Hepatitis+B+and+Liver+Cancer

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 * Introduction **

include component="page" wikiName="islaslab" page="HTML_p_indent" Liver cancer is one of the top leading causes of death among cancer patients in the United States. It is estimated that in 2012 more than 21,000 men and 7,000 women will be diagnosed with primary liver cancer (1). Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is often secondary to hepatitis B infection, hepatitis C virus (HCV) infection, or cirrhosis. include component="page" wikiName="islaslab" page="HTML_p_indent" Globally, hepatitis B is major risk factor for HCC. However, in the United States, where hepatitis B virus vaccines are common, cirrhosis from alcoholism and hepatitis C infection are the leading predictors of HCC (2). The focus of our project will be on the latter .The relationship between HCV and HCC is unique because a large majority of individuals with HCV are asymptomatic. As a result, they are not often aware of the disease until there are symptoms such as liver cancer. Furthermore, those who are diagnosed with liver cancer only have a 14% survival rate (3). include component="page" wikiName="islaslab" page="HTML_p_indent" From a public health perspective, eliminating HCV will limit the number of deaths from liver cancer. But as public health efforts are in progress, it will be important for researchers to understand HCV induction of HCC. By understanding the mechanism, we can potentially develop targeted treatments that will hopefully increase the survival rate of liver cancer. include component="page" wikiName="islaslab" page="HTML_p_indent" Currently, researchers have proposed a variety of mechanisms; however, it is not yet clear how hepatitis C infection induces HCC. We have examined the role HCV plays in hepatocarcinogenesis by analyzing the leading proposed mechanisms in academic literature, and believe that HCV induced HCC arises from a combination of three factors: 1) Inflammation 2) Oncogenic properties of the core protein and 3) Oxidative stress.

** Hepatitis C Infection Overview **
__// What is Hepatitis? //__ (3) include component="page" wikiName="islaslab" page="HTML_p_indent" Hepatitis is a term used to describe inflammation of the liver. It is caused by a variety of factors including toxins, certain drugs, alcohol, disease, and/or pathogens. Viral hepatitis is a subset of hepatitis most commonly caused by the hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV). include component="page" wikiName="islaslab" page="HTML_p_indent" Although HAV, HBV, and HCV infections cause similar symptoms in their human hosts, each virus has a different genetic makeup and different modes of transmission.

__// What causes hepatitis C infection? //__ (4) include component="page" wikiName="islaslab" page="HTML_p_indent" Hepatitis C infection is caused by the hepatitis C virus (HCV), which is an enveloped, single stranded, positive sense RNA virus within the //Flaviviridae// family. Hepatitis C infection is the third most common form of hepatitis infection. It begins as an acute infection, but in some cases may become chronic.

Acute hepatitis C infection:
include component="page" wikiName="islaslab" page="HTML_p_indent" Approximately 80% of acutely infected individuals are asymptomatic, making testing and diagnosis very difficult. In those individuals who do develop symptoms, the average time period from exposure to symptom onset is 4-12 weeks. Approximately 40% of individuals with acute HCV will fully recover.

Chronic hepatitis C infection:
include component="page" wikiName="islaslab" page="HTML_p_indent" Chronic disease is defined as continuing disease without improvement for at least 6 months. More specifically, the immune system of a chronically infected individual has not been able to fully eradicate HCV. Unfortunately, 60%-80% of those with chronic hepatitis do not present any symptoms. A unique aspect about chronic hepatitis C is the high rate of chronic infection and slow lifelong progression, often leading to cirrhosis and liver cancer.

__// Symptoms of hepatitis C infection //__ (1) The following symptoms may be caused by hepatitis C infection (CITE): include component="page" wikiName="islaslab" page="HTML_p_indent" __// Diagnosis of hepatitis C infection //__ (5) The following tests and procedures may be used to diagnose hepatitis C infection: > enzymes released by the liver. One protein is serum //alanine aminotransferase (ALT)//. Elevated amounts of ALT may be indicative of hepatitis C infection. include component="page" wikiName="islaslab" page="HTML_p_indent" __// How does hepatitis C infection spread? //__ (3) Hepatitis C is spread when blood from an infected individual enters the body of an uninfected individual. This can happen during: include component="page" wikiName="islaslab" page="HTML_p_indent" __ // Treatment of hepatitis C infection // __ (5) include component="page" wikiName="islaslab" page="HTML_p_indent" Currently, there are only vaccines to prevent against hepatitis A and hepatitis B infection. There is no vaccine for hepatitis C. Prior infection with any type of hepatitis virus does not protect against later infection with the same or different type of the hepatitis virus. The following treatment may be used to manage hepatitis C infection: include component="page" wikiName="islaslab" page="HTML_p_indent" include component="page" wikiName="islaslab" page="HTML_p_indent"
 * pain or tenderness in the upper, right abdomen
 * pain around the right shoulder blade
 * fever
 * nausea
 * vomiting
 * loss of appetite and feelings of fullness
 * unusual weakness or fatigue
 * // Jaundice // (yellow skin and eyes, pale stool, dark urine)
 * ** Liver enzymes (liver function tests) ** : One specific type of blood test measures the amount of certain
 * Injection drug use
 * Recipient of donated blood, blood products, and organs
 * Needle stick injuries in healthcare settings
 * Birth to an HCV-infected mother
 * Sex with an infected individual
 * Sharing of personal items contaminated with infectious blood
 * ** Interferon Alpha: ** Interferon alpha is a recombinant protein that stimulates an anti-viral response in most cells. This injection is often used in combination with Ribavirin, an anti-viral drug. Interf eron alpha can have bad side effects including flu-like symptoms, and it is also very expensive.

** Hepatitis C Virus (HCV) Overview **
__//Hepatitis C Virus//__ include component="page" wikiName="islaslab" page="HTML_p_indent" HCV was identified in 1988 - at the time, it was called non-A, non-B hepatitis (6). HCV is classified in the genus //Hepacivirus// within the family //Flaviviridae//.There are eleven major genotypes associated of HCV and approximately 100 different strains (7).

__// HCV Structure and Genome //__ (8) include component="page" wikiName="islaslab" page="HTML_p_indent" HCV is a positive-sense, single-stranded RNA virus. include component="page" wikiName="islaslab" page="HTML_p_indent" The HCV genome is 9.6kB. It codes for 10 viral proteins -- 3 structural proteins and 7 non-structural proteins. H CV has one large open reading frame that allows the translation of one large polyprotein, encoding all 10 viral proteins, that later undergoes post-translational cleavage by host and viral proteases. include component="page" wikiName="islaslab" page="HTML_p_indent" The 3 structural proteins form the 3D structure of the viral particle. All 3 structural proteins as well as the first non-structural protein, p7, are freed from the polyprotein chain by liver cell enzymes. //Structural Proteins://
 * 1) //core// - icosahedral shell
 * 2) //E1// - the first HCV envelope glycoprotein
 * 3) //E2// - the second HCV envelope glycoprotei

include component="page" wikiName="islaslab" page="HTML_p_indent" The 7 non-structural (NS) proteins have roles in viral replication. The remaining 6 non-structural proteins are freed from the polyprotein chain by viral proteases. // Non-structural Proteins: // include component="page" wikiName="islaslab" page="HTML_p_indent" Considering that the HCV genome is relatively small (less than 10kB) and expresses only 10 mature proteins, and yet the virus itself has to figure out how to enter a host, replicate, make viral particles, exit a host, and all the time evade and escape the immune system – it’s probably very likely that each of the 10 HCV proteins carries out multiple functions. include component="page" wikiName="islaslab" page="HTML_p_indent" It is interesting to note that t he HCV genome is highly mutable because its RNA-dependent RNA polymerase lacks proof-reading function. Therefore, mutations that occur during replication are not properly corrected. In particular, the genomic region that codes for envelope proteins undergoes rapid mutation, allowing the virus to escape immune surveillance.
 * 1) //p7// - ion channel
 * 2) //NS2 -// cysteine protease
 * 3) //NS3// - serine protease & RNA helicase activity
 * 4) //NS4a// - RNA helicase activity
 * 5) //NS4b// - creates a site for RNA replication
 * 6) //NS5a// - creates a site for RNA replication
 * 7) //NS5b// - RNA-dependent RNA polymerase

__// HCV Replication //__ (9) include component="page" wikiName="islaslab" page="HTML_p_indent" HCV targets hepatocytes which are the predominant cell type in the liver (see “Hepatocytes”). Hepatocytes are the only cell type HCV is known to affect. Below is a simplified version of HCV replication : **Binding of virus to cell-surface receptors:** HCV interacts with proteins on the surface of hepatocytes such as GAGs, CD81, SR-BI, Claudin-1, LDL-receptor, and Occludin-1 (see below). include component="page" wikiName="islaslab" page="HTML_p_indent" //__ HCV Cell-Surface Proteins that Mediate Viral Binding and Entry __// (10) // GAGs (Glycosaminoglycans // // CD81 //  // SR-B1 (Scavenger Receptor B1) //  // Claudin-1 //  // LDL-R (Low Density Lipoprotein Receptor) //  include component="page" wikiName="islaslab" page="HTML_p_indent"  include component="page" wikiName="islaslab" page="HTML_p_indent"  ** Liver and HCC Overview **
 * 1) **Internalization & Uncoating of virus:** HCV enters the hepatocytes and is uncoated in the cytoplasm to expose RNA genome.
 * 2) **Expression of viral proteins:** Viral RNA is translated to form a polyprotein that is then cleaved by proteases to form individual proteins. Protein synthesis occurs in the cytoplasm and endoplasmic reticulum.
 * 3) **Replication of viral genome:** The viral genome is transcribed to a negative-sense RNA via RNA dependent RNA polymerase. The negative-sense RNA can then be used as a template to replicate positive-sense RNA (original viral genome).
 * 4) **Assembly & Release of virus:** Replicated RNA and proteins are assembled and released from the cell.
 * linear polysaccharides that are present on proteins of human cell surfaces
 * provides an immediate way for virus to stick to cells
 * a member of the tetraspanin superfamily
 * has 2 extracellular domains, 4 transmembrane domains, and 3 intracellular signaling domains
 * expressed on all nucleated cells
 * expression confers susceptibility to virus attack
 * a high-density lipoprotein receptor
 * expressed on hepatocytes
 * expression confers susceptibility to virus attack
 * forms the backbone of tight junction strands in epithelial tissues
 * highly expressed on hepatocytes
 * expression confers susceptibility to virus attack
 * viral RNA accumulation increased or decreased in parallel with LDL-R mRNA expression and LDL entry

