heat-inactivated fetal calf serum and 0.2% sodium bicarbonate and antibiotics in a .... Nuclear extracts from 1- to 2Ã107 cells were prepared according to the method of ..... Effect of stress treatments on vimentin mRNA stability. Actinomycin D ..... thymocytes undergoing apoptosis after heat-shock or dexamethasone treatment.
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Journal of Cell Science 110, 201-207 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS9384
Heat-shock and cadmium chloride increase the vimentin mRNA and protein levels in U-937 human promonocytic cells Nuria E. Vilaboa1,4,*, Laura García-Bermejo1,3, Concepción Pérez1,2, Elena de Blas1, Consuelo Calle4 and Patricio Aller1,† 1Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006-Madrid, Spain 2Instituto de Química Médica, Madrid, Spain 3Departamento de Biológía Celular y Genética, Universidad de Alcalá, Madrid, Spain 4Departamento de Bioquímica y Biología Molecular, Facultad de Medicina Universidad
Complutense (CC), Madrid, Spain
*Present address: Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, 1011 NW 15th Street, Miami, FL 33136-1019, USA †Author for correspondence
SUMMARY Heat-shock for 2 hours at 42°C, or the administration for 3 hours of 100 or 150 µM cadmium chloride, inhibited the subsequent proliferation activity, induced the expression of functional differentiation markers, and caused an increase in the amount of the stress-responsive HSP70 protein in U937 human promonocytic cells. In addition, both heat and cadmium produced an increase in the amount of the intermediate filament protein vimentin, as determined by immunoblot and immunofluorescence assays. By contrast, the amounts of actin and β-tubulin were not significantly altered. The amount of vimentin mRNA was also increased during recovery from stress, indicating that vimentin expression was not exclusively regulated at the protein level. Although cadmium caused an early, transient stimu-
lation of c-jun and c-fos expression and AP-1 binding activity, heat-shock failed to alter both protooncogene expression and transcription factor binding, indicating that the stress-induced vimentin increase was not the result of AP-1-mediated transcriptional activation. Finally, it was observed that the rate of decay of vimentin mRNA upon actinomycin D administration was decreased in heat- and cadmium-pretreated cells in comparison to untreated cells. These results indicate that stress treatments cause an increase in vimentin levels in promonocytic cells, which may be explained at least in part by transcript stabilization.
INTRODUCTION
shock decreases the phosphorylation degree of the nuclear lamins A and C, which are intermediate filament-like proteins (Krachmarov and Traub, 1993). By contrast, very little is known about the effects of stress treatments on the synthesis of cytoskeletal proteins. In the present report we analyze the effect of two typical stress agents, namely heat-shock and cadmium chloride, on the expression of the major cytoskeletal proteins in U-937 cells, a human promonocytic leukemia cell line (Sundström and Nilsson, 1976). The results indicate that the stress treatments do not only alter vimentin organization, but they also cause an increase in the amount of this intermediate filament protein.
A common response of all living organisms to abnormally high temperatures or other environmental or metabolic insults is the stimulation of the expression of a group of proteins termed heat-shock proteins (HSPs), which may serve to protect the cells from the lethal consequences of stress (Lindquist and Craig, 1988). The stress response is accompanied by other cellular phenomena which include the inhibition of the expression of normally synthesized proteins (Lindquist, 1986), the inhibition of DNA synthesis and cell proliferation (Roti Roti et al., 1992) and the induction of apoptosis (Sikora et al., 1993). One of the best known morphological effects of heat-shock is the production of alterations in the cytoskeleton structure. For instance, it may cause disorganization of the network of cytoplasmic intermediate filaments, to form juxtanuclear or perinuclear structures (Falkner et al., 1981; Welch and Suhan, 1985; Collier et al., 1993); destruction of the microtubule network (Lin et al., 1982); disintegration of normal actin structures including stress fibers (Iida et al., 1986); and appearance of intranuclear actin rod-shaped bodies (Welch and Suhan, 1985; Iida et al., 1986). In addition, it has been shown that heat-
Key words: Vimentin, Heat-shock, Cadmium, Promonocytic cell
MATERIALS AND METHODS Cell culture and treatments The U-937 cells used were mycoplasma-free. The cells were grown in suspension in RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum and 0.2% sodium bicarbonate and antibiotics in a humidified 5% CO2 atmosphere at 37°C. Cells were seeded in 100 mm plastic dishes at a concentration of 105 cells/ml and maintained in continuous logarithmic growth by passage every 2-3
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days. Actinomycin D (Sigma Química, Madrid, Spain) was dissolved in absolute ethanol at 5 mM and applied to the culture at of 5 µM. For cadmium treatments, cadmium chloride (Merck, Darmstad, FRG) was freshly dissolved in distilled water at 100 mM and applied to the culture at the desired final concentrations. For heat-shock, the cultures were transferred to an oven at 42°C. For recovery after the treatments, the cells were collected by centrifugation, washed once (in the case of heat-shock) or three times (in the case of cadmium treatment) with pre-warmed (37°C) RPMI medium, and resuspended in pre-warmed cadmium-free medium. As controls, unheated cadmium-untreated cells were subjected to the same manipulations as treated cells. Inhibition of cell proliferation and permeability to trypan blue were used as criteria to evaluate the toxicity of the treatments.
