Mei Hui Tai

A Human Breast Epithelial Cell Type with Stem Cell Characteristics as Target Cells for Carcinogenesis

Abstract Chang, C. C., Sun, W., Saitoh, M., Tai, M-H. and Trosko, J. E. A Human Breast Epithelial Cell Type with Stem Cell Characteristics as Target Cells for Carcinogenesis. Two types of human breast epithelial cells (HBEC) have been characterized. In contrast to Type II HBEC, which express basal epithelial cell phenotypes, Type I HBEC are deficient in gap junctional intercellular communication and are capable of anchorage-independent growth and of expressing luminal epithelial cell markers, estrogen receptors, and stem cell characteristics (i.e. the ability to differentiate into other cell types and to form budding/ductal organoids on Matrigel). A comparative study of these two types of cells has revealed a high susceptibility of Type I HBEC to immortalization by SV40 large T antigen, although both types of cells are equally capable of acquiring an extended life span (bypassing senescence) after transfection with SV40. The immortalization was accompanied by elevation of a low level of telomerase activity in the parental cells after mid-passage (∼60 cumulative population doubling levels). Thus HBEC do have a low level of telomerase activity, and Type I HBEC with stem cell characteristics are more susceptible to telomerase activation and immortalization, a mechanism which might qualify them as target cells for breast carcinogenesis. The immortalized Type I HBEC can be converted to highly tumorigenic cells by further treatment with X rays (2 Gy × 2) and transfection with a mutated ERBB2 (also known as NEU) oncogene, resulting in the expression of p185ERBB2 which is tyrosine phosphorylated.

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

Objectives: The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-l-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. Methods: Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. Results: Human pancreatic cell (HPC) cultures coexpressed &agr;-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. Conclusion: These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

Cigarette smoke components inhibited intercellular communication and differentiation in human pancreatic ductal epithelial cells

Smoking is a well‐documented risk factor for the development of pancreatic adenocarcinoma. Although the most abundant polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are methylated anthracenes and phenanthrenes, the epigenetic toxicity of these compounds has not been extensively studied. We previously showed that methylanthracenes, which possess a bay‐like structure, affect epigenetic events such as an induced release of arachidonic acid, inhibition of gap junctional intercellular communication (GJIC) and induction of mitogen‐activated protein kinases in a pluripotent rat liver epithelial stem cell line. Anthracenes with no bay‐like structures were inactive. These biological effects are all molecular events associated with the promotional phase of cancer. A human immortalized, nontumorigenic pancreatic ductal epithelial cell line, H6c7, was examined to study the epigenetic toxicity of PAHs related to pancreatic cancer by using scrape‐loading dye transfer, immunostaining, RT‐PCR and telomerase assay methods. H6c7 cells were GJIC‐incompetent and exhibited high telomerase activity when grown in growth factor and hormone‐supplemented medium. In the presence of the cAMP elevating drugs (forskolin and IBMX) the cells became GJIC competent and expressed connexins. Telomerase activity was also decreased by cAMP elevating drug treatment. After induction of cAMP, 1‐methylanthracene with bay‐like structures inhibited GJIC, whereas the 2‐methylanthracene lacking a bay‐like structure had no effect on GJIC. Telomerase activity remained high in 1‐methylanthracene treatment but not with 2‐methylanthracene. These results indicate that a prominent component of cigarette smoke, namely methylanthracenes with distinct structural configurations, could be a potential etiological agent contributing to the epigenetic events of pancreatic cancer.

Characterization of gap junctional intercellular communication in immortalized human pancreatic ductal epithelial cells with stem cell characteristics.