__// What is the Liver? //__ (11) include component="page" wikiName="islaslab" page="HTML_p_indent" The liver is the largest organ in the abdomen, and one of the largest in the human body. It fills the upper, right quadrant of the abdomen. It is located near the stomach, intestines, gallbladder, and pancreas. The liver has four lobes – two in the front, and two small lobes in the back. The liver has many vital functions, including: include component="page" wikiName="islaslab" page="HTML_p_indent" The liver receives blood from two vessels – the hepatic portal vein delivers //un//filtered blood from the stomach and intestine, and the hepatic artery delivers oxygenated blood from the heart. The hepatic vein takes //filtered// blood back to the heart.
 * filtration of harmful substances from the blood to be passed on to the urine and stool
 * production of enzymes and bile to help digest food and fats, respectively
 * storage of glycogen to be used as energy

__// Anatomy of a Liver Lobe //__ (12) include component="page" wikiName="islaslab" page="HTML_p_indent" In each liver lobe, blood enters through the portal vein and hepatic artry and is distributd by smaller vessels to enter the sinusoidal spaces. The sinusoidal spaces are lined by fenestrated endothelial cells and macrophages, called Kupffer cells. The fenestrated endothelial cells are perforated, allowing for free diffusion between blood and hepatocytes. Blood flows through this space and is then collected in the central vein and exits the liver. include component="page" wikiName="islaslab" page="HTML_p_indent" As stated earlier, the liver is responsible for the breakdown of old red blood cells. During the catabolism of the red blood cell’s heme, bile is formed and secreted into bile canaliculi. The bile canaliculi are small channels formed at the junctions of hepatocytes. Bile travels in the opposite direction from blood, flowing through a region called the canal of Herring and into the bile ducts. From the bile ducts, bile will continue into larger ducts and is ultimately transported to the gallbladder and intestine. include component="page" wikiName="islaslab" page="HTML_p_indent" The distance from the portal vein to the central vein measures 20-30 hepatocytes.

__//Hepatocytes//__ (12) include component="page" wikiName="islaslab" page="HTML_p_indent" Hepatocytes are the principal cell type of the liver, making up 70% of the organ. Under normal conditions the average half-life of a hepatocyte is 6 to 12 months. include component="page" wikiName="islaslab" page="HTML_p_indent" Hepatocytes are terminally differentiated cells. Typical post-mitotic cells have exited the cell cycle permanently and no longer undergo cell division. However, being “terminally differentiated” hepatocytes are unique in that they actually retain the ability to undergo extensive proliferation in response to liver injury. This means when necessary, hepatocytes can re-enter the cell cycle and divide. For example, in an individual who has had 70% of their liver surgically removed, almost every hepatocyte will complete the cell cycle once, allowing the liver cell mass to be restored within just a few days. include component="page" wikiName="islaslab" page="HTML_p_indent" As mentioned earlier (see "HCV Replication"), hepatocytes are the only confirmed site of replication for all members of the hepatitis virus family.

__// What is hepatocellular carcinoma? //__ (13) include component="page" wikiName="islaslab" page="HTML_p_indent" //Primary// liver cancer is cancer that begins in the liver. //Metastatic// liver cancer begins in other parts of the body and spreads to the liver. For the purposes of our report, we will focus on primary liver cancer and metastatic liver cancer will not be discussed further. include component="page" wikiName="islaslab" page="HTML_p_indent" Primary liver cancer can occur in both adults and children. For the purposes of our report, we will focus on adult primary liver cancer and childhood liver cancer will not be discussed further. include component="page" wikiName="islaslab" page="HTML_p_indent" Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. We will be focusing on HCC for the remainder of our project.

//__ Symptoms of __// __//hepatocellular carcinoma//__ (13) The following symptoms may be caused by hepatocellular carcinoma: include component="page" wikiName="islaslab" page="HTML_p_indent" __// Diagnosis of //__ __//hepatocellular carcinoma//__ (1, 14) include component="page" wikiName="islaslab" page="HTML_p_indent" Because most patients with HCC are asymptomatic until the cancer is in its late stages, only a small number of individuals are diagnosed early. The following tests and procedures may be used to diagnose adult primary liver cancer: // Medical imaging is non-invasive, and often the first strategy physicians will use to detect liver problems: // //Blood tests are another routine test used to detect liver problems:// //Surgical removal of a small sample of cells or tissue is needed when imaging and blood tests are inconclusive. Although mildly-invasive, the biopsy provides a definitive diagnosis:// include component="page" wikiName="islaslab" page="HTML_p_indent" //__ Stagin __////__ g of __// __//hepatocellular carcinoma//__ (14) The following stages are used for adult primary liver cancer: Key for HCC staging codes: include component="page" wikiName="islaslab" page="HTML_p_indent" __// Treatment of hepatocellular carcinoma //__ (1) include component="page" wikiName="islaslab" page="HTML_p_indent" Treatment of adult primary liver cancer depends on disease state, including tumor size, liver function, and other medical conditions. As mentioned earlier, most patients with HCC are asymptomatic until the disease is in its advanced stages making most therapies unsuccessful. There is no standard, universal treatment for adult primary liver cancer. The following treatments may be used to treat adult primary liver cancer: // Surgery: Surgery may be done using different techniques: // // Chemotherapy: // //A palliative form of care. Chemotherapy is usually systemic, in which anti-cancer drugs are taken by mouth or injected intravenously, entering the bloodstream and reaching cancer cells throughout the body. When used together with surgical resection or liver transplantation, the response rate is very low (less than 20%).// // Radiation therapy: A procedure that uses high-energy x-rays, gamma rays, neutrons, protons, and other sources to kill cancer cells and shrink tumors. Requires total doses of ionizing radiation greater than 30 Gy, which can result in radiation hepatitis and death. //  include component="page" wikiName="islaslab" page="HTML_p_indent" New treatments on the rise includes, **ethanol injection** directly into a tumor, **hyperthermia therapy**, in which body tissue is exposed to high temperatures in order to damage or kill cancer cells or to make cancer cells more susceptible to radiation and other anti-cancer drugs.
 * pain or tenderness in the upper, right abdomen
 * a lump or feeling of heaviness in the upper, right abdomen
 * pain around the right shoulder blade
 * swollen abdomen (bloating)
 * loss of appetite and feelings of fullness
 * unexplained weight loss
 * unusual weakness or fatigue
 * // Jaundice // (yellow skin and eyes, pale stool, dark urine)
 * ** Computer Tomography (CT) ** : Takes a series of detailed images of areas inside the body from various angles. A computer linked to an x-ray machine then creates an overall picture. Contrast material (iodinated dye, ethiodized oil emulsion, etc.) may be injected into a liver vessel or swallowed to help cancerous liver cells show up clearly.
 * ** Ultrasound ** : Uses high-energy soundwaves to reflect off internal tissues or organs, creating a pattern of echoes. The echoes create an image of the liver. Tumor tissue produces echoes that are different from the echoes made by non-cancerous tissues.
 * ** Magnetic Resonance Imaging (MRI ** ): Uses a magnet, radiowaves, and a computer to create a series of detailed images of areas inside the body. Contrast material (dyes) may be injected or swallowed to help the liver and other areas show up clearly.
 * It is important to note that an MRI is more accurate than a CT, but more expensive.
 * Positron emission tomography (PET) scans (similar to the one Dr. Islas showed in class) are not sensitive enough to detect HCC and are therefore not used.
 * ** Liver enzymes (liver function tests) ** : One specific type of blood test measures the amount of certain enzymes released by the liver. These proteins include serum //bilirubin//, //prothrombin time//, serum //albumin//, //alkaline phosphatase//, serum //alanine aminotransferase (ALT)//, and //aspartate aminotransferase (AST)//. Elevated amounts of a substance may be indicative of liver cancer.
 * ** Serum //alpha-fetoprotein// ** : Another specific type of blood test measures the levels of a specific tumor marker. Tumor markers are molecules associated with specific types of cancer when found in increased levels in the blood. The primary marker used for HCC is alpha-fetoprotein (AFP). Elevated levels of AFP may be indicative of liver cancer. It is important to note that AFP levels may also be high in non-cancerous conditions, such as cirrhosis and hepatitis.
 * ** It is important to note that presence of a hepatic mass in the CT, ultrasound, or MRI scan in addition to an AFP level greater than 500ng/mL is diagnostic of HCC. No tissue diagnosis is necessary. **
 * ** Liver biopsy ** : A procedure in which a doctor inserts a thin needle into the liver to remove a sample oftissue. CT and ultrasound may be used to help guide the needle.
 * ** Laparoscopic surgery ** : A procedure in which a surgeon makes small incisions in the wall of the abdomen. A laparoscope (thin, lighted tube) is inserted through the incisions to remove a sample of tissue.
 * ** Stage I ** : T1, N0, M0
 * ** Stage II ** : T2, N0, M0
 * ** Stage III ** :
 * ** Stage IIIA ** : T3, N0, M0
 * ** Stage IIIB ** : T4, N0, M0
 * ** Stage IIIC ** : any T, N1, M0
 * ** Stage IV ** : any T, any N, M1
 * ** T1 ** : solitary tumor without vascular invasion
 * ** T2 ** : solitary tumor with vascular invasion //or// multiple tumors smaller than 5cm
 * ** T3 ** : tumor involving a major branch of the portal or hepatic vein(s) //or// multiple tumors larger than 5cm
 * ** T4 ** : tumor(s) with direct invasion of adjacent organs other than the gallbladder //or// with perforation of //visceral peritoneum\//
 * ** N0 ** : no nodal involvement
 * ** N1 ** : regional nodal involvement
 * ** M0 ** : no distant metastasis
 * ** M1 ** : metastasis beyond the liver
 * ** Surgical resection ** (hepatectomy): The primary treatment option. May be used if the HCC tumor is localized in one hepatic lobe and in patients with __no__ cirrhotic disease. Recurrence rates after surgical resection are high.
 * ** Liver transplantation ** : Limited to select patients. May be used if the HCC tumor is localized and in patients with __no__ cirrhotic disease. Recurrence rates after liver transplantation are high. Liver recipients are required to take immunosuppressant drugs for the remainder of their life.
 * ** Radiofrequency ablation ** : Uses a probe with small electrodes to send heat directly into or nearby cancer cells. May be used if the HCC lesion is less than 5cm in diameter, localized, and unresectable.
 * ** Percutaneous ablation ** : May be used if the HCC lesion is less than 5cm in diameter, localized, and unresectable. Patients are required to have adequate coagulation and platelet counts.
 * ** Cryosurgery ** : Uses liquid nitrogen or liquid carbon dioxide to freeze and destroy cancer cells. May be used if the HCC lesion is less than 5 cm in diameter, localized, and unresectable.
 * ** It is important to note that complete hepatectomy and liver transplantation are the //only// curative options for HCC. The other treatment types listed below and above are used to manage and control the disease only. **
 * ** Chemoemoblization of the hepatic artery: ** A tiny catheter is placed into the hepatic artery. Chemotherapeutic drugs are injected into the catheter, followed by the infusion of substances that block the hepatic artery. The particles cut off blood flow to the tumor, preventing oxygen and nutrients from reaching the tumor and subsequently causing cancer cells to die without oxygen. Decreased blood flow also allows anti-cancer drugs to remain in the liver longer. Healthy liver tissue continues to receive blood from the hepatic portal vein. Can be used if the HCC lesion is less than 5cm in diameter, localized, and unresectable.
 * ** Radiolabled monoclonal antibodies ** : Radioactive material is attached to antibodies made in the laboratory. The antibodies, which target liver cancer cells, are injected into the body.

