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Biomedical subjects

L J Erkell

Publications and source records attributed to L J Erkell.

At least 19 recordsLinked to original sources
PubMed2000

Radiation-induced double-strand breaks in mammalian DNA: influence of temperature and DMSO.

PURPOSE: To investigate the effects of subphysiological irradiation temperature (2 28 degrees C) and the influence of the radical scavenger DMSO on the induction of double-strand breaks (DSB) in chromosomal DNA from a human breast cancer cell line (MCF-7) as well as in intact cells. The rejoining of DSB in cells irradiated at 2 degrees C or 37 degrees C was also investigated. MATERIALS AND METHODS: Agarose plugs with [14C]thymidine labelled MCF-7 cells were lysed in EDTA-NLS-proteinase-K buffer. The plugs containing chromosomal DNA were irradiated with X-rays under different temperatures and scavenging conditions. Intact MCF-7 cells were irradiated in Petri dishes and plugs were made. The cells were then lysed in EDTA-NLS-proteinase-K buffer. The induction of DSB was studied by constant field gel electrophoresis and expressed as DSB/100/Mbp, calculated from the fraction of activity released into the gel. RESULTS: The induction of DSB in chromosomal DNA was reduced by a decrease in temperature. This protective effect of low temperature was inhibited when the DNA was irradiated in the presence of DMSO. No difference was found when intact cells were irradiated at different temperatures. However, the rapid phase of rejoining was slower in cells irradiated at 37 degrees C than at 2 degrees C. CONCLUSIONS: The induction of DSB in naked DNA was reduced by hypothermic irradiation. The temperature had no influence on the induction of DSB in the presence of a high concentration of DMSO, indicating that the temperature effect is mediated via the indirect effects of ionizing radiation. Results are difficult to interpret in intact cells. Rejoining during irradiation at the higher temperature may counteract an increased induction. The difference in rejoining may be interpreted in terms of qualitative differences between breaks induced at the two temperatures.

DNA Damage↗
PubMed1999

Influence of temperature on radiation-induced inhibition of DNA supercoiling.

The influence of subnormal temperatures (2, 15 and 28 degrees C) on the effects of radiation in MCF-7 cell cultures was studied using the fluorescent (halo) nucleoid assay. Increasing the propidium iodide (PI) concentration (0.5-7.5 microgram/ml PI) resulted in relaxation, i.e. in increasing nucleoid area; higher concentrations up to 50 microgram/ml caused rewinding that resulted in nucleoid contraction. Rewinding was inhibited by X irradiation (2, 4 and 8 Gy) in a dose-dependent way. Incubation at subnormal temperature did not influence the nucleoid area but did reduce radiation-induced inhibition of rewinding after 4 Gy. The low temperature (2 degrees C) during rather than prior to irradiation appeared to protect from radiation-induced inhibition of nucleoid rewinding. Decreased temperature during irradiation may change the conditions so as to reduce DNA- matrix damage induced by radiation.

DNA Damage↗
PubMed1998

Sulfated glycosaminoglycans enhance tumor cell invasion in vitro by stimulating plasminogen activation.

Metastasizing tumor cells invade host tissues by degrading extracellular matrix constituents. We report here that the highly sulfated glycosaminoglycans, heparin and heparan sulfate, as well as the sulfated polysaccharide, fucoidan, significantly enhanced tumor cell invasion in vitro into fibrin, the basement membrane extract, Matrigel, or through a basement membrane-like extracellular matrix. The enhancement of tumor cell invasion was due to a stimulation of the proteolytic cascade of plasminogen activation since the effect required plasminogen activation and was abolished by inhibitors of urokinase-type plasminogen activator (uPA) or plasmin. Sulfated polysaccharides enhanced five reactions of tumor-cell initiated plasminogen activation in a dose-dependent manner. They amplified plasminogen activation in culture supernatants up to 70-fold by stimulating (i) pro-uPA activation by plasmin and (ii) plasminogen activation by uPA. (iii) In addition, sulfated polysaccharides partially protected plasmin from inactivation by alpha 2-antiplasmin. Sulfated polysaccharides also stimulated tumor-cell associated plasminogen activation, e.g., (iv) cell surface pro-uPA activation by plasmin and (v) plasminogen activation by cell surface uPA. These results suggest that sulfated glycosaminoglycans liberated by tumor-cell mediated extracellular matrix degradation in vivo might amplify pericellular plasminogen activation and locally enhance tumor cell invasion in a positive feedback manner.

