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Results for “clonal modeling”

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PubMed2006

Expression of feline immunodeficiency virus Vif is associated with reduced viral mutation rates without restoration of replication of vif mutant viruses.

The vif gene of lentiviruses has been demonstrated to be essential for efficient viral replication in many cell types. Although the Vif protein of feline immunodeficiency virus (FIV) displays limited homology to HIV-1 Vif, the role of vif in FIV replication is not known. We have examined the requirements of vif for replication of a FIV strain isolated from a non-domestic felid, Otocolobus manul (FIV-Oma). In agreement with others, we find that replication of FIV vif mutant molecular clones in CrFK cells is highly attenuated. Initial attempts to rescue vif mutant viruses in trans were limited by lack of detectable wild-type Vif expression from DNA constructs. We demonstrate that FIV-Oma Vif expression can be increased by re-synthesis of the gene to remove splice donor and acceptor sites as well as improving codon usage to a mammalian codon optimized model. Cellular localization of resynthesized Vif (Vif-RS) is cytoplasmic. Clonal stable transfectants expressing HA-tagged Vif-RS do not restore replication levels of vif mutant virus. However, in such cell lines, G-to-A mutation rates in replicating wild-type viruses are reduced.

Amino Acid Sequence↗

PubMed2000

Chromosomal imbalances in Barrett's adenocarcinoma and the metaplasia-dysplasia-carcinoma sequence.

To characterize cytogenetic alterations found in Barrett's adenocarcinoma (BA) and, more importantly, its premalignant stages, we studied chromosomal imbalances in various lesions in the histologically proposed metaplasia-dysplasia-carcinoma sequence using comparative genomic hybridization (CGH). Using 30 esophageal adenocarcinoma resection specimens, we were able to study 30 areas of Barrett's adenocarcinoma and 8 lymph node metastases (LN). In addition, we investigated 25 premalignant lesions adjacent to BA derived from a subset of 14 resection specimens including 11 areas of high grade dysplasia (HGD), 8 areas of low grade dysplasia (LGD), and 6 areas of intestinal metaplasia (IM), which were laser-microdissected and studied with CGH. To validate the CGH findings, fluorescence in situ hybridization analysis on 13 BA with probes specific for HER-2/neu and 20q13.2 were performed. The chromosomal alterations most often identified in BA were: gains on 8q (80%), 20q (60%), 2p, 7p and 10q (47% each), 6p (37%), 15q (33%) and 17q (30%). Losses were observed predominantly on the Y-chromosome (76%), 4q (50%), 5q and 9p (43% each), 18q (40%), 7q (33%) and 14q (30%). High-level amplifications were observed on 8q23-qter, 8p12-pter, 7p11-p14, 7q21-31, 17q11-q23. Recurrent chromosomal changes were also identified in metaplastic (gains on 8q, 6p, 10q, losses on 13q, Y, 9p) and dysplastic epithelium (gains on 8q, 20q, 2p, 10q, 15q, losses on Y, 5q, 9p, 13q, 18q). Novel amplified chromosomal regions on chromosomes 2p and 10q were detected in both Barrett's adenocarcinoma and premalignant lesions. An increase of the average number of detected chromosomal imbalances from IM (7.0 +/- 1.7), to LGD (10.8 +/- 2.2), HGD (13.4 +/- 1.1), BA (13.3 +/- 1.4), and LN (22 +/- 1.2) was seen. Although the detection of common chromosomal alterations in premalignant lesions and adjacent carcinomas suggest a process of clonal expansion, the occurrence of several chromosomal changes in an apparently random order relative to one another is striking evidence that clonal evolution is more complex than would be predicted by linear models. This is probably a reflection of the existence of many divergent neoplastic subpopulations and highlights one of the main problems associated with surveillance of Barrett's patients, namely sampling error.

