Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Small cell lung cancer (SCLC) accounts for nearly 15% of human lung cancers and is one of the most aggressive solid tumors. The SCLC cells are thought to derive from self-renewing pulmonary neuroendocrine cells by oncogenic transformation. However, whether the SCLC cells possess stemness and plasticity for differentiation as normal stem cells has not been well understood thus far. In this study, we investigated the expressions of multilineage stem cell markers in the cancer cells of SCLC cell line (NCI-H446) and analyzed their clonogenicity, tumorigenicity, and plasticity for inducing differentiation. It has been found that most cancer cells of the cell line expressed multilineage stem cell markers under the routine culture conditions and generated single-cell clones in anchorage-dependent or -independent conditions. These cancer cells could form subcutaneous xenograft tumors and orthotopic lung xenograft tumors in BALB/C-nude mice. Most cells in xenograft tumors expressed stem cell markers and proliferation cell nuclear antigen Ki67, suggesting that these cancer cells remained stemness and highly proliferative ability in vivo. Intriguingly, the cancer cells could be induced to differentiate into neurons, adipocytes, and osteocytes, respectively, in vitro. During the processes of cellular phenotype-conversions, autophagy and apoptosis were two main metabolic events. There is cross-talking between autophagy and apoptosis in the differentiated cancer cells. In addition, the effects of the inhibitor and agonist for Sirtuin1/2 on the inducing osteogenic differentiation indicated that Sirtuin1/2 had an important role in this process. Taken together, these results indicate that most cancer cells of NCI-H446 cell line possess stemness and plasticity for multilineage differentiation. These findings have potentially some translational applications in treatments of SCLC with inducing differentiation therapy.     

Lead-haematoxylin as a stain for endocrine cells

Summary A modification of MacConaill's lead-haematoxylin has been found to stain several endocrine cells producing polypeptides and monoamines, particularly A and D cells of the pancreatic islet, thyroid C cells, gastro-intestinal enterochromaffin cells, gastric G and X cells, pituitary ACTH and MSH cells, adrenal medullary cells, and chemoreceptive cells of the carotid body. A careful comparison of the results of this method with those of HCI-basic dye method and of monoamine methods suggested that carboxyl groups of proteins may be the main binding site of lead-haematoxylin. Experiments with various pretreatments of tissue sections support such a hypothesis. The possibility that biogenic amines take also some part in the staining cannot be ruled out.     

Teratocarcinoma stem cells as a model for differentiation in the mouse embryo

Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.     

Embryonic stem cells as a model for studying regulation of cellular differentiation

Mouse embryonic stem (ES) cells can be differentiated in vitro into near homogeneous populations of both neurons and skeletal muscle as well as other cell types. We previously showed that treatment of pluripotent ES cells with retinoic acid (RA) induced differentiation into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons. The reasons for generation of only GABA neurons as opposed to other neuronal cell types were not known. We have extended our previous work and now show that with RA induction of ES cells we not only obtain GABA neurons, but also dopaminergic neurons. Critical for the production of dopaminergic neurons after RA induction was the post-induction plating conditions used. No dopaminergic neurons were detected if cells were plated in serum-free media optimized for neuronal survival. However, significant numbers of dopamine neurons could be detected when cells were plated in media containing fetal calf serum. These observations support the conclusion that RA acts as a general neural inducing agent and that conditions post-induction either selectively support survival of a particular class of neuronal cells or that the conditions post-induction actually further instruct cells to differentiate into different types of neurons.     

Selected tetrapeptides lead to a GLP-1 release from the human enteroendocrine cell line NCI-H716

Enteroendocrine cells in the intestine sense the luminal contents and have been shown to respond to not only fatty acids, proteins, and monosaccharides but also artificial sweeteners and bitter compounds. Secretion of hormones such as CCK and GLP-1 from these cells is often associated with a rise in intracellular calcium concentration [Ca2+](i). The human NCI-H716 enteroendocrine cell line has been described as a proper model to study GLP-1 secretion in response to amino acids and protein hydrolysates. Here, we describe that NCI-H716 cells specifically respond to selective tetrapeptides such as tetra-glycine, tetra-alanine and Gly-Trp-Gly-Gly with a dose-dependent [Ca2+](i) response and a GLP-1 secretion, whereas selected free amino acids, dipeptides, tripeptides and pentapeptides failed to elicit such a response. Hormone secretion was not associated with changes in cAMP levels in the cells. The calcium-dependence of hormone secretion appears to involve store-operated calcium channels (SOCCs), since the SOCC blocker 2-APB abolished both the [Ca2+](i) response and GLP-1 release upon tetra-glycine stimulation. The nature of the sensor currently remains elusive, and no obvious common structural pattern in tetrapeptides eliciting GLP-1 secretion was identified. This tetrapeptide sensing in NCI-H716 cells may be underlying the effective stimulation of hormone secretion shown for various protein hydrolysates, and could involve a novel G-protein-coupled receptor (GPCR).     

