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H E Rugstad

Publications and source records attributed to H E Rugstad.

At least 73 records · Page 4Linked to original sources
PubMed1986

Cultured human cells with high levels of gold-binding cytosolic metallothionein are not resistant to the growth inhibitory effect of sodium aurothiomalate.

We have previously shown that cells with a high content of the gold-binding protein metallothionein (MT) are resistant to the growth inhibitory effects of gold(III) chloride and auranofin. To investigate whether MT confers resistance to sodium aurothiomalate two cell lines of cultured human epithelial (HE) cells were used; the parental cell line (HE) and a substrain (HE100) containing high levels of cytosolic MT. Sodium aurothiomalate and thiomalic acid without gold both caused a dose-dependent growth inhibition of both cell lines when used in the concentration range 25-300 mumol/l culture medium and for four days' exposure. MT, despite binding about one third of the cytosolic gold, did not protect against the antiproliferative effect of sodium aurothiomalate. The gold and the thiomalate moieties were distributed differently within the cells; 30% of the cellular gold and 80% of the thiomalate were recovered in the cytosol. Gold was mainly protein bound in both cell lines, as shown by G75 Sephadex gel filtration of the cytosols. In the HE100 cells about 30% of the gold eluted with MT. The thiomalate eluted mainly with substances with molecular weights less than 1000. Cellular synthesis of MT was not observed during sodium aurothiomalate treatment. The results indicate that the sodium aurothiomalate molecule dissociates and support the suggestion that the thiomalate moiety is, in part, responsible for the antiproliferative effect of the drug.

Cadmium↗
PubMed1986

Cultured human cells can acquire resistance to the antiproliferative effect of sodium aurothiomalate.

Cultured human epithelial cells (HE), grown as monolayers, acquired resistance to otherwise lethal concentrations (300 mumol/l, culture medium) of sodium aurothiomalate during five months' exposure to stepwise increased concentrations of the drug. The resistance acquired was shown by exposure to drug concentrations ranging from 25 to 300 mumol/, resulting in 100% of the resistant cells (HeMyo) surviving compared with controls. Only 13% of the sensitive parent cells survived when exposed to 300 mumol/l for four days. The HeMyo cells were also resistant to the antiproliferative effects of equimolar concentrations of thiomalic acid without gold. The cytosolic gold concentration and the association of 199Au with cytosolic proteins after gel filtration were similar in both cell lines after sodium aurothiomalate exposure to the exponentially growing cells. No synthesis of gold binding proteins of metallothionein character was observed in the HEMyo cells. The concentration of free thiomalate in the sonicates and cytosols of the HeMyo cells was decreased to 25-30% of the concentration found in the HE cells. Comparison with previous data for the cytosolic concentration of total thiomalate in the HE cells suggests that most of the cytosolic thiomalate present was free thiomalate. We conclude that the cells can develop resistance to the antiproliferative effect of sodium aurothiomalate, and that the resistance may be due to their capacity to maintain low concentrations of free thiomalate in the sonicates and cytosols. The results support previous findings that sodium aurothiomalate appears to dissociate within cells.

Cell Division↗
PubMed1985

Ploidity and cell cycle progression during treatment with gold chloride, auranofin and sodium aurothiomalate. Studies on a human epithelial cell line and its sub-strains made resistant to the antiproliferative effects of these gold compounds.

