RNA synthesis in Chinese hamster cells. II. Increase in rate of RNA synthesis during G1.

Cultures of mitotic Chinese hamster cells, prepared by mechanical selection, were pulse-labeled with methionine-methyl-(14)C or with uridine-(3)H at different stages in the life cycle. The rate of (14)C incorporation into 18S RNA was measured, as was the rate of uridine-(3)H incorporation into total RNA for both monolayer and suspension cultures. The rate of incorporation increased continuously throughout interphase in a fashion inconsistent with a gene-dosage effect upon RNA synthesis.

RNA synthesis in Chinese hamster cells. I. Differential synthetic rate for ribosomal RNA in early and late interphase.

The incorporation of methionine-methyl-(14)C into 18S ribosomal RNA of cultured Chinese hamster ovary cells in early and late interphase has been determined by zone-sedimentation analysis of phenol-extracted RNA preparations. Synchronized cell cultures were prepared for these studies by thymidine treatment and by mechanical selection of mitotic cells. The specific activity of 18S RNA labeled in late interphase was found to be 1.1-1.2 times that of 18S RNA labeled in early interphase. Upon correction for increase in RNA mass, the rate of methylation of 18S RNA in late interphase is about 1.9 times that in early interphase.

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.

Zinc-induced resistance to alkylating agent toxicity.

Suspension cultures of Chinese hamster ovary cells and three derived cadmium-resistant variants were exposed to 100 microM ZnCl2 prior to treatment with the alkylating agent, melphalan, and cytotoxicity was then determined by measuring colony-forming ability. A 10-fold or greater enhancement in survival of all zinc-pretreated cultures subsequently exposed to melphalan was observed which was unrelated to metallothionein induction capacity. Although the maximum achievable protection afforded by zinc occurred in cultures receiving 100 microM ZnCl2, concentrations of zinc only slightly in excess of levels found in human serum were shown to provide a 4.5-fold enhancement of protection, indicating that the phenomenon can also be induced at physiologically reasonable levels. These results suggest the existence of a novel zinc-inducible mechanism which protects cells against the toxic effects of alkylating agents.

Informosomal and polysomal messenger RNA. Differential kinetics of polyadenylation and nucleocytoplasmic transport in Chinese hamster ovary cells.

The relative kinetics of cytoplasmic appearance and polyadenylation were determined for informosomal (ribosome-free) and polysomal (ribosome-associated) mRNAs of cultured Chinese hamster cells. Label appeared in polysomal mRNA 17--20 min and into informosomal mRNA 2--5 min after addition of radio-labelled uridine. Adenosine appeared in polysomal mRNA 4--5 min before uridine. In contrast, adenosine label preceded uridine into informosomal mRNA by less than 1 min. About one-third of newly formed informosomal and two-thirds of newly formed polysomal mRNA are poly(A+). The data indicate that newly formed informosomal mRNA cannot be simple precursor to polysomal mRNA. Further, the pronounced difference in time required for polyadenylation and cytoplasmic appearances of these messenger ribonucleoproteins suggests that there may be fundamental differences in their mode of processing.

The isolation from ribonucleic acid of substituted uridines containing alpha-aminobutyrate moieties derived from methionine.

The RNA of an established line of Chinese hamster cells growing in cell culture contains a small number of uridines substituted at the 3-position with a gamma-linked alpha-aminobutyrate residue. Structure has been ascertained by: (a) examination of incorporation of isotopic labels from precursors; (b) degradation with anhydrous hydrazine and comparison of the products with synthetic material or with hydrazinolysis products of known uridines; and (c) comparison of the unknowns as their hydantoin derivatives with the 5-beta-(bromoethyl)hydantoin alkylation products of uridine and of 1- and 3-methylpseudouridine. In this manner it is shown that 18-S RNA of ribosomes contains a single residue of a nucleoside which we tentatively identify as 1-methyl-3-gamma-(alpha-amino-alpha-carboxypropyl)pseudouridine per molecule. RNA isolated from the supernatant fraction, sedimenting at 4 S and co-electrophoresing with transfer RNA on polyacrylamide gels, contains several similar bases, one of which is identified as 3-gamma-(alpha-amino-alpha-carboxypropyl)uridine. Each of the above nucleosides derives its alpha-aminobutyrate residue from methionine.

RNA synthesis in Chinese hamster cells. III. Non-coordinate increases during interphase in synthesis rates for informosomal, polysomal and heterogeneous nuclear RNAs.

Cultured Chinese hamster ovary cells were synchronized by mitotic selection. Relative synthesis rates for informosomal messenger-like RNA (mlRNA), polysomal messenger RNA (mRNA), and heterogeneous nuclear RNA (HnRNA) were estimated from the amount of labeled adenosine or uridine incorporated into these species in early and late interphase. The amounts of uridine incorporated into HnRNA, mRNA, and mlRNA during a pulse administered 9.75-10.75 h post-mitosis were 3.48 4.64, and 2.82 times the amounts incorporated 1.5-2.5 h post-mitosis. Adenosine incorporation values 9.5-11.0 h post-mitosis were 1.64 (HnRNA), 2.49 (mRNA), and 1.18 (mlRNA) times the 1.5-3.0 h values. The realitive incorporation into MRNA of large polysomes corresponded to incorporation into mRNA of smell polysomes. Thus, the synthesis rates of mRNA, mlRNA, and HnRNA increase during interphase in a noncoordinate fashion.

