stages could mitigate or even reverse the progress of the disease. This provides hope for the future to patients that are ravaged by this disease and for their caregivers alike.  +++++  Researchers from the University of Toronto’s Faculty of Applied Science & Engineering have found a way to bring together music, art, and science to provide better ways to stimulate and understand medical imaging of aneurysms in the brain.  Currently, if a patient comes into a medical clinic with an unruptured brain aneurysm, a clinician’s decision to operate or leave it depends on risk factors related to the patient’s medical history, as well as the aneurysm’s shape, size and location in the brain.  Aneurysms in the back of the brain, for example, are more likely to rupture than those at the front. However, many large aneurysms don’t ever rupture, and many small aneurysms that are normally left alone, do rupture.  So, the question remains, how to researchers treat riskier aneurysms?  David Steinman, a professor of mechanical engineering at UofT, uses an approach that fuses biomedical engineering and the arts to discover a solution to this issue. Collaborating with Toronto Western Hospital’s Aneurysm Clinic, as well as Peter Coppin, an assistant professor in the Faculty of Design at OCAD University, his lab is taking fresh insights from visual artists and sound designers to improve visual and audio communication in medical imaging, starting with aneurysms.  Using graphics and sound to amplify key features, while suppressing irrelevant information, would allow a user to visually concentrate on one field, while listening to the other. Certain aspects of complexity can be heard better than it can be seen.  This would then allow a clinician to more easily and efficiently decide whether to operate on an aneurysm.  If the simulated blood flow of the aneurysm were to show a very strong and unstable ‘jet’ coming into the aneurysm and against the aneurysm wall that might be a hint that that wall is more likely to be aggravated.  Steinmann hopes this innovative approach can help reduce the number of unnecessary treatments and the number of accidental ruptures.   To work alongside Coppin’s team at OCAD University, he has recruited post-doctoral researcher Thangam Natarajan to translate CFD visually, and master’s student Dan MacDonald to translate CFD into sounds. They are both in the department of mechanical and industrial engineering.  Steinman is optimistic that his work will lead to a consistent, new way of representing and understanding how to treat aneurysms in medical clinics.  +++++  Have you ever known anyone who has severe anxiety and feels like they are having a heart attack? A study at the University of Toronto has uncovered that the ‘invisible’ pain someone feels could actually be linked to the frontal lobe and pain transmission to the spine.  For 20 years, Min Zhuo, a professor of physiology, Faculty of Medicine, University of Toronto, has been enticed by chronic invisible pain with no obvious cause – no inflammation, no injury – and understanding how to treat it.  Zhuo used his suspicions about the frontal lobe in mice and rat models to prove that treating this area could be effective at preventing chronic pain. The results were published in Nature Communication.  Zhou explains, “When doctors can’t see anything wrong to cause chronic pain, often they think patients are making it up. But pain that originates in the frontal lobe would be very different from pain that comes from a physical injury, like a herniated disc. There wouldn’t necessarily be any injury to see. That’s because our personality and emotions live in this region. If the frontal lobe can produce physical pain, that pain would be deeply tied to emotions like anxiety.”  Scientists already knew that the prefrontal cortex was in some way involved in pain because it would light up in scans of people in pain. But that activity was always thought to be a symptom not cause.  When someone has extreme anxiety, more neurotransmitters are released that end up causing pain in the spine. This flood of neurotransmitters sends the spine into hyperdrive, and it starts treating ordinary sensations like pain. This could explain why anxiety can cause chest pain and make an individual think they are having a heart attack, or why some people experience pain when you touch them.   On the bright side, pain from the frontal lobe seems to be transmitted in a simple, direct way to the spine – making it relatively easy to shut down. Neurons in the frontal cortex send signals all the way down the spinal cord, whereas pain signals from other areas of the brain are mediated by a complex network.  In animals, Zhuo found that pain was associated with increased neurotransmitters released from the frontal cortex. He was able to lessen pain by reducing the amount released. His next step is to test this process in people.  This research will likely benefit those who suffer from anxiety coupled with neuropathic pain by using a painkiller that targets the frontal lobe.  The research was supported by the National Key Basic Science Research Project and the Natural Science Foundation of China.  +++++  Considering the many issues at the moment with opioid misuse, a biotechnology company from Calgary has found a way to mitigate some of the problems associated with manufacturing strong painkillers by using sugar instead of field grown opium poppies.   Epimeron Inc. has taken a major step toward that goal with its discovery of the isolation of a novel gene from the opium poppy (Papaver somniferum). The gene encodes the enzyme thebaine synthase, which had previously been hypothesized, but never found, until now. Thebaine is the essential starting point in the synthesis of widely-prescribed pharmaceuticals, including the analgesics oxycodone and hydrocodone and the addiction treatments buprenorphine and naltrexone.  