HT Cells Research Articles

Osmoadaptation-related genes in inner medulla of mouse kidney using microarray

To distinguish biological molecular processes of osmotic stress occurring in inner medulla, we utilized microarrays to monitor expression profiles. RNAs from three segments (cortex, outer medulla, and inner medulla) of mouse kidney were isolated and applied to microarrays. We found 35 genes expressed highly in inner medulla. Next, microarrays for the RNAs from mouse medullary collecting duct cell line (mIMCD) cells and osmotically adapted mIMCD cells (HT cells) were performed (designed as resistant to 1270 mOsm/H 2O). Of 35 genes highly expressed in inner medulla, 6 genes such as; B-cell translocation gene protein (BTG), myc-basic motif homologue, gelsolin, cell surface glycoprotein, laminin β2, and tubulo-interstitial nephritis antigen, were also expressed highly in HT cells. Using real-time PCR, we confirmed the expression of six genes. Additionally acute osmotic stress induced the BTG. By comparing the inner medulla to a mIMCD3, we identified genes which respond to acute and chronic hyperosmotic stress.

Ascorbate and glutathione metabolism in two sunflower cell lines of differing α-tocopherol biosynthetic capability

The natural antioxidants, tocopherols and ascorbate (ASC), are of great interest in terms of human health, because of their role in the prevention of chronic diseases. In cell metabolism, tocopherols are the major lipid-soluble antioxidants, whereas ASC and glutathione (GSH) are hydro-soluble antioxidants. These three metabolites cooperate in scavenging for oxygen radicals and protecting cell membranes. ASC and GSH are required in the process of regeneration of tocopherol from its α-cromanoxyl radical, while, GSH donates electrons for the reduction of dehydroascorbate (DHA), the fully oxidised form of ASC. Two cell lines of sunflower ( Helianthus annuus L. cv Gloriasol) with differing capability to synthesise α-tocopherol were identified. In spite of the differing content of α-tocopherol (almost threefold higher in the high synthesising cell line, HT, than in the low synthesising one, LT), the cell lines have comparable growth curves. In the cells collected in the stationary phase, the ASC and GSH pools are also significantly higher in the HT cells than in the LT cells. On the other hand, the enzymes responsible for H 2O 2 scavenging and ASC and GSH recycling had higher activity in the LT than in the HT cells. The cooperation between the three antioxidant systems in the maintenance of the cellular redox balance is discussed, as well as the possible utilisation of the HT cell line for the in vitro production of natural antioxidants.

Characterization of Hydrogenobacter thermophilus cytochromes c(552 )expressed in the cytoplasm and periplasm of Escherichia coli.

Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).

Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients.

The role of c-Src in growth signaling by angiotensin (Ang) II was investigated in vascular smooth muscle cells (VSMCs) from arteries of hypertensive patients. c-Src and extracellular signal-regulated kinase 1/2 (ERK1/2) activity, proto-oncogene expression, activating protein-1 (AP-1) DNA-binding activity, and DNA and protein synthesis were studied in Ang II-stimulated VSMCs derived from small peripheral resistance arteries of normotensive subjects (NTs, n=5) and age-matched untreated hypertensive patients (HTs, n=10). Ang II type 1 (AT(1)) and type 2 (AT(2)) receptor status was also assessed. Ang II dose-dependently increased the synthesis of DNA and protein, with enhanced effects in VSMCs from HTs. PD 098,059, a selective inhibitor of the ERK1/2 pathway, attenuated Ang II-stimulated growth in HTs. The effects of PD 098,059 were greater in HTs than in NTs. In NTs, Ang II transiently increased ERK1/2 phosphorylation, whereas in HTs, Ang II-stimulated actions were augmented and sustained. PP2, a selective Src inhibitor, reduced ERK1/2 activity and normalized ERK1/2 responses in HTs. Ang II-induced c-Src phosphorylation was 2- to 3-fold greater in HTs than in NTs. In HTs but not NTs, kinase activation was followed by overexpression of c-fos and enhanced AP-1 DNA-binding activity. PD 098,059 and PP2 attenuated these responses. AT(1) receptor expression was similar in NTs and HTs. In HT cells transfected with c-fos antisense oligodeoxynucleotide, Ang II-stimulated growth was reduced compared with sense oligodeoxynucleotide. Our findings suggest that augmented Ang II-stimulated VSMC growth is mediated via hyperactivation of c-Src-regulated ERK1/2-dependent pathways, leading to overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. Because AT(1) receptor expression was unaltered in HTs, increased Ang II signaling may be a postreceptor phenomenon. These data define a signal transduction pathway whereby Ang II mediates exaggerated growth in VSMCs from HTs.