Cell Adhesion↗
PubMed1995

Structural and functional analysis of the rainbow trout (Oncorhyncus mykiss) metallothionein-A gene.

In the present study, the distal part of the 5'-flanking region of the rainbow trout metallothionein-A promoter was sequenced in order to identify cis-acting regulatory elements. Analysis of this sequence combined with that previously reported for the 5'-flanking region directly proximal to the start of transcription revealed several putative regulatory sequences. In total, six metal-responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500-700 bp upstream from the start of transcription. In addition, the 5'-flanking region contained sequences sharing high similarity with the activator protein 1 consensus sequence as well as one nuclear-factor-interleukin-6-responsive element. Functional analysis of the promoter was performed by introducing deletion mutants of the 5'-flanking region into the vector pGL-2, directly upstream from the luciferase reporter gene. Both MRE clusters were needed for maximal metal inducibility in both rainbow trout hepatoma (RTH-149) and human hepatoblastoma (Hep G2) cell lines. Furthermore, the distal region was found to be functional in promoting gene transcription following exposure of RTH-149 cells to hydrogen peroxide.

Amino Acid Sequence↗
PubMed1991

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells.

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.

Collagen↗
PubMed1990

Lectin binding in murine tumour lines with different malignant characteristics. Effect of enzyme (pronase, neuraminidase) treatment on lectin labelling.

We used 5 syngeneic murine tumour systems for studying quantitative lectin surface binding of intact viable tumour cells. We also investigated, for 3 of the tumours, whether the lectin binding sites were susceptible to proteolytic enzyme (pronase) or neuraminidase treatment. There were significant differences between two of the tumour lines in the binding of wheat germ agglutinin (WGA), concanavalin A (ConA). peanut agglutinin (PNA), soybean agglutinin (SBA) and Ulex europeus agglutinin (UEA). There were also variations in lectin binding between the other tumor lines, but these differences were not statistically significant. Lectin binding showed no evident relationship to the malignancy or the metastasis-forming capacity of the respective tumour cell line. Proteolytic treatment, which drastically affects intravenously induced experimental metastasis formation by one of the tumours, caused a decreased binding of SBA, ConA and WGA. Neuraminidase treatment increased both PNA and SBA binding in three different cell lines, presumably by removing sialic acid masking D-galactose and N-acetyl-D-galactose-amine groups.

Animals↗
PubMed1989

Induction of urokinase activity and malignant phenotype in bladder carcinoma cells after transfection of the activated Ha-ras oncogene.

In order to characterize further the previously observed induction of a highly metastatic phenotype in mouse bladder carcinoma cells by Ha-ras transfection, we studied production of plasminogen activator, in vitro invasiveness, and the potential for lung colonization of these cells. The parent carcinoma cells produced predominantly tissue-type plasminogen activator. Out of 13 clones of ras-transfected cells tested, 8 secreted quantitatively elevated levels of plasminogen activator (up to 3.5-fold) as compared to the control transfectants. The plasminogen activator activity in cell lysates was maximally increased 3-fold, the surface-associated activity increased 2.5-fold. The secreted plasminogen activator of cloned ras-transfected cells was characterized to be predominantly of the urokinase type (71.3% compared to 20.5% with the parental BL cells). Thus, in addition to the quantitative augmentation of plasminogen activator production and secretion in a large fraction of the ras-transfected cell population, a significant qualitative shift from tissue-type to urokinase-type has been observed. In addition, ras-transfection augmented the capacity of the cells for invasion into Matrigel in a double-filter in vitro assay as well as their ability to colonize the lungs of syngeneic animals. These malignant properties of the transfected cells might be responsible for their highly metastatic behaviour induced by ras transfection.

Animals↗
PubMed1988

Quantitative in vitro assay for tumor cell invasion through extracellular matrix or into protein gels.