CACTAG-3′ were also synthesized and used to prepare an AP-1 mutant oligoprobe. The binding reaction was carried out for 15 minutes at room temperature in 20 µl of binding buffer (25 mM NaCl, 60 mM KCl, 2 mM 1,4-dithiotreitol, 2% Ficoll, 0.1% (v/v) Nonidet P-40, 0.1 mg/ml bovine serum albumin, 25 mM Hepes, pH 7.4) containing 5 ng of the labeled probe, 8 µg of total nuclear proteins, 2 µg of poly(dI:dC), and 2 µg of salmon sperm DNA. To examine the specificity of binding, the reaction was carried out in the presence of excess wild-type or mutated AP-1 unlabelled probes, and in the presence of 1 µl of a polyclonal anti-jun antibody or pre-immune serum (obtained from Dr R. Bravo, Bristol-Myers Squibb, Princeton, NJ, USA). The samples were electrophoresed in 4% polyacrylamide gels, and the gels dried and autoradiographed.
Measurement of differentiation markers Nitro blue tetrazolium (NBT, Sigma Química) reduction was measured by incubating 106 cells for 30 minutes at 37°C in 1 ml of PBS containing 0.1% NBT and 0.15 µM 12-O-tetradecanoylphorbol13-acetate (TPA, Sigma Química), after which 0.2 ml of 5 N HCl was added and the cells kept for 1 hour at room temperature. Upon centrifugation, the cell pellet was resuspended in 0.5 ml dimethyl sulfoxide and the absorbance recorded by spectrometry at 560 nm. The surface expression of CR3 (CD11b/CD18) leukocyte integrin was determined by indirect immunofluorescence combined with flow cytometry using the Bear 1 (anti-CD11b) monoclonal antibody, as previously described (Aller et al., 1992).
RNA blot assays Total RNA was extracted using an Ultraspect-II RNA isolation kit (Biotecx Laboratories, Inc. Houston, TX, USA), following the procedure described by the manufacturer. RNA samples were denatured, electrophoresed in 1.1% agarose-formaldehyde gels containing 0.1 µg/ml ethidium bromide and blotted onto nylon membranes (Hybond-N, Amersham). Ethidium bromide staining of 28 S and 18 S ribosomal RNAs was routinely checked before blotting as a control of sample loading and after blotting as a control of RNA transfer. RNA blots were prehybridized, hybridized with excess 32Plabeled probes, washed under highly stringent conditions and autoradiographed. The probes used were: the 1.1 kb human vimentinspecific XhoI fragment of p4F1 plasmid (Ferrari et al., 1986); the 2.7 kb fos-specific XhoI-NcoI fragment of pc-foshuman clone (Van Straaten et al., 1983); and the 1.8 plus 0.8 kb human jun-specific EcoRI-PstI fragments of AH119 clone (Ryseck et al., 1988). The fragments were labeled to approximately 109 cpm/µg of DNA with [α-32P]dCTP (3,000 Ci/mmol, New England Nuclear, Boston, MA, USA) by random hexanucleotide priming (Feinberg and Vogelstein, 1984).
