Effects of retinoic acid on plasminogen activator and mitogenic responses of cultured mouse cells.

Treatment of simian virus 40-transformed 3T3 cells with 1 microM beta-all-trans-retinoic acid (RA) resulted in a 5- to 6-fold enhancement of plasminogen activator (PA) release. Intracellular PA levels rose to twice control levels. Confluent 3T3 cells were less responsive to RA. In 6 of 10 experiments, no increase in 3T3 cell PA levels was noted, although up to a 2.5-fold enhancement of PA elaboration has been observed in some experiments at a dose of 10 microM RA. Simultaneous treatments of 3T3 cells with 10 microM RA and 2.1 to 9.3 mM Ca2+, 5 to 40 ng phorbol myristate acetate per ml, or 150 to 600 ng Fraction I from lactalbumin hydrolysate (Fl) protein per ml indicated that RA potentiated the PA stimulatory activities of these agents. Extracellular PA levels of RA-treated cells increased by 4 to 10 times the amount of increase observed for Ca2+, PMA, or Fl alone. A potentiating activity of RA was also evident when quiescent 3T3 cells were pretreated with 10 microM RA and then stimulated to synthesize DNA with Ca2+, PMA, or Fl. For RA-pretreated cells, an increased percentage of nuclei was labeled with [3H]thymidine (24 hr) in response to doses of the three mitogens which were ineffective without RA pretreatment (2.4mM Ca2+, 5 ng PMA per ml, or 150 ng Fl protein per ml). Additional experiments have indicated that, like platelet extracts, Ra renders quiescent 3T3 cells competent to synthesize DNA in response to the progression factors of human plasma as defined by Pledger et al. (Proc. Natl. Acad. Sci. U. S. A., 74: 4481-4485, 1977). Pretreatment of quiescent 3T3 cells for 6 hr with 10 microM RA resulted in a greater than 4-fold increase in the percentage of cells which incorporated [3H]thymidine in response to a 36-hr treatment with 10% human plasma, as compared to cells treated with human plasma alone. Thus, under certain conditions, RA may have cell-activating properties, and caution should be exercised with regard to its suggested use as an antitumor agent.

Microtubule network and microtubule-associated proteins in leukemic T lymphocytes.

Cytoskeletal changes have been known to occur in cell transformation. Vinca alkaloids which bind to the tubulin dimer and inhibit microtubule (MT) assembly as well as disrupting the MT network and mitotic spindle, have been used as cancer chemotherapeutic agents. It has been proposed that apart from their anti-mitotic activity, these drugs act on the peripheral MT of malignant cells to produce their cytolytic effects. In this paper we demonstrate the presence of an altered cytoplasmic MT network in MOLT-4 and HuT-78 leukemic cells (human T-cell leukemic lines) compared to normal human peripheral blood lymphocytes stimulated with mitogens. In addition, using a selective extraction protocol we have compared microtubule-associated proteins (MAPs) profiles of G1/S synchronized leukemic human T-cells and 20 h mitogen-stimulated human peripheral blood T-cells. We observed a dramatic decrease in the expression of a MAP of apparent molecular weight 52 kDa and pI 5.2 in the leukemic cells synchronized at the G1/S border of the cell cycle. These results suggest that altered MT network morphology and MAP synthesis may be components of the malignant phenotype in the T-lymphocytic leukemias studied here.

Growth restraint and differentiation by LPS/TNF-alpha/IFN-gamma reorganization of the microtubule network in human leukemia cell lines.