Estrogen receptor knockout mice as a model for endocrine research

The biological effects of estrogen in mammalian target tissues are important for multiple organ systems including the male and female reproductive tract and the neuroendocrine, skeletal, and cardiovascular systems. Numerous physiological effects of estradiol are modulated by the estrogen receptor (ER), a Class I member of the nuclear receptor superfamily. However, more recent studies have also implicated nongenomic effects of estrogen, which may involve a membrane-binding site. The two forms of the ER are the classical estrogen receptor-alpha (ERalpha) and the more recently discovered estrogen receptor-beta (ERbeta). Gene-targeting techniques were used to generate mice lacking either functional ERalpha (alphaERKO), ERbeta (betaERKO), or both ERs (alphabetaERKO) to provide a model for evaluating estrogen receptor action. These knockout models provide a unique tool to study the effects of estrogen in the context of the whole animal and to discern the role of each ER in various tissues. The reproductive phenotypes as well as some of the nonreproductive phenotypes of the different ERKO models are summarized.     

Conversion of iris epithelial cells as a model of differentiation control

Abstract. It has been shown that, upon lentectomy or in culture, iris epithelial cells (IECs) of adult newts become converted into lens cells, and this conversion is the basic event of lens regeneration in newts. Whether in situ or in cell culture, the conversion requires the passage of a specific number of cell cycles. The progeny of IECs which fails to traverse this cell-cycle number redifferentiates as IECs in situ. The passage through cell cycles of IECs is associated with progressive alterations of cytoplasm and cell surface, during which the original state of differentiation disappears (dedifferentiation). It is speculated that the altered state of cells caused by proliferation leads to the appearance of factors which interact with the genome and switch the gene activation pattern to that of the lens cell. In this model, developmental controls are geared to the cell-cycle progression and not directly to the activation of lens-characteristic genes. A number of points are raised which speak against the long-held idea that a factor from neural retina induces lens differentiation in IECs. It is proposed that the retinal factor plays the role of growth factor which is essential in the conversion in situ, but not required in the conversion in cell culture. The proposed model is compared with reprogramming of differentiation of some cell lines by cytidine analogs and with ontogenic systems of differentiation control.     

Nodulation as a model for studying differentiation in higher plants

The stages of the legume-rhizobial symbiosis and nodule structure in various legume plants are briefly reviewed. Modern data on the mechanisms involved in the control of nodule initiation and morphogenesis are considered.     

Establishment of a Tcrb and Trp53 genes deficient mouse strain as an animal model for spontaneous colorectal cancer.

A congenic C57BL/6JJcl-Tcrbtm1MomTrp53tm1 (Tcrb-/-:Trp53-/-) mouse lacking T-cell receptor beta chain (TCR beta) and transformation related protein 53 (p53) has been established at the N8th generation of backcrossing male Tcrb-/-:Trp53-/- mice, which had been obtained by mating a Tcrb-/- mouse with a Trp53-/- mouse, with female C57BL/6JJcl mice. In the mice deficient for the both genes, occurrence of tumor masses was observed mostly in the cecum with high frequency as examined at 3 months of age. The majority of the masses had histologic features of hyperplasia or dysplasia while occasional lesions were noted to be adenocarcinomas invading the submucosa (invasive adenocarcinoma). As examined at 4 months of age and thereafter, all mice had 4-5 colorectal tumors per animal, the lesions being located mainly in the cecum and, histopathologically, all the obvious neoplastic growths in the regions examined were invasive adenocarcinomas. The Tcrb and Trp53 genes deficient mouse strain which develops spontaneous colorectal carcinoma with fairly high frequency at early age would be useful as an animal model for colorectal cancer.     

Placental villous stroma as a model system for myofibroblast differentiation

Different subtypes of myofibroblasts have been described according to their cytoskeletal protein patterns. It is quite likely that these different subtypes represent distinct steps of differentiation. We propose the human placental stem villi as a particularly suitable model to study this differentiation process. During the course of pregnancy, different types of placental villi develop by differentiation of the mesenchymal stroma surrounding the fetal blood vessels. In order to characterise the differentiation of placental stromal cells in the human placenta, the expression patterns of the cytoskeletal proteins vimentin, desmin, - and -smooth muscle actin, pan-actin, smooth muscle myosin, and the monoclonal antibody GB 42, a marker of myofibroblasts, were investigated on placental tissue of different gestational age (7th–40th week of gestation). Proliferation patterns were assessed with the proliferation markers MIB 1 and PCNA. Additionally, dipeptidyl peptidase IV distribution was studied in term placenta and the ultrastructure of placental stromal cells was assessed by electron microscopy. Different subpopulations of extravascular stromal cells were distinguished according to typical co-expression patterns of cytoskeletal proteins. Around the fetal stem vessels in term placental villi they were arranged as concentric layers with increasing stage of differentiation. A variable layer of extravascular stromal cells lying beneath the trophoblast expressed vimentin (V) or vimentin and desmin (VD). They were mitotically active. The next layer co-expressed vimentin, desmin, and -smooth muscle actin (VDA). More centrally towards the fetal vessels, extravascular stromal cells co-expressed vimentin, desmin, - and -smooth muscle actin, and GB 42 (VDAG). Cells close to the fetal vessels additionally co-expressed smooth muscle myosin (VDAGM). Ultrastructurally, V cells resembled typical mesenchymal cells. VD cells corresponded to fibroblasts, while VDA and VDAG cells developed features of myofibroblasts. Cells of the VDAGM-type revealed a smooth muscle cell-related ultrastructure. In earlier stages of pregnancy, stromal cell types with less complex expression patterns prevailed. The media smooth muscle cells of the fetal vessels showed a mixture of different co-expression patterns. These cells were separated from extravascular stromal cells by a layer of collagen fibres. The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessels in the centre of the stem villi. The spatial distribution of the various stages of differentiation suggests that human placental villi could be a useful model for the study of the differentiation of myofibroblasts.     

Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.     

Screening for colorectal cancer.

In the United Kingdom there are almost 31,000 new cases of colorectal cancer each year and nearly 20,000 deaths. It is the second most common cause of all deaths from cancer in the United Kingdom. The efficacy of screening with faecal occult blood tests to detect and treat tumours at an early stage, and thereby to reduce mortality, is currently being assessed in several randomised controlled trials. In general, faecal occult blood tests have a low sensitivity and only moderate levels of uptake among the population invited for screening. Until the studies in Denmark, Sweden, and Nottingham report their findings, there is no case for providing such screening routinely to an asymptomatic population. Detecting and removing premalignant adenomas by flexible sigmoidoscopy could be more effective than detecting early localised, asymptomatic cancers in reducing deaths from colorectal cancer. It is estimated that screening people at about the age of 60 by a single flexible sigmoidoscopy examination with appropriate colonoscopic surveillance could prevent 5500 cases of colorectal cancer and 3500 deaths in the United Kingdom each year. A randomised controlled trial to confirm these observations should be seen as a priority.     

Calcium prevents tumorigenesis in a mouse model of colorectal cancer

Background and Aim

Calcium has been proposed as a mediator of the chemoprevention of colorectal cancer (CRC), but the comprehensive mechanism underlying this preventive effect is not yet clear. Hence, we conducted this study to evaluate the possible roles and mechanisms of calcium-mediated prevention of CRC induced by 1,2-dimethylhydrazine (DMH) in mice.

Methods

For gene expression analysis, 6 non-tumor colorectal tissues of mice from the DMH + Calcium group and 3 samples each from the DMH and control groups were hybridized on a 4×44 K Agilent whole genome oligo microarray, and selected genes were validated by real-time polymerase chain reaction (PCR). Functional analysis of the microarray data was performed using KEGG and Gene Ontology (GO) analyses. Hub genes were identified using Pathway Studio software.

Results

The tumor incidence rates in the DMH and DMH + Calcium groups were 90% and 40%, respectively. Microarray gene expression analysis showed that S100a9, Defa20, Mmp10, Mmp7, Ptgs2, and Ang2 were among the most downregulated genes, whereas Per3, Tef, Rnf152, and Prdx6 were significantly upregulated in the DMH + Calcium group compared with the DMH group. Functional analysis showed that the Wnt, cell cycle, and arachidonic acid pathways were significantly downregulated in the DMH + Calcium group, and that the GO terms related to cell differentiation, cell cycle, proliferation, cell death, adhesion, and cell migration were significantly affected. Forkhead box M1 (FoxM1) and nuclear factor kappa-B (NF-κB) were considered as potent hub genes.

Conclusion

In the DMH-induced CRC mouse model, comprehensive mechanisms were involved with complex gene expression alterations encompassing many altered pathways and GO terms. However, how calcium regulates these events remains to be studied.     

Micrasterias cells as a model system for research on morphogenesis.

Micrasterias species have been the subject of numerous experimental studies on cell shape formation in the last 40 years. Chemical and physical treatment during different developmental stages, as well as investigations of ultrastructure by means of various different preparation methods, have yielded information about some principles of morphogenesis in the symmetric, highly ornamented Micrasterias cell. The basic symmetry of a Micrasterias cell is determined prior to mitosis and is established without nuclear control thereafter. Normal cell development, however, may occur only under the conditions of continuous protein synthesis throughout the cell cycle. A prepattern for the later cell shape seems to be present at the plasma membrane at the early stages of septum formation. It is realized by a local, patterned distributed incorporation of cell wall material that is delivered by Golgi-produced vesicles. The areas where fusions take place between the primary wall material containing vesicles and the plasma membrane are defined by inward ionic currents that are carried at least in part by calcium. These areas develop into lobes during the following course of cell growth. Cell shaping in Micrasterias cells is thus mediated by both an enhanced extension of the cell wall and an additional incorporation of wall material in the areas of the lobes. Numerous studies have indicated that actin plays an important role in morphogenesis, whereas microtubules do not participate in this process but are involved mainly in nuclear migration. The present review shows that although a wealth of details concerning Micrasterias morphogenesis has already been elucidated, two main questions, i.e., the method of septum formation and the splitting of the lobes, remain to be answered.