Adenocarcinoma↗

PubMed1993

The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of a clonal plasma cell population in the bone marrow (BM). It is of particular interest that the tumor plasma cells appear to be restricted to the microenvironment of the BM and are rarely detected in the peripheral system, yet the disease is found widely disseminated throughout the axial skeleton. Furthermore, isolation of MM tumor cell lines has proven to be quite problematic due to their slow growth rate. These observations have instigated the search for earlier cells in the B cell lineage that are clonally related to the plasma cell tumor and that may represent the growth fraction of the tumor. We used allele-specific oligonucleotides (ASO) derived from the third complementarity determining region of the rearranged tumor immunoglobulin heavy chain gene to detect isotypes clonally related to the plasma cell tumor. By reverse transcribing RNA from the BM with a panel of CH primers (mu, delta, alpha, and gamma), followed by ASO-polymerase chain reaction amplification, we demonstrate the existence of preswitch isotype species that are clonally related to the myeloma tumor. Furthermore, we show that separation of the BM cells into CD45+ and CD38+ cell populations results in a lineage-specific expression of the clonally related RNA molecules, with the C mu and C delta in the CD45+, and C gamma in the CD38+ population. Interestingly, clonally related C alpha transcripts are also derived from the CD45+ fraction. These results confirm the presence of B cell populations clonally related to the plasma cell tumor and are consistent with models that propose the existence of myeloma precursors.

ADP-ribosyl Cyclase↗

PubMed1984

Gene-regulated expression of glycolipids: appearance of GD3 ganglioside in rat cells on transfection with transforming gene E1 of human adenovirus type 12 DNA and its transcriptional subunits.

Malignant transformation is known to be associated with characteristic changes of cell surface glycolipids, but the genetic background and underlying molecular mechanism of these changes are still unknown. Incorporation of oncogenic elements into cultured cells should provide a useful model for studies on this problem. We analyzed gangliosides of a clonal and fibroblastic rat cell line, 3Y1, and its transformed counterparts, E1Y, HY1, and CY1 cell lines. E1Y cells were established by transfection of 3Y1 cells with the transforming gene E1, minimum DNA fragment of human adenovirus type 12 (Ad 12) required for complete transformation of cells. HY1 cell line is the cells established with E1A which is a transcriptional subunit of the E1 and can transform incompletely cells. CY1 was established by co-transfection with parts of non-transforming gene E3 and E4 of Ad 12 DNA in addition to the E1. GD3 ganglioside (NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) appeared in all these transfection-transformed cells but was undetectable in untransformed control cells. The appearance of GD3 was undoubtedly due to expression of E1A gene in association with oncogenic cell transformation. The expression of other gangliosides, particularly GM2 and GM1, were also influenced by transfection, but their expressions depended on the kind of oncogenic DNA fragments used. These results all suggest that ganglioside metabolism is under unexpectedly complicated gene control.

Adenoviruses, Human↗

PubMed1991

Effects of volatile anesthetics on cytoplasmic Ca2+ signaling and transmitter release in a neural cell line.

To provide new insights into the effects of volatile agents on the basic regulatory events involved in cytoplasmic free Ca2+ ([Ca2+]i) and stimulus-secretion coupling, the well-characterized clonal rat pheochromocytoma cell line PC12 was chosen as an experimental model. This cell line possesses nicotinic and muscarinic receptors, L-type voltage-operated channels (VOCs), and receptor-operated Ca2+ channels (ROCs). A PC12 variant, defective in nicotinic response, made it possible to study the influx-independent inositol trisphosphate-mediated intracellular Ca2+ release that is triggered by muscarinic receptor stimulation. [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2. Dopamine and norepinephrine secretion were determined by high-performance liquid chromatography. High K+ and nicotinic-receptor-induced [Ca2+]i increase and catecholamine secretion were inhibited by halothane, enflurane, isoflurane, and methoxyflurane in a dose-dependent manner; half-maximal inhibition (IC50) occurred within the clinically relevant concentration range. The inhibition was reversible after wash-out of anesthetic; was not restricted to dihydropyridine-sensitive L-type VOCs; and could not be overcome by increasing extracellular Ca2+. The inhibitory mechanisms of volatile anesthetics therefore differed from those of classical organic Ca2(+)-channel blockers, a difference also reflected by the differing Hill coefficients found for both substance groups. In contrast, the muscarinic-receptor-evoked internal Ca2+ release remained unimpaired, and secretion even increased under anesthetic exposure. In conclusion, the current study provides evidence that volatile anesthetics depress the Ca2+ influx through at least two independent Ca2+ channels, one of which proved insensitive to the dihydropyridine Ca2(+)-channel blocker nifedipine. This is particularly noteworthy, since dihydropyridine-insensitive N-type VOCs, so far found exclusively in neurons, are assumed to play a dominant role in synaptic transmission, which, although resistant to dihydropyridine inhibition, is effectively blocked by volatile anesthetics.

