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Biomedical subjects

K Dutt

Publications and source records attributed to K Dutt.

At least 19 recordsLinked to original sources
PubMed2000

Cell fate decisions in a human retinal precursor cell line: basic fibroblast growth factor- and transforming growth factor-alpha-mediated differentiation.

The purpose of this study was to determine if immortalized human retinal precursor cells could serve as a model to investigate cues that modulate cell fate and differentiation. We investigated the effects of a variety of growth factors broadly but specifically tested the effects of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)a in retinal cell differentiation and commitment. To determine the role of exogenously added growth factors in a human retinal precursor cell line (KGLDMSM), established from a first-trimester retina, cells were adapted to grow in a defined medium and exposed to a variety of trophic factors (epidermal growth factor [EGF], neuron growth factor [NGF], TGFalpha, TGFbeta, acidic FGF, and bFGF). Dose-response curves were developed to arrive at optimal concentrations. The neurotrophic potential of growth factors was determined by 3H-thymidine incorporation and bromodeoxyuridine (BrdU) labeling. The identity of the emerging neuronal phenotypes were determined by phase-contrast microscopy, immunolabeling for the neuron-specific antigens neurofilament protein (NF) and neuron-specific enolases (NSE), and photoreceptor-specific antigens (Rho1D4, 7G6) using immunocytochemistry and Western blot analysis. To identify some of the early response genes (c-fos, c-myc) expressed in response to growth factors, Northern blot analysis was performed. Almost all of the factors tested increased the total number of cells with a neuronal phenotype. Potency of growth factors to generate neurons was TGFalpha > bFGF > EGF > NGF. Both TGFalpha and bFGF, alone or in combination, increased the total number of neurons. Most of the neurons generated were photoreceptors, as depicted by the polarized phenotype, expression of photoreceptor-specific antigens, and processes resembling rudimentary outer segments. The increase in photoreceptor-like neurons is possibly attributable to an increase in numbers rather than greater survival. Additionally, the majority of the photoreceptors generated labeled with BrdU and for photoreceptor-specific antigens, suggesting that an inductive effect of bFGF and TGFalpha could occur in the cell cycle or shortly thereafter. Both bFGF and TGFalpha induced the expression of the early response gene c-fos while not altering the expression of c-actin or c-myc. The emergence of a photoreceptor phenotype was confirmed by both immunocytochemistry and Western blot analysis. The immortalized retinal precursor cell line could prove valuable in determining the role of exogenously added growth factors in retinal development and differentiation. Both bFGF and TGFalpha enhance the photoreceptor phenotype in medium-density cultures under conditions of defined medium. The same was confirmed by phase-contrast microscopy, immunocytochemistry, and Western blot analysis. Furthermore, cell fate determination in cultured precursor cells could occur during the late part of the cell cycle or shortly after completion of cell division. The effects of TGFalpha and bFGF seem to be slightly additive. The cell line will be extremely valuable in studying mechanisms of cell commitment and generation of retinal cell types, which could be tested for their potential for transplantation.

Cell Differentiation↗
PubMed1998

Expression of the protective proteins hemopexin and haptoglobin by cells of the neural retina.

The blood-retinal barrier, consisting of retinal pigment epithelial cells and retinal endothelial cells, prevents hemopexin and haptoglobin, anti-oxidant protective plasma proteins normally synthesized by the liver, from entering the neural retina. If present, these proteins must, therefore, be made locally. The cell types within the retina in which hemopexin and haptoglobin mRNAs are made have been investigated. RNA was extracted from both the neural retina and pigment epithelium obtained by dissection of human donor eyes as well as from cultured pigment epithelial and photoreceptor cells. The mRNAs for both haptoglobin and hemopexin were detected, using reverse-transcriptase polymerase chain reaction, in the neural retina and cultured photoreceptors but not in pigment epithelial cells. The cellular location of these mRNAs was determined using in situ hybridization of sections of human retina which revealed that haptoglobin mRNA was located principally in the photoreceptor cells, cells of the inner nuclear layer and some cells of the ganglion cell layer. Hemopexin mRNA, previously shown to be made in the human neural retina (Hunt et al., 1996. Journal of Cellular Physiology 168: 71-80), is expressed by most of the cells of neural retina including the photoreceptors and, notably, the ganglion cells.