THE ROLE OF CASPASE 3 AND BclxLIN THE ACTION OF INTERLEUKIN 7 (IL-7): A SURVIVAL FACTOR IN ACTIVATED HUMAN T CELLS

The effects of interleukin 7 (IL-7) on apoptosis in interleukin 2 (IL-2)-dependent, activated, primary, human T lymphocytes (hT cells) was examined. IL-7 (like IL-2) rescued cells from apoptosis, as measured by their cellular DNA profile and fragmentation. IL-2 also acted as a mitogen in these T cells. Both cytokines abrogated the dexamethasone-induced stimulation of Caspase 3 and prevented the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate for the Caspase 3. IL-7 upregulated the expression of BclxLand counteracted the downregulation of this anti-apoptotic protein by the synthetic glucocorticoid, dexamethasone. Bcl-2 protein expression was uupregulated by IL-7 with or without dexamethasone, but Bcl02 was expressed at a much lower level than BclxLin these cells. Levels of Bax did not markedly change on either cytokine stimulation or dexamethasone treatment. An unidentified 23-kDa band, which was recognized by the anti-Bcl-2 antibody, was induced by dexamthasone and suppressed by IL-7 and IL-2. This protein was subject to independent regulation as compared to the p26 Bcl-2 protein, suggesting that it may be a novel factor, possibly involved in the regulation of apoptosis. A clear role for IL-7 as a survival factor for cytokine withdrawal and glucocorticoid induced apoptosis in activated primary hT cells is implicated. In addition, regulation of BclxLand downstream inhibition of Caspase 3 activity may mediate this rescue signal.

Tamoxifen-mediated anti-cellular effect against a choriocarcinoma cell line.

The present study was undertaken to evaluate the efficacy of using steroid hormone antagonists tamoxifen and Ru486 for chemotherapy or chemoprevention of choriocarcinoma or other less malignant gestational trophoblastic diseases (GTDs) such as invasive mole. Using 4 trophoblast cell lines, we have shown that tamoxifen (>/= 2 microM) has anti-growth activity on the choriocarcinoma cell line BeWo but not on the other cell lines in a time and dose dependent manner while Ru486 invariably had no detectable effect. Based on a radioimmunoassay, we have been able to detect low levels of estrogen receptors on BeWo (6 +/- 0.4 fm/mg; Kd=438+/- 73 pM) and JEG-3 (6.55 +/- 1.2 fm/mg; Kd=710 +/- 42 pM) cells and progesterone receptors on HT (48.62 fm/mg; Kd=1,690 +/- 182 pM) and TL (8.46 fm/mg; Kd=1,540 +/- 115 pM) cells. However, there is no definite correlation between steroid responsiveness and the presence of the receptors. The mechanism of our observed tamoxifen-mediated anti-cellular effect is uncertain and characteristics commonly associated with apoptotic cell death were not observed. The level of neither wild-type nor mutant forms of the p53 protein correlated with sensitivity to tamoxifen. Our results suggest that estrogen may be a growth hormone for some trophoblasts and tamoxifen may be potentially useful for the treatment of selected cases of choriocarcinoma or other trophoblastic diseases.

Selectively enhanced cellular signaling by Gi proteins in essential hypertension. G alpha i2, G alpha i3, G beta 1, and G beta 2 are not mutated.