The possible influence of gold(III) chloride and the two gold(I)-containing anti-arthritic drugs, auranofin and sodium aurothiomalate, on cellular ploidity and cell cycle progression was investigated on cultured human epithelial cells. Four different cell lines were used: the parent line (HE) and three sub-strains which previously had acquired resistance to the antiproliferative effects of either 350 mumol gold chloride/l culture medium (HEAu350), 2 mumol auranofin/l (HEAF) or 300 mumol sodium aurothiomalate/l (HEMyo). DNA-histograms were obtained by flow cytometry examinations during a 9-days' exposure to either of these gold-containing compounds and concentrations. The HE, HEAF and HEMyo cells had similar ploidities, close to tetraploid. The HEAu350 cells had altered ploidity to distinct tetraploid. The distribution of the resistant cells with the cell cycle phases was not different from that of untreated HE cells. The HE cells, when treated with auranofin or sodium aurothiomalate, accumulated in the G2-phase of the cell cycle. In addition, a new cedecoploid peak appeared. No such changes were observed on gold chloride exposure or in HE controls grown without drug supplement. The effects of auranofin and sodium aurothiomalate on cell cycle progression of the HE cells possibly indicate a tendency to polyploidity, and furthermore that inhibition of cellular mitosis is one mechanism of the antiproliferative effect common to the two drugs.

Auranofin↗
PubMed1985

Chemosensitivity measurements of human tumour cells by soft agar assays are influenced by the culture conditions.

To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity.

Agar↗
PubMed1985

Mapping of proteins from cultured fibroblasts of synovial and subcutaneous origin by high resolution two-dimensional polyacrylamide gel electrophoresis.

Nine different cell lines of human (A) rheumatoid and (B) normal synovial fibroblasts and of (C) normal skin fibroblasts were obtained from tissue explants and grown as monolayers. The cellular protein synthesis was studied by high resolution two-dimensional polyacrylamide gel electrophoresis. Between the fourth and 10th passage of the cells in culture apparently no consistent differences were found which could be ascribed to the origin of the cells, to the rheumatoid state of the synovial tissue, or to the increasing age of the cultured fibroblasts.

Adult↗
PubMed1985

Acquired resistance to auranofin in cultured human cells.

A substrain (HEAF) of cultured human epithelial cells, grown as monolayers, was selected for resistance to auranofin (AF), a gold-containing anti-arthritic drug, by growing the parental HE cells with stepwise increased concentrations of AF in the medium. HEAF cells acquired resistance to 2 mumol AF/l, twice the concentration tolerated by the sensitive HE cells. Resistance to AF was also demonstrated in another substrain (HE100) originally selected for by its cadmium resistance, and characterized by a high cytosolic metallothionein (MT) content. Following continuous exposure to 2 mumol AF/l for 4 days, 58% of the HEAF cells, 67% of the HE100 cells, and 16% of the HE cells remained adherent to the flasks, compared with non-treated controls. Following 24 h AF exposure to living cells, HEAF cells had one-half and HE100 cells twice the cellular and cytosolic gold concentration per mg protein, as compared with HE cells. Gel filtration of cell cytosols revealed gold-binding proteins with a mol. wt. of about 10 000 apparently occurring on AF exposure in HEAF and HE cells. They bound 10-15% of cytosolic gold. MT in HE100 cells bound AF-gold to about the same extent. We suggest that the ability of cells to maintain the gold concentration at a low level (HEAF) and trapping of gold by MT (HE100) or low molecular weight proteins occurring on AF treatment (HEAF) may be mechanisms contributing to the observed cellular resistance to AF.

Auranofin↗
PubMed1985

Acquired resistance against gold(III)-chloride in cultured human cells.

Acquired resistance against gold(III)-chloride was developed in cultures of human epithelial cells derived from normal skin, by growing them with stepwise increased concentrations of the compound in the medium. Resistance to 350 mumol gold-chloride/l, about twice the commonly tolerated concentration and an otherwise lethal concentration to the cells, was attained after an adaptation period of 2-3 months. These cells had preserved their normal appearance except from small inclusion bodies in the cytoplasm. The effect of 350 mumol gold-chloride/l culture medium on cell growth of the sensitive (HE) and resistant (HEAu350) cell strain, was tested over a period of 9 days. On day 9, 2% of the HE cells and 80% of the HEAu350 cells remained adherent to the flasks compared with their nontreated control cells. The doubling time of the HE cells grown without gold was 24 h, that of the HEAu350 cells grown continuously on 350 mumol/l was 36 h. The HE cells died on prolonged exposure to this concentration. Measurement of the cellular content of Au, Cu and Zn by atomic absorption spectrophotometry revealed that the HEAu350 cells contained 1.8 times as much cellular and cytosolic Au per mg cell protein as the HE cells. Sixty per cent of the cytosolic gold in the HEAu350 cells was bound to high molecular weight proteins; no metallothionein was detected. Both strains contained minor amounts of zinc and copper compared with gold. The HEAu350 cells contained twice as much cellular Cu/mg cell protein as the HE cells, whereas the zinc content remained unchanged. The mechanism of acquired resistance against gold-chloride in these cells remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1985

Heart transplantation in Norway. One-year experience.

