Mechanisms of resistance to apoptosis in the human acute promyelocytic leukemia cell line NB4.

Current frontline therapies have improved overall survival in acute promyelocytic leukemia (APL) patients to exceptional rates; however, relapse is still a problem among high-risk and old patients. Therefore, the development of better and safer therapies continues to be a goal in the treatment of this disease. In the present work, we examined three different pathways that hinder cell death in the APL cell line NB4, shedding light on the mechanisms that underlie resistance to apoptosis in these cells and that might help provide them with a proliferative advantage. We found that the proteasome inhibitor MG-132 specifically induces in NB4 cells an Nrf2-mediated antioxidant response which counteracts mitochondria-dependent apoptosis induced by the lipophilic cation dequalinium. More importantly, we also demonstrated that high basal autophagy levels and the gain-of-function of mutant p53 are intrinsic mechanisms of resistance to apoptosis in this cell line. According to our results, the pharmacological inhibition of autophagy and p53 mutants are useful tools to explore resistance to apoptosis in APL and other types of cancer and could be the bases of new therapeutic approaches that improve the efficiency and allow dose reduction of the current treatments.

P38 MAPK protects against TNF-alpha-provoked apoptosis in LNCaP prostatic cancer cells.

PURPOSE: One of the most relevant aspects in cell death regulation is the signalling of apoptosis by the serine/threonine kinases MAPKs. The aim of this study was to investigate the effects of TNF-alpha stimulation on MAPK activation, and the pro- or anti-apoptotic role of these kinases in LNCaP and PC3 cells. MATERIAL AND METHODS: Treatments were carried out using several TNF-alpha concentrations, as well as specific pharmacological inhibitors of MAPKs. Apoptosis rates were evaluated by DAPI staining and flow cytometry. MAPK phosphorylation/activation was measured by Western blot. RESULTS: TNF-alpha induced apoptosis in a dose-dependent manner in LNCaP but not in PC3 cells. The MAPK inhibitors revealed that the apoptotic rate in LNCaP cells increased significantly following p38 inhibition. The kinase inhibitors failed to cause changes in apoptosis in PC3 cells. CONCLUSIONS: The potentiation of apoptosis by p38 inhibition points to this kinase as a possible target for the treatment of androgen-dependent prostatic cancer.

Regulation of apoptosis/necrosis execution in cadmium-treated human promonocytic cells under different forms of oxidative stress.

Pulse-treatment of U-937 human promonocytic cells with cadmium chloride followed by recovery caused caspase-9/caspase-3-dependent, caspase-8-independent apoptosis. However, pre-incubation with the glutathione (GSH)-suppressing agent DL-buthionine-(S,R)-sulfoximine (cadmium/BSO), or co-treatment with H2O2 (cadmium/H2O2), switched the mode of death to caspase-independent necrosis. The switch from apoptosis to necrosis did not involve gross alterations in Apaf-1 and pro-caspase-9 expression, nor inhibition of cytochrome c release from mitochondria. However, cadmium/H2O2-induced necrosis involved ATP depletion and was prevented by 3-aminobenzamide, while cadmium/BSO-induced necrosis was ATP independent. Pre-incubation with BSO increased the intracellular cadmium accumulation, while co-treatment with H2O2 did not. Both treatments caused intracellular peroxide over-accumulation and disruption of mitochondrial transmembrane potential (delta psi m). However, while post-treatment with N-acetyl-L-cysteine or butylated hydroxyanisole reduced the cadmium/BSO-mediated necrosis and delta psi m disruption, it did not reduce the effects of cadmium/H2O2. Bcl-2 over-expression, which reduced peroxide accumulation without affecting the intracellular GSH content, attenuated necrosis generation by cadmium/H2O2 but not by cadmium/BSO. By contrast, AIF suppression, which reduced peroxide accumulation and increased the GSH content, attenuated the toxicity of both treatments. These results unravel the existence of two different oxidation-mediated necrotic pathways in cadmium-treated cells, one of them resulting from ATP-dependent apoptosis blockade, and the other involving the concurrence of multiple regulatory factors.

The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells.

