We have identified a novel protein in bone marrow-derived insulin-producing cells. Here we characterize this protein, hereby named islet homeostasis protein (IHoP), in the pancreatic islet.
Detection ...of IHoP mRNA and protein was performed using reverse transcriptase-polymerase chain reaction, immunocytochemistry, and in situ hybridization. Islet homeostasis protein functions were utilizing proliferation, insulin secretion by in vitro assays, and following small interfering RNA protocols for suppression of IHoP.
We found that IHoP did not homolog with known pancreatic hormones. Islet homeostasis protein expression was seen in both bone marrow-derived insulin-producing cells and isolated pancreatic islets. Immunohistochemistry on pancreatic islet revealed that IHoP localized to the glucagon-synthesizing α cells. Inhibition of IHoP by small interfering RNA resulted in the loss of glucagon expression, which induced low blood glucose levels (63-85 mg/dL). Subsequently, cellular apoptosis was observed throughout the islet, including the insulin-producing β cells. Islets of preonset diabetic patients showed normal expression of IHoP and glucagon; however, IHoP was lost upon onset of the disease.
These data suggest that IHoP could be a new functional protein in the islet and may play a role in islet homeostasis.
To date the molecular signals regulating activation, proliferation, and differentiation of hepatic oval cells are not fully understood. The Wnt family is essential in hepatic embryogenesis and ...implicated in hepatic carcinogenesis. This study elucidates novel findings implicating Wnt1 in directing oval cell differentiation during the rat 2-acetylaminofluorene (2AAF) and ⅔ partial hepatectomy (PHx) liver regeneration model. Proteins of Wnt family members were predominantly localized in pericentral hepatocytes during liver injury, oval cell activation, and hepatocyte regeneration. In addition, Wnt message increased coinciding with the rise in oval cell number, whereas protein levels peaked immediately after the height of oval cell proliferation. Immunohistochemical analysis demonstrated nuclear translocation of β-catenin within oval cells throughout the 2AAF/PHx protocol. Furthermore, RNA interference was used in vivo to confirm the physiological requirement of Wnt1 during the oval cell induction. Ultimately, inhibition of Wnt1 resulted in failure of oval cells to differentiate into hepatocytes and alternatively induced atypical ductular hyperplasia. Taken together, these data indicate that in vivo exposure to Wnt1 shRNA inhibited rat oval cell liver regeneration. In the absence of Wnt1 signaling, oval cells failed to differentiate into hepatocytes and underwent atypical ductular hyperplasia, exhibiting epithelial metaplasia and mucin production. Furthermore, changes in Wnt1 levels are required for the efficient regeneration of the liver by oval cells during massive hepatic injury.
The cellular origin of tumors remains as one of the unanswered, fundamental questions of cancer biology. The notion that tumors may arise from tissue stem cells is supported by phenotypic ...similarities between these two cell types, such as proliferative potential and expression of onco-fetal proteins. Liver stem cells, or oval cells, have been put forth as a possible target for hepatocarcinogens. Genetically modified and in vitro transformed oval cells have been shown to form tumors in transplantation to animals. Chemical carcinogenesis models in the liver demonstrate varying degrees of oval cell proliferation. There is also preliminary evidence that hepatocellular carcinoma may maintain a bipotential phenotype consistent with an oval cell origin. Whereas definitive proof of an oval cell origin of hepatocellular has yet to be presented, the current circumstantial evidence justifies continued research on this subject.
The overarching goal of this work is to design and develop organic self-assembled monolayer (SAM) based cell culture platforms (SCCPs) to provide an appropriate microenvironment that promotes cell ...attachment, growth and functionality with the ultimate goal of developing cell-based bioreactors for rapid drug toxicity screening. We describe proliferation of a model cell line, HepG2, and primary rat hepatocytes for culture periods up to 3 days, on a model peptide, Gastrin Releasing Peptide (GRP), and a Growth Factor, Epidermal Growth Factor (EGF), that is covalently coupled to the amine end group of 3-aminopropyl triethoxysilane (APTES)-Self-Assembled Monolayer (SAM) on conducting indium tin oxide (ITO) substrates. The scaffolds were characterized using contact angle and surface-IR techniques. The cells, HepG2 or primary hepatocytes, were cultured on GRP- and EGF-immobilized scaffolds for 24, 48, and 72 hrs. MTT (3-methyltetrazoliumbromide) cell proliferation and Lactose Dehydrogenase (LDH) cytotoxicity assays were performed on HepG2 cells and primary hepatocytes cultures on peptide and growth factor modified scaffolds to evaluate cellular heath and toxicity. Cell proliferation analysis indicated that the HepG2 cells cultured on EGF- and GRP-immobilized substrates showed increased cell viability with time from 24 to 72 hours. The LDH production after 48 hours was reduced in cells cultured on GRP and EGF immobilized surfaces in comparison to the cells cultured on ITO and ITO-APTES substrates. The results overall showed that cell viability increased and cytotoxicity decreased for both HepG2 cells and primary hepatocytes cultured on GRP- and EGF-modified scaffolds. Furthermore, the increase of cell viability with reduced cytotoxicity is extended to 72 hrs with good biocompatibility.