Introduction Gap junctional intercellular communication has been implicated in the homeostatic regulation of cell growth, differentiation, and apoptosis. Cancer cells, which have been viewed as “partially blocked stem cells,” and which lack the ability for growth control, terminal differentiation, and apoptosis, also lack functional gap junctional communication. Aims and Methodology A clone of a human pancreatic ductal epithelial cell line, H6c7, derived after immortalization with human papilloma virus, was used to examine gap junctional intercellular communication and the ability to differentiate under different growth conditions. Results The cells showed characteristic epithelial morphology on standard tissue culture dishes. When placed on Matrigel they showed phenotypical changes with extensive ductal organization and budding structures. In growth medium containing hormones and growth factors, these cells were gap junctional intercellular communication (GJIC)–incompetent. In the presence of c-AMP elevating agents, isobutylmethylxanthine, and forskolin, in basal medium that did not contain the hormones and growth factors, the cells became GJIC-competent and expressed connexin43 gap junction protein within 48 hours after treatment. RT-PCR analyses of the cells under different growth conditions showed that the cells expressed connexin 32, 36, and 43 genes when cultured in the basal medium with c-AMP elevating agents. They also expressed the connexin 45 gene that did not change with c-AMP treatment. H6c7 cells also have the capacity to turn on an ectopic insulin promoter reporter gene. Conclusion Our data suggest that the immortalized H6c7 cells retain stem-like characteristics and have the potential to differentiate into duct-like structures and perhaps insulin-producing cells.

Mannitou Monoclonal Antibody Uniquely Recognizes Paucimannose, a Marker for Human Cancer, Stemness, and Inflammation

The trimannosyl structure, also called “paucimannose,” is characteristic for all eukaryotic N-glycans. The unmodified structure is expressed abundantly in plants and invertebrates, whereas in normal mammalian tissue it has been detected in only very small amounts. We have developed a monoclonal antibody called Mannitou that specifically recognizes paucimannose and clearly stains all human cancer tissues analyzed, human adult pancreatic stem cells, and inflamed mouse pancreata. Normal tissues/cells exhibited only weak or no staining. The up-regulation of paucimannose under tumorigenic and inflammatory conditions makes the Mannitou antibody a promising tool for diagnosis and potential therapeutic applications.

Melatonin decreases estrogen receptor binding to estrogen response elements sites on the OCT4 gene in human breast cancer stem cells.

Cancer stem cells (CSCs) pose a challenge in cancer treatment, as these cells can drive tumor growth and are resistant to chemotherapy. Melatonin exerts its oncostatic effects through the estrogen receptor (ER) pathway in cancer cells, however its action in CSCs is unclear. Here, we evaluated the effect of melatonin on the regulation of the transcription factor OCT4 (Octamer Binding 4) by estrogen receptor alpha (ERα) in breast cancer stem cells (BCSCs). The cells were grown as a cell suspension or as anchorage independent growth, for the mammospheres growth, representing the CSCs population and treated with 10 nM estrogen (E2) or 10 μM of the environmental estrogen Bisphenol A (BPA) and 1 mM of melatonin. At the end, the cell growth as well as OCT4 and ERα expression and the binding activity of ERα to the OCT4 was assessed. The increase in number and size of mammospheres induced by E2 or BPA was reduced by melatonin treatment. Furthermore, binding of the ERα to OCT4 was reduced, accompanied by a reduction of OCT4 and ERα expression. Thus, melatonin treatment is effective against proliferation of BCSCs in vitro and impacts the ER pathway, demonstrating its potential therapeutic use in breast cancer.

Leech photoreceptors project their galectin‐containing processes into the optic neuropils where they contact AP cells

We characterized a subset of leech sensory afferents, the photoreceptors, in terms of their molecular composition, anatomical distribution, and candidate postsynaptic partners. For reagents, we used an antiserum generated against purified LL35, a 35 kD leech lactose‐binding protein (galectin); monoclonal antibody (mAb). Lan3‐2, which is specific for a mannose‐containing epitope common to the full set of sensory afferents; and dye injections. Photoreceptors differ from other types of sensory afferents by their abundant expression of galectin. However, photoreceptors share in common with other sensory modalities the mannose‐containing epitope recognized by mAb Lan3‐2. Photoreceptors from a given segment project their axons directly into the CNS ganglion innervating the same segment. They assemble in a target region, the optic neuropil, which is separate from the target regions of other sensory modalities. They also extend their axons as an optic tract into the connective to innervate optic neuropils of other CNS ganglia, thereby providing extensive intersegmental innervation for the 33 CNS ganglia comprising the leech nerve cord. Because of its intimate contact with the optic neuropil, a central neuron, the AP effector cell, is a strong candidate second order visual neuron. In confocal images, the AP cell projects its primary axon for about 100 μm alongside the optic neuropil. In electron micrographs, spines emanating from the axon of the AP cell make contact with vesicle laden nerve terminals of photoreceptors. Leech photoreceptors and their second order visual neurons represent a simple visual system for studying the mechanisms of axonal targeting.

