Biomedical subjects

J Skouv

Publications and source records attributed to J Skouv.

14 recordsLinked to original sources

PubMed1995

Potent inhibitory effects of transplantable rat glucagonomas and insulinomas on the respective endogenous islet cells are associated with pancreatic apoptosis.

Effects of transplantable rat insulinomas (IN) and glucagonomas (GLU) on the endogenous pancreas were analyzed using morphometry, immunocytochemistry, in situ hybridization, and staining for apoptotic cells. Hyperinsulinemia (IN-rats) and hyper-GLP-1/glucagonemia (GLU-rats) were both associated with marked islet atrophy (67 and 76% of control average planimetrical islet area, respectively). Selective islet B cell inhibition of proinsulin (I and II) genes as well as of expression of the insulin gene transcription factor, IPF1/STF1, was found in IN-rats. Moreover, these islets were characterized by significant B cells apoptosis in the absence of infiltrating lymphocytes. In GLU-rats selective islet A cell inhibition was observed at the level of glucagon mRNA. These islets contained small, highly condensed but clearly active B cells with prominent IPF1/STF1-positive nuclei, surrounded by densely packed glucagon-negative cells with reduced cytoplasm. Furthermore, an active apoptotic process was found exclusively in the exocrine pancreas of GLU-rats. Thus, in IN-rats, islet B cell mass reduction is distinguished by non-immune-mediated programmed cell death, while GLU-rats exhibit A cell mass reduction by cytoplasmic retraction and selective exocrine apoptosis.

Animals↗

PubMed1994

Tumor-promoting phorbol ester transiently down-modulates the p53 level and blocks the cell cycle.

Activation of the protein kinase C signaling pathway by tumor-promoting phorbol esters, such as 4 beta-phorbol 12-myristate 13-acetate (PMA), induced a decrease in the level of p53 mRNA in several serum-starved human cell lines. Also, the tumor-promoting phosphatase inhibitor okadaic acid induced a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 mRNA was minimal after 9 h of exposure to PMA. After approximately 120 h, the p53 mRNA level was similar to the pretreatment level. PMA induced a similar transient decrease in the level of p53 protein in the A549 cell line. The decrease in the p53 mRNA level could not be explained by changes in the transcriptional rate or the p53 mRNA stability. The protein synthesis inhibitor cycloheximide completely abolished the PMA-induced down-modulation of the p53 mRNA, suggesting that a short-lived protein was involved in the down-modulation. Flow cytometric cell cycle analysis showed that the phorbol ester treatment induced a block in the late G1 phase. The blockage was transient, and its duration correlated with the level of p53 protein in the two cell lines. We propose that the protein kinase C-catalyzed phosphorylation of p53 may be a key event in the down-modulation of p53 expression as well as in the induced blockage of the cell cycle.

Cell Cycle↗

PubMed1993

Two phorbol ester receptor affinities in partially transformed human urothelial cells and decrease of receptor binding in desensitized cells.

The presence of specific binding sites for phorbol esters was studied in a transformed but non-tumorigenic human urothelial cell line HCV-29 by assay of specific binding of 3H-phorbol-12,13-dibutyrate (3H-PDBu) to intact living cells. 3H-PDBu bound specifically to HCV-29 cells in a saturable and competitive manner. Scatchard plot analysis of specific binding yielded a curved plot consistent with two binding sites with Kd of 11 nM and 102 nM, respectively. At saturation the corresponding PDBu binding capacities (Bmax) were 8.8 pmol/10(6) cells (5.2 x 10(6) molecules bound per cell) and 2.8 pmol/10(6) cells (1.7 x 10(6) molecules bound per cell). 3H-PDBu binding was displaced by biologically active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein, but not by tumor promoters such as L-tryptophan, anthranilic acid and sodium saccharin. In cells desensitized by pretreatment with 1 microgram/ml (2 microM) TPA or PDBu for 24 h the level of binding was reduced to 28% of the level in non-exposed cells. The ability of desensitized cells to bind 3H-PDBu was gradually restored within 5-6 days. At the same time the cells became sensitive to the morphological alteration induced by PDBu. This suggests that desensitization of HCV-29 cells is due to a decreased receptor-ligand binding capacity probably associated with down regulation of the phorbol ester receptors.

Caenorhabditis elegans Proteins↗

PubMed1990

Ribosomal protein S1 of Escherichia coli is the effector for the regulation of its own synthesis.