Dr. John Wilson, director, Physical and Life Sciences, Innovate Calgary says, “The discovery of the thebaine synthase gene is significant. This work has unlocked the path to transforming the commercial production of opiates and indicates the very real potential of developing non-addictive opioids. It is satisfying to witness how Dr. Faccini’s comprehensive research has transitioned into creating a considerable impact.”  The breakthrough enables the completion of commercial, non-plant based biosynthetic manufacturing systems for active opioid agents and intermediates. It also opens the door to the creation of new opioid molecules, some with new characteristics such as reduced addictiveness. The discovery was published in a Nature Chemical Biology article titled “A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis“.  Peter Facchini, PhD, chief scientific officer of Epimeron comments that their demonstrated expertise in research and entrepreneurship will continue to drive this important endeavour. That they have made great progress so far, and this announcement is an indication of the potential that lies ahead in developing new drugs to manage pain.  Microbial production of active pharmaceutical ingredients from sugar as source material could potentially replace current opioid manufacturing methods that rely on opium poppies for raw ingredients. Current regulatory requirements and commercial practices require the importation of crushed poppy straw from producer countries such as India and Turkey. The imported ingredients are then processed locally to the final pharmaceutical products. Legal opium poppy farming in producer countries is plagued by diversion of legitimately made controlled substances into the illicit drug trade.  Local biosynthetic manufacturing directly from sugar will eliminate the need for opium poppy raw materials and will decrease or eliminate diversion as a source of illicit ingredients. Moving away from outmoded plant ingredient purification techniques also enables improved quality and consistency, simplified logistics compared to moving narcotic raw materials around the world and avoids using fertile land that could better be employed for food production.  In addition, the microbial manufacturing strains will provide a basis from which to develop novel less addictive opioids not currently accessible from the plant or traditional chemistries. Until now it was not commercially viable to attempt to make certain modifications to the opioid molecule, but microbial biosynthesis now makes them possible. From these, numerous candidate pharmaceuticals will arise for testing, and some of these may well be superior to currently marketed products.  +++++  Well that wraps up another episode of Biotechnology Focus radio! Thanks for listening in! Check out the articles in full at biotechnologyfocus.ca. Hope you all have a great week ahead! From my desk to yours – this is Michelle Currie.    

Wed, 24 Sep 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17521/ https://edoc.ub.uni-muenchen.de/17521/1/Schaeffauer_Achim_J.pdf Schäffauer, Achim ddc:610, ddc:600, Medizinisch

Neuroendocrine tumors are a heterogeneous group of malignancies with an increasing prevalence. Since there is not much progress in therapy, model systems are urgently needed. We have a CEA424-SV40 TAg transgenic mouse model which develops spontaneous tumors in the antral region of the stomach. In addition, several cell lines derived from the tumor were established. Gene expression analysis of the tumor tissue as well as cell lines revealed neuroendocrine markers. Therefore we further characterized this model with special emphasis on the cells of origin and used it for testing new targeted treatment protocols. To analyze CEA424-SV40 TAg mouse model in more detail, tumor tissue as well as the cell lines derived from the primary tumor were investigated by immunohistochemistry, immunofluorescence, western blot, and ELISA. Antibodies used were directed at SV40 TAg, Ki-67, chromogranin A, chromogranin B, secretin, H+-K+-ATPase, glucagon, and transcription factors NeuroD1 and Nkx2.2. Plasma hormone levels of serotonin and secretin were measured by ELISA. Immunostainings of SV40 TAg and Ki-67 revealed highly proliferative tumors cells. The tumors stained intensively for the neuroendocrine markers chromogranin A, chromogranin B, secretin and glucagon. The tumor tissue as well as the cell lines expressed transcription factors NeuroD and Nkx2.2, which are involved in the differentiation of the neuroendocrine lineage. Hormone levels of serotonin and secretin in the plasma of the transgenic mice were dramatically elevated when compared with normal littermates, thus supporting the neuroendocrine phenotype. As the neuroendocrine phenotype of CEA424-SV40 TAg transgenic mouse was confirmed, molecularly targeted therapies were tested in this model system both in vitro and in vivo. Cell lines were tested for drug sensitivity with mTOR inhibitors (RAD001, NVP-BEZ235), paclitaxel, E2F inhibitor, HSP90 inhibitor, and p53 stabilizer Nutlin-3a. All the drugs tested in vitro could efficiently inhibit cell proliferation in a dose dependent manner. From these drugs the mTOR inhibitor RAD001 was chosen for the in vivo experiment. Daily feeding of 10 mg/kg RAD001 inhibited the tumor development and prolonged the survival time of the CEA424-SV40 TAg transgenic mice dramatically. The effects of the RAD001 treatment on tumor cells were achieved mainly through inactivating mTOR-p70S6K and mTOR-4EBP1 signaling as proven by western blot and immunohistochemistry. Still, some cells must develop escape mechanisms, since the tumor tend to grow. To gain a better understanding of the T antigen transforming mechanisms as well as the possible escape mechanisms, some efforts were made on the tumor originating cells in the CEA424-SV40 