The microtubule (MT) network of the cytoskeleton has been implicated as a mediator of cellular signal transduction; disorganization of this network may allow for mitogenesis. In previous work, loss of MT network organization in human MOLT4 and HUT78 T-cell leukemias was demonstrated in contrast to an organized "spoke-wheel-like arrangement" in normal human T-lymphocytes. In this study, loss of MT network organization was shown in several representative acute myeloid leukemia (AML) cell lines: KG1 myeloblastic, HL60 promyelocytic, and U937 myelomonocytic cells. Re-organization of the MT network was observed in HL60 and U937 AML cells treated with combined lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). This re-organization paralleled earlier work which showed this combination was effective in inducing monocytic pathway differentiation and growth restraint in HL60 cells, and growth restraint in U937 cells. In contrast, KG1 cells exhibited growth restraint, but did not re-organize with LPS/TNF-alpha/IFN-gamma treatment. These results are consistent with a role for the MT network in mitogenesis. Loss of MT network organization appeared to parallel the neoplastic phenotype in three AML cell lines, whereas MT network re-organization accompanied recovery of growth control in 2 of 3 AML cell lines.

Environmental wavelengths of ultraviolet light induce cytoskeletal damage.

The ultraviolet component of sunlight is the major cause of skin cancer and is responsible for accelerating the aging of human skin. It is therefore important to determine the mechanisms by which ultraviolet light alters normal cellular functions. The potential importance of ultraviolet light-induced damage to non-DNA targets has received little attention. Since the cytoskeleton is an important participant in the control of normal cell growth, the microfilaments and microtubules of UV irradiated human skin fibroblasts have been studied using fluorescence microscopy. Polychromatic ultraviolet light, composed of environmentally relevant wavelengths, was found to disrupt the cytoplasmic microtubule complex in a dose dependent manner. The induction of microtubule disassembly did not correlate with the cytotoxicity of ultraviolet light of varying composition.

An immunofluorescence study of the calcium-induced coordinated reorganization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the coordinated reorganization of microtubules, microfilaments, and keratin intermediate filaments in cultured human epidermal keratinocytes following a switch from low-Ca++ (0.15 mM) medium to high-Ca++ (1.05 mM) medium. A dramatic reorganization occurs concurrently in the three major cytoskeletal components shortly after the calcium switch. The most prominent features are the alignment of keratin filaments at the plasma membranes of apposed cells, the induction of microfilament rings, the restriction of microtubules to the area within the boundaries of the microfilament rings, and the alignment of actin bundles at cell borders. Additional changes are observed in terminally differentiated cells. This is the first report that describes simultaneous changes in the organization of the three major cytoskeletal components of epidermal keratinocytes. Cytochalasin D and demecolcine (colcemid) studies were performed to determine whether the organization of microtubules, microfilaments, and keratin filaments, as well as the calcium-induced reorganization of these cytoskeletal elements, may be dependent on the existence of structural relationships between them. These studies demonstrate that the disruption of microfilaments results in the formation of a latticelike keratin network, with a close association of actin and keratin being maintained. The formation of keratin filament alignments occurs even in the absence of intact microfilaments. In addition, it was found that the Ca(++)-induced reorganization of microfilaments and keratin filaments is not dependent on an intact microtubule network. Furthermore, the reorganization of actin into concentric rings can be dissociated from changes in the organization of keratin filaments.

Disruption of keratin intermediate filaments by ultraviolet radiation in cultured human keratinocytes.

Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect.

Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions.

To understand the mechanisms of Cd2+ and Ni2+ cytotoxicity, we have studied the effects of these two metal ions on the organization of cytoskeletal elements, microtubules (MT) and microfilaments (MF), cytoskeletal protein sulfhydryls and cellular glutathione (GSH) in cultured 3T3 cells. At a metal ion dose that caused 95% inhibition of DNA synthesis, Cd2+ (10 microM, 16 h exposure) induced MT depolymerization whereas Ni2+ (2 mM, 20 h exposure) elicited MT aggregation and bundling. Under these conditions, Cd2+ and Ni2+ also caused MF aggregation and redistribution. Furthermore, exposure of cells to Cd2+ resulted in a dose-dependent increase in cytoskeletal protein sulfhydryls and cellular GSH levels. In contrast, treatment of cells with Ni2+ resulted in a dose-dependent decrease in cytoskeletal protein sulfhydryls as well as cellular GSH content. Time course studies showed that cells exposed to 10 microM Cd2+ exhibited a biphasic response in regulating their cytoskeletal protein sulfhydryls and cellular GSH, e.g. an initial decrease followed by a steady recovery and overshooting upon prolonged incubation. However, restoration of cytoskeletal protein sulfhydryls occurred approximately 2 h after commencement of cellular GSH recovery in Cd(2+)-treated cells. These results suggest that cellular GSH may play an important role in regulating cytoskeletal protein sulfhydryls. On the other hand, decrease of cellular GSH induced by Ni2+ might facilitate oxidation of cytoskeletal protein sulfhydryls and formation of disulfide bonds between individual MT polymers which would favor MT aggregation in Ni(2+)-exposed cells. In addition, we also demonstrated that elevation of cellular GSH in Cd(2+)-treated cells probably resulted from new GSH synthesis.