__// Prognosis for hepatocellular carcinoma //__ (14) include component="page" wikiName="islaslab" page="HTML_p_indent" Prognosis of adult primary liver cancer depends on disease state, including tumor size, liver function, and other medical conditions. Typically though, prognosis for HCC is very poor. HCC is almost always fatal within 2 years of diagnosis. Only 10-20% of HCCs can be removed completely using surgery. If the cancer cannot be entirely removed, the disease is usually fatal within 6 months.

** Hepatitis C-Induced Hepatocellular carcinoma **



include component="page" wikiName="islaslab" page="HTML_p_indent" The picture above shows the progression of hepatitis C infection to hepatocellular carcinoma (see picture for a description of each disease state). In the U.S., 1-5% of the 3.2 million persons with chronic hepatitis C infection will develop hepatocellular carcinoma. This ends up being 32,000 to 160,000 people that will develop this form of liver cancer. include component="page" wikiName="islaslab" page="HTML_p_indent" The two studies below provide evidence that hepatitis C infection leads to hepatocellular carcinoma in transgenic mice models. In the first study, expression of the entire HCV polyprotein leads to hepatic steatosis and eventually, hepatocellular carcinoma in transgenic mice. In the second study, expression of the HCV core protein only also leads to steatosis and hepatocellular carcinoma. An analysis comparing the two experiments is provided after both studies.

** Steatosis and liver cancer in transgenic mice expressing structural and nonstructural proteins of hepatitis C virus ** (15) Herve Lerat, Masao Honda, Michael R. Beard, Kim Loesch, Jiaren Sun, Yan Yang, Michiari Okuda, Rainer Gosert, Shu-yuan Xiao. Steven A. Weinman, and Stanley M. Lemon [GASTROENTEROLOGY 2002, 122:352-365]

** // Aim: // ** Researchers sought to determine whether expression of hepatitis C virus proteins alters hepatic morphology or function, in the absence of inflammation. ** // Methods: // ** Researchers used mice from the C57BL/6 strain to create two transgenic lineages with liver-specific expression of RNA encoding the complete HCV polyprotein (FL-N transgene) or HCV __structural__ proteins only (S-N transgene). Transgenic mice were compared to non-transgenic littermates for altered hepatic morphology or function. ** // Results: // ** FL-N transcripts were detectable by reverse-transcriptase polymerase chain reaction only. S-N transcripts were detectable by Northern blot analysis. The abundance of viral proteins was sufficient for detection in S-N transgenic mice only. There was no inflammation in the livers of transgenic mice. Mice expressing either transgene (FL-N or S-N) developed hepatic steatosis that was more severe with age and in males. Apoptotic or proliferating hepatocytes were not significantly increased. Mice expressing either transgene (FL-N or S-N) developed hepatic steatosis that was more severe with age and in males. Also, older, male mice expressing either transgene (FL-N or S-N) developed hepatocellular adenoma or carcinoma, but their incidence reached statistical significance in FL-N transgenic mice only. Steatosis, hepatocellular adenoma, or hepatocellular carcinoma was not seen in non-transgenic mice. ** // Conclusion: // ** Constitutive expression of HCV proteins at low levels leads to common pathologic features of hepatitis C in the absence of inflammation. Expression of HCV structural proteins induced steatosis in mice, while expression of the entire HCV polyprotein (additional expression of HCV __non-structural__ proteins) increases the risk of cancer.

__ // Definitions // __ ** Alanine aminotransferase (ALT) ** – A liver enzyme. Elevated amounts of ALT in blood may be indicative of liver damage or liver cancer.

** Albumin ** – A protein made by the liver. It is the most abundant protein in human blood plasma. Albumin is water soluble and therefore acts to transport many small, hydrophobic molecules through the blood, including [|bilirubin], calcium, progesterone, and medications (16).

** Bromodeoxyuridine (BrdU) ** – A synthetic nucleoside that is an analogue of thymidine. During DNA replication, BrdU can be incorporated into the newly synthesized strand of DNA in place of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues. Antibodies specific for BrdU are used to detect the incorporated chemical, thereby locating cells that are actively replicating their DNA (17).

** Hepatic steatosis ** – Fatty liver disease is a form of chronic hepatitis. The condition is characterized by hepatocytes with fat droplets of various sizes in the cytoplasm (18, SAME AS LAST OF "CORE"). The spectrum of hepatic steatosis is wide and encompasses mild, moderate, and severe steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis.

__ // Production of Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers used a plasmid containing the murine albumin enhancer/promoter with a downstream Simian virus 40 (SV40) intron/polyadenylation cassette. Complementary DNA corresponding to the complete HCV polyprotein (core to NS5B) // OR // the HCV structural proteins only (core to E2/p7) was inserted into the plasmid – in between the albumin enhancer/promoter and SV40 intron/polyadenylation cassette (15). Therefore, in each transgene the HCV open reading frame was under the transcriptional control of the liver-specific murine albumin promoter/enhancer. include component="page" wikiName="islaslab" page="HTML_p_indent" The two resulting plasmids were inserting into mouse embryos from the C57BL/6 strain. Transgenic F 0 founder mice (15 FL-N founder mice, 12 S-N founder mice) were mated with normal C57BL/6 mice to produce F 1 and subsequent generations of mice. The mice were cared for a according to institutional guidelines, fed an ordinary, sterile chow diet, and maintained in pathogenic-free conditions. include component="page" wikiName="islaslab" page="HTML_p_indent" Non-transgenic littermates of FL-N and S-N transgenic mice were used as controls.

__ // Transgene Expression in FL-N and S-N Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers used two methods to confirm the expression of RNA in 2-month-old transgenic mice: Northern blot analysis and the more sensitive technique of reverse-transcriptase polymerase chain reaction. include component="page" wikiName="islaslab" page="HTML_p_indent" In 0 out of 15 FL-N (complete HCV polyprotein) transgenic mice, intrahepatic RNA transcripts were not detectable by Northern analysis. Conversely, in 2 out of 12 S-N (HCV structural proteins only) transgenic mice, intrahepatic RNA transcripts were readily detectable by Northern analysis (15). include component="page" wikiName="islaslab" page="HTML_p_indent" In 4 out of 15 FL-N transgenic mice, intrahepatic full-length RNA transcripts were detectable after reverse-transcriptase polymerase chain reaction. In addition to the 2 S-N lineages for which RNA was detectable by Northern blot, liver samples from mice in 3 other S-N lineages also contained RNA transcripts detectable by reverse transcriptase polymerase chain reaction (15).

__ // Phenotypes of FL-N and S-N Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Of the 9 transgenic lineages (4 FL-N lineages and 5 S-N lineages) that showed positive HCV-specific RNA transcripts, only 2 lineages were capable of breeding in large numbers (FL-N/35 and S-N/863) (15). include component="page" wikiName="islaslab" page="HTML_p_indent" The transgenic mice appeared normal and did not have any obvious defects in growth and development. There was no inflammation in the livers of transgenic mice. There was also no significant difference in serum alanine aminotransferase levels between transgenic mice compared to non-transgenic littermates.

__ // Hepatic Steatosis // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Researches determined that hepatic steatosis was present in transgenic mice from the FL-N/35, FL-N/963, and S-N/863 lineages. Particularly, mice from the FL-N/35 and S-N/863 lineages developed __ severe __ steatosis. include component="page" wikiName="islaslab" page="HTML_p_indent" To determine whether steatosis was correlated with age, researchers used oil red O to stain fat droplets and quantify the degree of steatosis in individual liver sections. In 9-15-month-old Fl-N/35 mice, the area occupied by oil red O-stained fat droplets was significantly greater compared to age-matched, non-transgenic mice ( // P // <0.02). Similarly, in 12-16-month-old S-N/863 mice, the area occupied by oil red O-stained fat droplets was significantly greater compared to age-matched, non-transgenic mice ( // P // <0.02). Researchers also discovered that incidence of steatosis was significantly higher in male transgenic mice compared to female transgenic mice (19.3% ± 4.4% // versus // 8.4% ± 1/1%, // P // <0.05) (15). include component="page" wikiName="islaslab" page="HTML_p_indent" Past studies have indicated that hepatic steatosis can result from mitochondrial injury and increased lipid peroxidation (19). To ensure that oxidative stress was not a contributing factor for the development of steatosis, researchers measured the levels of lipid peroxides in transgenic versus non-transgenic mice. In >8-month-old FL-N/35 and S-N/863 mice, there was no significant difference in lipid peroxides compared to age-matched, non-transgenic mice (55.6 ± 8.9 pmol/mg and 38.9 ± 8.9 pmol/mg // versus // 51.1 ± 4.5 pmol/mg) (15). include component="page" wikiName="islaslab" page="HTML_p_indent" The results indicate that hepatic steatosis increased with age in the transgenic mice. Additionally, the fact that S-N transgenic mice developed steatosis, suggests that its occurrence is related to the expression of one or more of the HCV structural proteins.