Anesthetics↗

PubMed1993

Opposing effects of tumor necrosis factor alpha and leukemia inhibitory factor in lipopolysaccharide-stimulated myelopoiesis.

Three myelopoietically active, lipopolysaccharide (LPS)-stimulated monokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and leukemia inhibitory factor (LIF), were tested for effect in an in vitro model for LPS-induced inflammatory murine monocytopoiesis. Neither cytokine stimulated clonal proliferation of marrow-derived progenitors; however, both IL-1 alpha and TNF-alpha enhanced macrophage colony-stimulating factor (M-CSF)-dependent colony formation. The additional progenitors stimulated by IL-1 alpha and TNF-alpha to form colonies in response to M-CSF were equivalent to the precommitment, transitional progenitors stimulated by M-CSF and bacterial LPS. In addition, the additional colonies elicited by IL-1 alpha and TNF-alpha were not additive in cultures containing both M-CSF and LPS, indicating these colonies arose from the same LPS-responsive, two-signal-dependent transitional progenitors. Leukemia inhibitory factor did not influence M-CSF-stimulated colony formation; however, LIF effected a dose-dependent inhibition of colony formation by transitional progenitors responding to combinations of M-CSF and LPS, IL-1 alpha, TNF-alpha, or an additional transitional cell costimulant, substance P. Neutralizing anti-murine TNF-alpha antibodies abrogated transitional cell colony formation stimulated by combinations of M-CSF and TNF-alpha, IL-1 alpha, LPS, or substance P but had no effect on colony formation stimulated solely by M-CSF. The results indicate that TNF-alpha may be an important positive stimulus for commitment of progenitors to the mononuclear phagocyte lineage and that TNF-alpha may be the endogenous regulator of the costimulatory effects of LPS, IL-1, and substance P. In addition, the results indicate that LIF may play an opposing negative regulatory role acting to inhibit LPS and TNF-alpha stimulation of the transitional progenitors.

Animals↗

PubMed1996

Rapid clearance of the bacterial superantigen staphylococcal enterotoxin B in vivo.

Following bolus injection of the superantigen Staphylococcus enterotoxin B (SEB) into mice, ligand-reactive T cells are triggered to release toxic amounts of lymphokines. Subsequently, within 6 to 10 h ligand-reactive Vbeta8+ T cells become anergic and clonally expand and thereafter are deleted via apoptosis. Since the binding affinities of SEB to major histocompatibility complex (MHC) class II molecules are as low as 1.7 microM, the concentration of SEB in the fluid phase dictates the presentation via MHC class II molecules. Here, we study the pharmacokinetics of SEB in vivo and correlate pharmacokinetics with immunogenicity. We describe here how after a bolus injection of SEB, the superantigen becomes systemically distributed, with peak levels within 5 to 30 min in blood and in lymph nodes. Clearance occurs within 10 to 24 h, with the kidneys being a major route. To induce T-cell activation in vivo, SEB must be present in concentrations above 10(-4) microg/ml. These concentrations exist for only 15 h. Manifestation of functional outcomes such as anergy, clonal expansion, and clonal deletion begins after 24 h. We conclude that the SEB model system can be used to separate the phase of T-cell receptor ligation from the phase of manifestation of functional outcomes.

Animals↗

PubMed1983

Preneoplastic and neoplastic progression during hepatocarcinogenesis in mice injected with diethylnitrosamine in infancy.

