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M D Enger

Publications and source records attributed to M D Enger.

At least 19 recordsLinked to original sources
PubMed1993

Cd(2+)-induced c-myc mRNA accumulation in NRK-49F cells is blocked by the protein kinase inhibitor H7 but not by HA1004, indicating that protein kinase C is a mediator of the response.

Cd2+ is a toxic cation that, at sublethal and marginally lethal levels, modifies cell growth and metabolism. Cd2+ exposure of NRK-49F cells results in inhibition of early EGF-induced DNA synthesis, but induction of delayed DNA synthesis; in stimulation of anchorage independent growth; in accumulation of specific oncogene mRNAs; and in an hypertrophic response. Determining whether specific signal transduction pathways (STPs) are involved in specific gene deregulation by cadmium in NRK-49F cells is important to defining possible mechanisms by which Cd2+ elicits these physiological responses. In this study it is shown that Cd2+ induces delayed myc (8-10 h) and jun (12 h) mRNA accumulation, as well as both early (0.5-1 h) and late (12 h) fos but not TGF beta mRNA accumulation. The times of appearance of Cd(2+)-induced c-fos, c-myc and c-jun expression are dose dependent. The Cd2+ induced accumulation of these specific mRNAs is insensitive to cycloheximide and therefore not due to preinduction of TGF beta or other gene-activating growth factors, but rather to direct induction of oncogene expression and/or mRNA stabilization. Accumulation of c-myc mRNA is shown further to be inhibited by the protein kinase inhibitor H7 but not HA1004, indicating a role for one or more protein kinases C in the STPs by which Cd2+ induces oncogene expression. Thapsigargin, a compound which stimulates increased cytosolic [Ca2+], induces c-myc expression also by an H7 sensitive, HA1004 insensitive pathway. These results suggest that Cd2+ acts through one or more defined signal transduction pathways involving specific protein kinases C to induce the accumulation of c-fos, c-myc and c-jun messenger RNAs.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Cadmium's action on NRK-49F cells to produce responses induced also by TGF beta is not due to cadmium induced TGF beta production or activation.

Transforming growth factor beta (TGF beta) is a multifunctional regulator of cell growth that has either a stimulatory or inhibitory effect on cell proliferation, depending on TGF beta concentration and on cell type, history and culture conditions. Cadmium mimics some of the effects of TGF beta in cultured cells. In this study the effects of Cd2+ and TGF beta on EGF-induced DNA synthesis in a clonal subpopulation (N1) of NRK-49F cells were compared. It was found that TGF beta 1 and cadmium both inhibit EGF-induced DNA synthesis and cell proliferation in a dose-dependent fashion, but that neither inhibits EGF-induced myc oncogene accumulation. TGF beta 1 and cadmium added at the same time as EGF or several hours after EGF addition showed similar inhibitory effects on EGF-induced [3H]Tdr incorporation, indicating that the inhibitory effect of TGF beta 1 and cadmium on EGF-induced DNA synthesis does not involve early G1 events. Rather, they occur in late G1, at the G1/S boundary or during S phase. Because of the similarities in nature and timing of the Cd2+ and TGF beta responses, the possibility that Cd2+ acts through stimulation of TGF beta production and/or activation was explored. It is shown in this paper however that TGF beta neutralizing antibody blocks the effects of TGF beta 1, but not the cadmium effects, on EGF-induced DNA synthesis, suggesting that cadmium is not functioning through activation or preinduction of TGF beta.

Cadmium↗
PubMed1992

Buthionine sulfoximine-induced cytostasis does not correlate with glutathione depletion.