Tag transgenic mouse model. Possible candidates for these tumor originating cells in the stomach are the newly described epithelial as well as mesenchymal stem cells. In a first attempt, the expression feature of epithelial and mesenchymal stem cell markers were analyzed. Established cell lines as well as tumor tissue from the tumor bearing mice were investigated by reverse transcription PCR (RT-PCR), immunohistochemistry, immunofluorescence, western blot, and microarray analysis. From several markers analyzed, the tumor cell lines showed a high expression level of the potential epithelial stem cell marker Bmi1 in RT-PCR and cDNA expression array. This could be further substantiated by western-blotting and immunostaining. Consequently, Bmi1 message could also be found in the growing tumors both in mRNA and protein levels. Experiments using siRNA to knock down the SV40-TAg expression showed that the Bmi1 expression went down in the cell lines thus showing the interrelationship. On the other hand, the mesenchymal stem cell marker Etv1 was also found to be expressed in the tumor tissue and cell lines derived from the tumor. More interestingly, Etv1 expression level was up-regulated over the time course of the tumor development. From these, an Etv1 positive mesenchymal cell could be a possible candidate for transformation. Since the CEA-promoter used for the generation of the T-antigen transgenic animals contains Etv1 binding sites, it is tempting to speculate, that this may drive the transcription of the T antigen. In conclusion, our data provide convincing evidence that CEA424-SV40 TAg mice are a clinically relevant model for neuroendocrine tumor. Testing of molecularly targeted therapies both in vitro and in vivo offered promising candidates for further clinical evaluation. Thus, this new model system could be of great value not only for studies on the mechanisms of how SV40 TAg induces neuroendocrine tumors but also for exploring novel targeted therapy in a preclinical setting.

A growth signal can mediate feedback that reduces cell proliferation and stem cell self-renewal.

The t(10;11)(p13;q14) is a recurring translocation resulting in the fusion of the CALM and AF10 genes. The leukemogenic CALM/AF10 fusion genes codes for a 1595 amino acids protein. This translocation was first identified in a patient with hystiocytic lymphoma and was subsequently found in patients with AML, T-ALL and malignant lymphoma. This translocation is found in younger patients and is associated with a poor prognosis. The CALM/AF10-associated leukemias can exhibit myeloid, lymphoid or mixed lymphoid-meyloid features, indicating a stem cell or an early commited progenitor as the target cell of leukemic transformation. At the present time the target cells in CALM/AF10-associated leukemogenesis are unknown. It is also not known which target genes are up or downregulated by the presence of the CALM/AF10 fusion protein. To answer these questions, the following experiments were performed: 1) Five transgenic mouse lines, two expressing CALM/AF10 under the control of the immunoglobulin heavy chain enhancer promoter and three under the control of the murine proximal Lck promoter were generated. Although the CALM/AF10 expression was confirmed to be present and specific to the cells targeted by the promoters used (B- and T-cell progenitors for IgH and Lck promoters, respectively), the transgenic animals did not show a phenotype that could be detected after meticulous clinical, haematological, immunological, flow cytometrical and immunohistopatological analysis . 2) We performed molecular characterization of several CALM/AF10 patient samples: A group of 13 patients with different types of leukemia: case 1 (AML M2), case 2 (Acute Biphetnotypic leukemia), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), cases 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), case 14 (T-ALL), case 15, 16 and 17 (AML) with a t(10;11) translocation detected by cytogenetic analysis suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM fusion transcripts by RT-PCR and sequence analysis. All these patients were found to be positive for the CALM/AF10 fusion. In addition, we analyzed a series of twenty-nine patients with T-ALL with T-cell receptor ≥¥ rearrangement. Among these patients, four (case 10 to 13) were positive for the CALM/AF10 fusion transcript, indicating a high incidence of CALM/AF10 fusions in this group of leukemia. Three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4 were found. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In patient 16 we found an extra exon before nt 424 of AF10. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 14). There was no correlation between disease phenotype and breakpoint location. Ten CALM/AF10 positive patients were analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the polycomb group gene BMI1, the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent to the pattern seen in leukemias with rearrangements of the MLL gene, normal karyotypes and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. In addition, the general pattern of gene expression of CALM/AF10 patients when compared to other leukemia subtypes and to normal bone marrow was dominated by a global downregulation of genes some of them with function identified as related to important molecular mechanisms, such as membrane trafficking, cell growth regulation, proliferation, differentiation and tumor suppression. 3) We cloned CALM/AF10 fusion gene into a vector that allowed us to induce the expression of CALM/AF10 using doxycycline in transiently and stably-transfected NIH3T3 and HEK293 cells. This system will be an important tool to identify direct CALM/AF10 target genes and to answer the question whether continued CALM/AF10 expression is necessary to maintain the CALM/AF10-associated expression pattern.