Calmodulin modulation of adverse effects of Cd(2+) on microtubules and tubulin polymerization in vitro.

The contribution of calmodulin to the effects of Cd(2+) on microtubules has been investigated in the detergent-extracted cytoskeletons of cultured cells and on tubulin polymerization in vitro. When incubated with micromolar concentrations of Cd(2+) or Ca(2+) alone, or a mixture of both ions, microtubules in the extracted cytoskeletons disassembled. This disassembly was prevented by compound 48/80, a potent calmodulin inhibitor. In bovine brain microtubule protein containing microtubule-associated proteins, calmodulin also enhanced the Cd(2+)-mediated inhibition of tubulin polymerization; further, this enhancing effect of calmodulin was reversed by compound 48/80. These results demonstrate that Cd(2+) can affect microtubules by binding to and activating calmodulin.

Cytoskeletal injury induced by hexavalent chromate.

To understand the mechanisms of toxicity of hexavalent chromate (K(2)CrO(4), Cr(6+)), the effects of this metal ion on the organization of microtubules (MTs) and microfilaments (MFs), DNA synthesis, cytoskeletal protein synthesis, cytoskeletal protein sulphhydryls (-SH groups), and cellular glutathione (GSH) levels in 3T3 cells were examined. Exposure of cells to Cr(6+) for 16 hr resulted in a dose-dependent inhibition of DNA synthesis with 50% inhibition occurring at 16.6 mum. Treatment of cells with 3.13 mum-Cr(6+) for 16 hr resulted in a slight cell retraction and, in some cells, MT bundling, without much effect on the morphology of dense MFs. At 25 mum, Cr(6+) caused disruption of the cell sheet, depolymerization of MTs, particularly those in the peripheral areas, and redistribution of MFs, which assumed a smearing morphology. Exposure to 100 mum-Cr(6+) induced severe thinning of MTs and loss of MFs. Although doses of 3.13 mum-Cr(6+) or less slightly enhanced the level of cytoskeletal protein synthesis (e.g. 38% increase at 3.13 mum-Cr(6+)), Cr(6+) at 6.25 mum or more produced a dose-dependent inhibition of cytoskeletal protein synthesis (50% inhibition at 11.25 mum). Exposure of cells to Cr(6+) for 16 hr resulted in a dose- and time-dependent increase of cellular GSH level. Furthermore, the elevated cellular GSH induced by Cr(6+) was diminished by treatment with buthionine sulphoximine (BSO), a potent inhibitor of GSH biosynthesis. In addition, depletion of GSH by BSO increased cell sensitivity to Cr(6+) insult and aggravated Cr(6+)-induced cytoskeletal perturbation. However, Cr(6+) treatment of cells did not significantly affect the amount of cytoskeletal protein sulphhydryls. These results suggest that cytoskeletal injury may be an important part of the mechanism for Cr(6+) toxicity. The cytoskeletal damage may result directly from the inhibition of cytoskeletal protein synthesis rather than from the interaction between Cr(6+) and cytoskeletal protein sulphhydryls.

Microtubule disassembly induced by sensitizing halogenated nitrobenzene derivatives.