The effects of L-buthionine-(S,R)-sulfoximine (BSO) on the proliferation of normal rat kidney fibroblasts (NRK-49F) were determined and compared with the effects of BSO on cellular glutathione (GSH) content. The proliferation rate of exponentially growing NRK-49F cells was found to be slowed in 0.01 and 0.1 mM BSO and arrested in 1.0 and 10 mM BSO. There is no retardation in the proliferation of cells cultured in 0.001 mM BSO. However, varying BSO concentrations at and above 0.1 mM did not result in concordant differences in the rate and extent of GSH depletion. A dose-dependent effect of BSO on GSH levels was observed at BSO concentrations less than or equal to 0.01 mM. BSO was found also to inhibit epidermal growth factor (EGF)-induced DNA synthesis in NRK-49F cells arrested by serum deprivation in a dose-dependent pattern dissimilar to that of BSO-induced cellular GSH depletion. Removal of BSO allowed cells to resume proliferation. Further, growth-arresting BSO treatments were found to affect neither cell viability nor colony-forming efficiency. Addition of exogenous GSH or cysteine overcame BSO inhibition of EGF-induced DNA synthesis but not BSO depletion of cellular GSH levels. BSO was further found to inhibit the uptake of cysteine, cystine, and alpha-[1-14C]-methylaminoisobutyric acid (MeAIB) by the EGF-stimulated quiescent cells in a dose-dependent fashion. The results presented here thus demonstrate that BSO inhibits the proliferation of NRK-49F cells. This effect, however, does not correlate with BSO-induced cellular GSH depletion and is not due to an overt toxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Cadmium inhibits EGF-induced DNA synthesis but increases cellular glutathione levels in NRK-49F cells.

The effects of cadmium (CdCl2) on epidermal growth factor (EGF) induced DNA synthesis and on cellular glutathione (GSH) content in growth-arrested NRK-49F cells were studied. The cadmium effects were compared with those of L-buthionine-(S,R)-sulfoximine (BSO). EGF at a concentration of 10 ng/ml was found to stimulate DNA synthesis (as judged by [3H]thymidine incorporation) in growth-arrested NRK-49F cells. CdCl2 inhibited this EGF-induced DNA synthesis in a dose-dependent fashion. It also increased significantly cellular GSH content in both growth arrested and EGF-stimulated NRK-49F cells. This effect of CdCl2 was contrary to that of BSO, which depleted cellular GSH. Although BSO both inhibited EGF-induced DNA synthesis and decreased cellular GSH content in EGF-stimulated NRK-49F cells, these two BSO effects showed dissimilar dose dependencies. BSO and CdCl2 together inhibited EGF-induced DNA synthesis in NRK-49F cells in an additive fashion. These results demonstrate that cadmium inhibition of EGF-induced DNA synthesis in NRK-49F cells is not due to an effect on cellular GSH content. Both cadmium and BSO inhibit EGF-induced DNA synthesis in NRK-49F cells, but probably through different mechanisms. Although GSH may be involved in regulation of DNA synthesis, BSO-induced inhibition of EGF-stimulated DNA synthesis in NRK-49F cells does not in its dose-dependency correlate with GSH depletion.

Animals↗
PubMed1991

Increased glutathione levels in quiescent, serum-stimulated NRK-49F cells are associated not with a response to growth factors but with nutrient repletion.

Treatment of quiescent cells with serum results concomitantly in an increase in cellular glutathione (GSH) content and growth stimulation. A possible association between the GSH increase and the growth response was examined by studying separately the effects of nutrients and growth factors on the levels of cellular GSH and proliferation of quiescent NRK-49F cells. The addition of fresh medium with 10% calf serum was found to result in both a twofold increase in cellular GSH and growth stimulation (DNA synthesis and cell proliferation). 10% calf serum alone, without fresh medium, stimulated cell growth but failed to cause a comparable increase in cellular GSH. The addition of fresh medium without 10% serum, and of 0.5 mM cysteine and glutamate, resulted in both instances in a marked increase in cellular GSH, but failed to stimulate cell growth. EGF, in contrast, induced a complete mitogenic response but did not increase cellular GSH. Finally, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GSH synthesis, decreased cellular GSH and inhibited EGF-induced DNA synthesis, but these two responses do not, in their dose dependency, correlate. The results obtained thus show that the increase in cellular GSH that occurs in quiescent, serum-stimulated NRK-49F cells is a result of nutrient repletion rather than mitogenic stimulation, and increased GSH levels do not necessarily precede DNA synthesis and mitosis.

Animals↗
PubMed1991

Cd++ inhibits EGF induced DNA synthesis but not EGF induced myc mRNA accumulation in serum starved NRK-49F cells.