Immunoblot and immunofluorescence assays For immunoblot assays, cells were washed with Ca2+- and Mg2+-free phosphate buffered saline (PBS) and lysed in 62.5 mM Tris-HCl, pH 6.8, containing 2% SDS, 5% (v/v) β-mercaptoethanol, and 10% (v/v) glycerol. After boiling for 2 minutes at 98°C, the protein extracts were separated on SDS-polyacrylamide (10%) slab minigels (Laemmli, 1970). Electrophoretic blotting and immunological detection of proteins were carried out essentially as described by Towbin et al. (1979). The following mAbs were used as first antibodies: a mouse anti-human HSP70 which specifically recognized the stress-inducible form (clone C92F34-5, Stress Gene, Biotechnologies Corp, Victoria, Canada); a mouse anti-chicken actin (Amersham International, Little Chalfont, UK); a mouse anti-chicken β-tubulin (Amersham); and a mouse anti-porcine vimentin (Amersham). As the second antibody, horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts, Copenhagen, Denmark) was used. The filters were developed with an enhanced chemiluminiscence western blotting detection kit (Amersham), following the procedure described by the manufacturer. For immunofluorescence assays, cells were washed with PBS and collected on glass slides using a cytospin. Upon fixation and permeabilization for 15 minutes with cold absolute methanol, the cells were firstly incubated for 1 hour at room temperature with the anti-vimentin mAb in the presence of 1% bovine serum albumin, then washed three times with PBS, and incubated again for 45 minutes at room temperature with fluorescein-conjugated sheep anti-mouse IgG (Amersham) in the presence of 1% bovine serum albumin. The cells were examined by fluorescence microscopy. Gel retardation assays Nuclear extracts from 1- to 2×107 cells were prepared according to the method of Schreibert et al. (1988) and stored at −70°C. The partially complementary oligonucleotides 5′-GGCTAGTGATGAGTCAAGCCGGATC-3′ and 5′-GGGATCCGGCTTGACTCATCACTAG-3′ were synthesized in a Gene Assembler Plus (Pharmacia LKB, Uppsala, Sweden) and used to prepare a double-strand oligonucleotide containing an AP-1-recognizing sequence (wild type AP-1). The probe was labeled with [α-32P]dCTP (3,000 Ci/mmol, New England Nuclear, Boston, MA, USA) following the method of Sambrook et al. (1989). The oligonucleotides 5′-GGCTAGTGATGCTGCAAGCCGGATC-3′ and 5′-GGGATCCGGCTTGCAGCAT-
RESULTS Cell growth, cell differentiation and HSP70 expression We firstly investigated the capacity of pulse treatments with heat (2 hours at 42°C) or cadmium chloride (3 hours at concentrations ranging from 25 to 150 µM) to affect the subsequent proliferation activity of U-937 cells, since proliferation inhibition is a criterium of stress (Roti Roti et al., 1992). The treatments transiently decreased proliferation, which was gradually reinitiated at hour 36 of recovery in the case of heatshock and 100 µM cadmium, and at hour 60 in the case of 150 µM cadmium (Fig. 1A). The treatments caused a slight increase in the number of non viable cells, as measured by trypan blue penetration at hour 12 of recovery. In addition heatshock and cadmium caused a transient increase in the expression of differentiation markers, namely the surface accumulation of the CD11b/CD18 leukocyte integrin (Hickstein et al., 1989) and the capacity to reduce NBT (Collins et al., 1980) (Fig. 1B). This suggests that stress treatments induce the functional differentiation of myeloid cells, as earlier observed by Richards et al. (1988) using HL-60 human promyelocytic cells. As a criterium of stress at the molecular level, the amount of the stress-responsive protein HSP70 was measured at different times of recovery by immunoblot assays. Both heat-shock (Fig. 2) and 100 µM cadmium chloride (result not shown) produced a transient increase in HSP70 which was already detected at the end of the treatment (hour 0 of recovery), reached the maximum at hours 12 to 24, and decreased thereafter to control
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Fig. 2. Changes in HSP70 protein content. U-937 cells were heated for 2 hours at 42°C (HS) or treated for 3 hours with 150 µM cadmium chloride (Cd), and then allowed to recover for the indicated time periods at 37°C in cadmium-free medium. As control (Cont), unheated cadmium-untreated cells were used. Protein samples (5 µg per lane) were assayed by immunoblot, using an antibody which specifically recognized the HSP70 stress-inducible form. Reference proteins were rabbit muscle phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa) and hen egg white ovoalbumin (42.7 kDa).