__ // Hepatocellular Proliferation and Apoptosis // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Past studies have indicated that HCV proteins may alter cell signaling and regulate apoptotic pathways (20, 21, 22, and 23). To investigate this, researchers measured apoptotic hepatocytes and hepatocytes proliferation rates in transgenic versus non-transgenic mice. include component="page" wikiName="islaslab" page="HTML_p_indent" There was no significant increase or decrease in numbers of apoptotic hepatocytes in both FL-N and S-N transgenic mice compared to age-matched non-transgenic littermates (15, SAME AS PAPER). include component="page" wikiName="islaslab" page="HTML_p_indent" To measure basal hepatocytes proliferation rates, researchers measured the frequency of hepatocytes incorporating BrdU over a 5-hour period before necropsy in 9-11-month-old transgenic and non-transgenic mice. Although in FL-N/35 male mice proliferation was highest, the difference compared to non-transgenic mice was not significant (3.1 ± 1.3 cells per 40x field // versus // 0.82 ± 0.7 cells) (15). Similarly in S-N/863 mice proliferation was also high; however once again, the difference compared to non-transgenic mice was not significant.

__ // Hepatocellular Carcinogenesis // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Researches determined that transgenic mice from the FL-N/35 and S-N/863 lineages developed hepatocellular carcinoma. include component="page" wikiName="islaslab" page="HTML_p_indent" At necroscopy, hepatic adenomas or hepatocellular carcinomas ranged from a few millimeters to more than 2cm in diameter. Adenomas or hepatocellular carcinoma was seen in 5 out of 37 (17 male, 20 female) FL-N and 1 out of 42 (24 male, 18 female) S-N/863, >13-month-old, transgenic mice (15). include component="page" wikiName="islaslab" page="HTML_p_indent" A ll of the tumors identified occurred in males. Adenomas or hepatocellular carcinoma was present in 5 out of 17 male FL-N/35 transgenic mice, >13-months-old, compared to 0 out of 20 age-matched female FL-N/35 mice. Similarly, adenomas or hepatocellular carcinoma was present in 1 out of 24 male S-N/863 transgenic mice, >13-months-old, compared to 0 out of 18 female S-N/863 mice (15). include component="page" wikiName="islaslab" page="HTML_p_indent" No tumors were seen in non-transgenic mice (0 out of 24) (15). include component="page" wikiName="islaslab" page="HTML_p_indent" T he development of adenomas or hepatocellular carcinoma in FL-N/35 male mice (5 out of 17) was significantly higher compared to S-N/863 male mice (1 out of 24) (15). include component="page" wikiName="islaslab" page="HTML_p_indent" The greater frequency of liver cancer in FL-N/35 lineage along with the researchers earlier findings that HCV structural proteins are expressed at a higher level in the S-N/863 lineage (detectable by Northern blot) than the FL-N/35 lineage (not detectable by Northern blot), suggest that the __ non-structural __ proteins of HCV may contribute to hepatic carcinogenesis, even when they are expressed at low levels.

__ // Conclusion // __ include component="page" wikiName="islaslab" page="HTML_p_indent" In this study, researchers demonstrated that liver-specific expression of HCV structural genes induces hepatic steatosis, while expression of the entire HCV genome at low levels, results in progressive morphological and biochemical changes that ultimately leads to the development of HCC. This finding suggests that the additional expression of the __ non-structural __ proteins of HCV may contribute significantly to hepatic carcinogenesis. Additionally, the results indicate that a chronic state of inflammation from hepatitis C infection is not an absolute pre-requisite for the development of HCC. Moreover, the findings suggest that HCV itself is directly involved in the development of HCC.

** The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice ** (24) Kyoji Moriya, Hajime Fujie, Yoshizumi Shintani, Hiroshi Yotsuyanagi, Takeya Tsutsumi, Kotaro Ishibashi, Yoshiharu Matsuura, Satoshi Kimura, Tatsuo Miyamura, and Kazuhiko Koike [NATURE MEDICINE 1998, 9:1065-1067]

** // Aim: // ** Researchers sought to determine whether expression of hepatitis C virus core protein alters hepatic morphology or function, in the absence of inflammation. ** // Methods: // ** Researchers used mice from the C57BL/6 strain to create two transgenic lineages, C21 and C49, each with expression of RNA encoding the core HCV protein. Transgenic mice were compared to non-transgenic littermates for altered hepatic morphology or function. ** // Results: // ** The abundance of the viral protein was similar to core protein levels found in the liver of chronic hepatitis C patients. There was no inflammation in the livers of transgenic mice. Young mice from both transgenic lineages developed hepatic steatosis as a histological feature, characteristic of chronic hepatitis C. After the age of 16 months, mice from both lines developed hepatic tumors that first appeared as adenomas containing fat droplets in the cytoplasm. Then, once again mice from both lines developed hepatocellular carcinoma within the adenomas, presenting in a “nodule-in-nodule” manner – a histopathological feature of early hepatocellular carcinoma in chronic hepatitis C patients. Steatosis, hepatocellular adenoma, or hepatocellular carcinoma was not seen in non-transgenic mice. ** // Conclusion: // ** Constitutive expression of the HCV core protein leads to common pathologic features of hepatitis C in the absence of inflammation. Expression of HCV core protein induced steatosis, adenomas, and hepatocellular carcinoma in mice, increasing the risk of cancer.

__ // Definitions // __ ** Alanine aminotransferase (ALT) ** – A liver enzyme. Elevated amounts of ALT in blood may be indicative of liver damage or liver cancer.

** Eosinophilic cells ** -- White blood cells that act to combat multi-cellular parasites and infection (25).

** HCV core protein ** -- The core protein makes up the viral capsid. It is approximately 22kDa and is encoded by the 5’-proximal region of the HCV genome. The gene that encodes the core protein is highly conserved amongst various HCV strains, suggesting its importance in HCV pathogenesis (26).

** Hepatic adenoma – ** Uncommon benign epithelial liver tumors that develop in an otherwise normal appearing liver. Polyps range in size from 1cm to 30cm. (27)

** Hepatic carcinoma ** – P rimary tumor of the liver, which usually develops in the setting of chronic liver disease, particularly in patients with chronic hepatitis B and C (28).

** Hepatic steatosis ** – Fatty liver disease is a form of chronic hepatitis. The condition is characterized by the excessive accumulation of triglycerides in the form of lipid droplets in the liver (18). The spectrum of hepatic steatosis is wide and encompasses mild, moderate, and severe steatosis, non-alcoholic steatohepaitits (NASH), fibrosis, cirrhosis.

__ // Production of Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" To ensure high-level expression in the liver without interfering with normal mouse development, researchers used a plasmid containing the hepatitis B virus (HBV) enhancer/promoter with a downstream intron/polyadenylation cassette. Complementary DNA corresponding to the HCV core protein was inserted into the plasmid in between the HBV enhancer/promoter and intron/polyadenylation cassette. Therefore, the HCV core gene was under the transcriptional control of the HBV regulatory region. include component="page" wikiName="islaslab" page="HTML_p_indent" The resulting plasmid was inserted into mouse embryos from the C57BL/6 strain. The mice were cared for according to institutional guidelines, fed an ordinary, sterile chow diet, and maintained in pathogenic-fee conditions. Non-transgenic littermates of C21 and C49 transgenic mice were used as controls (24).

__ // Hepatic Steatosis & Adenoma in Younger Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Researches determined that hepatic steatosis was present in transgenic mice from 12 months of age. include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers killed transgenic mice from both lineages at 3, 6, 9, 12, 16, and 19 months of age and pathologically examined liver tissue samples. C21 and C49 transgenic mice, 3, 6, 9, and 12-months-old, had marked hepatic steatosis. However, 16-month-old transgenic mice had gross hepatic nodules. Further microscopic examination of these hepatic nodules showed that they compressed neighboring normal hepatocytes and that they consisted of eosinophilic cells with fat droplets in the cytoplasm. The histological examination of these nodules was characteristic of hepatocellular adenoma (24).

__ // Alanine aminotransferase in Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Although the mean alanine aminotransferase (ALT) levels in 3-9-month-old transgenic mice (n=12), was higher compared to age-matched non-transgenic mice (n=6), the difference was not significant (90.4 ± 83.3 // versus // 80.3 ± 48.7 IU/L). Similarly, although the mean ALT levels in 16-19-month-olf transgenic mice (n=18), was higher compared to age-matched non-transgenic mice (n=10), the difference was still not statistically significant (136.2 ± 116.3 // versus // 121.9 ± 91.9 IU/L) (24).

