Bioartificial Liver Being Studied at the Center. Inside this Issue. research center for alcoholic liver and pancreatic diseases.

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1 research center for alcoholic liver and pancreatic diseases The Center is supported by a grant from the National Institute on Alcohol Abuse and Alcoholism, P50 AA11999 Fall 2002 A publication of the Research Center for Alcoholic Liver and Pancreatic Diseases Inside this Issue Bioartificial Liver Being Studied at the Center By Anne Taguchi There are no effective medical treatments for cirrhosis except liver transplantation. With limited donor livers available, many cirrhosis patients die while waiting for liver donors or because they cannot afford the only life-saving surgery. In collaboration with a Japanese biotech company, Able Corp., the Center s Cirrhosis Research Program has acquired a radial flow bioartifical liver system to test whether it optimizes reconstitution of functional liver tissues Bioartificial Liver Being Studied at Center Ron Thurman Remembered Spotlight on Anna Gukovskaya Center Supports the American Liver Foundation 2002 Lee Summer Student Research Fellowship Award Center Still Matching Donations for Thurman Lectureship Award New Center Publications The USC Cirrhosis Research Program has begun growing hepatocytes and nonparenchymal liver cells isolated from rats to experimentally test the central concepts in the radial-flow bioreactor system. The program currently has two 30ml bioreactor systems and four 5ml systems being used for on-going studies. The director of the program, Dr. Hide Tsukamoto, has a long-standing interest in studying the pivotal role of hepatic stellate cells (HSC) in both the maintenance of normal liver cell functions and the genesis of cirrhosis. His team, including Drs. Shigang Xiong and Jiaohong Wang, is incorporating HSC and other non-parenchymal liver cells into the system to promote growth and Managing Editor Anne Taguchi Science Editor Maria Runnegar Contributors Rosy Macias Hide Tsukamoto Liver cells are grown onto hydroxyapatite beads. Unique from other systems, the beads allow the cells to grow three dimensionally rather than on a two-dimensional flat surface. Photo courtesy of Able Corp. Please send submissions or queries regarding this publication to ataguchi@usc.edu or fax to (323) differentiation of hepatocytes, the parenchymal cells. Says Tsukamoto, There has been a surging interest in the development and application of bioartificial liver for the treatment of patients with cirrhosis or acute Continued on page 2

2 page 2 Bioartifical Liver, Continued from page 1 fulminant hepatitis as a conjunctive measure to support these patients while waiting for liver transplantation. Even though several clinical trials have been under way, the outcomes of these studies are in general limited due mainly to fundamental problems. L-R: Dr. Michael Lai of USC; Mr. Yohichi Ishikawa, President of Able Corp.; Dr. Hideki Aizaki Postdoctoral Fellow in Dr. Lai s laboratory; Mr. Tomokatsu Hongo of Able Corp.; and Dr. Hide Tsukamoto, Director of the Center. These problems include use of cancerous liver cells which are not fully functional liver cells or cells derived from other species such as pig. Even when hepatocytes freshly derived from human livers are used, they quickly become de-differentiated because the device cannot provide the best environment for the cells to maintain their liver-specific functions. Continued on page 5 What Is Cirrhosis? Cirrhosis is a chronic liver disease in which normal liver cells are damaged and replaced by scar tissue. This scar tissue prevents the liver from performing important functions such as processing nutrients, hormones, and detoxifying drugs and poisons, including alcohol. Excessive alcohol intake is one of the two most common causes of cirrhosis. Liver cirrhosis is ranked as the 5th to 7th leading cause of death among adults years old. It is responsible for approximately 20,000 deaths per year in the United States, a mortality rate which is roughly equivalent to that caused by stroke, HIV or type I diabetes. Many deaths attributed to cirrhosis preventable since excessive alcohol intake is a common cause of cirrhosis. However, it is also estimated that 40-60% of chronic liver disease is related to hepatitis C virus (HCV) infection, the most common chronic bloodborne infection in the United States, with approximately 4 million people infected. About 20% of the HCV infected individuals develop chronic liver disease and cirrhosis. Chronic liver disease often shows no symptoms, and many patients are found to have the disease during the course of physical examination for an unrelated illness. No curative therapy is currently available for cirrhosis except for liver transplantation. Due to an insufficient pool of donors, many patients die while they are on the donor waiting list. Ron Thurman Remembered at RSA Meeting Joined by Many Center Members By Rosy Macias In honor of Ronald G. Thurman, Professor of Toxicology and Pharmacology at the University of North Carolina, the Bowles Center for Alcohol Studies of UNC hosted a symposium entitled, Alcohol and the liver: a memorial for Ron Thurman on June 28, 2002 at the Research Society on Alcoholism ISBRA meeting in San Francisco. The symposium was chaired by Dr. Fulton Crews, Bowels Center Director, Professor Nobu Sato of Juntendo University, Dr. David Brenner from UNC, and Dr. Hide Tsukamoto, our Center director. Dr. Tsukamoto presented on Ron Thurman and the Tsukamoto- French model of alcohol liver injury. Dr. Samuel French spoke on The pathogenesis of alcoholic liver disease. We are pleased that the Center was well represented at this meeting. Dr. Jose Fernandez-Checa spoke on Regulation of cell death by mitochondrial glutathione at the symposia. Posters from Center members were: The effect of CYP2E1 Continued on page 6 Center investigators, Nico Tao and Dr. Peter Deng present posters at the RSA meeting.