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Fig. 1. Effect of heat-shock and cadmium chloride on U-937 cell proliferation and expresion of differentiation markers. Cells were heated for 2 hours at 42°C (squares) or treated for 3 hours with 25 µM (triangles), 100 µM (open circles) or 150 µM (filled circles) cadmium chloride, and then allowed to recover at 37°C in cadmiumfree medium. Unheated cadmium-untreated cells were used as control (crosses). (A) Increase in the number of viable cells in control and treated cultures. The inset indicates percentages of nonviable cells (cells permeable to trypan blue) at hour 12. (B) Surface expression of the CD11b/CD18 leukocyte integrin, as determined by indirect immunofluorescence combined with flow cytometry using the Bear 1 (anti-CD11b) antibody; and reduction of NBT, as determined by spectrometry. The values are representative of one of two experiments with similar results.
levels. However, the increase in HSP70 produced by 150 µM cadmium persisted even at 120 hours of recovery (Fig. 2). Expression of cytoskeletal proteins To analyze possible effects of the stress treatments on the expression of cytoskeletal proteins, the accumulation levels of actin, β-tubulin and the intermediate filament protein vimentin were measured at different times of recovery by immunoblot assays. It was found that the levels of both actin and tubulin, which were already elevated in non-stressed cells, remained unaltered during recovery from heat-shock (Fig. 3) and cadmium treatments (results not shown). By contrast the level of vimentin, which was low in non-stressed cells, underwent a
Fig. 3. Relative levels of actin, β-tubulin and vimentin. The experimental conditions were the same as in Fig. 2, except that antiactin, anti-β-tubulin and anti-vimentin antibodies were used instead of anti-HSP70, and that 20 µg protein samples per lane were used for vimentin assays.
significant increase during recovery from treatments. In the case of heat-shock (Fig. 3) and 100 µM cadmium chloride (result not shown), the increase reached the maximum at hour 24 of recovery and decreased thereafter to control levels. However, in the case of 150 µM cadmium the amount of vimentin remained elevated even at 120 hours of recovery (Fig. 3).
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N. E. Vilaboa and others Fig. 4. Changes in cytoskeleton-associated vimentin level and organization as a consequence of heat-shock (2 hours at 42°C) or treatment with cadmium chloride (3 hours at 150 µM). Following formaldehyde fixation and Triton X-100 permeabilization, the reactivity with an anti-vimentin antibody was examined by fluorescence microscopy. (A,E) Unheated cadmium-untreated cells; (B,H) 24 hours recovery from heat-shock; (C) 72 hours recovery from heat-shock; (D) 72 hours recovery from cadmium treatment; (F) 3 hours recovery from heat-shock. (G) 12 hours recovery from cadmium treatment. Left column for A-D, phase contrast. Bars, 15 µm.
Fig. 5. Changes in vimentin mRNA level. U-937 cells were heated for 2 hours at 42°C (HS) or treated for 3 hours with 150 µM cadmium chloride (Cd), and then allowed to recover for the indicated time periods at 37°C in cadmium-free medium. Total RNA samples (15 µg per lane) were examined by northern blot using a vimentin specific cDNA probe. As control, unheated cadmium-untreated cells were used. Ethidium bromide staining of ribosomal RNAs in the gel is shown at the bottom, as a control of sample loading.
The fluorescence microscopy assays also revealed changes in vimentin organization. The well-organized vimentin network present in unstressed cells (Fig. 4E) disappeared 3 hours after heat-shock (Fig. 4F) or 12 hours after cadmium treatment (Fig. 4G), having the appearance of diffuse fluorescence. The network was partially formed again at 24 hours of recovery after heat-shock (Fig. 4H).
The changes in vimentin content were corroborated by fluorescence microscopy. The frequency of cells with detectable amounts of cytoskeleton-associated vimentin was increased at hour 24 of recovery from heat-shock (Fig. 4B) in relation to untreated cells (Fig. 4A), to decrease again at hour 72 (Fig. 4C). However, a considerable fraction of cells was still positive for vimentin at hour 72 of recovery from treatment with 150 µM cadmium (Fig. 4D).
