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Physiological stress conditions associated with the tumor microenvironment play a role in resistance to anticancer therapy. In this study, treatment of EMT6 mouse mammary tumor cells with hypoxia or the chemical stress agents brefeldin A (BFA) or okadaic acid (OA) causes the development of resistance to the topoisomerase II inhibitor etoposide. The mechanism of physiological stress-induced drug resistance may involve the activation of stress-responsive proteins and transcription factors. Our previous work shows that treatment with BFA or OA causes activation of the nuclear transcription factor NF-kappa B. Pretreatment with the proteasome inhibitor carbobenzyoxyl-leucinyl-leucinyl-leucinal inhibits stress-induced NF-kappa B activation and reverses BFA-induced drug resistance. To test whether NF-kappa B specifically mediates stress-induced drug resistance, an inducible phosphorylation site-deficient mutant of I kappa B alpha (I kappa B alpha M, S32/36A) was introduced into EMT6 cells. In this study, we show that I kappa B alpha M expression inhibits stress-induced NF-kappa B activation and prevents BFA-, hypoxia-, and OA-induced resistance to etoposide. These results indicate that NF-kappa B activation mediates both chemical and physiological drug resistance to etoposide. Furthermore, they imply that coadministration of agents that inhibit NF-kappa B may enhance the efficacy of topoisomerase II inhibitors in clinical cancer chemotherapy.

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

A variety of key events in the molecular apoptotic pathway involve the mitochondria. Cyclosporin A (csA) affects the mitochondria by inhibiting the mitochondrial permeability transition (MPT), thereby preventing disruption of the transmembrane potential. The role of the MPT in apoptosis is not fully understood, but inhibition of the MPT may prevent the release of mitochondrial caspase activators, such as cytochrome c (cyt c), into the cytosol. Certain hexavalent chromium [Cr(VI)] compounds are known occupational respiratory tract toxins and carcinogens. We have previously shown that these compounds induce apoptosis as a predominant mode of cell death and that this effect can be observed in cell culture using soluble Cr(VI). We show here that Cr(VI)-induced apoptosis in Chinese hamster ovary (CHO) cells involves disruption of mitochondrial stability. Using a cyt c-specific monoclonal antibody, we observed a dose-dependent release of mitochondrial cyt c in cytosolic extracts of CHO cells exposed to apoptogenic doses of sodium chromate. Co-treatment of these cells with csA inhibited the release of cyt c and abrogated Cr(VI)-induced apoptosis as determined by a reduction in internucleosomal DNA fragmentation. Co-treatment with csA also markedly increased clonogenic survival of Cr(VI)-treated CHO cells. In contrast, the general caspase inhibitor Z-VAD-FMK markedly inhibited most of the morphological and biochemical parameters of apoptosis but did not prevent cyt c release and did not increase clonogenic survival. These results suggest that the MPT plays an important role in the regulation of mitochondrial cyt c release and that this may be a critical point in the apoptotic pathway in which cells are irreversibly committed to death.

Essential and non-essential metal ions were compared on the basis of their growth-inhibitory potency and their mediation of metallothionein induction in a Chinese hamster ovary cell line resistant to cadmium. Cadmium-resistant cells were found to be 20-fold and 6-fold more resistant than wild-type Chinese hamster ovary cells to the non-essential metals CdCl2 and HgCl2, respectively. In contrast, cadmium-resistant cells showed 2-fold or less resistance to growth inhibition due to the metals with known or possible biological essentiality, ZnCl2, CuSO4, CoCl2, and NiCl2. Resistance to either cadmium or mercury was not due to decreased uptake as measured isotopically or by X-ray fluorescence. At concentrations near the threshold of growth inhibition, CdCl2 and ZnCl2 induced metallothionein 8- to 10-fold above background levels in cadmium-resistant cells within 8-10 hr. A 2- to 3-fold induction of this protein was produced in resistant cells by levels of HgCl2, CuSO4, and CoCl2 near the threshold of growth inhibition whereas NiCl2 produced no measurable elevations of metallothionein at concentrations below, near, and above those that inhibit cell growth. Induction of metallothionein was measured by a modified 203Hg binding assay and by [35S]cysteine incorporation. No measurable induction of metallothionein was evident in wild-type cells with any metal treatment using a reasonable quantity of cells consistent with our assay. These results in cadmium-resistant cells demonstrate selective induction of metallothionein by various metals and suggest that induction of this protein alone is not solely responsible for differences in the growth-inhibitory potential of these elements.