Cd++ inhibits EGF-induced 3H-thymidine incorporation in serum deprived NRK-49F cells in a dose dependent pattern. The underlying mechanisms for this inhibition are largely unknown. EGF-induced myc mRNA accumulation in NRK-49F cells and the effects of Cd++ on this response were examined under conditions that result in partial or complete inhibition of EGF-induced DNA synthesis. It was found that doses of Cd++ that inhibit EGF-induced DNA synthesis do not inhibit EGF-induced protein synthesis and myc mRNA accumulation. Cd++ doses of 0.5 microM and 1 microM were found actually to increase EGF-induced myc mRNA accumulation and amino acid incorporation. These results show that the effect of Cd++ on EGF-induced DNA synthesis is not due to inhibition of entrance into G1, but rather that Cd++ acts on events subsequent to myc accumulation; that is, events associated with either G1 progression, entry into S or DNA synthesis.

Amino Acids↗
PubMed1991

Cadmium induces hypertrophy accompanied by increased myc mRNA accumulation in NRK-49F cells.

Previous studies showed that Cd++ inhibits EGF-induced DNA synthesis but not EGF-induced myc mRNA accumulation or amino acid incorporation into protein in serum-starved NRK-49F cells. In this study, flow cytometry was used to analyze the DNA and protein content of individual cells stimulated with Cd++ and/or epidermal growth factor (EGF). myc oncogene expression in these cells was also measured. It was found that, in both parental NRK-49F cells and in a clonal subpopulation, N1, Cd++ induces an hypertrophic response. In parental NRK-49F cells, however, lower doses of Cd++ (0.5 microM) induced more pronounced hypertrophic responses than did higher doses (4 microM); whereas in N1 cells, the Cd+(+)-induced hypertrophic response shows a pattern of increasing response with doses of Cd++ from 0.5 to 4 microM. myc mRNA accumulation measured 2 hours after stimulation correlated with the hypertrophic responses in both NRK-49F cells and in N1 cells. The results show that Cd+(+)-induced hypertrophy in NRK-49F cells is associated with increased myc oncogene mRNA accumulation, indicating that cell proliferation and cell hypertrophy may in part share common activation pathways.

Animals↗
PubMed1991

Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion.

Treatment of A549 human lung carcinoma cells with L-buthionine-[S,R]-sulfoximine (BSO) results concomitantly in cellular glutathione (GSH) depletion and growth inhibition. The nature of BSO effects on cell growth and the relationships between BSO inhibition of cell growth and BSO effects on cellular GSH levels were determined in this study. A dose dependent effect of BSO on cell growth was observed, but this effect was found not to correlate with BSO effects on cellular GSH levels. Treatment with BSO for 60 h at concentrations of 5 and 10 mM was found to deplete cellular GSH at similar rates and to an undetectable level (below 0.5 nmol/mg protein). However, cessation of growth occurred in 10 mM BSO whereas growth continued at better than one half the control rate in 5 mM BSO. The results suggest there may be a distinct threshold level of intracellular GSH (on the order of or less than 0.5 nmol/mg protein) required for cell growth and for cells to protect themselves from the antiproliferative effects of BSO. At a concentration of 10 mM, BSO inhibited both DNA and protein synthesis and arrested growth of A549 cells throughout rather than at a specific phase of the cell cycle. BSO inhibition of growth was not, as indicated by colony-forming efficiency (CFE) and electron microscopy studies, accompanied by indications of cytotoxic effects. A stimulatory effect of 0.1 mM BSO on the growth of A549 cells was found also.

Buthionine Sulfoximine↗
PubMed1991

TGF beta elicits opposite responses in clonal subpopulations of NRK-49F cells.

Clonal subpopulations of NRK-49F cells were isolated and characterized for their responses to transforming growth factor beta (TGF beta). Two fibroblastic clones, N1 and N4, were found to have opposite TGF beta responses. TGF beta inhibits EGF-induced proliferation in growth-arrested, subconfluent monolayer cultures of N1 but not N4 cells. In contrast, TGF beta stimulates DNA synthesis and an increase in cell number in N4 but not N1 cells. The inhibitory effect of TGF beta on DNA synthesis in N1 cells is due not to modulation of the EGF receptor or other early G1 events. EGF-induced myc mRNA accumulation is not inhibited, and the action point for TGF beta inhibition of the entry into S of N1 cells is at the G1-S boundary.