Vimentin mRNA accumulation It is known that HSPs may interact with and stabilize other proteins, including cytoskeletal components (Nishida et al., 1986; Lee et al., 1993). Since the kinetics of accumulation of HSP70 (Fig. 2) and vimentin (Fig. 3) during recovery from stress treatments overlapped, we asked whether the increased expression of the intermediate filament during stress could be exclusively regulated at the protein level. To investigate this question, the vimentin mRNA content was measured by northern blot assays. Some of the obtained results are shown in Fig. 5. It was found that the amount of vimentin mRNA was increased during recovery from heat-shock and cadmium treatments, indicating that the stress agents also stimulated vimentin expression at the RNA level. c-jun and c-fos expression, and AP-1 binding activity Earlier works indicated that stress agents stimulated c-jun and
Heat- and cadmium-induced vimentin expression
c-fos expression as well as AP-1 binding activity in some cell types (Bukh et al., 1990; Jin and Ringertz, 1990; Sikora et al., 1993). In addition, we previously observed that the increase in vimentin expression caused by differentiation inducers in U937 cells seemed to be mediated by the stimulation of AP-1 binding (Pérez et al., 1994). Hence, we found it of interest to measure the binding of this transcription factor in stressed U937 cells. It was found that 150 µM cadmium chloride transiently stimulated AP-1 binding at 2 to 5 hours of recovery, to decrease thereafter (Fig. 6). The treatment also stimulated the expression at the RNA level of c-jun and c-fos protooncogenes, which encode proteins constituent of AP-1 (Fig. 7). Similar results were obtained using 100 µM cadmium chloride (results not shown). By contrast, heat-shock failed to significantly stimulate both AP-1 binding activity (Fig. 6) as well as c-jun and c-fos expression (result not shown). Vimentin mRNA stability It has been reported that stress treatments caused stabilization of specific gene transcripts (Andrews et al., 1987; Bukh et al., 1990; Edington and Hightower, 1990). For this reason, we wanted to determine whether the stress treatments could affect the vimentin mRNA stability. With this aim, actinomycin D was applied for increased time periods to untreated cells, as well as to cells allowed to recover for 24 hours after heat-shock and cadmium treatments. Incubations longer than 5 hours were omitted, since actinomycin D caused significant cell damage. The results in Fig. 8 indicate the rate of vimentin mRNA decay
Fig. 7. Changes in c-jun and c-fos mRNA levels. Total RNA samples (15 µg per lane) extracted from untreated cells (Cont), from cells treated with cadmium chloride (3 hours at 150 µM) for the indicated time periods (left panel), and from cells allowed to recover from the cadmium treatment for the indicated time periods (right panel), were examined by northern blot using c-fos and c-jun specific probes. All other conditions, as in Fig. 5. 10
Absorbance (A.U.)
Fig. 6. Changes in AP-1 binding activity. Nuclear extracts obtained from untreated cells (Cont) and cells allowed to recover for the indicated time periods after cadmium chloride (Cd, 2 hours at 150 µM) or heat-shock (HS, 2 hours at 42°C) treatments were analyzed by gel retardation assay for AP-1 binding. As controls (right panel), nuclear extracts from cells allowed to recover for 2 hours from cadmium treatment were assayed under the following conditions: in the absence of competitor (−), in the presence of 50-fold molar excess of either wild-type (WT) or mutant (Mut) AP-1 unlabelled probes, and in the presence of either anti-Jun antibody (Jun) or a preimmune serum (PS). The position of free oligoprobe (filled arrowhead) and oligoprobe-AP-1 complexes (open arrowhead) is indicated.
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Fig. 8. Effect of stress treatments on vimentin mRNA stability. Actinomycin D (5 µM) was applied for the indicated time periods to untreated cells (Cont), as well as to cells allowed to recover for 24 hours after heat-shock (HS, 2 hours at 42°C) and cadmium (Cd, 3 hours at 150 µM) treatments. Total RNA was extracted, and samples (35 µg per lane) were assayed by northern blot. (A) Autoradiographs corresponding to two representative experiments. Ethidium bromide staining of 28 S ribosomal RNA in the gel is shown, as a control of sample loading. The autoradiographs were arbitrarily exposed for better comparison of the rates of DNA decay in untreated versus stress-treated cells. Hence, they do not reveal the stress-induced increase in vimentin RNA content. (B) Values obtained by densitometric reading of all autoradiograms. The signal corresponding to hour 5 in control cells was below detection levels. Each point represents the mean of at least three determinations with similar results (differences were below 12%). All results at hour 0 were given the arbitrary value of 10. Crosses, untreated cells; squares, heat-shocked cells; circles, cadmium-treated cells.