Alkaline elution studies demonstrated CaCrO4-induced DNA single strand breaks and DNA-protein crosslinks. DNA single strand breaks increased following treatment with 10-400 microM CaCrO4 in Chinese hamster ovary cells maintained with a minimal salts/glucose medium. DNA single strand breaks were rapidly repaired when extracellular CaCrO4 was removed even following exposure levels of CaCrO4 (200 microM for 2 hr) which reduced survival to 0.6%. Under these exposure conditions the trypan blue exclusion was greater than 80%, whereas cell growth was inhibited by 46% within 24 hr. The DNA-protein crosslinks induced by 10 microM CaCrO4 were repaired in the absence of metal within 24 hr. In contrast, the amount of DNA-protein crosslinks measured 24 hr after a 2-hr treatment with 50, 100, and 200 microM CaCrO4 remained unchanged at the 50 microM level or increased at the two higher concentrations. Thus, at concentrations of 50 microM or greater there was no repair of the DNA protein crosslinks, and this may have been due to cytotoxicity of the metal. CaCrO4 at 10 or 25 microM exposure for 6 hr also induced DNA-protein crosslinking in Chinese hamster ovary cells maintained in normal tissue culture growth media. The lack of repair of DNA-protein crosslinks at the 25 microM level, which did not substantially reduce cell survival, indicated the persistence of these lesions in a noncytotoxic form. Uptake of CaCrO4 was linear with all of the concentrations tested. Analysis of the cell cycle sensitivity to CaCrO4 revealed that cells in early S phase were the most sensitive to the cytotoxic and strand breaking activity of CaCrO4. Compared with other phases of the cell cycle, there was also an elevated level of DNA-protein crosslinks when cells were treated in early S phase and incubated 24 hr without CaCrO4. These results implicate the DNA-protein crosslink as an important lesion that may be responsible for the cytotoxic and carcinogenic properties of chromate.

Previous studies have shown that in vitro treatment of a synthetic double-stranded DNA template with chromium(III), or chromium(VI) in the presence of ascorbate, resulted in guanine-specific DNA polymerase arrests that correlated strongly with DNA-DNA cross-linking. In vivo chromium(VI) undergoes a more complicated intracellular cascade of reductive metabolism than is achievable in an in vitro model. Moreover, in living cells, DNA is highly packaged in the form of chromatin which may alter the accessibility of DNA to chromium. A repetitive primer-extension assay was employed to determine whether chromium forms polymerase-arresting lesions in vivo. Normal human lung fibroblasts treated with chromium(VI) exhibited adduct levels of 0.13-0.92 mmol Cr/mol DNA-nucleotides in the total genome (0.26-1.84 Cr adducts/Kbp DNA) and DNA interstrand cross-links. Genomic DNA was isolated and alphoid sequences (1-5% of the genome) were used as a substrate for repetitive primer extension using Taq polymerase. The results showed a dose-dependent, guanine-specific, replication termination, even at low doses resulting in greater than 90% survival. The same treatment resulted in dose-dependent suppression of thymidine incorporation into DNA immediately after treatment. Thymidine incorporation increased during the first 6 h after the 2-h exposure, probably related to the repair of the single strand breaks, but then returned to high suppression levels at 24 h. The chromate treatments inhibited cell growth by specific blocking of the progression of cells through S-phase of the cell cycle. The results confirmed our studies in cell-free systems and taken together they strongly indicate that guanine-guanine DNA interstrand cross-links induced by chromate in living cells is the lesion responsible for blocking DNA replication processivity.