Antioxidants↗
PubMed1998

Establishment and characterization of a retinal Müller cell line.

PURPOSE: Primary cultures of Müller cells have proven useful in cell biologic, developmental, and electrophysiological studies of Müller cells. However, the limited lifetime of the primary cultures and contamination from non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized cell line that exhibits characteristics of Müller cells. METHODS: Primary Müller cell cultures were prepared from retinas of rats exposed to 2 weeks of constant light. Cells were immortalized by transfection with simian virus 40. Single clones were obtained by repeatedly passaging cells using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial fibrillary acidic protein (GFAP)-specific and cellular retinaldehyde-binding protein (CRALBP)-specific antibodies. Transient transfections with CRALBP-luciferase constructs were performed by electroporation. RESULTS: Oncogene transformation resulted in the establishment of a permanent cell line that could be readily propagated. Immunocytochemical and immunoblotting studies demonstrated that the Müller cell line, rMC-1, expressed both GFAP, a marker for reactive gliosis in Müller cells, and CRALBP, a marker for Müller cells in the adult retina. Transient transfection assays showed that promoter-proximal sequences of the CRALBP gene were able to stimulate reporter gene expression in rMC-1. CONCLUSIONS: Viral oncogene transformation has been successfully used to isolate a permanent cell line that expresses Müller cell phenotype. The rMC-1 cells continue to express both induced and basal markers found in primary Müller cell cultures as well as in the retina. The availability of rMC-1 should facilitate gene expression studies in Müller cells and improve our understanding of Müller cell-neuron interactions.

Animals↗
PubMed1996

Proto-oncogene expression in cAMP and TPA-mediated neuronal differentiation in a human retinal cell line KGLDMSM.

PURPOSE: A human retinal cell line, KGLDMSM, developed by SV-40T antigen gene transfection, is stable in culture for a long period, unlike the primary cells. The cell line shows some degree of morphological differentiation with limited extension of stublike neurites upon transfer to defined medium. In our effort to explore genes implicated in neuritic extension and neuronal differentiation seen in response to cAMP and TPA, we have analyzed time dependent induction of a variety of proto-oncogenes: c-myc, H-ras, c-ras, and c-fos. METHODS: Cells were adapted to grow in defined media and exposed to differentiation inducing agents cAMP, TPA, Retinoic Acid, and sodium butyrata. Cells were assessed for phenotypic changes and altered expression of proto-oncogenes as evaluated by Northern Blot analysis and immunocytochemistry. RESULTS: Exposure of the cells to cAMP and TPA induced dramatic changes, with 100% of the cells extending neuritic processes. However, other differentiation inducing agents such as retinoic acid and sodium butyrata failed to elicit any response. We report that agents that promote neuritic extension also induce expression of c-fos. Transcriptional activation of c-fos in response to cAMP (30 min) and TPA (1hr) is also accompanied by expression of fos gene product as evaluated by using fos antibody. No fos expression was seen in uninduced cells. CONCLUSION: In retinal cell line KGLDMSM, agents that enhance neuronal differentiation (cAMP, TPA) also induce c-fos expression. Expression of c-fos may be a necessary prerequisite in neuronal differentiation and the established retinal cell line offers an excellent cell model for dissecting the molecular events underlying neuronal differentiation.

Cell Differentiation↗
PubMed1995

Vasculotropin/vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultured in vitro.