__ // Hepatocellular Carcinoma in Older Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers killed transgenic mice from both lineages at 3, 6, 9, 12, 16, and 19 months of age and pathologically examined liver tissue samples. Approximately one-fourth of 16-19-moth-old, male transgenic mice had hepatic nodules. Further microscopic examination of these hepatic nodules showed that they were well-differentiated hepatocellular carcinomas with few cells containing fat droplets in the cytoplasm. Both normal cells and hepatocellular adenomas had numerous fat droplets in the cytoplasm of cells, whereas hepatocellular carcinomas had few fat droplets (24). include component="page" wikiName="islaslab" page="HTML_p_indent" Most of the hepatic nodules had distinctive “nodule-in-nodule” characteristic, in which hepatocellular carcinoma developed within an adenoma. The histological examination of these “nodules-in-nodules” was characteristic of the development of hepatocellular carcinoma in humans with chronic hepatitis C – where small well-differentiated hepatocellular carcinomas showed fatty changes and more poorly-differentiated hepatocellular carcinomas without fatty changes developing within them. include component="page" wikiName="islaslab" page="HTML_p_indent" Microscopic examination of hepatic carcinomas showed that they compressed neighboring normal hepatocytes and that they consisted of eosinophilic cells with mitotic features (24). include component="page" wikiName="islaslab" page="HTML_p_indent" In C21 and C49 transgenic mice between the ages of 16 and 19 months, 7 out of 27 (25.9%) and 4 out of 13 (30.8%) mice, respectively, developed hepatocellular carcinoma. These incidences of hepatocellular carcinoma was significantly higher in male transgenic mice compared to their male non-transgenic mice counterparts ( // P // <0.01 and // P // <0.05, respectively). The low frequency of hepatocarcinogenesis in female transgenic mice is consistent with the epidemiological data that men chronically infected with hepatitis C are more likely to develop hepatocellular carcinoma than woman who are similarly infected (24). include component="page" wikiName="islaslab" page="HTML_p_indent" To ensure that the HCV core protein was indeed a contributing factor for the development of hepatocellular carcinoma in transgenic mice compared to non-transgenic littermates, researchers compared livers of HCV __ core __ transgenic mice // versus // transgenic mice expressing the HCV __ envelope __ protein under the same hepatitis B virus regulatory region. include component="page" wikiName="islaslab" page="HTML_p_indent" HCV envelope gene transgenic mice did not develop hepatic adenoma or hepatocellular carcinoma during their entire lifespan of >24 months (24).

__ // Localization of HCV Core Protein in Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" To determine the cellular location of HCV core protein, researchers used immunofluorescence. Electron microscopy comparing HCV core transgenic mice to non-transgenic controls showed accumulation of the core protein in the nuclei and mitochondria. include component="page" wikiName="islaslab" page="HTML_p_indent" The localization of the core protein in the nuclei suggests that the HCV core protein may act as a transcriptional regulator, which may lead to cancer. The localization of the core protein in the mitochondria suggests that it may play a role in the disappearance of the mitochondria double membrane, which may lead to steatosis through impairment of fatty acid oxidation in the mitochondria (CITE, SAME AS PAPER).

__ // Conclusion // __ include component="page" wikiName="islaslab" page="HTML_p_indent" In this study, researchers demonstrated that expression of the HCV core gene results in progressive morphological and biochemical changes that ultimately leads to the development of HCC. The results indicate that a chronic state of inflammation from hepatitis C infection is not an absolute pre-requisite for the development of HCC. Moreover, the findings suggest that HCV itself is directly involved in the development of HCC.

include component="page" wikiName="islaslab" page="HTML_p_indent" The HCV-transgenic mice used in each study were significantly different from each other. Comparing the two studies, specifically the mice models, there are several reasons that suggest the transgenic mice used by Lemon et al. may be more physiologically relevant to the effects of hepatitis C in human patients. include component="page" wikiName="islaslab" page="HTML_p_indent" First, although both studies observed steatosis and HCC in transgenic mice, the mice used by Lemon et al. expressed the entire HCV polyprotein while the mice used by Moriya et al. (24) expressed only the core protein, and not the other structural or non-structural proteins. Studies have shown the HCV core protein interacts with the HCV envelope proteins and also one or more non-structural proteins (39), these interactions of which could significantly modulate the effects of HCV // in vivo //. The interplay between various HCV proteins highlights the importance of examining the activities of HCV proteins within the context of the entire HCV polyprotein like Lemon et al. rather than in isolation like Moriya et al. include component="page" wikiName="islaslab" page="HTML_p_indent" A second and more obvious reason as to why Lemon et al.’s mice may be more physiologically relevant is that viruses with RNA encoding the full complement of HCV proteins are more than likely the causative agent in human hepatitis C infection. In other words “normal” HCV and not HCV with only the core protein is probably the infectious agent in human hepatitis C infection. include component="page" wikiName="islaslab" page="HTML_p_indent" Third, the level of core protein was grossly overexpressed in mice described by Moriya et al. and easily detectable by Western blot (24). In contrast, the expression of HCV proteins in the FL-N/35 lineage mice used by Lemon et al. was not detectable in immunoblots, and only detectable by reverse transcriptase polymerase chain reaction (RT-PCR) (15). In the majority of patients with chronic hepatitis C infection, viral proteins are typically not present at detectable amounts. Furthermore, in most patients, RT-PCR, the same method used by Lemon et al., is necessary for detection of the low levels of HCV RNA transcripts. Therefore, Lemon et al.’s mice express viral proteins at levels similar to those seen in many infected humans. include component="page" wikiName="islaslab" page="HTML_p_indent" There were many differences in rates of steatosis and HCC development in transgenic mice used by Lemon et al. compared to transgenic mice used by Moriya et al. Despite the absence of detectable levels of HCV protein expression in Lemon et al.’s mice, HCC occurred in FL-N/35 mice as young as age 13 months (15). In contrast, Moriya et al.’s HCV core transgenic mice did not develop HCC until age 16 months (24). However, steatosis developed in Moriya et al.’s HCV core transgenic mice by age 6 months and was frequently seen in females (24). In contrast, steatosis rarely occurred in Lemon et al.’s FL-N/35 transgenic mice before age 10 months and was not see in females (15). The fact that Moriya et al.’s mice developed steatosis faster than Lemon et al.’s mice is most likely due to the significantly increased levels of core protein expression in mice studied by Moriya et al. At the same time, a delayed incidence of HCC with a quicker incidence of steatosis in Moriya et al.’s mice, suggests that there is no link between steatosis and HCC. It also indicated that HCV proteins other than core (other structural and non-structural HCV proteins) may accelerate the process of hepatocarcinogenesis, similar to that observed by Lemon et al. include component="page" wikiName="islaslab" page="HTML_p_indent" Based on the reasons laid out above, the mice studied by Lemon et al. most likely mimics the effects of HCV in humans better than the mice studied by Moriya et al. Regardless of their differences, both studies provide strong evidence that hepatitis C infection contributes to the development of hepatocellular carcinoma in mice.
 * Comparison of Lemon et al.** (15) **and Moriya et al.** (24)

** How does HCV cause Hepatocellular carcinoma? **

include component="page" wikiName="islaslab" page="HTML_p_indent" Although the precise mechanism of hepatocarcinogenesis in HCV is still undefined, we propose three possible modes as to how HCV causes HCC (29): **1. HCV causes chronic inflammation in hepatocytes** include component="page" wikiName="islaslab" page="HTML_p_indent"

** 2. HCV RNA polymerase affects hepatocellular tumor suppressor proteins ** include component="page" wikiName="islaslab" page="HTML_p_indent" The interaction between the HCV RNA polymerase and the tumor suppressor protein, Rb, leads to the disruption of mechanisms that regulate cell-cycle progression, which overtime leads to hepatocellular carcinoma.

** 3. HCV core protein causes o ****xidative stress** include component="page" wikiName="islaslab" page="HTML_p_indent" Studies show that expression of the HCV core protein in mice, leads to oxidative stress that results in the accumulation of genetic damage, which overtime leads to hepatocellular carcinoma.

** 1. HCV causes chronic inflammation in hepatocytes **
** NF-kB and STAT 3- key players in liver inflammation and cancer **(30) Guobin He, Michael Karin [CELL RESEARCH 2011, 21:159-168]

__ //Introduction// __ include component="page" wikiName="islaslab" page="HTML_p_indent" Inflammation and cancer and closely linked. Inflammatory cells are recruited into the tumor due the the "necrotic cell death in the core of rapidly growing tumor mass due to lack of oxygen and nutrients." The continual death of cells and inflammatory cell infiltration produces a high number of cytokines, chemokines, and growth factors that promote cell proliferation. Furthermore, oxygen and nitrogen species created by inflammatory cells can damage host DNA leading the the activation of oncogenes, inactivation of tumor suppressing genes, or epigenetic effects.

__ //Background// __ include component="page" wikiName="islaslab" page="HTML_p_indent" __ // NF-kB Background // __ include component="page" wikiName="islaslab" page="HTML_p_indent" __ //STAT3 Background// __ include component="page" wikiName="islaslab" page="HTML_p_indent" __ //IKK/NF-kB and STAT3 signalling pathway in HCC development// __ include component="page" wikiName="islaslab" page="HTML_p_indent" __ //Implications// __
 * Chronic HCV infection causes hepatocyte death and recruitment of inflammatory cells. Over time, continual cell death and liver repair leads to severe liver cirrhosis.
 * The IKKb-dependent classical NF-kB pathway is an important signaling pathways in the injury-inflammation response. It is a regulator of cell survival, immunity, and inflammation.
 * In addition to this pathway, STAT 3 is a transcriptional factor involved HCV induced HCC. It plays a role in inflammation and is critical for liver regeneration and chemically induced HCC.
 * NF-kB is the name for a group of dimeric transcription factors that promotes cell proliferation and survival
 * In non-stimulated cells, NF-kB dimers are bound to inhibitory IkB proteins.
 * When there is a pro-inflammatory signal (tumor necrosis factor (TNF), cytokines etc.), the IkB kinase complex (IKK), phosphorylates the IkB protein, and activates the NF-kB dimer.
 * Downstream products promote cell survival and proliferation.
 * Belongs to the Signal Transducer and Activation of Transcription protein family.
 * STAT3 is inactive in unstimulated cells but is activated by cytokines and growth factors (includes hepatocyte growth factor)
 * When cytokines or growth factors bind to cell receptor, JAK2 is mediates the phosphorylation of STAT3. STAT 3 dimerizes and enters the nucleus where it binds to DNA.
 * There are negative feedback loops (phophatases, suppressor of cytokine signalling proteins, etc.) that regulate STAT3. However, STAT3 is constitutively active in many cancer cells.
 * Under low grade inflammation, hepatocytes communicate to Kupffer cells via the IKK/NF-kB pathway. The pathway is meant to lead to the destruction of infected cells and the proliferation of healthy cells. Cytokines and chemokines are released.
 * In response to this signal, Kupffer cells initiate inflammatory response to repair injuries.
 * IL-6, a major STAT3 activator, is also produced by the IKK/NF-kB pathway. IL-6 affect the original hepatocytes leading to cell survival and proliferation
 * Researchers knocked out STAT3 gene in mice and found a reduction in HCC development. Results show that STAT3 is an oncogenic transcription factor and is a possible target for cancer therapy.
 * Although the IKK/NF-kB pathway is intended to promote cell survival and proliferation after damage, it can play a tumor-promoting role by protecting tumor cells from death.
 * The liver's mechanism for protecting itself from damage often does the opposite. In cases of chronic inflammation (i.e. HCV), hepatocytes activate the IKK/NF-kB pathway//.// Even though this is intended to destroy infected cells and promote growth in healthy cells, tumor cells are often protected and allowed to proliferate.
 * The release of IL-6, a pro-inflammatory cytokine, by the IKK/NF-kb pathway activate STAT3 and maintains a inflammatory microenvironment.