Eight patients underwent orthotopic heart transplantation in Norway during 1984, with retransplantation in one case. The age range of the 5 men and 3 women was 19-53 years. The preoperative diagnosis was cardiomyopathy in 6 patients, ischaemic heart disease in one, and a combination of the two disorders in one patient. The immunosuppressive regimen, with cyclosporin A and low-dose prednisolone, and the treatment of graft rejection, followed the Stanford University protocol. There was no operative mortality. Three patients died shortly after the transplantation, 2 of them after about a week from acute rejection; in one of these 2 cases a second transplant was made, but was followed by pulmonary and renal complications. The third death occurred about 10 weeks postoperatively, from donor heart failure due to Toxoplasma myocarditis. The 5 survivors are clinically in good condition.

Adult↗
PubMed1984

Protein mapping of two metallothionein-rich cell strains and their parent lines, using high-resolution two-dimensional electrophoresis.

A high-resolution two-dimensional gel electrophoresis (2-D) technique was used to characterize one human and one murine cadmium-resistant substrain and their parental wild-type lines. The substrains are cultured on 100 microM cadmium and contain high levels of the cysteine-rich protein metallothionein (MT). All four cell lines were labeled with [35S]methionine during growth. A remarkable consistency was found in the protein maps of the resistant strains compared to those obtained from their corresponding wild-type lines. Thus, in the maps from the human substrain only two spots were detected which were not found in the parent cells. In the murine substrain, two spots were more abundant and two diminished compared to the parent cells. No distinct spots corresponding to authentic MT were detected in any of the autoradiographs from the cadmium-resistant cells. The reason for this was found to be failure of the protein to focus in the first dimension. Purified [35S]cystine-labeled MT appeared as a diffuse labeling over the entire gel, and subsequently as wide horizontal bands in the second dimension. These bands were also clearly visible in the protein maps when MT-rich cells had been labeled with [35S]cysteine. This study shows that the standardized 2-D gel system used in many laboratories cannot be used to screen cell populations for MT.

Animals↗
PubMed1984

Tumours from a cell strain with a high content of metallothionein show enhanced resistance against cis-dichlorodiammineplatinum.

Cultured cells with a high content of the extremely cysteine-rich protein metallothionein (MT) have previously been shown to exhibit resistance when exposed to otherwise lethal doses of cis-dichlorodiammineplatinum (cis-DDP), chlorambucil or prednimustine, and to have a significant proportion of intracellular drug associated with MT after such treatment. In order to study this protective mechanism in vivo, cells from a MT-rich variant of a murine fibroblast line resistant to cis-DDP and its parent sensitive line with only trace amounts of MT, were injected subcutaneously into nude mice (24 animals in each group), and tumour growth was compared during cis-DDP treatment. Animals in the treatment groups received 3 intravenous doses of either 4 mg/kg or 8 mg/kg of cis-DDP on day 12, 26 and 33 after inoculation of cells, whereas the control groups received saline. The 8 mg/kg dose produced an almost complete growth inhibition of the tumours derived from the parent cells, as well as from the MT-rich variant. However, following the injections with 4 mg/kg of cis-DDP, tumour volume was reduced by approximately 80% in tumours from the parent cells, whereas the tumours from MT-rich cells were almost completely resistant. This study provides for the first time evidence that metallothionein-conferred protection against cis-DDP toxicity also is mediated in tumours in vivo.

