Participation of liver stem cells in cholangiocarcinogenesis after aflatoxin B1 exposure of glutathione S-transferase A3 knockout mice.

Aflatoxin B1, arguably the most potent human carcinogen, induces liver cancer in humans, rats, trout, ducks, and so on, but adult mice are totally resistant. This resistance is because of a detoxifying enzyme, mouse glutathione S-transferase A3, which binds to and inactivates aflatoxin B1 epoxide, preventing the epoxide from binding to DNA and causing mutations. Glutathione S-transferase A3 or its analog has not been detected in any of the sensitive species, including humans. The generation of a glutathione S-transferase A3 knockout (represented as KO or -/-) mice has allowed us to study the induction of liver cancer in mice by aflatoxin B1. In contrast to the induction of hepatocellular carcinomas in other species, aflatoxin B1 induces cholangiocarcinomas in GSTA3-/- mice. In other species and in knockout mice, the induction of liver cancer is preceded by extensive proliferation of small oval cells, providing additional evidence that oval cells are bipolar stem cells and may give rise to either hepatocellular carcinoma or cholangiocarcinoma depending on the nature of the hepatocarcinogen and the species of animal. The recent development of mouse oval cell lines in our laboratory from aflatoxin B1-treated GSTA3-/- mice should provide a new venue for study of the properties and potential of putative mouse liver stem cells.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.

Bronchial lesions of mouse model of asthma are preceded by immune complex vasculitis and induced bronchial associated lymphoid tissue (iBALT).

We systematically examined by immune histology the lungs of some widely used mouse models of asthma. These models include sensitization by multiple intraperitoneal injections of soluble ovalbumin (OVA) or of OVA with alum, followed by three intranasal or aerosol challenges 3 days apart. Within 24 h after a single challenge there is fibrinoid necrosis of arterial walls with deposition of immunoglobulin (Ig) and OVA and infiltration of eosinophilic polymorphonuclear cells that lasts for about 3 days followed by peribronchial B-cell infiltration and slight reversible goblet cell hypertrophy (GCHT). After two challenges, severe eosinophilic vasculitis is present at 6 h, increases by 72 h, and then declines; B-cell proliferation and significant GCHT and hyperplasia (GCHTH) and bronchial smooth muscle hypertrophy recur more prominently. After three challenges, there is significantly increased induced bronchus-associated lymphoid tissue (iBALT) formation, GCHTH, and smooth muscle hypertrophy. Elevated levels of Th2 cytokines, IL-4, IL-5, and IL-13, are present in bronchial lavage fluids. Sensitized mice have precipitating antibody and positive Arthus skin reactions but also develop significant levels of IgE antibody to OVA but only 1 week after challenge. We conclude that the asthma like lung lesions induced in these models is preceded by immune complex-mediated eosinophilic vasculitis and iBALT formation. There are elevations of Th2 cytokines that most likely produce bronchial lesions that resemble human asthma. However, it is unlikely that mast cell-activated atopic mechanisms are responsible as we found only a few presumed mast cells by toluidine blue and metachromatic staining limited to the most proximal part of the main stem bronchus, and none in the remaining main stem bronchus or in the lung periphery.

Tumor-derived mesenchymal stem cells and orthotopic site increase the tumor initiation potential of putative mouse mammary cancer stem cells derived from MMTV-PyMT mice.

The ability to transplant mammary cancer stem cells, identified by the phenotype CD24(+)CD29(+)CD49f(+)Sca-1(low), is dependent on the microenvironment in which the cells are placed. Using the MMTV-PyMT mouse model of mammary cancer, we now report two methods of tumor growth enhancement: contributions of tumor stroma in the form of tumor-derived mesenchymal stem cells and orthotopic vs. heterotopic transplantation sites. To support evidence of stem cell function, tumor-derived mesenchymal stem cells differentiated into adipocyte- and osteocyte-like cells after culture in specific medium. Co-injection of tumor-initiating cells with tumor-derived mesenchymal stem cells significantly increased tumor initiation compared to subcutaneous injection of TICs alone; co-injection also allowed tumor initiation with a single TIC. Interestingly, we observed the formation of sarcomas after co-injections of tumor-derived mesenchymal stem cells or mouse embryonic fibroblasts with TICs; sarcomas are not observed in spontaneous MMTV-PyMT tumors and rarely observed in injections of TICs alone. Tumor initiation was also significantly increased in the orthotopic injection site compared to heterotopic injections. We conclude that tumor stroma and orthotopic sites both enhance tumor initiation by mammary cancer stem cells.

Characterization of mammary cancer stem cells in the MMTV-PyMT mouse model.