3 page 3 Spotlight on By Maria Runnegar Anna Gukovskaya Anna is a Researcher at the UCLA Department of Medicine and West Los Angeles VA Medical Center. Anna s research focuses on the mechanisms of pancreatic diseases. It was not always so. Anna was born in the Ukraine and her academic training was in Moscow and Leningrad. During her years in Russia the main focus of Anna s research was the study of calcium signaling in lymphocytes leading to many publications. Till 1991 Anna was not able to travel or attend meetings in the West. When Gorbachev came in control there was a thaw in East-West relations. As funds became available, Anna was invited by Sergio Grinstein to join his laboratory in the Division of Cell Biology at the famous Hospital for Sick Children in Toronto, Canada, to study aspects of the mechanism of ion signaling in neutrophils. Anna and Sergio showed that the proton pumps that play a role in maintaining cytosolic ph in activated neutrophils are regulated by protein kinase C (PKC). The work resulted in a major publication in the Journal of Biological Chemistry. From Toronto, Anna applied for a position in the US. The posting of her CV in Science and Nature resulted in queries from a number of labs. Amongst those, Anna chose the possibility to work with Steve Pandol in San Diego. Steve was sufficiently impressed with Anna s CV to travel to Toronto to interview her as her visa precluded any travel outside Canada. After returning to Moscov, Anna, her husband Ilya and daughter Sonya were allowed to emmigrate to the US in Anna joined the laboratory of Steve Pandol in the Department of Medicine, at the Veterans Affairs Medical Center in San Diego, while Ilya joined the laboratory of Michael Geoff Rosenfeld at UCSD. Steve Pandol and his group had been investigating calcium homeostasis and transport in pancreas. Anna was well suited to join these studies because of her many years experience in the study of calcium regulation in thymocytes. Many novel observations resulted from this work that helped define the role of calcium in the secretory process. In 1996, Anna decided to move to Los Angeles with Steve Pandol to the Veterans Affairs Greater Los Angeles Healthcare System/UCLA. The subsequent year Ilya also joined Steve s group. What has followed is an extremely productive research program that continues today. The next research challenge for Anna was to begin to understand factors that cause pancreatitis or contribute to the severity of this disease. The hallmarks of pancreatitis are loss of pancreatic cells and inflammatory cell infiltration. In animal models of pancreatitis (rat, mouse and even opossum). Anna and coworkers found that the relative proportion of necrotic and apoptotic cell death varied in the three species and correlated with the degree of the inflammatory response. This was the first indication that cell death and inflammation are connected in this disease. The regulation of inflammation and cell death is complex with many cell processes and components contributing to it. Anna s work has resulted in a series of elegant papers that established the involvement in pancreatitis of inflammatory mediators: cytokines and chemokines (such as interleukins and tumor necrosis factor (TNFalpha)), and platelet-activating factor. Anna first showed that the exocrine pancreas expressed TNFalpha and its receptors. She went on to further show that pancreatic acinar cells themselves release and respond to TNFalpha and that this cytokine regulates apoptosis in isolated pancreatic acini and in experimental pancreatitis. Nuclear factor-kappab (NF-kappaB) activation via its nuclear translocation is one of the first steps in intracellular TNF signaling. Since the critical indicators of pancreatitis: inflammation and cell death, have been shown in other diseases to be regulated by NF-kappaB activation, it was important to show that it was also true in pancreatitis. In a rat model of pancreatitis (induced by infusion of cerulein), Anna and Ilya, and their coworkers found that activation of NF-kappaB was an early event. It correlated directly with the establishment of the disease since, if pancreatitis was prevented, so was NF-kappaB activation and correspondingly, if the activation was blocked by N-acetyl cysteine (NAC) the severity of the disease was