Recent studies have shown an enhanced signaling capacity of receptors coupled to pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G proteins) in immortalized B lymphoblasts from patients with essential hypertension. In the present study, we analyzed (1) whether such alterations would also be expressed in nontransformed cells of these individuals and (2) whether other G protein-mediated signaling pathways were also altered. Therefore, we established primary cultures of skin fibroblasts from previously characterized normotensive and hypertensive individuals (NT and HT cells, respectively). [Ca2+]i rises induced by lyso-phosphatidic acid (LPA), thrombin, and sphingosine-1-phosphate as well as the formation of inositol 1,4,5-trisphosphate and [3H]thymidine incorporation evoked by LPA were PTX sensitive and enhanced twofold in HT fibroblasts. In contrast, cellular responses induced by bradykinin, endothelin-1, and angiotensin II (all PTX insensitive) were similar in NT and HT cells. Formation of cAMP induced by stimulation of Gs with isoproterenol was identical in NT and HT cells. Western blot analysis yielded no evidence for an overexpression of G alpha i2, G alpha i3, G beta 2, and G beta 4. Furthermore, sequencing of cDNAs encoding for the ubiquitously expressed PTX-sensitive G protein subunits G alpha i2, G alpha i3, G beta 1, and G beta 2 from NT and HT cell lines yielded no evidence for mutations in these genes. Although the molecular mechanisms remain to be defined, these data support the concept of a selective enhancement of signal transduction via PTX-sensitive G proteins in essential hypertension.

Production of a Monoclonal Antibody to an Antigen Present on Both Trophoblasts and Leukocytes

In the present study, we report the establishment of a monoclonal antibody (Mab) designated F10 that recognized an antigen commonly shared by human trophoblasts and leucocytes. F10 MAb was obtained using cell membrane components from a trophoblast cell line HT as immunogen. Based on immunochemical studies, the F10 reactive antigen (F10-Ag) could be located on both villous and nonvillous trophoblasts from early and term placental tissues and on all trophoblastic cell lines. In addition, flow cytometry revealed that most ( > 95%) peripheral blood lymphocytes, monocytes, as well as polymorphonuclear leukocytes (PMN) were positively stained with F10 MAb. Immunoblotting with F10 MAb identified two protein bands with apparent molecular mass of 62 and 56 kDa. Furthermore, the antigens were glycoproteins and were glycosylated via the O-linkage. Scatchard plot analyses of the binding data between 125I-labeled MAb F10 IgG and HT cells revealed a single class of F10 binding sites with an apparent dissociation constant (Kd) of 10.54 +/- 2.03 pM and maximum binding-site (Bmax) value of 2.1 +/- 0.11 x 10(6) sites per cell. We suggest that F10 may be useful for the identification of a novel epitope that is commonly shared by all trophoblasts and leukocytes and such an epitope may be potentially active in maternal-fetal interactions.

Growth inhibition of human lymphoma cell lines by the marine products, dolastatins 10 and 15.

Dolastatins 10 and 15 are small peptides isolated from the marine sea hare Dolabella auricularia. In vitro studies of these peptides have demonstrated antimitotic and antiproliferative activity and growth inhibition in hematopoietic progenitor cells. The purpose of our in vitro study was to determine the biological effects of these marine peptides on growth of human lymphoma cell lines and to investigate mechanisms by which the dolastatins may act. Cell lines DB, HT, RL, and SR were grown from the ascites or pleural effusion of four patients with lymphoma. The DB, HT, and RL cell lines are of B-cell origin, and the SR cell line appears to be a less differentiated lymphoid cell type. Cells from these lines were cultured in the presence of vincristine or dolastatin 10 or 15. [3H]Thymidine-uptake assays were used to measure effects on DNA synthesis. Cell cycle analysis using propidium iodide was performed to measure drug-induced cell-cycle arrest. DNA fragmentation was used as an assay for drug-induced apoptosis and was measured by agarose gel electrophoresis. In the three B cell lines, dolastatin 10 was more effective than dolastatin 15. Values for concentrations required for inhibition of proliferation by 50% (IC50) were .00013-.0013 nM for dolastatin 10 in each cell line; values for dolastatin 15 were approximately .13 nM in DB and HT cells and .0013-.013 nM in RL cells. SR cells were more sensitive to dolastatin 15 than to dolastatin 10 (IC50 = .00013-.0013 nM versus .0013-.013 nM). Both dolastatins arrested more than 70% of cells in mitosis in all cell lines. This effect was reversed if the drug was removed by 4 hours, but by 8 hours of exposure, reversal was not possible. Both dolastatins 10 and 15 produced apoptosis in DB and HT cells but not in the other two cell lines. We have demonstrated that dolastatins 10 and 15 have a profound antiproliferative effect on four different human lymphoma cell lines and that the dolastatins are approximately 3-4 logarithms more effective as antiproliferative compounds, on a molar basis, than vincristine--a clinically useful, antiproliferative agent. These data support the hypothesis that apoptosis, as measured by DNA fragmentation, appears to be a cell-specific response and may not be directly related to the antimitotic effect of the dolostatins. Our results suggest that these compounds may be good candidates for development as antineoplastic agents.