Animals↗
PubMed1990

Diamide reduces cadmium accumulation by human lung carcinoma A549 cells.

Human lung carcinoma A549-T27 cells were used to determine the effect of diamide on cadmium accumulation. Treatment of the cells with diamide decreased their cellular glutathione content to 51.6 +/- 7% of control and significantly decreased their cadmium accumulation both as a function of time and as a function of Cd2+ concentration. Verapamil also decreased cadmium accumulation. Its effect compares well in magnitude with that which resulted from diamide treatment. No additive effect was observed when the cells were simultaneously treated with diamide and verapamil. The results suggest that a change in the GSH/GSSG ratio affects cadmium uptake. Further, calcium channels may be involved in cadmium uptake by A549-T27 cells in a fashion that is dependent on sulfhydryl status.

Azo Compounds↗
PubMed1990

Cellular cadmium responses in subpopulations T20 and T27 of human lung carcinoma A549 cells.

Subpopulations T20 and T27, cloned from the human lung carcinoma line A549, differ significantly in their Cd2+ cytotoxic response. T27 has an LC50 of 31 microM Cd2+ and a cytotoxic response threshold of 5 microM Cd2+, whereas the T20s LC50 is 15 microM Cd2+ and there is no observed threshold for cytotoxicity. Cadmium-induced metallothionein (MT) synthesis, cadmium accumulation, glutathione (GSH) content, and Cd2(+)-induced changes in GSH content were studied in T20 and T27 in an attempt to determine the mechanism(s) causing differential cytotoxic response. MT synthesis measured by following Cd2(+)-induced [35S] incorporation into MT was found not to differ between T20 and T27. There is, however, a difference in Cd2+ accumulation between the two subclones. T20 and T27 cells were exposed to 5 microM Cd2+ for different times or to different concentrations of Cd2+ for 8 h. The T27 subline, which is the more Cd2+ resistant, was found to accumulate significantly more Cd2(+)-both as a function of time exposed to Cd2+ and as a function of Cd2+ concentration. The two subpopulations were found to have comparable initial GSH contents, but showed different Cd2(+)-induced changes in [GSH] when the cells were exposed to 5 microM Cd2+. T27 cells maintained their GSH content following Cd2+ exposure but T20 cells showed a Cd2(+)-induced decrease in GSH content. The results indicate that the difference in Cd2+ cytotoxic response between A549--T20 and A549--T27 cells is not attributable to alterations in MT synthesis nor to a difference in initial GSH content. Relative Cd2+ cytotoxicity also does not in these cells correlate with relative Cd2+ accumulation. The fact that T27 cells accumulate more Cd2+ and yet are more Cd2+ resistant than T20 cells suggests that T27 cells have a much more effective non-MT mechanism to handle intracellular Cd2+. This may involve different GSH metabolism and/or yet undefined molecular factors.

Cadmium↗
PubMed1990

Glutathione content and growth in A549 human lung carcinoma cells.

The relationship between glutathione content and cell growth was investigated in A549 human lung carcinoma cells. A decreased cellular glutathione content was achieved by exposing the cells to L-buthionine-SR-sulfoximine (BSO). It also occurred in these cells as they approached their plateau phase of growth. During exponential growth, a lower initial glutathione content correlated with a longer lag phase in subcultured cells. Further, depletion of cellular glutathione by BSO inhibited cell growth. This inhibition became apparent 36 h after the addition of BSO. These observations raise the possibility that a critical concentration of GSH may be required for optimal growth of A549 human lung carcinoma cells.

Antimetabolites↗
PubMed1990

Cadmium cytotoxicity correlates with the changes in glutathione content that occur during the logarithmic growth phase of A549-T27 cells.