Basophilic hepatic foci, nodules, and trabecular hepatocellular carcinomas, collectively referred to as focal hepatic lesions, were induced by single injections of 5.0 micrograms of diethylnitrosamine (DEN) per gram body weight in 15-day-old C57BL/6J X C3HeB/FeJ F1 (B6C3 F1) mice. Groups of eight experimental and eight control mice were killed at 3 days and at 1, 2, 4, 10, 20, 28, 36 and 41 weeks after injection. The only observable acute hepatic toxic effect of DEN, a mild steatosis, was noted at 3 days, but this had disappeared by 7 days following injection. Basophilic foci, composed entirely of altered hepatocytes, were first noted, when very small, at 10 weeks. At later times, some of the foci also contained small collections of proliferated ductules, apparently a result of secondary ingrowth from nearby interlobular bile ducts. The hepatocytes within basophilic foci were characterized by their abundant cytoplasmic RNA, a high nuclear to cytoplasmic ratio (two times greater than normal), which gave them a "crowded appearance," and decreased glucose-6-phosphatase activity. During the course of the study, basophilic foci appeared to increase in size and number. Cytologic anaplasia also became more evident, ultimately culminating in the development of typical trabecular hepatocellular carcinomas by 44 weeks. Invasion of hepatic veins by basophilic foci, first noted at 10 weeks, was prominent by 20 weeks and indicated that many of the lesions manifested this characteristic of malignancy well in advance of the anaplastic features that are also diagnostic of hepatocellular carcinoma. The high growth rates of basophilic foci were confirmed by their greatly increased 3H-thymidine labeling indices, which were 20 times greater than background hepatocytes at 20 weeks following DEN injection. Tumor progression during the course of the study was also suggested by a doubling of labeling indices of hepatocytes in the basophilic foci between 20 to 28 weeks. (The term tumor progression is used in a broad biological sense to encompass any or all of the qualitative and quantitative changes describing the stepwise development of initiated cells to highly malignant neoplasms. This definition differs from the more clinical usage which restricts the process to qualitative changes during the late stages in the development of fully autonomous neoplasms.) An analysis of the number and size of transections through basophilic foci and in some cases, actual reconstructions of the foci from serial sections, indicated that, in aggregate, they grew exponentially between 10 to 36 weeks, with a volume doubling time of 2.5 weeks. The combined morphologic and kinetic data support the view that trabecular hepatocellular carcinomas develop from basophilic foci. Because of their ease of quantitation on conventional H&E stained sections, their rather uniformly spherical shapes, and the high probability of their clonal origin, the induced focal hepatic lesions should provide a useful model for studying tumor growth kinetics during carcinogenesis.

Age Factors↗

PubMed1990

Analysis of the autoimmune response in lupus mice: the behaviour and lifespan of anti-DNA-secreting B-cell clones.

We present the results of a study of the physical, haematological and serological features of the progress of the SLE-like syndrome in MRL/Mp-lpr/lpr and (NZB x NZW)Fl mice. As part of this study, we have analysed the IEF spectrotypes of anti-ssDNA antibodies in the sera of these mice and shown that the anti-ssDNA response is clonally restricted, as we have previously shown in a mouse chimaera model and in human SLE. Sequential qualitative and quantitative analysis of anti-ssDNA clonotypes has revealed that the lupus mouse anti-ssDNA clones are relatively short lived, having a lifespan of only 6 to 8 weeks, contrasting sharply with the much longer lifespan previously reported for a mouse anti-DNP-secreting clone and the exceptionally long lifespan of most anti-ssDNA-secreting clones of SLE patients. The implications of these observations for our understanding of the regulation of the autoimmune response are discussed.

Age Factors↗

PubMed1992

Spectrotypes of anti-DNA antibodies show that anti-DNA-secreting B-cell clones of SLE patients are restricted in number, stable and long lived.