Hypertetraploid cells in vocal cord epithelia.

DNA measurements yield information about the nature of cells and may provide diagnostic and prognostic information. Static cytofluorometry was performed on smears removed at microlaryngoscopy from 107 vocal cord lesions (96 patients). All stem cell lines were diploid except 3; 2 carcinomas and 1 severe dysplasia were polyploid. The mean proliferative activity (percentage of nuclei greater than diploid peak) was 2.1% for the group of epithelia with hyperplasia and mild dysplasia, 3.1% for those with moderate dysplasia, 4.0% for severe dysplasia, and 6.8% for carcinomas. Hypertetraploid cell nuclei (HT cells) were not found in epithelia with hyperplasia and mild dysplasia. Seven out of 15 patients with epithelia showing moderate dysplasia had HT cells; 5 of these patients developed a carcinoma. One of 8 without HT cells developed a severe dysplasia. Nine patients with severe dysplasia had HT cells; 4 had recurrences and 4 developed carcinoma within 4 years. In 14 patients without HT cells, 3 had recurrences and 1 developed a carcinoma 6 years later. HT cells were found in 15 patients with T1 & T2 carcinomas; residual carcinoma was present in 2 after radiotherapy and 4 had recurrences within 11 months. Fourteen patients with T1 & T2 carcinoma did not have any HT cells; one had residual carcinoma after radiotherapy and 3 had recurrences between 18 months and 4 years. DNA measurements and, especially, the demonstration of epithelia with HT cells prove to be of prognostic importance.(ABSTRACT TRUNCATED AT 250 WORDS)

T2-T5 spinothalamic neurons projecting to medial thalamus with viscerosomatic input.

Spinothalamic tract neurons projecting to medial thalamus (M-STT cells), ventral posterior lateral nucleus (VPL) of the thalamus (L-STT cells), or both thalamic regions (LM-STT cells) were studied in 19 monkeys anesthetized with alpha-chloralose. Twenty-seven M-STT cells were antidromically activated from nucleus centralis lateralis, nucleus centrum medianum, or the medial dorsal nucleus. Stimulation of VPL elicited antidromic responses from 22 cells and 13 cells were activated from both VPL and medial thalamus. Antidromic conduction velocities of M-STT cells were significantly slower than those of L-STT or LM-STT cells. M-STT cells were located in laminae I, IV, V, and VII with greater numbers found in the deepest laminae. L-STT cells were located mostly in lamina IV, whereas most LM-STT cells were found in lamina V. Twenty-four of 27 M-STT cells, all L-STT cells, and all LM-STT cells received input from both cardiopulmonary sympathetic and somatic afferent fibers. WDR cells were most common among the L-STT and LM-STT groups, whereas HT cells were the most common class in the M-STT cell group. Excitatory receptive fields of M-STT cells were large, and often bilateral. Receptive fields of L-STT cells were simple and never bilateral. Receptive fields of LM-STT cells could be similar to M-STT or L-STT cells. Thirty-three percent of the M-STT cells, 37% of the L-STT cells, and 62% of the LM-STT cells had inhibitory receptive fields. Inhibition was elicited most often by a noxious pinch of the hindlimbs. Sixteen of 23 (70%) M-STT cells received C-fiber cardiopulmonary sympathetic input in addition to A-delta-fiber input. The other 7 cells received only A-delta-fiber input. Only 45% of the L-STT cells and 38% of the LM-STT cells received both A-delta- and C-fiber inputs. The maximum number of spikes elicited by A-delta-input was related to segmental locations for L-STT cells with greatest responses in T2 and lesser responses in more caudal segments; however, no such trend was apparent for M-STT cells or for responses to C-fiber input for either group. Electrical stimulation of the left thoracic vagus nerve inhibited 7 of 18 M-STT cells, 10 of 16 L-STT cells, and 6 of 12 LM-STT cells. These results are the first description of visceral input to cells projecting to medial thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Thymus-histone treated BHK21 cells; malignant characteristics in vitro but reduced tumorigenicity