Animals↗
PubMed1984

Metallothionein--a unique protein with multiple detoxifying properties?

Metallothioneins (MT) are a group of proteins of very peculiar structure, in that 15-20 of its amino acid residues are cysteinyl. We have studied possible functions of the SH-rich MT as a detoxifying molecule by using human and murine cell lines with high amounts of Cd-induced MT. Such cells show radioresistance, they are resistant to Cd, cisplatinum, chlorambucil and gold, and the MT binds these substances firmly. The clinical relevance may be twofold. Firstly, MT may confer resistance to useful drugs such as alkylating agents. Secondly, MT may be a resistance factor to harmful effects of xenobiotics.

Animals↗
PubMed1984

Cadmium uptake and metabolism in cultured cells.

Cultured cells have been made resistant to otherwise lethal concentrations of the toxic Cd ion, probably by induction of metallothionein (MT) synthesis and binding of Cd to the MT. One human epithelial cell line (HE) and two enzyme-deficient mutants of mouse fibroblasts (L-cells) (Cl 1D and A9) and their Cd-resistant substrains with a high content of MT, have been used to study cellular Cd uptake and metabolism. For cell survival of "wild type" cells, the critical level of intracellular Cd is determined to be around 6 nmole Cd/mg cell protein. Resistant cells can tolerate Cd levels several times above this concentration, if the major part of Cd is bound to MT. The technique of perturbed angular correlation spectroscopy (PAC) has been applied to living Cd-resistant cells. It was shown that greater than 66% of Cd in the resistant strains was bound to MT, and that MT is apparently freely suspended in the cell cytoplasm. Chelating agents differ in toxicity and Cd-releasing effect on the cells, but apparently remove the non-MT-bound Cd pool. After various periods of Cd omission, either in vitro or in vivo, growing the cells as tumors in athymic nude mice, the stability of Cd resistance in these cells seems to be dependent on the capacity of cells for de novo synthesis of MT shortly after re-exposure to the metal.

Animals↗
PubMed1983

[Piroxicam (Felden)].

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Anti-Inflammatory Agents↗
PubMed1983

Pharmacokinetics and protein binding of prednisolone after oral and intravenous administration.

The pharmacokinetics of prednisolone after oral and intravenous administration of 10 and 20 mg have been studied. Serum protein binding of prednisolone was also measured after the i.v. injections. The bioavailability after oral administration was 84.5% after 10 mg and 77.6% after 20 mg (p greater than 0.05). Dose dependent pharmacokinetics were found, the VDss and Clt being significantly larger (p less than 0.01) after 20 mg i.v. than after 10 mg i.v. The protein binding of prednisolone in all subjects was non-linear, and is the most likely cause of the dose dependent pharmacokinetics, as there was no dose dependent variation in elimination half-time.

Administration, Oral↗
PubMed1983

Stability of cadmium resistance and metallothionein levels in vitro and in vivo.

Cultured cells can be adapted to large concentrations of the toxic cadmium ion, apparently by induction of synthesis of the Cd-binding protein metallothionein (MT). One human epithelial line derived from normal skin (HE100) and one murine fibroblast line, derived from L cells (C1 1D100), were used to study the stability of Cd resistance, the MT levels following omission of Cd, and the inducibility of MT synthesis in cells on reexposure to Cd. In the murine cells there was no significant loss of resistance during a 4-week period either after cultivation in vitro or after growing the cells in nude mice. In the human cells a decrease (50%) in resistance was noted the first week after Cd omission. After removing Cd from the cells, a rapid decrease in MT content was demonstrated. After 3 weeks of cultivation only trace amounts were left in both cell lines. However, approximately 60% (HE100) and 80% (C1 1D100) of the previous levels were demonstrated after reexposure to maximum tolerable doses for 24 hr. The data indicate that the degree of stability of Cd resistance is dependent on the capacity in cells for an immediate de novo synthesis of large amounts of MT on reexposure to Cd. The animal experiments demonstrate that Cd resistance is maintained even after growing the cells in vivo.

Animals↗