greatly reduced. This work and work from other groups greatly furthered understanding of the events triggering the inflammatory process in pancreatitis. In contrast, the mechanism and pathways involved in cell death in pancreatitis remained unexplored. Very recently Anna set out to rectify this. Central to apoptosis (programmed cell death) is the activation of specific serine proteases (caspases). To test whether this is true in pancreatitis, Anna turned to a well-characterized in vitro model: pancreatic acini treated with high doses of cholecystokinin (CCK-8). She found that CCK treatment caused increased apoptosis and it also increased the activation of several caspases (caspase 3, 8 and 9). This was already detectable 30 minutes after addition of CCK. This caspase activation was prevented by zietd-fmk, a Continued on page 4

4 page 4 Anna Gukovskaya, Continued from page 3 specific inhibitor of caspase-8. CCK increased the cytochrome c release from mitochondria into the cytosol. As described in other systems, this cytochrome release was associated with the loss of the mitochondrial membrane potential. Cytochrome c release from mitochondria can be mediated by the opening of PTP (permeability transition pore), which was shown by using the PTP inhibitor cyclosporin A. Intriguingly, cyclosporin A prevented the CCKinduced release of cytochrome c but not the mitochondrial depolarization. All mitochondrial alterations were prevented by the addition of caspase inhibitors indicating that upstream activation of caspases is the most likely trigger for the sequence of events that follows CCK treatment. This work has been published in the Journal of Biological Sciences of June this year. These studies provide many answers but also lead to new questions and hence the need for new studies to fully explain pancreatitis. It is only with a full understanding of the disease that successful therapy can be achieved. In humans, alcohol is a major cause of pancreatitis. In ongoing studies funded by the Research Center for Alcoholic Liver and Pancreatic Diseases, Anna and coworkers have elucidated the metabolism of ethanol in pancreas. The pancreas and acinar cells in particular metabolize ethanol by both oxidative and non-oxidative pathways. It is the balance between the two pathways that determines whether the key pro-inflammatory transcription factors NFkappaB or AP-1 are induced or inhibited in the presence of alcohol. It is not clear yet whether in alcoholic pancreatitis another insult is needed to trigger the disease. In experimental studies from Steve Pandol s group it had been found that ethanol sensitizes rats in the development of CCK-induced pancreatitis. The current studies on the effect of alcohol in the pancreas by Anna and coworkers, which they have reported at this year AGA meeting in San Francisco, focus on its modulation and potentiation of the inflammatory response. Does alcohol influence the signaling cascades that are triggered by CCK? These include PKC and calcium. Ethanol by modifying these cellular responses could increase NF-kappaB activation and hence inflammation. Another aspect of the effect of alcohol on the pancreas that Anna is actively investigating is the mechanism by which alcohol sustains the activation of NFkappaB. The work so far indicates that alcohol interferes with the silencing of NF-kappaB by inhibiting an inhibitor of NF-kappaB activation. The most promising candidate appears to be the transcription factor PPAR. At present, there is no successful therapy for pancreatitis be it from alcohol, duct obstruction or from unknown causes. It often causes death. The pancreas itself stops secreting normally. Because of the lack of amylase and Center Supports the American Liver Foundation: Greater Los Angeles Chapter By Hide Tsukamoto In support of the American Liver Foundation: Greater Los Angeles Chapter, the Center donated the $25 registration fee for each participating staff member in their annual Liver Walk. The 10K run and 5K walk was held on June 1, 2002 at Griffith Park in Los Angeles. In addition to walking for a great cause, we enjoyed the entertainment, food, beverages, prizes and awards. The Laker Girls also made an appearance and signed many autographs. The event raised $90,000, and Center members raised approximately $1,000 for the American Liver Foundation! Animal Core Manager, Dr. Peter Deng s daughter, Jiahan Deng, won a pass to an upcoming movie premier. Her name was drawn out of the hundreds of participants at the walk. Congratulations, Jiahan! We look forward to next year s walk at Griffith Park on Saturday, May 31, 2003! Continued on page 5

