Evidence for role of transforming growth factor-beta in RRR-alpha-tocopheryl succinate-induced apoptosis of human MDA-MB-435 breast cancer cells

MDA-MB-435 human breast cancer cells treated with 10 micrograms/ml of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for one, two, three, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respectively. Likewise, cells cultured for one, two, and three days with conditioned media (CM) obtained from MDA-MB-435 cells treated with VES exhibit 10%, 36%, and 74% apoptosis, respectively. A quantitative luciferase-based assay showed CM from VES-treated cells collected at 24 and 48 hours after treatment initiation to contain 75 and 32 pg of active transforming growth factor-beta (TGF-beta), respectively, per 10(6) cells. Although purified TGF-beta 1 is not an effective apoptotic agent for MDA-MD-435 cells, cotreatment of the cells for three days with suboptimal levels of VES (2.5 and 5 micrograms/ml) + 10 ng/ml of purified TGF-beta 1 enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF-beta-signaling pathway by transient transfection of MDA-MB-435 cells with antisense oligomers to TGF-beta type II receptor (TGF-beta R-II) blocked VES-induced apoptosis. Likewise, addition of neutralizing antibodies to TGF-beta 1 or to all three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) blocked VES- and CM-induced apoptosis. Furthermore, inhibitors of TGF-beta conversion from an inactive latent form to a biologically active form inhibited VES-induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF-beta or the TGF-beta receptor-signaling pathway with antisense oligomers or ligand-neutralizing antibodies or prevention of activation of TGF-beta indicates a role for TGF-beta signaling in VES-induced apoptosis.     

Advantages of a two-chamber culture system to test drug nephrotoxicity: the example of cephaloridine

BACKGROUND: The adult prostate is maintained through an equilibrium between cell growth and death rates. Androgen deprivation induces an increase in intracellular Ca++, AP-1 gene expression of androgen-inducible genes. METHODS: Northern blot analysis, band-shift assays, and transient cotransfection assays were used to study the effects of Ca++ mobilizer A23187 on gene expression in human prostate cancer cells. RESULTS: A23187 repressed androgen-upregulated mRNAs for prostate-specific antigen (PSA) and hKLK2, and rapidly induced mRNA levels for c-fos and c-jun. AP-1 protein-DNA binding activities were elevated after A23187 treatments. Androgen receptor (AR)-mediated induction of chloramphenicol acetyltransferase (CAT) reporter was repressed by AP-1 proteins. CONCLUSIONS: The repression of AR-mediated induction of PSA and hKLK2 genes by Ca++ mobilizers is due to the interference of AR transactivation activity by AP-1 proteins.     

A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes

RRR-alpha-tocopheryl succinate (VES) was studied for effects on murine EL-4 cell proliferation and production of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta). VES was biphasic in its actions: 0.1 microgram/ml enhanced EL-4 cell proliferation, whereas 10-20 microgram/ml inhibited cellular proliferation. Cell-conditioned media (CM) from EL-4 cells treated with 0.2 ng/ml phorbol myristate acetate (PMA) + 0.1 microgram/ml VES contained increased amounts of IL-2, as determined by the murine cytotoxic T cell IL-2-dependent CTLL-2 bioassay. VES at 0.1 microgram/ml or 0.1 microgram/ml VES + 0.2 ng/ml PMA induced the expression of IL-2 mRNA by EL-4 cells three to nine hours after treatment. CM from EL-4 cells treated with VES at 10-20 microgram/ml exhibited potent antiproliferative activity when tested in the TGF-beta-responsive mink lung cell (Mv1Lu) bioassay and showed reduced inhibitory effects when tested on TGF-beta receptor-negative mink lung (DRA-27) cells. CM from control-treated EL-4 cells exhibited no antiproliferative activity. The VES-induced antiproliferative activity was characterized as TGF-beta by neutralization analyses and immunoprecipitation of metabolically labeled proteins with TGF-beta-specific reagents. VES treatment of EL-4 cells had no effect on TGF-beta 1 mRNA expression while downregulating TGF-beta 3 mRNA expression. In summary, these studies showed that 0.1 microgram/ml VES enhanced cellular proliferation, in part, via increased IL-2 production, whereas 10-20 micrograms/ml VES inhibited cellular proliferation, in part, via the secretion of biologically active TGF-beta.     