BHK 21/C 13 cells have been treated in maintenance medium with bullock thymus histones. This has produced a line of cells, which we have designated BHK 21/C 13/HT, capable of continuous sub-culture, and which shows virtually all the in vitro characteristics associated with malignantly transformed cells. These include completely disordered growth pattern, growth in semi-solid agar, growth in the presence of low concentrations of serum, as well as high cloning efficiency. In spite of this, the cell line produced has been shown to be considerably less tumorigenic than the original BHK 21/C 13 cells. Its properties have been compared with BHK 21/C 13/Py 6 cells. The evidence presented suggests that the appearance of the HT cells is due to transformation and not to selection. Possible mechanisms, by which exogenous histone could interact with cultured cells, as well as reasons which lead to the breakdown in the dissociation between in vitro and in vivo malignancy are discussed.

Properties of cells derived from adenovirus-induced hamster tumors by long-term in vitro cultivation : II. Nature of the restricted response to type 2 adenovirus

The response of cultured cells derived from type 12 adenovirus-induced hamster tumors (HT cells) to superinfection with type 2 adenovirus (Ad.2) has been studied in detail. The low yield of infectious Ad.2 from such cultures has been shown, by infectious center and mass yield analyses, to be due to the presence of a small number of virus-producing cells. However, all superinfected HT cells are capable of producing viral structural antigen(s), provided the input multiplicity of Ad.2 is at least of the order of 10–100 PFU per cell. The restricted production of infectious progeny is not mediated by a transmissible interfering factor such as interferon. HT cells are as competent to produce vesicular stomatitis virus as are normal hamster cells. SV40-transformed hamster cells and normal hamster cells produce comparable (large) amounts of type 2 adenovirus. The evidence is consistent with the hypothesis that the response of HT cells to superinfection with Ad.2 is controlled by persistent genetic material of type 12 adenovirus responsible for the initiation of the malignant change. Several considerations are discussed which suggest that the response to superinfection is a more specific indicator of genome persistence than other identified properties of HT cells and that, operationally, the relation of the Ad.12 genome to HT cells may be analogous to that of a defective prophage to its bacterial host.

Properties of cells derived from adenovirus-induced hamster tumors by long-term in vitro cultivation : I. Clonal stability of three biological characteristics

After several hundred generations in vitro, HT cells originally derived from type 12 adenovirus (Ad.12)-induced hamster tumors have retained their malignant character, the capacity to synthesize Ad.12-specific antigen(s), and a significantly reduced capacity, compared with that of normal hamster cells, to produce infectious type 2 adenovirus (Ad.2) in response to superinfection. Of four independently derived cell lines studied, one (HT2) consistently produces less Ad.2 than the other three lines. No evidence has been obtained for “rescue” of Ad.12 among the yields from Ad.2-superinfected HT cells. The low yield from such cells is not due to poor adsorption of inoculum virus. Thirteen clones of two cell lines (HT2 and HT8) have been established after microdrop isolation of single cells. The fact that all clones tested have retained the three characteristics suggests genetic homogeneity of the parental populations. In particular, the differing extent to which synthesis of Ad.2 is depressed in the two parental lines is reflected in the response of their derived clones. The evidence argues against the idea that HT cell populations consist of a majority of genetically stable “resistant” and a minority of stably “susceptible” cells.