The potency of certain halogenated nitrobenzene derivatives to induce allergic contact dermatitis has been demonstrated in guinea-pigs and in man. As the first step towards understanding the mechanisms of cellular injury induced by these sensitizers, we have studied by fluorescence microscopy the effects of 13 halogenated (nitro)benzene derivatives on microtubule organization in mouse 3T3 fibroblasts and human AG1522 skin fibroblasts. Untreated cells have numerous microtubules distributed in a network fashion throughout the cytoplasm and extending to the cell periphery. Exposing cells for 3 hr to micromolar doses of halogenated nitrobenzene derivatives, which induce allergic contact dermatitis in guinea-pigs and man, resulted in a dose-dependent disassembly of microtubules. In contrast, incubation with 10-70 times higher doses of seven halogenated (nitro)benzene derivatives that do not sensitize guinea-pigs or man, had no discernible effect on microtubule organization of both cell types under identical assay conditions. Thus, for the 13 halogenated (nitro)benzene derivatives tested in this study, a 100% positive correlation exists between the sensitizing capacity as determined by in vivo tests on guinea-pigs and man (Landsteiner and Jacobs, 1936) and the ability to cause microtubule disassembly in cultured cells. These results may provide the basis for developing an in vitro screening assay for identifying other potential halogenated nitrobenzene sensitizers. In addition, these studies represent a new approach to investigating the mechanisms of contact sensitivity induced by simple chemicals.

Distinct cytoskeletal injuries induced by As, Cd, Co, Cr, and Ni compounds.

The risks of metal compounds to human health are highlighted by the ubiquity of exposure and their persistence in the environment. Although compounds of As, Cd, Co, Cr, and Ni are known or "reasonably anticipated" to be carcinogenic to humans and/or experimental animals, the cellular targets of these health hazards and the underlying mechanisms of their carcinogenicity are still unclear. We show in this report that dramatic, time- and dose-dependent cytoskeletal perturbations, especially in the distribution and organization patterns of microtubules and microfilaments, two of the principal components of the cytoskeleton, occurred in 3T3 cells upon exposure to these metal salts. Each metal salt appeared to induce a different, typical pattern of cytoskeletal injury, probably reflecting the specificity of action of each metal ion. These results suggest that the cytoskeleton can indeed act as a target for injury by epigenetic carcinogenic metal compounds in the environment. These findings should help our efforts to understand the mechanisms of action of metal compounds at the subcellular and molecular levels.

Role of calmodulin in cadmium-induced microtubule disassembly.

Micromolar CdCl2 has been shown to cause disassembly of the cytoplasmic microtubule complex in cultured Swiss 3T3 cells. We show in this paper that Cd(II), an environmental and occupational health hazard, induces microtubule disassembly in an in situ cytoskeleton model system, and that the calmodulin inhibitors, trifluoperazine and Compound 48/80, prevent this Cd(II)-induced microtubule disassembly. Our results suggest that Cd(II) affects microtubules by activating calmodulin associated with the cytoskeleton. Furthermore, the fact that these two ions have very similar ionic radii (0.99A vs. 0.97A) supports our conclusion that Cd(II) acts similarly to Ca(II) in inducing microtubule disassembly. This may be relevant to the mechanism of Cd-mediated cellular injury.

Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts.

The relationship between total glutathione (GSH) content and cell growth was examined in 3T3 fibroblasts. The intracellular GSH level of actively growing cultures gradually decreases as these cells become quiescent by either serum deprivation or high cell density. Upon mitogenic stimulation of sparse, quiescent (G0/G1) cultures with serum, there is a rapid 2.3-fold elevation in intracellular GSH levels which is maximal by 1 h and returns to baseline by 2 h. This is followed by a more gradual increase in GSH content as cells enter the S phase. In addition, the elevation in GSH content is required for maximum induction of DNA synthesis. Treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose-dependent decrease in the percent of cells capable of entering S phase. These results indicate that GSH may be important in the regulation of cellular proliferation.

Effects of sodium arsenite on the cytoskeleton and cellular glutathione levels in cultured cells.