Hepatocellular carcinoma (HCC) was fifth in incidence among all cancers worldwide in the year 2000 (1). One of the major risk factors associated with the development of HCC is dietary exposure to the ...fungal metabolite and hepatocarcinogen aflatoxin B1 (AFB1). This molecule requires metabolic activation to an ultimate carcinogenic form in order to assert its maximum carcinogenic potential. Metabolism of AFB1 occurs in the liver where it undergoes an initial enzymatic oxidation, mainly by members of the cytochrome P450 superfamily, to the electrophilic intermediate AFB1-8,9-epoxide. The epoxide functional group is modified by glutathione-s-transferase (GST) mediated conjugation with glutathione (GSH) forming a water-soluble product (2,3). Unmodified AFB1-epoxide has the potential to react with any cellular macromolecule, including DNA, forming a covalently bound adduct. AFB1-DNA adduction is believed to be the source of point mutations that initiate AFB1 induced HCC (4,5). In the liver, GST level is the principal regulator of AFB1 sensitivity and is responsible for interspecies variations in AFB1 susceptibility (3,6–8). Previous studies in our laboratory and others demonstrate an increased sensitivity to AFB1 in mice with hyperproliferative livers (9–14). There is also a strong synergistic relationship between hepatitis B virus (HBV) infection and dietary AFB1 exposure (10,15–18). We hypothesize that hepatic GST is decreased during periods of hepatocyte proliferation rendering the liver more susceptible to AFB1-DNA adduction. The increased AFB1-DNA adduct load results in a greater number of tumor initiating point mutations. Herein, we examine hepatic GST levels and AFB1-DNA adduct formation in the context of proliferating liver associated with several mouse models of hepatocyte proliferation. We first look at two models for a rapidly proliferating liver, neonatal mouse liver and the proliferative response to 2/3 partial hepatectomy. We then examine two models of chronic hepatocyte proliferation, p53 deficient and hepatitis B surface antigen (HBsAg) expressing mice. We conclude that rapid hepatocyte proliferation associated with neonatal liver or 2/3 partial hepatectomy greatly reduces hepatic GST levels and increases AFB1-DNA adduct formation upon exposure to AFB1. This likely contributes to the increased hepatocarcinogenic potential of AFB1 reported in these systems. Chronic hepatocyte proliferation associated with loss of p53 also decreases hepatic GST and increase AFB1-DNA adduct formation whereas these parameters appear to be unaffected in HBsAg expressing mice.
It has been hypothesized that cancer stem cells result from the initiation of normal tissue stem cells by mutagens. These cells give rise to a population of growth and differentiation dysregulated ...transient amplifying cells that represent the bulk of the tumor. Fifty years of research has provided a relatively large knowledge base on adult liver stem cells termed “oval cells” in rodents and hepatic progenitor cells in humans. Despite this fact, information regarding liver cancer stem cells remains scarce. Abundant circumstantial evidence suggests that bipotential liver progenitor cells may act as targets for carcinogens, giving rise to liver cancer. Evidence is also beginning to indicate that these mutated progenitor cells, or their derivatives, may act as cancer stem cells. These cells maintain themselves through self-renewal and give rise to the transient amplifying cells that comprise a majority of the liver tumor volume. Putative liver cancer stem cells likely escape chemotherapeutic treatment, both by limiting time in the cell cycle, and by up-regulating membrane transporters. It is also likely that the ability to establish metastasis is limited to the liver cancer stem cell population. Because regrowth of tumors following unsuccessful cytoreductive therapy is mediated by tumor stem cells, careful consideration must be paid to this cell population when developing future liver cancer therapies. Several potential markers for the identification of liver cancer stem cells are currently under investigation. CD133 and CD90 show particular promise as discriminators of human liver cancer stem cells. These markers are being used to help unravel the mystery of tumor reoccurrence following treatment of the original tumor by surgery and cytoreductive drug/radio therapy, and may someday lead to a true cure for this ominous form of cancer. The following chapter presents evidence for both the stem cell origin of liver tumors, and the presence of altered stem cells within the tumors themselves.
Although the extracellular matrix (ECM) is only a minor constituent of the liver, it has a fundamental role, providing a structural framework and maintaining the differentiated state of all liver ...resident cells. The important role of the hepatic ECM was demonstrated in vitro, indicating that hepatocyte phenotype is dependent on the nature of the ECM upon which it is cultured 1. The ECM modulates repair in many tissues, including the liver. Therefore, defining the ECM distribution in the normal liver, its phenotypic expression in various regenerative states, and the cells responsible for its synthesis in vivo, is an important step in understanding its role in homeostasis and repair. In this chapter, we sought to provide a thorough description of the liver ECM and its intrinsic association with the cells that constitute the hepatic tissue, as well as their location (zonation) and associations in order to maintain hepatic function. Hepatic mechanobiology and ECM contribution is also described. Furthermore, we list important breakthroughs and shortcomings of current human liver cell therapies and how new approaches involving matrix carriers could impact the field.
PDF