include component="page" wikiName="islaslab" page="HTML_p_indent" include component="page" wikiName="islaslab" page="HTML_p_indent"

** 2. HCV RNA polymerase affects hepatocellular tumor suppressor proteins **
** Down-regulation of the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA polymerase ** (31) Tsubasa Munakata, Mitsuyasu Nakamura, Yuqiong Liang, Kui Li, and Stanley M. Lemon [PNAS 2005, 102, 18159-18164]

**// Aim: //** Researchers examined the interaction between hepatitis C virus RNA-dependent RNA polymerase (NS5B) and the tumor suppressor protein, retinoblastoma (Rb). **// Methods: //** Researchers created two cell lines, C-5B/2-3 and C-5B/2-3c, __with__ expression of RNA encoding the HCV RNA polymerase and __without__ expression of the HCV RNA polymerase, respectively. Cells with the HCV RNA polymerase were compared to cured C-5B/2-3c cells in which the HCV RNA polymerase had been eliminated by treatment with interferon. **// Results: //** Cells with the HCV RNA polymerase had decreased Rb abundance. When C-5B/2-3c cells were treated with interferon, C-5B/2-3c cells exhibited restored Rb abundance – indicating the HCV RNA polymerase is responsible for the negative regulation of Rb. The HCV RNA polymerase forms a complex with Rb, targeting the tumor suppressor protein for degradation, thereby resulting in decreased Rb abundance, activation of the E2F/DP transcription factor, and cell proliferation. The HCV RNA polymerase contains a conserved Leu-x-Cys/Asn-x-Asp motif that is homologous to Rb-binding domains in the oncoproteins of other DNA viruses. **// Conclusion: //** The interaction between the HCV RNA polymerase and Rb, and the subsequent disruption in cell-cycle progression, reveals a mechanism by which HCV may promote hepatocellular carcinoma.

__// Definitions //__ ** Retinoblastoma (Rb) ** and **E2F/DP** – Rb is a tumor suppressor protein. E2F/DP is a transcription factor that regulates genes encoding proteins that are necessary for S-phase of the cell cycle. Rb and E2F/DP play a major role in controlling the G1 to S-phase transition in the cell cycle. When Rb is present, E2F/DP is suppressed, and cells do not transition from G1 to S-phase/ do __not__ proliferate. When Rb is absent or at decreased levels, E2F/DP is activated, and cells transition from G1 to S-phase/ proliferate.

** NS5B ** – The HCV RNA-dependent RNA polymerase. NS5B is essential for HCV RNA replication that occurs in the cytoplasm. NS5B is one of the 7 non-structural HCV proteins.

__// Rb is Negatively Regulated in Cells Supporting HCV RNA Replication //__ include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers compared Rb abundance in cells containing the HCV RNA polymerase to cells in which the HCV RNA polymerase had been eliminated by treatment with interferon. They found that Rb levels in C-5B/2-3, the HCV RNA polymerase-containing cell line, was reduced compared with that in the cognate-cured cell line, C-5B/2-3c. Rb is a member of a family of three closely related proteins that include p107 and p130. The expression of p107 and p130 is regulated independently of Rb. include component="page" wikiName="islaslab" page="HTML_p_indent" To ensure the HCV RNA polymerase affects Rb specifically, researchers measured Rb, p107, and p130 abundance in cells containing the HCV RNA polymerase. In contrast to Rb, levels of p107 and p130 in the HCV RNA polymerase-containing cell line was not reduced. include component="page" wikiName="islaslab" page="HTML_p_indent" To determine whether reduction in Rb abundance is transcriptionally regulated, researchers used Northern blot analysis of Rb mRNA. They found that Rb mRNA levels in C-5B/2-3 cells were not reduced, but rather upregulated. The upregulation in Rb mRNA levels despite the decrease in Rb protein levels in the HCV RNA polymerase-containing cells, is due to the fact that Rb gene expression is negatively regulated by the Rb protein product itself. Therefore, low Rb levels in C-5B/2-3 cells causes only weak suppression of Rb gene expression, leading to increased levels of Rb mRNA. include component="page" wikiName="islaslab" page="HTML_p_indent" To determine whether Rb protein stability is affected by the HCV RNA polymerase, researchers used a Pulse-Chase labeling experiment to measure the half-life of Rb. They found that Rb half-life in the cured C-5B/2-3c cell line was ~2 hours, compared to only 1.2 hours in the HCV RNA polymerase C-5B/2-3 cell line. include component="page" wikiName="islaslab" page="HTML_p_indent" Together, the results provide strong evidence that Rb is down-regulated post-transcriptionally by the HCV RNA polymerase.

__// HCV RNA Polymerase (NS5B)-Mediates the Regulation of Rb //__ include component="page" wikiName="islaslab" page="HTML_p_indent" The life cycle of HCV is entirely cytoplasmic. The HCV RNA polymerase is essential for replication of the HCV RNA genome – a process that occurs in the cytoplasm. Rb, however, is predominantly nuclear. To examine the cellular localization of both the HCV RNA polymerase and Rb, researchers used immunofluorescence imaging. They found that Rb in cured C-5B/2-3c cells was primarily localized to the nucleus and on occasion was found in the cytoplasm. In contrast, Rb in C-5B/2-3 cells was reduced to the point of nondetection. HCV RNA polymerase (NS5B) in C-5B/2-3 cells was localized in the cytoplasm. include component="page" wikiName="islaslab" page="HTML_p_indent" The results suggest that the HCV RNA polymerase interacts with Rb and likely targets it for degradation before its transport to the nucleus.

__// HCV RNA Polymerase (NS5B) Active Site Residues Are Involved in Binding and Regulating Rb Abundance //__ include component="page" wikiName="islaslab" page="HTML_p_indent" Rb is a key target of many viral proteins expressed by many DNA tumor viruses, but RNA viruses, like hepatitis C, are not known to express viral proteins that regulate Rb function. Most of the viral proteins that bind to Rb and are expressed by DNA tumor viruses share a common Leu-x-Cys/Asn-x-Asp motif. include component="page" wikiName="islaslab" page="HTML_p_indent" To determine whether the HCV RNA polymerase has a similar binding motif for Rb, researchers sequenced the NS5B gene. They found that NS5B does indeed have a conserved Leu-Val-(Cys/Asn)-Gly-Asp sequence with homology to the Leu-x-Cys/Asn-x-Asp motif in DNA viral proteins. include component="page" wikiName="islaslab" page="HTML_p_indent" Next, to determine whether this Leu-Val-(Cys/Asn)-Gly-Asp motif contributes to the ability of the HCV RNA polymerase to bind and negatively regulate Rb, researchers constructed a series of point mutations, designated D318N/D319N, N316C, N316A, and L314A/N316A. Researchers expressed these modified HCV RNA polymerase proteins in cells and confirmed equivalent expression levels of the mutant proteins and wild-type HCV RNA polymerase proteins. They found that Rb abundance was negatively regulated by both the wild-type HCV RNA polymerase and the mutant HCV RNA Polymerase, N316C. Rb abundance was not significantly changed in L314A/N316A, D318N/D319N, or N316A mutants. include component="page" wikiName="islaslab" page="HTML_p_indent" The results suggest that the Leu-Val-(Cys/Asn)-Gly-Asp motif is essential for down-regulation of Rb in the HCV RNA polymerase-containing cells.

__// E2F/DP-Responsive Genes Are Activated by NS5B Expression //__ include component="page" wikiName="islaslab" page="HTML_p_indent" When Rb is present, E2F/DP is suppressed, and cells do not transition from G1 to S-phase/ do __ not __ proliferate. When Rb is absent or at decreased levels, E2F/DP is activated, and cells transition from G1 to S-phase/ proliferate. Therefore expression of the HCV RNA polymerase and the decreased abundance of Rb that follows, should lead to the activation of E2F/DP and the expression of the genes controlled by E2F/DP. include component="page" wikiName="islaslab" page="HTML_p_indent" To make this determination, researchers measured p107 and MAD2 abundance in cells containing the HCV RNA polymerase. p107 is an Rb family member and is expressed under the control of the E2F/DP transcription factor. MAD2 is a mitotic checkpoint protein also expressed under the control of the E2F/DP transcription factor. They found a 3- to 5-fold increase in both p107 and MAD2 levels in the HCV RNA polymerase-containing cell line compared with that in the cognate-cured cell line. Additionally, when researchers used a cell line containing the HCV RNA polymerase and a mutant Rb that does not bind E2F/DP, they found that p107 and MAD2 expression was not activated. include component="page" wikiName="islaslab" page="HTML_p_indent" The results suggest that the HCV RNA polymerase reverses Rb-mediated repression of E2F/DP transcription factors.