Vasculotropin (VAS), also called vascular endothelial growth factor (VEGF) or vascular permeability factor, is a secreted growth factor whose target cell specificity has been reported as restricted to vascular endothelium. Its effects are mediated by at least two distinct membrane-spanning tyrosine kinase receptors, KDR and flt-1; the expression of which also seems restricted to vascular endothelium. We describe here that cultured human retinal pigment epithelial (HRPE) cells express both KDR and flt-1 receptors, bind VAS/VEGF on two high affinity sites (apparent Kd of 9 and 210 pM corresponding to 940 and 18,800 sites per cell) and proliferate or migrate upon recombinant VAS/VEGF addition. HRPE cells also express the mRNA corresponding to the 121 and 165 amino acid forms of VAS/VEGF. HRPE cells release in their own culture medium and store in their extracellular matrix self-mitogenic and chemoattractant factors indistinguishable from 121 and 165 VAS/VEGF isoforms. The autocrine role of VAS/VEGF was confirmed by the inhibition of these bioactivities by neutralizing specific anti-VAS/VEGF antibodies.

Base Sequence↗
PubMed1994

Establishment of a human retinal cell line by transfection of SV40 T antigen gene with potential to undergo neuronal differentiation.

Recently, a number of laboratories have been interested in developing cell lines of ocular tissues to understand the pathogenesis of ocular diseases. Toward this end, we report here the generation of cell lines of human retina by transfection of simian virus SV40 T antigen gene. Established retinal cells grow as a monolayer and exhibit limited serum dependence. Phase-contrast and electron microscopic studies revealed distinct morphological cell types. Immunofluorescence studies showed that the established retinal cells were positive for neuron-specific enolase, neurofilament protein, glycine receptor, synaptophysin, and secretogranin. Cells were negative for glial fibrillary acidic protein, glutamine synthetase, galactocerebroside, and carbonic anhydrase II. In addition to neuronal features, a small percentage of flat cells were, however, positive for cellular retinaldehyde binding protein, and cells with the phenotype of rod and cone photoreceptor coexpressed opsin and interphotoreceptor retinoid-binding protein. An important feature of this cell line is that addition of phorbol ester and cAMP induced dramatic changes, with 100% of the cells extending long, thin neuritic processes. Thus, the established retinal cells would be useful for studies dealing with differentiation and plasticity of the cells of the nervous system.

Antigens, Polyomavirus Transforming↗
PubMed1994

Nucleoside transport sites in a cultured human retinal cell line established by SV-40 T antigen gene.

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.

Adenosine↗
PubMed1993

Transdifferentiation of adult human pigment epithelium into retinal cells by transfection with an activated H-ras proto-oncogene.

The identification of homologs to viral oncogenes in normal cells coupled with development of techniques for DNA transfer into cells offers a powerful approach to dissect the processes associated with differentiation-specific oncogenes. We have derived cell lines by transfection of viral DNAs and proto-oncogenes into primary retinal pigment epithelial (RPE) cells. Establishment of cell lines was successfully achieved with the SV40 large T-antigen gene activated form of Harvey (H)-ras proto-oncogene, c-myc, and adenovirus E1A. The cell lines derived using the H-ras oncogene appeared to contain cells with a neuronal phenotype. This feature was not observed in cell lines established with the other oncogenes. Characteristically, H-ras-transfected cells all exhibited features associated with neurons around 10-14 passages. The transdifferentiated cells were biochemically characterized and found to express neuronal markers, such as neurofilament protein and neuron-specific enolases. The specific neuronal changes were restricted to only two primary cultures of RPE derived from carcinoma donors. Although transdifferentiation of pigmented cells of iris, or the retina, into the lens has been demonstrated, our studies presented in this report provide evidence that RPE cells from adults can transdifferentiate into neurons under the influence of a specific oncogene. To the best of our knowledge, this is the first report on transdifferentiation of adult human pigment epithelium into a neuronal cell type.

Cell Differentiation↗
PubMed1991

Extracellular matrix mediated growth and differentiation in human pigment epithelial cell line 0041.