To facilitate the study of the regulation of the rpsA gene, a translational fusion between the rpsA gene and the lacZ gene was constructed. Synthesis of the fusion protein was repressed about 10-fold when rpsA was supplied in trans on a multicopy plasmid. This repression is similar to the post-transcriptional regulation previously found for the wild type rpsA gene. Addition of purified protein S1 to a coupled in vitro transcription-translation system caused a specific reduction in the synthesis of the rpsA-lacZ fusion protein. Addition of various subdomain fragments of protein S1 to the coupled in vitro system showed that the N-terminal fragment, possessing the ribosome binding domain of protein S1, was able to repress the synthesis of the rpsA-lacZ fusion protein. In contrast, fragments from the C-terminal region, containing the nucleic acid binding domain of protein S1, were inactive in this repression. Induction of truncated rpsA genes, coding for either the N-terminal 101 or 329 amino acids caused a reduction in the synthesis of the chromosomally encoded protein S1, thus confirming in vivo that the N-terminal part of protein S1 represses rpsA expression.

Amino Acid Sequence↗

PubMed1990

The sialosyl Lewis a ganglioside is present in tumorigenic human urothelial cell lines.

The sialosyl Lewis a antigen was identified in a ganglioside extract from a malignant human urothelial cell line (Hu 1703He) by fast atom bombardment mass spectrometry. The presence of this antigen in urothelial cell lines with varying tumorigenic properties was further studied using the 19-9 monoclonal antibody (MAb). The sialosyl Lewis a ganglioside was expressed only in the tumorigenic cell lines. Thus, the expression of this antigen is a marker of malignancy for human urothelial cell lines. Mass spectrometry also suggests that the fucosyl GM1 ganglioside was expressed in the Hu 1703He cell line. This was confirmed using the F12 MAb, specific for fucosyl GM1. However, the expression of this antigen was not confined to cell lines with tumorigenic properties.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1990

Deregulation in trans or c-myc expression in immortalized human urothelial cells and in T24 bladder carcinoma cells.

The expression of a number of cellular oncogenes was investigated in human urothelial cell lines with different in vitro growth properties. Constitutively elevated levels of expression of c-myc RNA were found in Hu609, an immortalized, nontumorigenic cell line that was derived from normal urothelium, and in the bladder carcinoma cell line T24. Potential mechanisms that might underlie deregulation of c-myc expression in these cells were investigated. It was found that the c-myc gene was apparently intact and not amplified in Hu609 and T24. No increased stability of c-myc RNA was detected. A c-myc-CAT fusion construct containing 2.5 kb of normal c-myc 5' sequences showed levels of expression that paralleled the overexpression of the endogenous gene, indicating that the high constitutive levels of c-myc expression were due, at least in part, to alterations in the activities of cellular trans-acting transcriptional regulators.

Animals↗

PubMed1990

Reduced HLA-A,B,C expression in tumourigenic v-raf transfected human urothelial cells.

Tumourigenic (TGrIII) human urothelial cells grown in vitro have previously been demonstrated to have a markedly decreased expression of beta 2-microglobulin and HLA-A,B,C antigens as compared to non-tumourigenic (TGrII) human urothelial cell lines. Furthermore, during 'spontaneous' in vitro transformation of a non-tumourigenic (TGrII) human urothelial cell line Hu609 into a tumourigenic (TGrIII) subline Hu609T/LLH, changes in morphology and tumourigenicity have been demonstrated to be accompanied by a decreased HLA-A,B,C expression. After malignant transformation of the non-tumourigenic (TGrII) human urothelial cell line HCV29 by DNA transfection with the v-raf oncogene, four sublines could be isolated. In this study we have investigated these sublines for their expression of membrane bound HLA-A,B,C antigens and provide further evidence that an inverse relationship exists between tumourigenicity and monomorphic HLA-A,B,C expression. Treatment of the cells with recombinant human interferon alpha for 3 days increased the expression of HLA-A,B,C antigens by 50-150% indicating that at least some of the reduced HLA-A,B,C expression could be due to decreased synthesis of HLA-A,B,C antigens. All the transfected cell lines overexpress v-raf and c-myc.

Cell Line↗

PubMed1989

Malignant transformation of human bladder epithelial cells by DNA transfection with the v-raf oncogene.

Transfection of the v-raf oncogene into immortalized, nontumorigenic human bladder epithelial cells resulted in the isolation of two tumorigenic transformants. Both were identified as human and of the same origin as the parent cell line by human leukocyte antigen typing and Southern blot analysis. Both the primary tumorigenic transfectants and the cell lines established from the induced tumors expressed v-raf mRNA and v-raf protein. In both tumorigenic transformants the level of c-myc mRNA was enhanced compared with that of the parent cell line.

Animals↗

PubMed1988

Plasminogen activator inhibitor type-1 protein, mRNA and gene transcription are increased by phorbol esters in human rhabdomyosarcoma cells.