We have investigated the number of B-lymphocyte clones secreting anti-ssDNA antibodies in SLE patients and a chronic active hepatitis patient by isoelectric focusing and reverse immunoblotting of serum antibodies. Individual clones can be identified by the unique pattern of bands produced by their antibodies (the clonotype). Using this technique, we have shown that the anti-DNA response of the majority of SLE patients is clonally restricted, in many cases only a single B-cell clone responding. We have also measured qualitative and quantitative changes in expression of B-cell clones and shown that these clones are remarkably stable with lifespans of up to six years or more. These results are in agreement with previous observations of clonal restriction of the anti-DNA response in three mouse models of SLE and in addition show that, unlike the mouse models, human anti-DNA-secreting B-cell clones are extremely stable and long-lived. The implications of these results for models of initiation and regulation of the autoimmune response are discussed.

Antibodies, Antinuclear↗

PubMed2003

Stromal cell heterogeneity in fibroblast growth factor-mediated stromal-epithelial cell cross-talk in premalignant prostate tumors.

Homeostasis of normal prostate and two-compartment nonmalignant prostate tumors is dependent on two-way communication between epithelial and stromal compartments. Independence of epithelial cells on controlling instructions from stroma is a hallmark of extremely malignant epithelial cell tumors. To better understand the evolution of stromal independence during malignant progression, we performed a clonal analysis of stromal cells derived from a well-defined model of two-way stromal-epithelial cell communication that loses response to stroma during prostate tumor progression. Directionally specific signaling from stroma to epithelium contributes to homeostasis between the two compartments. Stromal cells were characterized in respect to expression and activity of isotypes of the fibroblast growth factor (FGF) family of ligands and receptors in addition to morphology and cytoskeletal markers. One stromal subtype (DTS1) exhibited a fibroblast-like morphology and did not display smooth muscle cell (SMC) alpha-actin. The other (DTS2) exhibited SMC alpha-actin and an SMC-like morphology in vitro. Both subtypes expressed FGF7 and equally low levels of FGFR2IIIc mRNA, whereas fibroblast growth factor receptor (FGFR) 1 predominated in DTS1 cells. DTS1 cells also expressed FGF10 and no detectable FGFR3, whereas the absence of FGF10 and presence of FGFR3 distinguished DTS2 cells. Epithelial cell-derived FGF9 bound to FGFR and stimulated growth of specifically FGFR3-positive DTS2 cells, not the FGFR3-negative DTS1 cells. These results demonstrate stromal cell heterogeneity in signal reception of FGF from epithelium. This correlated with potential heterogeneity in the response back to epithelial cells. Epithelium-dependent control of a stromal cell phenotype within a tumor may be a determinant of whether tumors remain in nonmalignant homeostasis or progress to malignancy.

Adenocarcinoma↗

PubMed1992

Recent trends in research on differentiation of hematopoietic cells and lymphokines (hemopoietins).

A recent trend in hematological research fields has been to isolate and characterize hematopoietic stem cells/progenitors and their growth factors (hemopoietins) to gain a much better understanding of the nature of the stem cell and the mechanisms regulating its development. It is generally accepted that all the various types of blood cells develop from a single progenitor called a hematopoietic stem cell. Quantitative studies of the function of hemopoietic stem cells began two decades ago with the development of a spleen colony assay, and then, clonal cell culture techniques for committed progenitors were developed with several models for hematopoietic differentiation being proposed. Within the last few years, some hormones have been discovered that are known as hematopoietic growth hormones or hemopoietins, each of which is of protein nature and causes specific classes of blood cells to be made and primed. These hormones also enhance the function of the mature cells, the genes of which have recently been cloned. On the other hand, long-term bone marrow culture has recently permitted detailed investigations of the relationship between hematopoietic cells and the microenvironment in which they are found, e.g. stromal cells, in vitro, relating to the regulation of cell proliferation and differentiation. Further, in hematological fields, other bioactive factors including differentiation-inducing compounds, e. g. bioactive glycosphingolipids, and leukocyte-endothelial cell recognition molecules (adhesion receptors) have been discovered, the molecular mechanism(s) of which have yet to be elucidated. This communication focuses on recent advances in research on soluble hemopoietins and other bioactive factors relating to differentiation-induction and to cell-to-cell recognition.