Breast cancer stem cells, the root of tumor growth, present challenges to investigate: Primary human breast cancer cells are difficult to establish in culture and inconsistently yield tumors after transplantation into immune-deficient recipient mice. Furthermore, there is limited characterization of mammary cancer stem cells in mice, the ideal model for the study of breast cancer. We herein describe a pre-clinical breast cancer stem cell model, based on the properties of cancer stem cells, derived from transgenic MMTV-PyMT mice. Using a defined set of cell surface markers to identify cancer stem cells by flow cytometry, at least four cell populations were recovered from primary mammary cancers. Only two of the four populations, one epithelial and one mesenchymal, were able to survive and proliferate in vitro. The epithelial population exhibited tumor initiation potential with as few as 10 cells injected into syngeneic immune-competent recipients. Tumors initiated from injected cell lines recapitulated the morphological and physiological components of the primary tumor. To highlight the stemness potential of the putative cancer stem cells, B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) expression was knocked down via shRNA targeting Bmi-1. Without Bmi-1 expression, putative cancer stem cells could no longer initiate tumors, but tumor initiation was rescued with the introduction of a Bmi-1 overexpression vector in the Bmi-1 knockdown cells. In conclusion, our data show that primary mammary cancers from MMTV-PyMT mice contain putative cancer stem cells that survive in culture and can be used to create a model for study of mammary cancer stem cells.

Age dependence of oval cell responses and bile duct carcinomas in male fischer 344 rats fed a cyclic choline-deficient, ethionine-supplemented diet.

UNLABELLED: The age dependence of the oval cell response and bile duct carcinomas of male F344 rats exposed to a cyclic choline deficiency-ethionine (CDE) diet (2 weeks on, 1 week off) supports the concept of loss of potential of liver stem cells to form cancers with aging. Livers of rats exposed at 3 weeks of age demonstrated a robust and widespread oval cell proliferation followed by cholangiofibrosis and bile duct metaplasia with extensive mucinous cysts throughout all lobes, and induction of cholangiocarcinomas (CCAs) in seven of eight rats. Livers of rats exposed beginning at 8 weeks of age had much less oval cell response and cholangiofibrosis with only 1 of 15 rats developing a CCA. Livers in old (10-12 months when started) rats remained virtually unaffected, with minimal oval cell proliferation, only occasional and small foci of ductular dysplasia, and none of 16 rats developed CCAs. In contrast to most published studies using uninterrupted choline deficiency plus a carcinogen, hepatocellular carcinoma (HCC) was not observed under the conditions of this study. CONCLUSION: With aging, male F344 rats exposed to cyclic CDE diet display a diminished oval cell response and fewer CCAs. The absence of HCC is possibly due to the fact that during cyclic CDE, the week off may allow putative liver stem cells to avoid death or differentiation and survive to give rise to CCAs, whereas with continuous CDE exposure, the stem cells are forced to differentiate and develop into HCCs with relatively few CCAs.

Direct and indirect contribution of bone marrow-derived cells to cancer.

Stromal-epithelial interactions may control the growth and initiation of cancers. Here, we not only test the hypothesis that bone marrow-derived cells may effect development of cancers arising from other tissue cells by forming tumor stroma but also that sarcomas may arise by transformation of stem cells from the bone marrow and epithelial cancers may arise by transdifferentiation of bone marrow stem cells to epithelial cancers. Lethally irradiated female FVB/N mice were restored with bone marrow (BM) transplants from a male transgenic mouse carrying the polyoma middle T-oncoprotein under the control of the mouse mammary tumor virus promoter (MMTV-PyMT) and followed for development of lesions. All of 8 lethally irradiated female FVB/N recipient mice, restored with BM transplants from a male MMTV-PyMT transgenic mouse, developed Y-chromosome negative (Y-) cancers of various organs surrounded by Y+ stroma. One of the female FVB/N recipient mice also developed fibrosarcoma and 1, a diploid breast adenocarcinoma containing Y chromosomes. In contrast, only 1 of 12 control female mice restored with normal male BM developed a tumor (lymphoma) during the same time period. These results indicate not only that the transgenic BM-derived stromal cells may indirectly contribute to development of tumors in recipient mice but also that sarcomas may arise by transformation of BM stem cells and that breast cancers arise by transdifferentiation of BM stem cells, presumably by mesenchymal-epithelial transition.

Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice.

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

Downregulation of Bmi1 in breast cancer stem cells suppresses tumor growth and proliferation.

Targeting cancer stem cells during initial treatment is important to reduce incidence of recurrent disease. Bmi1 has been associated with cancer stem cell self-renewal and aggressive disease. The purpose of this study was to determine the effects of downregulation of Bmi1 in breast cancer stem cells in order to target and eliminate the stem cell population in the tumor mass. Bmi1 was downregulated using two approaches in the mouse breast cancer stem cell line FMMC 419II-a small molecule inhibitor (PTC 209) and stable transfection with a Bmi1 shRNA plasmid. The functional effect of Bmi1 downregulation was tested in vitro and in vivo. Each approach led to decreased Bmi1 expression that correlated with an inhibition of cancer stem cell properties in vitro including cell cycle arrest and reduced mammosphere forming potential, and a decrease in tumor mass in vivo after either intra-tumoral or systemic nanoparticle-targeted delivery of anti-Bmi1. These results show that inhibiting Bmi1 expression in breast cancer stem cells could be important for the complete elimination of tumor and potentially preventing disease relapse.