A new quantitative method for the study of tumor cell invasion in vitro is presented. It is intended to facilitate the study of the mechanisms of invasion using an isolated basement membrane without the involvement of stromal structures or using defined protein gels. Cells are allowed to migrate through the pores of a Nuclepore polycarbonate filter into a protein gel on a nitrocellulose filter, or they may have to penetrate a cell-derived extracellular matrix (ECM) to reach the gel. Experiments with a nonmetastatic mouse lymphoma (Eb) and its two metastatic variants (ESb and ESb-MP) showed that the metastatic lines penetrated a Matrigel (a gel containing the components of a basement membrane) much better than the nonmetastatic cell line, but only the most metastatic line (ESb) was able to penetrate into a native collagen I gel. The presence of an ECM on the polycarbonate filter reduced the number of cells invading a fibrin gel, demonstrating that the dense, fibrillar structure of the cell-derived ECM was a barrier to the tumor cells. The metastatic lines penetrated the ECM to a 4- to 6-fold higher extent than the nonmetastatic cell line. It is concluded that in order to metastasize efficiently, the tumor cells must be able to penetrate many different kinds of barriers.

Animals↗
PubMed1986

Effects of zinc on tumor transplantability.

The effects of zinc treatment on the transplantability of tumor cells were investigated in two syngeneic murine tumor systems. A 15-min incubation of tumor cells in 0.1 mM zinc caused a dramatic decrease in intravenous transplantability for melanoma B16 cells, while MCG 101-AA cells were unaffected. The subcutaneous transplantability remained unchanged in both cases. In order to elucidate the mechanism responsible for the effect on B16 cells, a number of factors of possible importance for the intravenous transplantability were investigated. Zinc treatment reduced the activity of extracellular neutral protease, and increased the cell rigidity and fragility in a similar way in both cell lines. However, zinc effects on cell adhesion and cell proliferation were different in the two cell lines. It is possible that zinc reduces the intravenous transplantability of melanoma B16 cells by impairment of cell adhesion.

Aminopeptidases↗
PubMed1986

Characterization of two murine tumor sublines selected purely in vivo for specific metastatic patterns.

We have attempted to clone an ascites tumor, MCB 21-AA, which was originally a fibrosarcoma, by transplantation of spontaneous metastases from the lungs or a lymph node to a subcutaneous site, and by repetition of this procedure ten times. We isolated two tumor sublines, LU (from lung) and LN (from lymph node), which differ from the original tumor in several cell characteristics. When tested for the formation of experimental metastasis, these sublines disseminate in the same manner as the original tumor. We interpret this finding as an argument against the widely held view that metastasis is a highly selective cloning process.

Animals↗
PubMed1984

The tumor-cell-releasing machine.

We describe the construction of a machine for the injection of 10- to 100-microliter volumes of viable cells in suspension. The cells are maintained in a tissue culture environment and can be injected according to a preprogrammed schedule. Preliminary results show that the tumor incidence and metastasis pattern of TA3Ha cells injected intravenously into mice are the same if the cells are injected in a single dose, or administered in 20 fractions during 3 h.

Animals↗
PubMed1981

Synthesis and binding to tubulin of an allocolchicine spin probe.

A spin probe for the colchicine binding site on tubulin has been synthesized from allocolchicine. The probe is competitive to colchicine with an apparent inhibition constant of 11 microM while allocolchicine has an inhibition constant of 2 microM. Microtubule assembly is inhibited to 50% by 7 microM of the spin probe. As a mitotic poison the spin probe is less potent than colchicine. These data suggest that the probe binds to the same site on tubulin as colchicine and in spite of the somewhat lower efficiency, it seems to be a valuable tool for the study of the microtubule system.

Animals↗
PubMed1980

Increased activity of a phospholipid base-exchange system by the differentiation of neoplastic cells from the nervous system.

The activity of a base-exchange enzyme system, specific for the polar head-groups of brain phospholipids, was measured in both C 1300 mouse neuroblastoma (clone 41A3) and human glioma 138 MG cell lines. This base-exchange is thought to occur between free nitrogenous bases and membrane-bound phospholipids. The incorporation of free 14C-labeled ethanolamine and L-serine was determined in undifferentiated and morphologically differentiated cells. Both cell lines showed a significant increase in ethanolamine and L-serine incorporation upon differentiation.

Animals↗
PubMed1980

Differentiation of mouse neuroblastoma cells under increased oxygen tension.

After exposure to increased oxygen tension for 3 days, cultured mouse neuroblastoma cells were found to undergo morphological differentiation and to significantly increase their ecto-acetylcholinesterase activity. Differentiation could not be blocked by KCN or lipid-soluble antioxidants, indicating that neither an increased respiration, nor an increased oxidation of membrane lipids was responsible for the effect.

Acetylcholinesterase↗