Treatment with 0.2 mM hydrogen peroxide (H(2)O(2)) or with 0.5 mM cisplatin caused caspase-9 and caspase-3 activation and death by apoptosis in U-937 human promonocytic cells. However, treatment with 2 mM H(2)O(2), or incubation with the glutathione suppressor DL-buthionine-(S,R)-sulfoximine (BSO) prior to treatment with cisplatin, suppressed caspase activation and changed the mode of death to necrosis. Treatment with 2 mM H(2)O(2) caused a great decrease in the intracellular ATP level, which was partially prevented by 3-aminobenzamide (3-ABA). Correspondingly, 3-ABA restored the activation of caspases and the execution of apoptosis. By contrast, BSO plus cisplatin did not decrease the ATP levels, and the generation of necrosis by this treatment was not affected by 3-ABA. On the other hand, while all apoptosis-inducing treatments and treatment with 2 mM H(2)O(2) caused Bax translocation from the cytosol to mitochondria as well as cytochrome c release from mitochondria to the cytosol, treatment with BSO plus cisplatin did not. Treatment with cisplatin alone caused Bid cleavage, while BSO plus cisplatin as well as 0.2 and 2 mM H(2)O(2) did not. Bcl-2 overexpression reduced the generation of necrosis by H(2)O(2), but not by BSO plus cisplatin. These results indicate the existence of different apoptosis/necrosis regulatory mechanisms in promonocytic cells subjected to different forms of oxidative stress.

Effect of glutathione depletion on antitumor drug toxicity (apoptosis and necrosis) in U-937 human promonocytic cells. The role of intracellular oxidation.

Treatment with the DNA topoisomerase inhibitors etoposide, doxorubicin, and camptothecin, and with the alkylating agents cisplatin and melphalan, caused peroxide accumulation and apoptosis in U-937 human promonocytic cells. Preincubation with the reduced glutathione (GSH) synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO) always potentiated peroxide accumulation. However, although GSH depletion potentiated the toxicity of cisplatin and melphalan, occasionally switching the mode of death from apoptosis to necrosis, it did not affect the toxicity of the other antitumor drugs. Hypoxia or preincubation with antioxidant agents attenuated death induction, apoptotic and necrotic, by alkylating drugs. The generation of necrosis by cisplatin could not be mimicked by addition of exogenous H(2)O(2) instead of BSO and was not adequately explained by caspase inactivation nor by a selective fall in ATP content. Treatment with cisplatin and melphalan caused a late decrease in mitochondrial transmembrane potential (DeltaPsim), which was much greater during necrosis than during apoptosis. The administration of the antioxidant agents N-acetyl-l-cysteine and butylated hydroxyanisole after pulse treatment with cisplatin or melphalan did not affect apoptosis but attenuated necrosis. Under these conditions, both antioxidants attenuated the necrosis-associated DeltaPsim decrease. These results indicate that oxidation-mediated alterations in mitochondrial function regulate the selection between apoptosis and necrosis in alkylating drug-treated human promonocytic cells.

Modulation of the stress response during apoptosis and necrosis induction in cadmium-treated U-937 human promonocytic cells.

Treatment for 2 h with 200 microM cadmium chloride, followed by recovery, caused apoptosis and induced heat-shock protein 70 (HSP70) expression in U-937 promonocytic cells. However, pre-incubation with the GSH depleting agent L-buthionine-[S,R]-sulfoximine (BSO, 1 mM for 24 h) caused necrosis instead of apoptosis and failed to induce HSP70 expression. This failure was a consequence of necrosis instead of GSH depletion, since BSO allowed or even potentiated HSP70 induction when used in combination with heat shock (2 h at 42.5 degrees C) or with 50 microM cadmium, which caused apoptosis. The administration of N-acetyl-L-cysteine (NAC) at the beginning of recovery after BSO/200 microM cadmium treatment prevented the execution of necrosis and restored the execution of apoptosis, but did not restore HSP70 induction, indicating that the inhibition by BSO of HSP70 expression is an early regulated event. This contrasted with the capacity of NAC to prevent the alterations caused by BSO/200 microM cadmium in other proteins, namely the suppression of Bax expression and the increase in Bcl-2 and HSP-60 expression. Finally, it was observed that treatment with 200 microM cadmium rapidly increased the HSP70 mRNA level and stimulated heat-shock factor 1 (HSF1) trimerization and binding, and that these effects were prevented by pre-incubation with BSO. Taken together, these results indicate that the stress response is compatible with apoptosis but not with necrosis in cadmium-treated promonocytic cells. The suppression of the stress response is specifically due to the early inhibition of HSF1 activation.