Correlation of cadmium cytotoxicity with cellular glutathione content as it changes during cell growth was examined in human lung carcinoma A549-T27 cells. Cellular glutathione content was found to increase rapidly during the first 24 h of subculture, which includes the lag and early log phases of growth, and to decrease continuously thereafter. Glutathione content reached its lowest level at 108 h of subculture. This period of glutathione decrease represented most of the logarithmic phase of cell growth. Cells exposed to cadmium at different times during the logarithmic growth phase showed differential sensitivity. Cells with the higher initial glutathione content that occurs at the early period of the logarithmic growth phase were cadmium-resistant relative to those of lower glutathione content found at the later period of the logarithmic phase. A high correlation (r = 0.82) between cadmium sensitivity and glutathione content was found, which suggests that intracellular glutathione content is an important determinant of overall cadmium cytotoxicity.

Cadmium↗
PubMed1989

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis.

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-T27) cells to the cytotoxic effects of Cd++. The effects of GSH depletion on Cd++ accumulation and Cd++-induced metallothionein (MT) content were investigated to determine the possible role of these Cd++ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd++-induced accumulation of exogenous 35s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd++ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd++ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd++, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd++-induced MT accumulation rates.

Buthionine Sulfoximine↗
PubMed1988

Glutathione is involved in the early cadmium cytotoxic response in human lung carcinoma cells.

Depletion of cellular glutathione (GSH) has been shown to sensitize A549-T27 human tumor cells to the cytotoxic effects of Cd2+. In this study the temporal and quantitative relationships between reduced cellular GSH levels and cadmium cytotoxic response in these cells were further investigated. Exposure of A549-T27 cells to 10 mM buthionine sulfoximine (BSO) for 8 h decreased their GSH level by 65%. This GSH level remained relatively constant for 8 h in the presence or absence of BSO, but recovered to 83% of the normal cellular level 24 h after removal of BSO. Exposure to 5 microM Cd2+ for 8 h did not significantly change cellular GSH levels. Pretreatment of the A549-T27 cells with 10 mM BSO for 8 h and subsequent exposure of the cells to Cd2+ for 10 days, with or without concurrent treatment of 10 mM BSO during the first 8 h of Cd2+ exposure, resulted in disappearance of the 5 microM Cd2+ threshold for cytotoxic response and reduction of the LC50 from 31 microM Cd2+ to 21 microM. Similar results were obtained when BSO pretreated cells were exposed to Cd2+ for 8 h. The threshold for cytotoxic response of 10 microM Cd2+ disappeared and the LC50 was reduced from 60 microM to 29 microM Cd2+ (with concurrent BSO treatment) and 30 microM (BSO pretreatment only). The results show that GSH plays an important role in early cellular protective responses to Cd2+.

Buthionine Sulfoximine↗
PubMed1987

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells.

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd++-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd++ cytotoxic effect and decreased the LD50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd++. Thus the results demonstrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd++ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd++ cytotoxic response. Comparison of reduced glutathione levels and of Cd++ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd++ resistance.

Buthionine Sulfoximine↗
PubMed1987

Cadmium produces a delayed mitogenic response and modulates the EGF response in quiescent NRK cells.

Recent studies have shown that cadmium, at subtoxic levels, may induce a response characteristic of that elicited by a type of growth factor that supports the anchorage independent growth of cells that are not fully transformed. That is, Cd++ was found to replace transforming growth factor beta in supporting soft agar growth of NRK-49F cells. To test the extent to which Cd++ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechanisms of action, we have determined the effects of Cd++ and/or epidermal growth factor (EGF) on DNA synthesis in quiescent NRK-49F cells. We found that subtoxic doses of Cd++ modulate EGF-induced DNA synthesis in a dose-dependent fashion. Although Cd++ effects on early (16-24 hr) EGF-induced DNA synthesis are primarily inhibitory, later effects involve stimulation as well. Subtoxic doses of Cd++ did not stimulate DNA synthesis in quiescent cells within 24 hr of addition. At later times (40 or 64 hr), however, an increase in DNA synthesis of up to threefold was induced by 0.25 microM Cd++. This pattern of mitogenic response, involving inhibition of early growth-factor induced DNA synthesis and stimulation of late DNA synthesis, is consistent with that reported to be effected in some instances by transforming growth factor beta. Because a defined pattern of gene expression also is associated with the mitogenic responses to transforming growth factor beta, future studies at the molecular level can definitively test the degree to which Cd++ and transforming growth factor beta effects are common.

Cadmium↗