5 page 5 Anna Gukovskaya, Continued from page 4 and lipase, patients become unable to digest foods resulting in a wasting disease. The inflammatory infiltration in the pancreas triggers necrosis of the organ. The standard treatment is to remove surgically as much of the necrotic tissue as possible. Unfortunately the benefit of the surgery is limited as pancreatic necrosis will recur and eventually no more pancreas can be resected. In addition there can be migration of inflammatory cells to the lung and liver resulting in multiple organ failure. We can see how the hallmarks of the disease: pancreatic cell death and inflammatory infiltration are the overall theme of Anna s research. Successful therapy will only be possible when the molecular mechanisms of these pancreatic changes are fully understood. Anna s beautiful work goes a long way toward achieving this goal. Pancreatic cancer is even more feared than pancreatitis because of the extremely poor prognosis. In almost a reversal from what is the case in pancreatitis, cancer cells become resistant to dying, this being the major mechanism of their resistance to chemotherapeutic drugs. Because the surrounding normal pancreatic cells remain exquisitely sensitive to these drugs the net result is even further loss of pancreatic function while the cancer remains unaffected. Anna through a grant from the VA is addressing this paradox: why are pancreatic cancer cells so resistant to apoptosis while normal cells are so susceptible? The aim is to develop strategies to overcome this therapeutic hurdle. This year Anna has renewed the VA grant. Steve and Anna s approach is a combination of in vitro studies that are then tested in an in vivo model of pancreatic cancer in nude mice. The latter is a collaboration with the world-renowned pancreatic surgeon, Dr. Howard Reber at UCLA. Continued on page 6 Bioartificial Liver, Continued from page 2 To this end, says Tsukamoto, there are two main problems. Firstly, the conventional systems do not incorporate non-parenchymal liver cells such as HSC, which produce unique micro-environment for optimal functions of hepatocytes. Secondly, most systems are based on hollow fibers as in a hemodialysis unit, which anatomically do not reproduce physiological blood flow and cell arrangement in the liver. It really makes sense to recapitulate the radial-flow arrangement and communication between different cell types seen in the liver in a bioartifical liver system to maximize its physiological functions. The bioreactor system recently acquired is currently the best prototype to meet these specific requirements. In conjuction with the bioartifical liver project, the Program is developing novel and innovative modalities for the treatment of cirrhosis with gene therapy Lee Summer Student Research Fellowship Awards By Anne Taguchi In the summer of 2001, the Center instituted the Lee Summer Student Research Fellowship Program to promote involvement of undergraduate and Master s students in research on our Center s theme. The program is named after Dr. S.P. Lee who made a donation toward this noble cause. This program entails a minimum of 8 weeks of research during the summer in the Center investigator s laboratory and a required presentation on his/her research at the Center s Progress Report Meeting which was held on August 20, Three of the four students who were awarded the fellowship presented in this meeting. The fourth student, Lucy Ngan (mentor Chin Sung, Ph.D.) will be presenting her work at a later time. Each student that presented received a certificate and a $500 check upon completion of their fellowship. All in attendance were very impressed with the student s work and with the quality of their presentations. Congratulations to all! Above: Student, Kim Jiramongkolchai with mentor, Hal Yee, M.D., Ph.D. of UCLA. Left: Student Yanyan Zheng with mentor, James Ou, Ph.D. of USC. Center: Student Kiran Mitha with mentor Anna Gukovskaya, Ph.D. of the VA and UCLA.