__// NS5B Regulates Cellular Proliferation and S-Phase Entry Through Its interaction with Rb //__ include component="page" wikiName="islaslab" page="HTML_p_indent" To determine the effects of the HCV RNA polymerase on cell-cycle progression, researchers used U2OS osteosarcoma cells that have wild-type p53 and Rb. Researchers confirmed that expression of the HCV RNA polymerase in these cells caused reduced Rb abundance. Researchers also confirmed that expression of the HCV RNA polymerase in these cells caused upregulation of p107, indicating activation of E2F/DP controlled genes. include component="page" wikiName="islaslab" page="HTML_p_indent" U2OS cells containing the HCV RNA polymerase had significantly greater number of cell colonies compared to U2OS control cells without the HCV RNA polymerase. They also found that expression of the HCV RNA polymerase resulted in a significant increase in the fraction of U2OS cells in S-phase. include component="page" wikiName="islaslab" page="HTML_p_indent" The results suggest that the HCV RNA polymerase, downregulates Rb abundance thereby, activating the E2F/DP transcription factor, stimulating the proliferation of U2OS cells, and enhancing the likelihood of malignant transformation.

__// Conclusion //__ include component="page" wikiName="islaslab" page="HTML_p_indent" Compared to cured C-5B/2-3c cells lacking the HCV RNA polymerase, cells containing the HCV RNA polymerase exhibit… include component="page" wikiName="islaslab" page="HTML_p_indent" Ultimately, the data reveals a mechanism by which hepatitis C may promote hepatocellular carcinoma. HCV RNA polymerase targets the tumor suppressor protein, Rb, for destruction, leading to activation of the E2F/DP transcription factor, hepatocyte proliferation and subsequently, the development of hepatocellular carcinoma.
 * ¯ Rb abundance
 * ↑ Rb mRNA abundance
 * Leu-Val-(Cys/Asn)-Gly-Asp motif to bind to Rb
 * ↑ expression of p107 and MAD2
 * ↑ number of cell colonies
 * ↑ fraction of cells in S-phase

** 3. HCV core protein causes oxidative stress **
** Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis ** (32) Kyoji Moriya, Kiyotaka Nakagawa, Tomofumi Santa, Yoshizumi Shintani, Hajime Fujie, Hideyuki Miyoshi, Takeya Tsutsumi, Teruo Miyazawa, Kotaro Ishibashi Toshiharu Horie, Kazuhiro Imai, Toru Todoroki, Satoshi Kimura, and Kazuhiko Koike [CANCER RESEARCH 61, 4365-4370, June 1, 2001]

**// Aim: //** Researchers sought to define the activity of hepatitis C virus core protein by determining whether expression of the core protein alters hepatic oxidant/anti-oxidant status, in the absence of inflammation. **// Methods: //** Researchers used mice from the C57NL/6 strain to create a transgenic lineage with expression of RNA encoding the core HCV protein. Transgenic mice were compared to non-transgenic littermates for hepatic oxidant/anti-oxidant status. **// Results: //** The abundance of the viral protein was similar to core protein levels found in the liver of chronic hepatitis C patients. There was no inflammation in the livers of transgenic mice. Mice expressing the transgene developed age-dependent increase in lipid peroxidation, as revealed by the determination of hydroperoxide levels. Antioxidant systems were also activated in accordance with the extent of lipid peroxidation. Mice expressing the transgene had significantly higher incidence of mitochondrial DNA deletion as well as aberrations in mitochondrial morphology. Transgenic mice fed alcohol had significantly increased hydroperodxide levels. However, hydroperoxide levels in CCl4-treated transgenic mice compared to CCl4-treated non-transgenic mice was not significantly increased. Increased hydroperoxide levels, mitochondrial DNA deletions, and disruption of mitochondria morphology was not seen in non-transgenic mice. **// Conclusion: //** Constitutive expression of the HCV core protein induces reactive oxygen species in the liver in the absence of inflammation, which may increase the risk for cancer.

__ //Definitions// __ ** Carbon tetrachloride (CCl4) ** – A well-investigated chemical that has cytotoxic effects. A study has shown that CCl4-treated mice exhibited hepatic pathological changes that included necrosis, inflammation, and fibrosis, along with increased serum alanine aminotransferase activities (33).

** Catalase ** – An anti-oxidant, and an enzyme common in all organisms exposed to oxygen. It catalyzes the conversion of hydrogen peroxide (H2O2), a harmful reactive oxygen specie, to water (H2O) and oxygen (O2) (34).

** Glutathione – ** An anti-oxidant that plays an important role in cellular defense against oxidative stress. It regulates the intracellular concentration of reactive oxygen species via a reaction catalyzed by the enzyme, glutathione peroxidase (GPx). Glutathione exists in two states: reduced glutathione (GSH) and oxidized glutathione disulfide ( //GSSG// ) (32).

** HCV core protein ** -- The core protein makes up the viral capsid. It is approximately 22kDa and is encoded by the 5’-proximal region of the HCV genome. The gene that encodes the core protein is highly conserved amongst various HCV strains, suggesting its importance in HCV pathogenesis (35).

** Reactive oxygen species (ROS) ** -- Endogenous oxidants that are produced by multiple intracellular pathways, one of which is aerobic respiration. They include radicals (superoxide, hydroxyl, and peroxyl) as well as a small subset of non-radicals (singlet-oxygen, hydrogen peroxide) that can be easily converted into radicals (32). Reactive oxygen species can induce genetic mutations and chromosomal alterations and are therefore, considered carcinogenic. Studies have also shown that reactive oxygen species can act as intra- and intercellular secondary messengers, thereby modulating various aspects of cellular functions, including those important in tumorigenesis -- proliferation, apoptosis, and gene expression (36).

** Lipid peroxidation ** -- The process by which free radicals steal electrons from the lipids that make up the cell membrane, resulting in an even greater increase in production of free radicals, as well as cellular and tissue damage (37). Studies have shown that lipid peroxidation is an accurate determinant of the extent of excessive reactive oxygen species (32,).

** Phosphatidylcholine hydroperoxides ( ** ** PCOOH) ** and **Phosphatidylet** **hanolamine hydroperoxides (PEOOH) ** – PCOOH and PEOOH are primary oxidation products of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. PCOOH and PEOOH are markedly accumulated in blood plasma of animals experiencing oxidative stress. PCOOH and PEOOH are reactive oxygen species, classified as hydroperoxides. Levels of PCOOH and PEOOH are an accurate determinant of extent of lipid peroxidation (32).

__ // Production of Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" To ensure high-level expression in the liver without interfering with normal mouse development, researchers used a plasmid containing the hepatitis B virus (HBV) enhancer/promoter with a downstream intron/polyadenylation cassette. Complementary DNA corresponding to the HCV core protein was inserted into the plasmid in between the HBV enhancer/promoter and intron/polyadenylation cassette. Therefore, the HCV core gene was under the transcriptional control of the HBV regulatory region. include component="page" wikiName="islaslab" page="HTML_p_indent" The resulting plasmid was inserted into mouse embryos from the C57BL/6 strain. The mice were cared for according to institutional guidelines, fed an ordinary, sterile chow diet, and maintained in pathogenic-fee conditions. Non-transgenic littermates of the transgenic mice were used as controls (32). include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers ran a two-by-two factorial design: non-transgenic // versus // transgenic mice and young (3-12 months old) // versus // old (>16 months old) mice.

__ // Lipid Peroxidation in HCV Core Gene Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" To investigate oxidative damage to membrane lipids, researchers analyzed lipid peroxidation by measuring the levels of two reactive oxygen species, phosphatidylcholine hydroperoxides ( PCOOH) and phosphatidylet hanolamine hydroperoxides ( PEOOH) in the livers of HCV core transgenic and non-transgenic mice. include component="page" wikiName="islaslab" page="HTML_p_indent" Results are shown in the table below: __ // Is the Transgenic Mouse Model Physiologically Relevant? // __ include component="page" wikiName="islaslab" page="HTML_p_indent" To confirm that the transgenic mice model was appropriate for studying the liver disease spectrum in human HCV infection, researchers compared core protein levels in transgenic mouse livers to those in liver samples from patients with chronic hepatitis C. Expression of HCV core protein in the transgenic mice was similar to core protein levels found in the livers of humans. include component="page" wikiName="islaslab" page="HTML_p_indent" Furthermore, to ensure that the plasmid used in their study was not a contributing factor for the difference seen in hydroperoxide levels between transgenic mice compared to non-transgenic littermates, researchers compared PCOOH levels in the livers of 16-month-old HCV __ core __ transgenic mice // versus // transgenic mice expressing the HCV __ envelope __ protein under the same hepatitis B virus regulatory region. include component="page" wikiName="islaslab" page="HTML_p_indent" Results are shown in the table below: include component="page" wikiName="islaslab" page="HTML_p_indent" The results indicate that not the regulatory feature of HCV core protein expression driven by the hepatitis B virus promoter/enhancer used in this study, but the expressed core protein itself is responsible for the excessive production of hydroperoxides in the mouse liver (32).
 * |||| ** Hydroperoxide ** ||
 * ^  || ** PCOOH ** || ** PEOOH ** ||
 * ** HCV core transgenic, Young **// versus //** Non-transgenic ** (control), **Young** ||  No significant difference  (0.89 ± 0.16 //versus// 0.83 ± 0.08nmol/g protein; //P//=0.46)   ||  No significant difference  (0.43 ± 0.11 //versus// 0.42 ± 0.14nmol/g protein; //P//=0.90)   ||
 * ** HCV core transgenic, Old **// versus //** Non-transgenic ** (control), **Old** ||   Significantly __higher__ in HCV-core Transgenic, Old mice //versus// Non-transgenic, Old mice   (2.30 ± 0.42 //versus// 0.83 ± 0.19nmol/g protein; //P//<0.01)   ||  Significantly __higher__ in HCV-core Transgenic, Old mice //versus// Non-transgenic, Old mice  (1.04 ± 0.32 //versus// 0.42 ± 0.15nmol/g protein; //P//<0.05)   ||
 * || ** PCOOH ** ||
 * ** HCV core transgenic, 16-month-old **// versus //** HCV envelope transgenic, 16-month-old ** || No significant difference  (0.83 ± 0.19 //versus// 0.79 ± 0.27nmol/g protein; //P//=0.82)   ||