The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays).

Treatment of U-937 human promonocytic cells with the stress inducers cadmium chloride (2 h at 200 microM), heat (2 h at 42.5 C) or X-rays (20 Gy), followed by recovery, caused death by apoptosis and stimulated caspase-3 activity. In addition, all stress agents caused intracellular oxidation, as measured by peroxide and/or anion superoxide accumulation. However, while pre-incubation with antioxidants (N-acetyl-L-cysteine or butylated hydroxyanisole) inhibited the induction of apoptosis by cadmium and X-rays, it did not affect the induction by heat-shock. Pre-incubation for 24 h with the GSH-depleting agent L-buthionine-[S,R]-sulfoximine (BSO) switched the mode of death from apoptosis to necrosis in cadmium-treated cells. By contrast, BSO only caused minor modifacions in the rate of apoptosis without affecting the mode of death in heat- and X-rays-treated cells. BSO potentiated peroxide accumulation in cells treated with both cadmium and X-rays. However, while the accumulation of peroxides was stable in the case of cadmium, it was transient in the case of X-rays. Moreover, the administration of antioxidants during the recovery period sufficed to prevent necrosis and restore apoptosis in BSO plus cadmium-treated cells. Cadmium and X-rays caused a decrease in intracellular ATP levels, but the decrease was similar in both apoptotic and necrotic cells. Taken together, these results demonstrate that (i) stress inducers cause intracellular oxidation, but oxidation is not a general requirement for apoptosis; and (ii) the duration of the oxidant state seems to be critical in determining the mode of death.

Differential redistribution of protein kinase C isoforms by cyclic AMP in HL60 cells.

In this study we have analyzed the distribution of protein kinase C isoforms in cytosol, membrane, and nucleus in HL60 cells. Furthermore, we have studied the redistribution of these isoforms after cyclic AMP treatment. Protein kinase C localization and cyclic AMP-induced translocation was demonstrated by Western blot analysis. Cytosol, membrane and nucleus in HL60 cells expressed the abundance of protein kinase C alpha, betaI, betaII, delta, lambda, and zeta isoforms. After cyclic AMP treatment, the amount of protein kinase C betaI and zeta increased only in the nucleus, while protein kinase C delta increased in the three fractions tested. These effects were dependent on the cyclic AMP concentration and duration of action. Our results suggest the existence of cross-talk between the cyclic AMP system and protein kinase C in HL60 cells. Taking into account the processes regulated by protein kinase C, these findings also suggest that cyclic AMP plays a regulatory role in various cellular responses in HL60 cells, such as differentiation and gene expression. The increase observed in PKC delta was due to cyclic AMP-dependent protein kinase C activation, and the synthesis of enzyme was probably activated by the nucleotide.

Etoposide stimulates 1,25-dihydroxyvitamin D3 differentiation activity, hormone binding and hormone receptor expression in HL-60 human promyelocytic cells.

The simultaneous administration of the DNA topoisomerase II inhibitor etoposide (0.15 mM) and 1,25-dihydroxyvitamin D3 (VD3) (10 nM) synergistically induced the differentiation of HL-60 human promyelocytic leukemia cells. Similar results were obtained using U-937 human promonocytic cells, or the topoisomerase II inhibitors doxorubicin (15 nM) and mitoxantrone (2.5 nM). When sequential treatments were used, pre-incubation with VD3 had little effect on the subsequent action of etoposide, while pre-incubation with etoposide greatly potentiated the subsequent action of VD3. In addition, etoposide treatment stimulated VD3 binding activity and increased VD3 receptor mRNA and protein levels. The increase in hormone receptor expression may explain, at least in part, the capacity of topoisomerase inhibitors to potentiate the differentiation inducing activity of VD3.