6 page 6 Anna Gukovskaya, Continued from page 5 Population studies indicate that a diet rich in fruit and vegetables affords some protection from cancer and that it is the polyphenols present that most likely have the preventative effect. Steve and Anna set out to investigate whether this held true in their model of pancreatic cancer and, if so, what was the mechanism behind it. In a recent joint publication they demonstrated that polyphenols can protect mice from pancreatic cancer through decreased growth of the tumor, increased apoptosis of cancer cells with sparing of normal surrounding cells, and prevention of metastases. In accompanying in vitro experiments they showed that polyphenols induce apoptosis of cancer cells by a mechanism that results in cytochrome c release from the mitochondria through the membrane permeability transition complex. Anna and coworkers went on to show that a combination of polyphenols is better at inducing apoptosis of cancer cells. The take home message is that a variety of fruit and vegetables as well as the quantity in the diet are likely to matter in cancer prevention. Finally, how best to describe Anna? What is striking is her meticulous planning, her rigorous approach to every detail of her studies, her elegant science, while still remaining very, very well aware of the ultimate goal: which is to achieve successful treatment strategies for these most intractable diseases. RSA Meeting, Continued from page 2 induction on the proteasomal chymotrypsin-like activity by Fawzia Bardag-Gorce, Model of choronic alcoholic pancreatitis by Ilya Gukovsky, Hepatic endoplasmic reticulum stress in the intragstric ethanol fed mice by Cheng Ji, Alcohol induces cell surface CXCR4 Co-receptor expression in normal human oral keratinocytes by Xuan Liu, Ethanol has differential effects on pathways mediating NF-kB activation in the pancreatic acinar cell by Saeed Hosseini, Genetics and alcoholism in Mexican Americans by Keh-Ming Lin and Yvonne Wan, Binge exacerbates alcoholic liver damage in intragastric infusion model by Qinggao Deng and Morbidity and mortality of alcoholic liver disease in Los Angeles County by Nico Tao. We are pleased that Center members made such excellent presentations and actively interacted with other RSA particpants! Center Still Matching Donations for Thurman Lectureship Award By Rosy Macias We have also had an excellent response from Center members taking advantage of our matching funds for the Ron G. Thurman Lectureship Award. So far, we have sent $2,800 to UNC. The contributors include Drs. Sam French, Steve Pandol, Hide Tsukamoto, Bruce Runyon, Shelly Lu, Tse- Ling Fong, Tim Moran and Kenga Morgan. Thank you for your contributions! The Center will continue to offer to match dollar-for-dollar all donations to this worthy cause until the end of 2002! Please take advantage of doubling your contribution and contact Anne Taguchi at the Center to make a donation. at (323) or ataguchi@usc.edu. New Center Publications Bardag-Gorce F, French BA, Li J, Riley NE, Yuan QX, Reitz R, Cai Y, Wan Y-JY, French SW. Shift in the redox to the reduced state during the urinary ethanol cycle in rats due to periodic hypoxia. Gastroenterology 123: , Colell A., Coll O, Mari M, Fernandez-Checa JC and Garcia-Ruiz C. Divergent role of ceramide generated by exogenous sphingomyelinases on NF-kB activation and apoptosis of human colon HT-29 cells. FEBS Lett 526, 15-20, Colell A., Morales A., Fernandez-Checa JC and Garcia-Ruiz C. Acidic sphingomyelinase contributes to tumor necrosis factor-a-mediated apoptosis in human colon HT-29 cells through glycosphingolipid generation. Possible role of ganglioside GD3. FEBS Lett 526, , Feng G and Kaplowitz N. Mechanism of staurosporine-induced apoptosis in murine hepatocytes. Am J Physiol. 282:G825-G834, Fernandez-Checa JC, Colell A, and Garcia-Ruiz C. A-adenosyl-L-methionine and mitochondrial GSH depletion in alcoholic liver disease. Alcohol 27, , Gukovskaya AS, Gukovsky I, Jung Y, Mouria M, Pandol SJ. Cholecystokinin induces cytochrome c release and apoptosis in pancreatic acinar cells. J Biol Chem 277: , Lu SC, Tsukamoto H, Mato JM. The role of abnormal methionine metabolism in alcoholic liver injury. Alcohol 27:155, McPhaul LW, Wang J, Hol EM, Sonnemans MAF, Riley N, Nguyen V, Yuan QX, van Leeuwen FW, French SW. Molecular misreading of the ubiquitin B gene and hepatic Mallory body formation. Gastroenterology 122: , Morgan K, French SW, Morgan TR. Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage. Hepatology, 36:122-34, Nagai H, Matsumaru K, Feng G, and Kaplowitz N. GSH depletion by acetaminophen or diethyl maleate causes oxidative stress-dependent necrosis and sensitization to TNF -induced, oxidative stress-independent apoptosis in cultured mouse hepatocytes. Hepatology 36:55-64, She H, Xiong S, Lin M, Zandi E, Giulivi C, Tsukamoto H. Iron activates NF-κB in Kupffer cells. Am J Physiol 283: G719-G726, 2002.