__ // Antioxidative State in HCV Core Gene Transgenic Mice // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Measurement of the two reactive oxygen species or hydroperoxides above (PCOOH and PEOOH), is not complete without the measurement of endogenous defense systems that protect against reactive oxygen species. In the assessment of reactive oxygen species, the balance between generation and elimination is more important than the measurement of any single component alone (32). include component="page" wikiName="islaslab" page="HTML_p_indent" To investigate the response of anti-oxidant systems, researchers measured the levels of: 1) catalase activity, 2) total glutathione (glutathione + glutathione disulfide) //(//GSH + GSSG), and 3) ratio of glutathione/total glutathione [GSH: (GSH+GSSG)] in the livers of __young__ (6-month-old) mice .  include component="page" wikiName="islaslab" page="HTML_p_indent" Results are shown in the table below:   include component="page" wikiName="islaslab" page="HTML_p_indent" The results suggest that reactive oxygen species are overproduced in the livers of HCV core transgenic mice even at a __ young __ age, but are promptly scavenged by activated antioxidants, such as catalase and the GSH system.
 * || ** Catalase activity ** || ** GSH+GSSG ** || ** GSH: (GSH+GSSG) ** ||
 * ** HCV core transgenic, 6-month-old **// versus //** Non-transgenic ** (control), **6-month-old** ||  Significantly __higher__ in HCV-core transgenic mice //verus// Non-transgenic mice  (149.8 ± 22.1 //versus// 104.2 ± 18.4units/100mg; //P//<0.01)   ||  Significantly __lower__ in HCV-core transgenic mice //versus// Non-transgenic mice  (2.62 ± 0.59 //versus// 3.89 ± 0.58 m mol/g; //P//<0.01)   ||  Significantly __lower__ in HCV-core transgenic mice //versus// Non-transgenic mice  (0.832 ± 0.051 //versus// 3.89 ± 0.016; //P//<0.05)   ||

__ // Mitochondrial Morphology and DNA Damage // __ include component="page" wikiName="islaslab" page="HTML_p_indent" To investigate the effects of oxidative stress on genome stability, researchers measured the incidence of mitochondrial DNA (mtDNA) deletion in hepatocytes of HCV core transgenic and non-transgenic mice. include component="page" wikiName="islaslab" page="HTML_p_indent" Researchers used polymerase chain reaction to screen for a large 3821-bp deletion associated with direct sequence repeats normally present in aging mice (38). include component="page" wikiName="islaslab" page="HTML_p_indent" Results are shown in the table below: include component="page" wikiName="islaslab" page="HTML_p_indent" The results suggest that increased production of reactive oxygen species in the livers of HCV core transgenic mice damages mtDNA. The weak detection of a band indicating the 3821-bp deletion in the 19-month-old non-transgenic control mice may be the result of aging (32). include component="page" wikiName="islaslab" page="HTML_p_indent" To further investigate the effects of oxidative stress on intracellular organelles, researchers observed the degeneration or swelling of the mitochondria using electron microscopy in HCV core transgenic and non-transgenic mice. include component="page" wikiName="islaslab" page="HTML_p_indent" Disruption of the mitochondrial double membrane was observed in hepatocytes of 3-month-old HCV core transgenic mice, but absent in non-transgenic mice of the same age. Additionally, scattered residual bodies or lipofuscin granules and secondary lysosomes carrying various cytoplasmic organelles were observed in hepatocytes from the livers of HCV core transgenic mice >12-month-old, but absent in non-transgenic control mice. Scattered residual bodies, lipofuscin granules, and secondary lysosomes carrying various cytoplasmic organelles are all structures found in senescent cells and considered morphological signs of damage induced by reactive oxygen species (32).
 * |||||| ** Age (months) ** ||
 * ^  || ** 2 ** || ** 9 ** || ** 19 ** ||
 * ** HCV core transgenic ** || deletion detectable || deletion detectable || deletion prominent ||
 * ** Non-transgenic ** (control) || deletion __not__ detectable || deletion __not__ detectable || deletion weak ||

__ // Effects of Alcohol and Inflammation of Lipid Peroxidation // __ include component="page" wikiName="islaslab" page="HTML_p_indent" In humans with chronic hepatitis C infection, alcohol intake is recognized as a synergistic factor for the acceleration of hepatocarcinogenesis (32). To investigate this, researchers fed alcohol to 6-month-old HCV core transgenic and non-transgenic mice and examined whether alcohol enhanced the effect of the HCV core protein on hydroperoxide levels in the liver. Mice were fed 5% ethanol for 3 weeks. include component="page" wikiName="islaslab" page="HTML_p_indent" Results are shown in the table below: include component="page" wikiName="islaslab" page="HTML_p_indent" In humans with chronic hepatitis C infection, inflammation is recognized as a synergistic factor for the acceleration of hepatocarcinogenesis (32). To investigate this, researchers injected CCl 4 into 6-month-old HCV core transgenic and non-transgenic mice and examined whether inflammation enhanced the effect of the HCV core protein on hydroperoxide levels in the liver. Mice were given 0.5ml of CCl 4 per kg body weight. include component="page" wikiName="islaslab" page="HTML_p_indent" Results are shown in the table below: include component="page" wikiName="islaslab" page="HTML_p_indent" Although CCl 4 administration increased PCOOH levels in mice, and levels were higher in HCV-core transgenic mice than in non-transgenic control mice, the difference was not statistically significant (32).
 * || ** PCOOH ** ||
 * ** HCV core transgenic, 6-month-old, fed 5% ethanol for 3 weeks **// versus //** Non-transgenic ** (control)**, 6-month-old, fed 5% ethanol for 3 weeks** ||  200% higher in HCV core transgenic mice //versus// Non-transgenic mice  (3.11 ± 0.54 //versus// 1.02 ± 0.27nmol/g protein; //P//<0.01)   ||
 * ** HCV core transgenic, 6-month-old, fed 5% ethanol for 3 weeks **// versus //** HCV core transgenic, 6-month-old, __not__ fed 5% ethanol for 3 weeks ** || 200% higher in HCV-core transgenic mice fed ethanol //versus// HCV core transgenic mice __not__ fed ethanol  (3.11 ± 0.54 //versus// 0.88 ± 0.28nmol/g protein; //P//<0.01)   ||
 * || ** PCOOH ** ||
 * ** HCV core transgenic, 6-month-old, CCl4 **// versus //** Non-transgenic ** (control)**, 6-month-old, CCl4** ||  No significant difference  (2.96 ± 1.03 //versus// 2.07 ± 0.48nmol/g protein; //P//=0.12)   ||

__ // Conclusion // __ include component="page" wikiName="islaslab" page="HTML_p_indent" Compared to old (>16 month) non-transgenic control mice, old transgenic mice expressing the HCV core protein exhibit…
 * ↑ PCOOH and PEOOH (indicating ↑ oxidative stress)
 * ↑ response in anti-oxidant systems (catalase and glutathione peroxidase)
 * ↑ mitochondrial DNA deletion
 * hepatocellular carcinoma

include component="page" wikiName="islaslab" page="HTML_p_indent" It is important to note that… include component="page" wikiName="islaslab" page="HTML_p_indent" Ultimately, the data reveals a mechanism by which hepatitis C may promote hepatocellular carcinoma. HCV core protein causes increased oxidative stress, leading to accumulation of genetic damage in hepatocytes and subsequently, hepatocellular carcinoma.
 * young (3-9 months) mice did __not__ develop hepatocellular carcinoma, regardless if they were transgenic core or non-transgenic mice
 * alcohol caused significantly increased PCOOH levels in 6-month-old transgenic core mice compared to non-transgenic mice
 * CCl4-induced inflammation did __ not __ significantly alter PCOOH levels in 6-month-old transgenic core mice compared to non-transgenic mice
 * all of these results were seen in the absence of inflammation – indicating that the HCV core protein // per se //, and not chronic inflammation, is sufficient to induce oxidative stress in mice liver

** Conclusion **
include component="page" wikiName="islaslab" page="HTML_p_indent" Based on the three academic studies examined, we believe the two most direct mechanisms of hepatitis C-indeced HCC is a combination of the second and third mechanisms, the HCV RNA polymerases’ affect on hepatocellular Rb, a tumor suppressor protein, and induction of oxidative stress by the HCV core protein, respectively. include component="page" wikiName="islaslab" page="HTML_p_indent" The first mechanism, chronic inflammation by HCV, is most likely not a ** direct ** mechanism of hepatocarcinogenesis. Given that only 1-5% of individuals with chronic HCV develop HCC, it is unlikely that inflammation directly leads to cancer. But that’s not to say that it does not contribute to the development of hepatocellular carcinoma. We believe inflammation can enhance the effects of the second and third mechanisms. Because a part of the inflammation response signals for cell proliferation, it is possible for the proliferation signal to accidentally promote tumor cells. These tumor cells exist in the liver in the first place because of the role of the viral proteins in 1) suppressing Rb, the tumor suppressor protein and 2) generating DNA damage through oxidative stress. Therefore, HCC in the presence of inflammation could be worse or more malignant than HCC in the absence of inflammation. include component="page" wikiName="islaslab" page="HTML_p_indent" Ultimately, preventing hepatitis C infection is the best way to prevent HCC. But until HCV diagnosis improves, it will be important for researchers to dissect the mechanism behind HCV-induced HCC. In understanding the how this virus works, we have the potential to change the prognosis of this disease-- a disease with merely a 14% survival rate. Our research sheds light on how targeted drug therapy may offer part of the solution.

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 * 1) Munakata, Tsubasa, Mitsuyasu Nakamura, Yuqiong Liang, and Stanley M. Lemon. "Down-regulation of the Retinoblastoma Tumor Suppressor by the Hepatitis C Virus NS5B RNA-dependent RNA Polymerase." //Proceedings of the National Academy of Sciences// 102.50 (2005): 18159-8164. Print.
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__// Disease spectrum of hepatitis viruses: //__ __//Composition of HCV://__ __//HCV genes and their gene products://__ __//HCV life cycle://__ __//HCV receptors://__ //__ Anatomy of the liver: __// __//Anatomy of a liver lobule://__ __//HCC://__ __//HCC detection by CT://__ __//Liver biopsy://__ __// Chemoembolization: //__ __//Three mechanisms://__
 * Images **
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