Regulation of multidrug resistance 1 (MDR1)/P-glycoprotein gene expression and activity by heat-shock transcription factor 1 (HSF1).

Infection of HeLa cells with adenovirus-carrying HSF1(+) cDNA, which encodes a mutated form of HSF1 with constitutive transactivation capacity, increased multidrug resistance 1 (MDR1) mRNA level and P-glycoprotein (P-gp) cell surface content and stimulated rhodamine 123 accumulation and vinblastine efflux activity. On the other hand, infection with adenovirus-carrying HSP70 and HSP27 cDNAs did not increase MDR1/P-gp expression. HSF1 regulates MDR1/P-gp expression at the transcriptional level, since HSF1(+) bound the heat-shock consensus elements (HSEs) in the MDR1 gene promoter and also activated the expression of an MDR1 promoter-driven reporter plasmid (pMDR1(-1202)). In addition, heat-shock increased pMDR1(-1202) promoter activity but not the activity of a similar reporter plasmid with point mutations at specific HSEs, and the heat-induced increase was totally inhibited by co-transfection with an expression plasmid carrying HSF1(-), a dominant negative mutant of HSF1. The stress inducers arsenite, butyrate, and etoposide also increased pMDR1(-1202) promoter activity, but the increase was not inhibited (in the case of butyrate) or was only partially inhibited (in the case of arsenite and etoposide) by HSF1(-). These results demonstrate that HSF1 regulates MDR1 expression, and that the HSEs present in the -315 to -285 region mediate the heat-induced activation of the MDR1 promoter. However, other factors may also participate in MDR1 induction by stressing agents.

Uncoupling of apoptosis and Jun/AP-1 activity in human promonocytic cells treated with DNA-damaging and stress-inducing agents.

Earlier studies have indicated that Jun/AP-1 activity is associated with, and probably required for apoptosis induction by DNA-damaging and stress-inducing agents in human myeloid cells. To investigate this possibility, we examined the capacity of continuous treatments with etoposide (10 microM) and camptothecin (0.4 microM), and pulse treatments with X-rays (20 Gy), heat (2 h at 42.5 C) and cadmium chloride (2 h at 200 microM) followed by recovery, to provoke apoptosis and to simulate c-jun and c-fos expression and AP-1 binding in U-937 human promonocytic cells. All these treatments generated apoptosis with similar efficacy (50-60% apoptotic cells at 6 h of treatment or recovery). However, the capacity to increase c-jun and c-fos mRNA levels and to stimulate AP-1 binding was very different, ranging from more than a twelve-fold increase in the case of cadmium, to almost no increase in the case of heat-shock and etoposide. When the cells were pre-conditioned with a soft heat shock (1 h at 42 degrees C) the cadmium-provoked apoptosis was greatly inhibited, but the stimulation of AP-1 binding was not affected. The administration of cAMP-increasing agents also reduced the etoposide- and cadmium-provoked apoptosis. However, cAMP greatly stimulated c-jun and c-fos expression and AP-1 binding when applied together with etoposide (which itself was ineffective), and potentiated the cadmium-induced AP-1 binding. Conversely, retinoic acid abrogated the cadmium-provoked stimulation of AP-1 binding and transactivation capacity, and greatly inhibited the stimulation of binding caused by camptothecin and X-rays. However, retinoic acid did not inhibit the induction of apoptosis by these agents. These results indicate that Jun/AP-1 activity is not necessarily coupled with apoptosis, nor required for apoptosis induction by DNA-damaging and stress-inducing agents in human promonocytic cells.

Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells.

Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 microM) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38(MAPK)) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38(MAPK)-specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H(2)O(2) accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38(MAPK) phosphorylation. In a similar manner, p38(MAPK) phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38(MAPK) activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells.

cAMP increasing agents attenuate the generation of apoptosis by etoposide in promonocytic leukemia cells.