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was slower in stressed cells than in untreated cells (Fig. 8). Under the assayed conditions (24 hours recovery from stress), transcript stabilization was considerably higher in cadmiumtreated cells than in heated cells. DISCUSSION The results in this work indicate that heat-shock and cadmium chloride do not only cause the disorganization of the vimentin network, but also induce an increase in the amount of this intermediate filament protein in U-937 human promonocytic cells. The treatments do not alter the accumulation of the other major cytoskeletal components, namely actin and tubulin. Earlier results by Courgeon et al. (1993) indicated that stress agents increased the synthesis of actin, tubulin, and a 46 kDa intermediate filament protein in Drosophila cells. In addition, Wachsberger and Coss (1993) showed that heat-shock increased the amount of nuclear C lamin in CHO hamster fibroblasts. In both cases, the overaccumulation of intermediate filaments was only demonstrated at the protein level. Our present results indicate that the stress-elicited vimentin increase in U-937 cells is regulated at least in part at the RNA level, excluding the possibility that it could be exclusively a consequence of protein stabilization. Stimulation of vimentin expression has been commontly observed upon mitogen activation of quiescent fibroblasts (Ferrari et al., 1986), as well as during the differentiation induction of myeloid leukemia cells (Rius and Aller, 1992). In both cases, vimentin stimulation seemed to be the result of increased transcription mediated by the AP-1 responsive elements present in the promoter of this gene (Rittling et al., 1989; Rius and Aller, 1992; Perez et al., 1994). This is not the case in stressed U-937 cells, even though the stress treatments induced the expression of differentiation markers. In fact, heatshock increased the vimentin mRNA level without significantly affecting AP-1 binding activity (a result which also contrasts with the reported effect of heat in other cell systems: Sikora et al., 1993). Cadmium chloride stimulated AP-1 binding, but there was a clear uncoupling between the time of AP-1 stimulation (from 2 to 5 hours of recovery) and the time of maximum increase in vimentin mRNA (from 24 hours of recovery onwards). The present observations suggest that the increase in vimentin mRNA content in stressed U-937 cells is the result, at least in part, of transcript stabilization. The stabilized RNA appears to be a mature, normally sized vimentin transcript (2.0 kb in size) capable of translation. Stressmediated stabilization has also been observed in the case of other gene transcripts, such as c-myc mRNA in Hyon pre-B lymphocytes (Bukh, 1990), c-fos mRNA in HeLa cells (Andrews et al., 1987), and HSP70 and HSP23 mRNAs in Drosophila cells (Peterson and Linquist, 1989) and chicken cells (Edington and Hightower, 1990), respectively. Nevertheless, it is probable that other mechanisms operating at the translational or transcriptional level may also contribute to the increase in vimentin expression. Actually, the human vimentin gene promoter possesses regulatory elements other than AP-1 which might modulate transcription in either a positive or negative fashion (Rittling and Baserga, 1987; Liliembaum and Paulin, 1993). The functional significance of vimentin stimulation by
heat-shock and cadmium chloride is unknown. Since the stress treatments induced the expression of differentiation markers, a possibility is that vimentin might form part of, and even regulate, the stress-induced differentiation process, as it was previously demonstrated using other inducers (Bernal and Chen, 1982; Rius and Aller, 1992). Nevertheless, it must be noted that the mechanism responsible for vimentin increase in cells treated with typical differentiation inducers is not the same as that operating in stressed cells, as commented above. An alternative explanation is that vimentin may be involved in the acquisition of stress tolerance, a function commontly atributed to HSPs (Li et al., 1982). In fact, our experiments showed that the expression of vimentin partially coincided with that of HSP70 in stressed U-937 cells. In addition, Lee et al. (1992) observed that Chinese hamster mutant cell lines with increased levels of vimentin exhibited increased heat-resistance in relation to the wildtype cells, although the mutant cells did not exhibit alterations in HSPs levels. Hence, it is possible that the overaccumulation of vimentin or other intermediate filaments is one of the factors which contribute to make the cells resistant to environmental stresses. This work was supported by DGICYT (Spain) Grant PB94-0063, FISss (Spain) Grants 94/0008-01 and 94/0276, and CAM (Spain) Grant AE00419/95. N.E.V. and L.G.B. were recipients of predoctoral fellowships from the Caja de Ahorros y Monte de Piedad de Madrid, and the Comunidad Autónoma de Castilla La Mancha, respectively.
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