Efforts to grow differentiated pigment epithelial cells have led to a characterization of the growth kinetics of spontaneously established, continuously growing, human retinal pigment epithelial (PE) cell line 0041 on several biomatrices. These substrates were prepared from (a) placental and amniotic membrane, (b) commercially available basement membrane matrix (Matrigel), (c) dishes coated with extracellular matrix secreted by endothelial cells (ECM), (d) dishes coated with collagen IV and/or laminin, (e) dishes coated with collagen I and/or fibronectin. Our findings suggest that tissue culture plastic and dishes coated with collagen IV alone promote higher cell densities, while highest plating efficiency (24 hrs) was seen on tissue culture plastic and Matrigel. The highest degree of differentiation (epithelioid appearance, apical villi and junctional complexes) was seen in cells plated on dishes coated with collagen IV and extracellular matrix secreted by endothelial cells. Cells were epithelioid and polarized on those two substrates; they expressed fine finger-shaped villi and the highest degree of cell contact (in the form of junctions). Cells grown on Matrigel looked like fibroblasts and became deeply pigmented; however, the nature of the pigment remains to be determined. Collagen IV and ECM coated dishes, therefore, are most suitable for cultures of human PE cell line 0041 because they provide higher cell densities while retaining the differentiated state. This is the first report where an established pigmented epithelial cell line has been induced to become differentiated by use of extracellular matrices and extracellular matrix components.

Adolescent↗
PubMed1990

Establishment of human retinal pigment epithelial cell lines by oncogenes.

The primary human retinal pigment epithelial cells were transfected with oncogenic sequences derived from viruses and cellular homologues of retroviral oncogenes 'protooncogenes' linked to simian virus 40 (SV-40) and retroviral promoters. Foci of cells were noted between 2 to 4 weeks after transfection. Individual colonies of cells were expanded from cultures transfected with SV-40 virion DNA, SV-40 large T antigen gene, Ha-ras oncogene, human and mouse c-myc and adenovirus E1A gene. Established cell lines tested were positive for the specific oncogene sequences by Southern hybridization and also expressed the protein as assayed by immunofluorescence and immunoblot analysis. Cell lines established with SV-40 large T antigen, and SV-40 virion DNA, exhibited epithelioid morphology up to the 25th passage and later became more rounded. However, all cell lines established with other oncogenes continued to retain epithelial morphology. Functional analysis of the cell lines demonstrated the presence of polarity and the ability to phagocytize rod outer segments, characteristics of retinal pigment epithelial cells. The use of oncogenes with immortalization/transformation potential may allow the establishment of cell lines from ocular tissues for analysing the biochemical basis of a disease like retinitis pigmentosa.

Antigens, Polyomavirus Transforming↗
PubMed1989

In vitro phenotypic and functional characterization of human pigment epithelial cell lines.

We have characterized human retinal pigment epithelium (HRPE) for the expression of cell surface antigens. Primary HRPE cultures, established cell lines, and freshly brushed pigment epithelial cells all express HLA-ABC but not HLA-DR antigens. However, both primary cultures and established cell lines can be induced by gamma interferon stimulation to express HLA-DR in a dose dependent manner. Only freshly brushed HRPE cells express Fc, and no cells demonstrated the presence of C3b. Our results show that HRPE cells change in culture, as reflected by the loss of Fc receptors, but retain the ability to synthesize HLA-ABC spontaneously and HLA-DR upon stimulation.

Adolescent↗
PubMed1989

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Adult↗
PubMed1981

Glutamine synthetase induction in chick embryo retina monolayers.

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.

Animals↗
PubMed1981

Comparative effects of adriamycin and N-trifluoroacetyladriamycin-14-valerate on cell kinetics, chromosomal damage, and macromolecular synthesis in vitro.

N-Trifluoroacetyladriamycin-14-valerate differs from Adriamycin in its rapid intracellular transport and lack of fluorescent binding to nuclei or chromosomes. Both of these anthracyclines cause inhibition in the incorporation of labeled precursors into nucleic acids, extensive chromosomal damage, and arrest of cell cycle traverse in G2. In human lymphoid cells, N-trifluoroacetyladriamycin-14-valerate, unlike Adriamycin, does not show cell cycle phase-specific or proliferation-related cytotoxic effects. In an L1210 soft-agar assay, both Adriamycin and N-trifluoroacetyladriamycin-14-valerate show no enhanced sensitivity of mid-S-phase cells to their cytotoxic action.

Animals↗