Cell Differentiation↗

PubMed2003

Viral delivery of P450 reductase recapitulates the ability of constitutive overexpression of reductase enzymes to potentiate the activity of mitomycin C in human breast cancer xenografts.

Indolequinones such as mitomycin C (MMC) require enzymatic bioreduction to yield cytotoxic moieties. An attractive approach to overcome the potential variability in reductive bioactivation between tumors is to exploit specific enzyme-bioreductive drug combinations in an enzyme-directed gene therapy (GDEPT) approach. To this end, human breast cancer cell lines (T47D, MDA468, and MDA231) that overexpress either DT-diaphorase (DTD) or NADPH:cytochrome P450 reductase (P450R) have been developed. Cytotoxicity of MMC was evaluated in the panel of cell lines following aerobic or anoxic exposure in vitro. DTD and/or P450R overexpression sensitized cells to MMC in air with no further increase in the cytotoxicity of MMC under anoxia. The most profound effect was seen in the MDA468 cells, where a 27-fold increase in potency was observed for MMC in the DTD-overexpressing cell line. The MMC sensitization achieved through DTD and P450R overexpression in MDA468 cells was maintained in vivo. Xenografts established from the clonal lines exhibited significant tumor control following MMC treatment (treated/control [T/C] 17% and 51% for DTD and P450R xenografts, respectively) that was not seen in wild-type tumors (T/C 102%). Delivery of a clinically relevant adenoviral vector encoding P450R to MDA468 wild-type tumors yielded comparable P450R activity to that seen in the P450R clonal xenografts and resulted in greater MMC sensitization (T/C 46%). The model systems developed will facilitate the identification of novel indolequinone agents that are targeted toward a specific enzyme for bioactivation and are consequently of potential use in a GDEPT approach.

Adenoviridae↗

PubMedDate not supplied

Interaction between clonal plasma cells and the immune system in plasma cell dyscrasias.

The term "monoclonal gammopathy" (MG) includes a group of clonal plasma cell disorders, which show heterogeneous clinical behavior. While multiple myeloma (MM) and plasma cell leukemia (PCL) are incurable malignant diseases, most patients with MG of undetermined significance (MGUS) show an indolent/benign clinical course. Evidence has accumulated which supports the role of the bone marrow microenvironment in MG. Accordingly, the survival, drug-resistance and proliferation of MM cells have been shown to be largely dependent on a supportive microenvironment. Among the different environment-associated parameters, those related to the status/activity of the immune system are particularly relevant. This review focuses on the different ways clonal plasma cells (PC) interact with the immune system in different models of MG, to characterize crucial events in the development and progression of MG. These advances may support the design of novel therapeutic approaches in patients with MG.

Bone Marrow↗

PubMed2005

[Molecular and genetic characterization of rifampicin- and/or izoniazid-resistent strains of M. tuberculosis, isolated in Novosibirsk and Tomsk regions].

The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.

Antitubercular Agents↗

PubMed1988

The influence of insulin and sorbinil on myoinositol uptake in peripheral nerve from normal and diabetic rats and a neuroblastoma cell line (N1E-115).