Effects of ionizing radiation of RNA metabolism in cultured mammalian cells. III. Specific reduction in mRNA synthesis during resumed division and plateau phases following exposure to 800 rads of x irradiation.

The cell growth response of cultured Chinese hamster ovary cells, line CHO, to 800 rads of X irradiation involves a period of division delay, followed by a period of resumed division which terminates in a plateau phase. Over 95% of the cells die eventually. No direct effects of RNA or protein metabolism are evident during the delay period. During the resumed division and beginning of plateau phases, however, a specific and relatively constant reduction in mRNA synthesis relative to messenger-like RNA and heterogenous nuclear RNA synthesis is evidenced. The ratio of mRNA to messenger-like RNA synthesis ranges from 0.8 to 0.65 during these phases. The effect is not due to altered cell-cycle distribution, and evidence is presented to indicate that it is probably not a compensatory response to the unbalanced growth that occurs during the division delay period.

Inducible protective proteins: a potentially novel approach to chemotherapy.

A number of toxic chemical and physical agents elicit the induction of a series of protein species, some of which react with the agents and render them nontoxic. A few of the induced species (such as metallothionein) are rich in thiol groups that might be expected to react with alkylating agents and render them nontoxic. If a safe means could be found for selectively enhancing the synthesis of alkylating-agent-reactive species in normal but not tumor cells, such a procedure would have ramifications in the area of cancer chemotherapy. In this report, we have utilized a variety of trace elements (Zn, Se, Cu, As) as inducers of synthesis of protective species in line CHO Chinese hamster cells and in a number of derived variants to determine whether this type of approach can be utilized to increase resistance to alkylating-agent toxicity. Our results indicate that Zn, Se and Cu elicit a protective response (increased survival, monitored by colony-forming ability) against the toxic effects of iodoacetate or melphalan, and, at least in the case of zinc, at levels that are physiologically reasonable. Arsenite appears to be a marginally effective inducer in the CHO cell and an ineffective inducer in the Cdr20F4 variant cell. The increased survival is not attributable to metallothionein inducibility, decreased availability of the alkylating agent in the medium, or decreased uptake of the drug into the trace-element-pretreated cells. The protective responses induced by zinc or selenite alone are additive in cells receiving both trace elements prior to exposure to alkylating agent, which suggests that different domains of response are elicited by the two metals. In view of reported differences in inducibility of protective proteins between normal and tumor cells, a possibility is raised for a novel approach to alkylating-agent chemotherapy that is somewhat analogous to the protocol utilized in high-dose methotrexate therapy.

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.

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+.

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(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.

Effects of cadmium on glutathione metabolism in cadmium sensitive and cadmium resistant Chinese hamster cell lines.

Three cadmium resistant sublines, Cdr20F4, Cdr30F9, and Cdr200T1, resistant to 26, 40, and 145 micron CdCl2, respectively, have been derived from the cadmium sensitive Chinese hamster cell line CHO (resistant to 0.2 microM CdCl2). The resistance appears to be largely a function of the increased ability of the variant cells to induce the synthesis of metallothioneins (MTs) in response to cadmium, as the incorporation of [35S]cysteine into MTs ranges from an undetectable level to nearly 60% of the total cysteine incorporation into proteins in the CHO and Cdr200T1 cell lines, respectively. Treatment of the cadmium resistant sublines and the parent line with maximal subtoxic levels of Cdcl2 produced increases in the concentration of glutathione and glutathione S-transferase activity. In the parent, cadmium-sensitive CHO cell, the glutathione concentration began to increase after 9 h of exposure to 0.22 microM CdCl2 to over 250% of control level by 12 h. In 1 cadmium resistant line (Cdr20F4) the increase, again at 9 h, was preceded by a decrease, possibly due to depletion of the cysteine pools by the cadmium-induced MT synthesis. The second cadmium-resistant cell line (Cdr30F9) displayed no decrease, and the most cadmium-resistant line (Cdr200T1) showed the decrease and a recovery, but no significant increase by 12 h. In all cell lines there was a small but significant increase in glutathione S-transferase activity by 9 or 12 h. These responses may be specific for the thiol-reactive metal, cadmium, or may represent more general responses to cellular toxicity.

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.

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 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.

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities.

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd(r)2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd(2+) compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd(2+)-resistant Cd(r)2C10 subline to induce synthesis of the Cd(2+)- and Zn(2+)-binding protein(s), metallothionein(s) (MT). Evidence that Cd(2+) behaves as an analog of the essential trace metal, Zn(2+), especially as an inducer of MT synthesis, suggested that the Cd(r) and CHO cell types could be employed to investigate cellular Zn(2+) metabolism. In the present study, measurements were made to compare CHO and Cd(r) cell types for (a) growth as a function of the level of ZnCl2 added to the culture medium, (b) uptake and subcellular distribution of Zn(2+), and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd(r) cell types grew normally (T d≊16-18 h) during exposures to Zn(2+) at levels up to 100 µM added to the growth medium, but displayed abrupt growth inhibition at higher Zn(2+) levels, (b) Cd(r) cells incorporate fourfold more Zn(2+) during a 24-h exposure to the maximal subtoxic level of Zn(2+) and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd(r) cell is proficient in this response during exposure to the maximal subtoxic Zn(2+) level. These findings suggest that (a) the CHO and Cd(r) cell systems will be useful in further studies of cellular Zn(2+) metabolism, especially in comparisons of Zn(2+) metabolism in the presence and absence of induction of the Zn(2+)-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn(2+) uptake.