The effects of As3+ (NaAsO2) on the microtubule and microfilament organization, cytoskeletal protein synthesis, cytoskeletal and cytosolic (soluble) protein sulfhydryls, and cellular glutathione (GSH) levels were examined in Swiss 3T3 mouse cells. Exposure of cells to 2.5 microM As3+ for 16 hr resulted in apparent cell retraction and loss of thick cables of actin filaments. However, the cells still retained numerous thinner microfilaments distributed in a disorganized manner. Microtubule organization was relatively undisturbed. At higher doses (greater than or equal to 20 microM), As3+ treatment caused a severe loss of microtubules and the remaining dense finer actin filaments formed smearing clusters in perinuclear areas. Treatment of cells with As3+ also induced a dose-dependent inhibition of cytoskeletal protein synthesis. Furthermore, As3+ exposure enhanced cellular GSH synthesis since the elevated cellular GSH content in As(3+)-treated cells could be abolished by treatment with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase required for GSH biosynthesis. As determined by the N-[3H]-ethylmaleimide binding assay, As3+ exposure also increased the amount of protein sulfhydryls in both the cytoskeletal and the cytosolic protein fractions. Moreover, a greater increase in protein sulfhydryls occurred in the cytoskeletal fraction than in the soluble fraction. These results indicate that the cytoskeleton could be a cellular target for injury by As3+ exposure. The elevated cellular GSH content induced by As3+ could provide a protective mechanism against further injury from this metal insult.

Microtubules and microtubule-associated proteins in resting and mitogenically activated normal human peripheral blood T cells.

The binding of an appropriate ligand to its specific receptor on the membrane of T cells triggers a cascade of events involved in T cell activation. An important yet unanswered question is how the mitogenic signals are transmitted through the cytoplasm and into the nucleus. The present study was carried out to determine changes in the microtubule (MT) system, following T cell activation. Fluorescence microscopy was employed to examine the organization of the microtubule network in human peripheral blood T cells in response to four different mitogens (phytohemagglutinin, concanavalin A, anti-CD3, and phorbol 12-myristate 13-acetate). The microtubules increase in length, number, and complexity of distribution 20 h after mitogenic stimulation. Using an in situ direct analysis protocol consisting of selective extraction of cells with detergent and Ca2+, 11 protein species, which fulfill the operational definition of microtubule-associated proteins (MAPs), were identified in resting human T cells. Alterations in the expression of these protein species were studied following mitogenic stimulation. These alterations in MAPs expression were also found in purified blast cell fractions indicating that they were specific changes occurring in activated T cell populations. These observations suggest a role for MT and MAPs in the cascade of human T cell activation.

Microtubule disassembly and morphologic alterations induced by 1-chloro-2,4-dinitrobenzene, a substrate for glutathione S-transferase.

1-chloro-2,4-dinitrobenzene (CDNB), a potent substrate for glutathione S-transferase, is known to rapidly deplete cellular glutathione (GSH) via conjugate formation. Treatment of quiescent 3T3 cells with 5 uM CDNB results in disassembly of microtubules (MT) within 1 hr as revealed by indirect immunofluorescence microscopy. In addition, CDNB treatment also induces dramatic morphologic alterations similar to those mediated by colchicine. Furthermore, taxol prevents both MT disassembly and morphologic changes normally occurring in CDNB as well as colchicine-treated cells. The mechanism of CDNB-mediated MT disassembly in vivo and its possible relationship to cellular GSH metabolism are under current studies.

Studies on the mechanisms of Ni2(+)-induced cell injury: I. Effects of Ni2+ on microtubules.