The uptake of myo-inositol was investigated in femoral nerve fascicular preparations taken from control and streptozotocin-diabetic rats and in a clonal murine neuro-blastoma cell line (N1E-115), as a model of the neuronal component of the nerve preparation. Uptake was investigated in medium containing glucose, 5.6-25 mmol/l and inositol, 4 x 10(-5) mol/l. In the presence of glucose (25 mmol/l) myo-inositol uptake was decreased in nerve taken from streptozotocin-diabetic animals when compared to control (26.4 +/- 2.2 pmol/100 micrograms protein/2 h vs 55.1 +/- 2.4 pmol/100 micrograms protein/2 h, p less than 0.005). Uptake in both preparations was higher in the presence of insulin added during the uptake experiment (73.4 +/- 5.7 pmol/100 micrograms protein/2 h and 64.4 +/- 3.9 pmol/100 micrograms/2 h, respectively). Prior treatment of the animals with insulin or with the aldose reductase inhibitor, sorbinil also resulted in an increase in myo-inositol uptake in streptozotocin diabetic nerve preparations. In control nerve preparations and in N1E-115 cells raising the glucose concentration from 5.6 through 25 mmol/l was associated with decreased myo-inositol uptake, with an inhibitory constant (Ki) of 21.4 mmol/l and 20.4 mmol/l for femoral nerve and N1E-115 cells respectively. An increase in myo-inositol uptake was found in N1E-115 cells, following pre-treatment of cells in culture with sorbinil or inclusion of insulin during the uptake experiment. Altered myoinositol metabolism may play a role in the functional and structural changes characterizing diabetic neuropathy. The effects of hyperglycaemia on myo-inositol uptake in experimental diabetes may be modified by insulin or by inhibition of sorbitol accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗

PubMed1993

Neoplastic transformation of human cells in vitro.

Efforts to investigate the progression of events that lead normal human cells in culture to become neoplastic in response to carcinogenic agents have been aided by the development of the suitable in vitro model systems. For initial human cell transformation studies, a flat, nontumorigenic clonal line, originally derived from a human osteosarcoma (HOS), was used. When treated with chemical carcinogens such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 3-methyl-cholanthrene (3MC), the HOS cells underwent morphological alterations and acquired tumorigenic properties. These cell lines were very useful inasmuch as a non-ras cellular transforming gene, met, and an activated H-ras oncogene have been isolated from MNNG-transformed and 3MC-transformed HOS lines, respectively, by DNA transfection procedure. Alteration of p53 gene in chemically transformed HOS cell lines has recently been shown. Although carcinogens cause human cancer, normal human cells in culture have proven difficult to achieve. Neoplastic transformation of human cells in culture has recently been achieved by a stepwise fashion-immortalization and conversion of the immortalized cells to tumorigenic cells. One of the critical initial events in the progression of normal human cells to tumor cells is the escape from cellular senescence. With few exceptions, normal human cells require immortalization to provide a practical system for transformation studies. Thus, the role of carcinogenic agents in the development of human cancers is now being defined using a variety of human cells. The neoplastic transformation in human cell cultures is reviewed. In doing so, this author attempts to put into perspective the history of human cell transformation by carcinogenic agents, and to discuss the current state of the art in transformation of human cells in culture; thus providing insight into the molecular and cellular mechanisms involved in the conversion of normal cells to a neoplastic state of growth.

Cell Line, Transformed↗

PubMed2005

Anorexigenic hormones leptin, insulin, and alpha-melanocyte-stimulating hormone directly induce neurotensin (NT) gene expression in novel NT-expressing cell models.

Neurotensin (NT) is implicated in the regulation of energy homeostasis, in addition to its many described physiological functions. NT is postulated to mediate, in part, the effects of leptin in the hypothalamus. We generated clonal, immortalized hypothalamic cell lines, N-39 and N-36/1, which are the first representative NT-expressing cell models available for the investigation of NT gene regulation and control mechanisms. The cell lines express the Ob-Rb leptin receptor neuropeptide Y (NPY)-Y1, Y2, Y4, Y5 receptors, melanocortin 4 receptor, insulin receptor, and the NT receptor. NT mRNA levels are induced by approximately 1.5-fold to twofold with leptin, insulin, and alpha-melanocyte stimulating hormone treatments but not by NPY. Leptin-mediated induction of NT gene expression was biphasic at 10(-11) and 10(-7) M. The leptin responsive region was localized to within -381 to -250 bp of the 5' regulatory region of the NT gene. Furthermore, we demonstrated direct leptin-mediated signal transducers and activators of transcription (STAT) binding to this region at 10(-11) m, but not 10(-7) m leptin, in chromatin precipitation assays. Leptin-induced NT regulation was attenuated by dominant-negative STAT3 protein expression. These data support the hypothesis that NT may have a direct role in the neuroendocrine control of feeding and energy homeostasis.

Animals↗