By use of an enzyme-linked immunosorbent assay, we have found that phorbol 12-myristate 13-acetate (PMA) causes an approximately 10-fold increase in the level of type-1 plasminogen activator inhibitor (PAI-1) accumulated in conditioned medium of the human rhabdomyosarcoma cell line. Half-maximal stimulation occurred at approximately equal to 15 nM PMA. The effect was only observed with phorbol esters that are tumor promoting. Maximal levels of secreted PAI-1 were observed 24 h after PMA addition. The increase in secreted PAI-1 was preceded by a transient approximately 10-fold increase in intracellular PAI-1 content, maximal at 8 h after PMA addition. There was a 20-fold increase in the cellular level of two 2.3- and 3.4-kilobase PAI-1 mRNAs and a more than 5-fold increase in the PAI-1 gene transcription rate. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increased the level of PAI-1 mRNA, and when both cycloheximide and PMA were used, an additive effect was observed. Cycloheximide changed the ratio between the two PAI-1 mRNAs in favor of the 3.4-kilobase species. Overall, the data show that transcriptional activation of the PAI-1 gene forms part of the pleiotropic responses to tumor-promoting phorbol esters.

Blotting, Northern↗

PubMed1987

Differential induction of transcription of c-myc and c-fos proto-oncogenes by 12-O-tetradecanoylphorbol-13-acetate in mortal and immortal human urothelial cells.

The effect of the skin tumor-promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on expression of cellular proto-oncogenes has been examined in cell lines derived from human urothelium. A single treatment with TPA (1 microgram/ml) increased the transcription of c-fos and c-myc proto-oncogenes at least 20-fold in the mortal cell line HU 1752. The induction was transient and was accompanied by a rapid but transient change in cell morphology. When immortalized cell lines were treated with TPA a similar rapid and transient morphological response was observed, but the TPA treatment only increased the level of c-fos mRNA, suggesting that the normal regulation of c-myc transcription is altered in immortalized cells irrespective of their tumorigenic properties. The levels of c-Ha-ras and c-Ki-ras mRNAs were unaffected by TPA treatment in all cell lines.

Cell Line↗

PubMed1986

The skin tumor-promoter 12-O-tetradecanoylphorbol-13-acetate induces transcription of the c-fos proto-oncogene in human bladder epithelial cells.

The effect of a single treatment with the skin tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of the cellular proto-oncogenes, c-myc, c-rasHa, c-rasKi and c-fos was examined in the non-tumorigenic human bladder epithelial cell line HCV 29. TPA (1 microgram/ml) increased the transcription of the c-fos gene of HCV 29 at least 50-fold, and this stimulation was observed within minutes. The response was transient, and was accompanied by a rapid and transient change in cell morphology. The expression of c-myc, c-rasHa and c-rasKi were not enhanced by the TPA treatment. These results show that human bladder epithelial cells respond to a known skin tumor-promoter, TPA, by altering the transcription of a specific proto-oncogene in these cells.

Cells, Cultured↗

PubMed1984

Transcriptional organization of the rpsA operon of Escherichia coli.

Three strong and two minor rpsA promoters were found by nuclease S1 mapping, promoter cloning and in vitro transcription. The longest transcript encodes a protein, located upstream from rpsA with a molecular weight of 25,000. The identity of this protein remains to be established. The other rpsA promoters are located within the gene for this 25 K protein. The rpsA leader region including the sequence of the 25 K protein and its promoter was DNA sequenced.

Base Sequence↗

PubMedDate not supplied

Gangliosides in human urothelial cell lines of different transformed phenotypes: the effect of v-raf-oncogene transfection.

In a previous study we have shown that established human urothelial cell lines, representing grade of transformation II (TGr II, non-tumorigenic, non-invasive cells), are characterized by accumulation of the GM2 ganglioside as compared to cell lines of TGr III with tumorigenic and invasive properties. In the present study, the analysis of gangliosides from two tumorigenic sublines obtained after transfection of the TGr II cell line HCV 29 with the v-raf-oncogene, provided further evidence for the inverse relationship between tumorigenicity and the GM2 ganglioside expression. The two transfected sublines: T112C1 and T112D1, representing TGr III, were characterized by a decreased level of GM2 which was accompanied by an increased content of the GM3 ganglioside as compared to the parental HCV 29 cell line.

Cell Line↗

PubMedDate not supplied

Detection of oncogene mRNA sequences in cultured cells by in situ hybridization.

The present study was undertaken to determine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultured cells. Following in situ hybridization with 32P-labeled v-src and v-Ha-ras DNA probes, src and Ha-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-Ha-ras transfected cell lines, the number of silver grains over individual cells were significantly higher (p less than 0.001, t-test) than in a non-transfected, non-tumorigenic, rat esophageal epithelial cell line. There was a highly variable number of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-Ha-ras DNA or that were pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times more oncogene related mRNAs than non-transfected cells as judged by the numbers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirmed that the expression of src and Ha-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of human cells and to human tumor cells in culture.

Animals↗