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and "early aging" mice.

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16-18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best.

Intraepithelial T-cell cytotoxicity, induced bronchus-associated lymphoid tissue, and proliferation of pneumocytes in experimental mouse models of influenza.

Immunopathologic examination of the lungs of mice with experimental influenza virus infection reveals three prominent findings. (i) There is rapidly developing perivascular (arterial) and peribronchial infiltration with T-cells and invasion of T-cells into the bronchiolar epithelium, separation of epithelial cells from each other and from the basement membrane, leading to defoliation of the bronchial epithelium. This reaction is analogous to a viral exanthema of the skin, such as measles and smallpox. This previously described but unappreciated reaction most likely is an effective way to eliminate virus-infected cells, but may contribute to acute toxicity and mortality. (ii) After this, there is formation of dense collections of lymphocytes adjacent to bronchi consisting mainly of B-cells, with a scattering of T-cells and macrophages. This is known as induced bronchial-associated lymphoid tissue (iBALT) and correlates with increased interleukin (IL)-17 in the lung. iBALT provides sites for a local immune reaction in the lung to both the original infection and related viral infections (heterologous immunity). (iii) Within the first 2-3 weeks, there is proliferation of type II pneumocytes and/or terminal bronchial epithelial cells extending from the terminal bronchioles into the adjacent alveoli, eventually leading to large zones of the lung filled with tumor-like epithelial cells with squamous metaplasia. The proliferation correlates with IL-17 and IL-22 expression, and the extent of this reaction appears to be determined by the availability of T-regulatory cells.

Preparation of epithelial and mesenchymal stem cells from murine mammary gland.

The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44, and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumor development.

Networks of intergenic long-range enhancers and snpRNAs drive castration-resistant phenotype of prostate cancer and contribute to pathogenesis of multiple common human disorders.

The mechanistic relevance of intergenic disease-associated genetic loci (IDAGL) containing highly statistically significant disease-linked SNPs remains unknown. Here, we present experimental and clinical evidence supporting the importantance of the role of IDAGL in human diseases. A targeted RT-PCR screen coupled with sequencing of purified PCR products detects widespread transcription at multiple IDAGL and identifies 96 small noncoding trans-regulatory RNAs of ~100-300 nt in length containing SNPs (snpRNAs) associated with 21 common disorders. Multiple independent lines of experimental evidence support functionality of snpRNAs by documenting their cell type-specific expression and evolutionary conservation of sequences, genomic coordinates and biological effects. Chromatin state signatures, expression profiling experiments and luciferase reporter assays demonstrate that many IDAGL are Polycomb-regulated long-range enhancers. Expression of snpRNAs in human and mouse cells markedly affects cellular behavior and induces allele-specific clinically relevant phenotypic changes: NLRP1-locus snpRNAs rs2670660 exert regulatory effects on monocyte/macrophage transdifferentiation, induce prostate cancer (PC) susceptibility snpRNAs and transform low-malignancy hormone-dependent human PC cells into highly malignant androgen-independent PC. Q-PCR analysis and luciferase reporter assays demonstrate that snpRNA sequences represent allele-specific "decoy" targets of microRNAs that function as SNP allele-specific modifiers of microRNA expression and activity. We demonstrate that trans-acting RNA molecules facilitating resistance to androgen depletion (RAD) in vitro and castration-resistant phenotype (CRP) in vivo of PC contain intergenic 8q24-locus SNP variants (rs1447295; rs16901979; rs6983267) that were recently linked with increased risk of PC. Q-PCR analysis of clinical samples reveals markedly increased and highly concordant (r = 0.896; p < 0.0001) snpRNA expression levels in tumor tissues compared with the adjacent normal prostate [122-fold and 45-fold in Gleason 7 tumors (p = 0.03); 370-fold and 127-fold in Gleason 8 tumors (p = 0.0001) for NLRP1-locus and 8q24-locus snpRNAs, respectively]. Our experiments indicate that RAD and CR phenotype of human PC cells can be triggered by ncRNA molecules transcribed from the NLRP1-locus intergenic enhancer at 17p13 and by downstream activation of the 8q24-locus snpRNAs. Our results define the IDAGL at 17p13 and 8q24 as candidate regulatory loci of RAD and CR phenotypes of PC, reveal previously unknown molecular links between the innate immunity/inflammasome system and development of hormone-independent PC and identify novel molecular and genetic targets with diagnostic and therapeutic potentials, exploration of which should be highly beneficial for personalized clinical management of PC.