Effects of ketanserin on DOI-, MCPP- and TRH-induced prolactin secretion in estrogen-treated rats

The effects of ketanserin (Ket), a serotonin (5-HT2) receptor antagonist, on DOI- and mCPP-, two 5-HT agonists, and TRH-induced PRL secretion were studied. Adult female Sprague-Dawley rats ovariectomized for two weeks and treated with a long-acting estrogen, polyestradiol phosphate for one week were used. Drug administration and serial blood sampling were accomplished through indwelling intraatrial catheters which were implanted two days before the experiment. Both DOI (0.5 mg/kg BW) and mCPP (1 mg/kg BW) stimulated prolactin secretion within 10 min after iv injection and the effects were diminished by 30 min. In animals pretreated with Ket (5 mg/kg BW, sc), the effect of DOI was blocked, while that of mCPP was augmented. Co-administration of Ket (1 mg/kg BW, iv) with DOI or mCPP produced similar effect. Pretreatment with Ket, similar to sulpiride (Sulp), a dopamine antagonist, potentiated the TRH-induced prolactin secretion. Co-administration of Ket and Sulp further potentiated the TRH action. It is concluded that Ket not only acts as a 5-HT2 receptor antagonist that blocks the action of DOI, but may also act on dopamine receptor(s) with lower sensitivity to Sulp.

Extracellularly applied horseradish peroxidase increases the number of dense core vesicles in leech sensory neurons

The uptake of horseradish peroxidase (HRP), applied as an extracellular tracer, is a classical method for studying endo/exocytosis of synaptic vesicles at the ultrastructural level. It is generally not considered that HRP may affect neuronal function. Reported here is the finding that extracellularly applied HRP (0.1%) perturbs dense core vesicles in the synaptic processes of leech neurons. The strength of the effect varies with neuronal class. In sensory afferents, the number of dense core vesicles increases 5-fold, while there is only a 2-fold increase in central neurons.

Diet/Nutrition, Inflammation, Cellular Senescence, Stem Cells, Diseases of Aging, and Aging

The complex process by which a multicellular organ, such as the human being, acquires the energy needed for life has been shaped by the evolutionary transition from a single-cell organism, which survived primarily by unlimited cell proliferation in an ever-changing oxygen-environment, using glycolysis. The transition to a multicellular state occurred in an oxygenated environment, which necessitated the development of a mitochondrial oxidative phosphorylation process and concomitant acquisition of new genotypes and phenotypes, including (1) the creation of germinal and somatic organ-specific adult stem cells; (2) their oxygen-protecting niches; (3) the ability to adaptively regulate symmetrical and asymmetrical cell division for either stem cell expansion or the differentiation of multiple cell types; (4) apoptosis; and (5) cellular senescence. Both endogenous and exogenous environmental and nutritional/dietary factors that can affect the quality and quantity of stem cells during embryonic/fetal development can alter the risk of stem cell-based diseases, such as cancer, later in life (the Barker hypothesis). Fundamental to both aging and the diseases of aging is the role of oxidative stress, which induces intracellular signaling and is critical to the behavioral choices that stem cells and life span-limited differentiated cells must make. It is here that oxidative stress-induced inflammatory processes can induce secreted factors that can affect multiple responses, i.e. proliferation, differentiation, apoptosis, in normal and initiated adult stem cells, normal progenitor, and terminally differentiated cells. It is here, also, where the nutritional status, directed by diet and exercise, can modulate (increase or decrease) the factors that can trigger oxidative stress-induced signaling in either the immune and other organ systems. In other words, these nutritional/dietary factors can either contribute to or protect against various inflammatory-related diseases.