Cytoskeletal perturbations have been associated with exposures to a variety of toxic agents as well as a number of human pathological conditions. We have observed dramatic alterations in the organization of microtubules (MT), a major component of the cytoskeleton, in 3T3 cells exposed to Ni2+. Severe perinuclear bundling and aggregation of MT occurred in both a time- and dose-dependent fashion, and this MT damage was reversible upon removal of Ni2+ from the culture media. To understand the mechanism of the Ni2(+)-induced MT change, we investigated the effect of Ni2+ (0.01 to 3.0 mM) on in vitro tubulin polymerization. Ni2+ at lower concentrations (0.01 to 1.0 mM) had little or no significant effect on the kinetics of MT polymerization. In contrast, in the presence of 1.5 to 2.0 mM Ni2+, a significant promoting effect on both the rate and the final extent of polymerization was observed. However, at Ni2+ concentrations higher than 2.0 mM, such stimulatory effect on the rate and the final extent of tubulin polymerization declined. Furthermore, the promoting effects of Ni2+ on MT polymerization were accompanied by a significant decrease in the lag period. Electron microscopic examination of samples of the polymerization product showed that MT, polymerized in the presence of 2.0 mM Ni2+, appeared more numerous and shorter (1.10 +/- 1.02 microns) than those of control (3.81 +/- 2.29 microns; p less than 0.005). This was probably a direct result of an increase in the number of initiation centers in the presence of Ni2+ as a consequence of the decreased critical concentration (7%, p less than 0.05) necessary for polymerization to occur. Our results suggest that Ni2+ may exert its toxic effect on MT in cultured cells by altering the normal kinetics of MT polymerization.

Relationship between 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione.

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthionine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 microM 1-chloro-2,4-dinitrobenzene and 250 microM buthionine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthionine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 microM 2-cyclohexene-1-one, which depletes free glutathione by conjugation, resulted in a complete depletion of total cellular glutathione content without altering the microtubule organization. These results suggest that the total glutathione content may be important for cellular recovery from 1-chloro-2,4-dinitrobenzene-mediated cytoskeletal injuries, and that microtubule disassembly observed in 1-chloro-2,4-dinitrobenzene-treated cells probably results from depletion of cellular glutathione coupled with binding to tubulin and/or other microtubule components.

Alterations in cytoskeletal organization and homeostasis of cellular thiols in cadmium-resistant cells.

To understand further the mechanisms of cadmium toxicity, cytoskeletal organization and homeostasis of cellular thiols were examined in cadmium-resistant cells isolated from Swiss mouse 3T3 cells by incubation in graded concentrations of CdCl2 (Cd2+) in the culture medium. Cd(2+)-resistant cells displayed profound alterations in their cytoskeletal organization characterized by the appearance of many elongated, tadpole-shaped cells with a high density of microtubules (MT) and microfilaments (MF), with the former being mainly distributed along the long axis of the cell. Exposure of Cd(2+)-resistant cells to 50 microM Cd2+ for 16 hr did not cause apparent cytoskeletal perturbations, whereas treatment of parental cells with 5 microM Cd2+ for the same duration produced a severe loss of MT and smeared patches of MF. Thus, the cytoskeleton of Cd(2+)-resistant cells is markedly more preserved and protected against Cd2+ damage than that of their parental counterparts. Cd(2+)-resistant cells contained a higher basal level of protein sulfhydryls (PSH) in both the cytoskeletal and cytosolic fractions than the parental cells. Exposure to 50 microM Cd2+ further increased cellular PSH contents, reaching 192 and 215% of the basal levels for the cytoskeletal and cytosolic fractions, respectively. Although 5 microM Cd2+ exposure also elevated the amounts of PSH in parental cells, the "absolute" values were still below the corresponding basal levels in Cd(2+)-resistant cells. Furthermore, Cd(2+)-resistant cells also exhibited enhanced basal levels of metallothionein and cellular glutathione (GSH), amounting to 19- and 2.1-fold of the parental basal levels, respectively. Thus, the Cd(2+)-resistant cells produced larger quantities of both protein and nonprotein thiol-containing elements than the parental cells. Interestingly, exposure of Cd(2+)-resistant cells to 50 microM Cd2+ also further increased metallothionein and cellular GSH to 178 and 138% of the basal levels, respectively. Based on the affinity of Cd2+ for sulfhydryls as a mechanism of Cd2+ toxicity, we propose that the coordinately increased levels of metallothionein, GSH, and PSH in Cd(2+)-resistant cells would provide a mechanistic basis for the homeostasis of cellular thiols which may collectively contribute to the cytoskeletal preservation by protecting the cytoskeleton from Cd2+ insult.