Evidence against a direct role for the induction of c-jun expression in the mediation of drug-induced apoptosis in human acute leukemia cells

Previous reports have demonstrated that a variety of anticancer drugs, e.g., 1-beta-D-arabinofuranosylcytosine (ara-C), mitoxantrone, etoposide, camptothecin, and cisplatin, induce the expression of c-jun oncogene in leukemic cells prior to producing internucleosomal DNA fragmentation and the morphological features of apoptosis. This has led to the impression that the induction of c-jun expression may be directly involved in the molecular signaling of the final common pathway of programmed cell death or apoptosis. In the present study, we examined the role of c-jun expression in three different settings of anticancer drug-induced apoptosis in human leukemic cells. First, exposure of human myeloid leukemia HL-60 cells to high-dose ara-C for 4 h produced internucleosomal DNA fragmentation preceded by c-jun induction. However, pretreatment of HL-60 cells with staurosporine, a protein kinase C inhibitor, repressed c-jun yet enhanced DNA fragmentation and apoptosis due to ara-C. Second, in human pre-B leukemia 697/BCL-2 cells which are transfected with the cDNA of the bcl-2 oncogene and overexpress p26BCL-2, although ara-C or mitoxantrone treatment caused greater c-jun induction than in the 697/neo cells, significantly reduced endonucleolytic DNA fragmentation and apoptosis was observed in 697/BCL-2 cells. Finally, taxol-induced internucleosomal DNA fragmentation and morphological features of apoptosis in HL-60 cells were not associated with the induction of c-jun expression. These lines of evidence indicate that the induction of c-jun expression may not have a direct role in the molecular signaling of anticancer drug-induced apoptosis, and that the anticancer drug-induced apoptosis can occur by a mechanism that does not involve the induction of c-jun expression.     

Ethanol exposure increases expression of c-jun and junD in human neuroblastoma cells

The aim of this study was to investigate the effect of ethanol exposure on the expression of fos and jun genes. Exposure of human neuroblastoma SH-SY5Y cells to ethanol for 2-4 days caused a dose-dependent increase in c-jun and junD mRNA levels, whereas mRNAs for c-fos, fosB and junB were not detectable in control or ethanol-treated cells. Four days of ethanol exposure also enhanced the AP-1 binding activity. Experiments with actinomycin D demonstrated that ethanol did not influence the degradation of c-jun and junD mRNAs. These results demonstrate that long-term exposure to ethanol increases c-jun and junD expression. This effect may be one of the mechanisms through which ethanol influences the gene regulatory system in neuronal cells.     

Activation of tissue-factor gene expression in breast carcinoma cells by stimulation of the RAF-ERK signaling pathway

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.     

Role of tyrosine kinases in induction of the c-jun proto-oncogene in irradiated B-lineage lymphoid cells

Exposure of B-lineage lymphoid cells to ionizing radiation induces an elevation of c-jun proto-oncogene mRNA levels. This signal is abrogated by protein-tyrosine kinase (PTK) inhibitors, indicating that activation of an as yet unidentified PTK is mandatory for radiation-induced c-jun expression. Here, we provide experimental evidence that the cytoplasmic tyrosine kinases BTK, SYK, and LYN are not required for this signal. Lymphoma B-cells rendered deficient for LYN, SYK, or both by targeted gene disruption showed increased c-jun expression levels after radiation exposure, but the magnitude of the stimulation was lower than in wild-type cells. Thus, these PTKs may participate in the generation of an optimal signal. Notably, an inhibitor of JAK-3 (Janus family kinase-3) abrogated radiation-induced c-jun activation, prompting the hypothesis that a chicken homologue of JAK-3 may play a key role in initiation of the radiation-induced c-jun signal in B-lineage lymphoid cells.