M. Duane Enger

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+.

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.

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.

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 occured 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 G 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.

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 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 tes the extent to which Cd++ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechnisms 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 μM 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 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.

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 µM and 1 µM 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.

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

Abstract Transforming growth factor beta (TGFβ) is a multifunctional regulator of cell growth that has either a stimulatory or inhibitory effect on cell proliferation, depending on TGFβ concentration and on cell type, history and culture conditions. Cadmium mimics some of the effects of TGFβ in cultured cells. In this study the effects of Cd 2+ and TGFβ on EGF-induced DNA synthesis in a clonal subpopulation (NI) of NRK-49F cells were compared. It was found that TGF β 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 β 1 and cadmium added at the same time as EGF or several hours after EGF addition showed similar inhibitory effects on EGF-induced [ 3 H]Tdr incorporation, indicating that the inhibitory effect of TGF β 1 and cadmium on EGF-induced DNA synthesis does not involve early G 1 events. Rather, they occur in late G 1 , at the G 1 /S boundary or during S phase. Because of the similarities in nature and timing of the Cd 2+ and TGFβ responses, the possibility that Cd 2+ acts through stimulation of TGFβ production and/or activation was explored. It is shown in this paper however that TGFβ neutralizing antibody blocks the effects of TGF β 1 , but not the cadmium effects, on EGF-induced DNA synthesis, suggesting that cadmium is not functioning through activation or preinduction of TGFβ.

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

Previous studies showed that Cd++ inhibits EGF-induced DNA synthesis that 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 μM) induced more pronounced hypertrophic responses than did higher doses (4 μM); whereas in N1 cells, the Cd++-induced hypertrophic response shows a pattern of increasing response with doses of Cd++ from 0.5 to 4 μM. 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.