Treatment of U-937 promonocytic cells with the DNA topoisomerase II inhibitor etoposide rapidly caused death by apoptosis, as determined by changes in chromatin structure, production of DNA breaks, nucleosome-sized DNA degradation, decrease in mitochondrial membrane potential and phosphatidyl serine translocation in the plasma membrane, and at the same time induced intracellular acidification. Both the execution of the apoptotic process and the intracellular acidification were reduced by the addition of forskolin plus theophylline or other cAMP increasing agents. These agents also attenuated the induction of apoptosis by camptothecin, heat-shock, cadmium chloride and X-radiation. Although etoposide slightly increased the production of reactive oxygen intermediates, this increase was not prevented by forskolin plus theophylline, and the addition of antioxidant agents failed to inhibit apoptosis. Etoposide caused a great increase in NF-(kappa)B binding activity, which was not prevented by forskolin plus theophylline, while AP-1 binding was little affected by the topoisomerase inhibitor. The treatments did not significantly alter the levels of Bcl-2 and Bax. By contrast, the expression of c-myc, which was very high in untreated U-937 cells and only partially inhibited by etoposide, was rapidly and almost totally abolished by the cAMP increasing agents. Finally, it was observed that etoposide caused a transient dephosphorylation of retinoblastoma (Rb), which was associated with cleavage of poly(ADP-ribose) polymerase (PARP). Both Rb dephosphorylation and PARP cleavage were inhibited by forskolin plus theophylline. The inhibition of Rb (type I) phosphatase and ICE/CED-3-like protease activities, and the abrogation of c-myc expression, are mechanisms which could explain the anti-apoptotic action of cAMP increasing agents in myeloid cells.

Epinephrine-induced reduction in insulin receptor mRNA level and stability in U-937 human promonocytic cells.

The administration of 10(-5) M epinephrine transiently decreased insulin receptor (IR) mRNA levels in U-937 human promonocytic cells, which reached their minimum value after 24 hours. Such a decrease seems to be due, at least in part, to a reduction in transcript stability, since the IR mRNA half-life was observed to decline from approximately 4h in untreated cells to 3 h in epinephrine-treated cells. Computer inspection of the sequence of the 3' untranslated region of the human IR mRNA showed nine AUUUA pentamers and three U-rich regions. These domains could be targets for a RNA-binding protein induced by treatment with epinephrine producing a destabilization of IR mRNA in U-937 cells.

Modulation of heat-shock protein 70 (HSP70) gene expression by sodium butyrate in U-937 promonocytic cells: relationships with differentiation and apoptosis.

The administration of sodium butyrate at 0.75 mM induced the functional differentiation of U-937 human promonocytic leukemia cells with negligible cell mortality. However, the drug rapidly caused cell death with characteristics of apoptosis when used at concentrations of 5 mM and above. In addition, butyrate stimulated the expression of the stress-responsive heat-shock protein 70 (HSP70) gene when applied at both differentiation-inducing and apoptosis-inducing concentrations. The induction of HSP70 by butyrate was inhibited by the simultaneous addition of cAMP-increasing agents (dibutyryl cAMP or the combination of forskolin plus theophylline). However, these agents did not prevent differentiation and only partially reduced apoptosis. Moreover, the DNA topoisomerase II inhibitor etoposide, which provoked U-937 cell differentiation and apoptosis with the same or greater efficiency than butyrate, failed to stimulate HSP70 expression. Finally, it was observed that cAMP-increasing agents also abrogated the induction of HSP70 and reduced the apoptosis caused by cadmium chloride, a typical inducer of the stress response. Taken together, these results indicate that HSP70 expression is not required for differentiation of promonocytic cells, as earlier proposed, and that butyrate probably triggers the stress response in these cells.

Tissue-specific modulation of insulin receptor mRNA levels in adrenaline-treated rats.

Insulin receptor (IR) gene expression at the mRNA level was investigated in liver, hindlimb skeletal muscle, and epididymal adipose tissue of rats exposed to prolonged in vivo administration of adrenaline in relation to control rats. In the liver of adrenaline-treated rats, there were no differences in relation to controls when DNA and protein content were measured. In skeletal muscle, only a slight decrease in protein concentration was detected. By contrast, a clear increase in both protein and DNA content was observed in the adipose tissue of treated animals. Northern blot assays revealed two IR mRNA species of approximately 9.5 and 7.5 Kb in the three tissues from controls. Adrenaline treatment induced an increase of approximately 60% in the levels of both RNAs in adipose tissue but not in liver or skeletal muscle. These results provide evidence for an in vivo tissue-specific regulation of IR gene expression at the mRNA level in rats under an experimental condition of excess of catecholamines.

Default cycle phases determined after modifying discrete DNA sequences in plant cells.

After bromosubstituting DNA sequences replicated in the first, second, or third part of the S phase, in Allium cepa L. meristematic cells, radiation at 313 nm wavelength under anoxia allowed ascription of different sequences to both the positive and negative regulation of some cycle phase transitions. The present report shows that the radiation forced cells in late G1 phase to advance into S, while those in G2 remained in G2 and cells in prophase returned to G2 when both sets of sequences involved in the positive and negative controls were bromosubstituted and later irradiated. In this way, not only G2 but also the S phase behaved as cycle phases where cells accumulated by default when signals of different sign functionally cancelled out. The treatment did not halt the rates of replication or transcription of plant bromosubstituted DNA. The irradiation under hypoxia apparently prevents the binding of regulatory proteins to Br-DNA.

Differentiation of U-937 promonocytic cells by etoposide and ICRF-193, two antitumour DNA topoisomerase II inhibitors with different mechanisms of action.

We have compared the action on U-937 human promonocytic leukemia cells of two DNA topoisomerase II inhibitors, namely the epipodophyllotoxin etoposide and the bisdioxopiperazine ICRF-193. One hour pulse-treatment with 3 microM etoposide caused topoisomerase associated, primary DNA breakage, which was rapidly followed by apoptosis. By contrast, these effects were not observed upon pulse-treatment with 6 microM ICRF-193. However, continuous treatments with subcytotoxic concentrations of etoposide (0.15 microM) and ICRF-193 (0.3 microM) produced several similar effects, namely decreased cell proliferation, accumulation of cells at G2, increase in cell mass, and induction of differentiation. Under these conditions, etoposide produced a biphasic activation of protein kinase C, which consisted in an early transient activation (from hours 1 to 6) of the membrane-bound enzyme followed by a later activation (hour 48) of the total, membrane-bound and cytosolic enzyme. By contrast, ICRF-193 only provoked a late activation (from hours 72 to 96) of the total enzyme. When used at differentiation-inducing concentrations, both topoisomerase inhibitors caused a great stimulation of AP-1 binding activity, with maximum value at hour 12 in etoposide-treated cells and at hour 48 in ICRF-193-treated cells. By contrast, the binding activity of the NF-kappa(B) and EGR-1 transcription factors was little affected. It is concluded that topoisomerase II inhibitors may induce the differentiation of promonocytic cells, independently of their capacity to cause DNA strand breaks. However, there are other effects, such as the early activation of protein kinase C, which are probably derived from the production of primary DNA breakage by some anti-topoisomerase drugs.

Heat-shock and cadmium chloride increase the vimentin mRNA and protein levels in U-937 human promonocytic cells.

Heat-shock for 2 hours at 42 degrees C, or the administration for 3 hours of 100 or 150 microM 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 U-937 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 beta-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 stimulation 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.

The accuracy of instrumental neutron activation analysis of kilogram-size inhomogeneous samples.

The feasibility of quantitative instrumental neutron activation analysis (INAA) of samples in the kilogram range without internal standardization has been demonstrated by Overwater et al. (Anal. Chem. 1996, 68, 341). In their studies, however, they demonstrated only the agreement between the "corrected" γ ray spectrum of homogeneous large samples and that of small samples of the same material. In this paper, the k(0) calibration of the IRI facilities for large samples is described, and, this time in terms of (trace) element concentrations, some of Overwater's results for homogeneous materials are presented again, as well as results obtained from inhomogeneous materials and subsamples thereof. It is concluded that large-sample INAA can be as accurate as ordinary INAA, even when applied to inhomogeneous materials.