Maximal adamantyl-substituted retinoid-related molecule-induced apoptosis requires NF-κB noncanonical and canonical pathway activation.

NF-κB transcription factors have a critical role in regulating cell survival and apoptosis. We have previously shown that 4-(3-Cl-(1-adamantyl)-4-hydroxyphenyl)-3-chlorocinnamic acid (3-Cl-AHPC), an adamantyl-substituted retinoid molecule, induced apoptosis and required NF-κB activation in prostate and breast carcinoma cells. Here, we show that 3-Cl-AHPC activated both IκB kinase (IKK)α and IKKß with subsequent activation of the canonical and noncanonical NF-κB pathways in the human breast carcinoma and leukemia cell lines. 3-Cl-AHPC-mediated activation of the NF-κB canonical pathway occurred within 6 h, whereas maximal activation of the NF-κB noncanonical pathway required 48 h. Knockout of IKKα or IKKß expression in mouse embryonic fibroblast cells and knockdown of IKKα or IKKß in MDA-MB-468 cells resulted in the inhibition of 3-Cl-AHPC-mediated apoptosis, indicating that activation of canonical and noncanonical pathways are required for maximal 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC activation of the noncanonical pathway was preceded by caspase-mediated decrease in the E3-ligase c-IAP1 with subsequent stabilization of NF-κB-inducing kinase (NIK) expression, increased binding of NIK by TRAF3, activation of IKKα, and the resultant increased levels of RelB and p52. Increased expression of c-IAP1 blocked 3-Cl-AHPC-mediated stabilization of NIK levels and 3-Cl-AHPC-mediated apoptosis. Cdc37 expression was required for activation of IKKα and IKKß by 3-Cl-AHPC. These findings suggest that NF-κB pathways have an important role in 3-Cl-AHPC-mediated apoptosis.

Treatment of volatile organic compounds in a biotrickling filter under thermophilic conditions.

The objectives of this research were to investigate the potential to biologically treat volatile organic compounds emitted by the forest products industry at thermophilic conditions and to examine the microbial community developed at high temperatures. Three biotrickling filters were run in parallel at temperatures ranging from 40 degrees C (mesophilic control) to 70 degrees C. The first phase involved treatment of methanol, for a 3-month run, and the second phase involved a 260-day run on the treatment of alpha-pinene. Methanol removal rates over 100 g m(-3) h(-1) where achieved at temperatures up to 70 degrees C. Alpha-pinene removal was achieved at temperatures up to 60 degrees C with optimal treatment occurring at 55 degrees C at rates up to 60 g m(-3) h(-1). The time for acclimation increased with increasing temperature and was longer for pinene than for methanol. Filter performance was also able to quickly recover from a shutdown period of up to 2 weeks due to the robustness of the microbial communities as determined by DNA fingerprinting analysis. The high-temperature communities treating methanol or pinene were more similar to each other than the mesophilic communities (i.e., 40 degrees C). The mesophilic methanol community had a high degree of functional redundancy, while the mesophilic pinene community was more unique and very distinct from the others. These results show that biofiltration at high temperatures is achievable and opens up a range of possibilities for applying biofiltration to hot gas streams.

Okadaic acid-mediated induction of the c-fos gene in estrogen receptor-negative human breast carcinoma cells utilized, in part, posttranscriptional mechanisms involving adenosine-uridine-rich elements.

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused approximately 3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr approximately 75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.

S-phase arrest and apoptosis induced in normal mammary epithelial cells by a novel retinoid.

The addition of all-trans-retinoic acid has been found to mediate a G1 cell cycle phase arrest but not apoptosis in normal mammary epithelial cells. We have now found that addition of the novel retinoid 6-[3-(1-adamantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which appears to function through a pathway independent of retinoic acid nuclear receptors, results in an S-phase arrest that is preceded by a 4-fold elevation in the levels of the cyclin-cyclin dependent kinase (cdk) inhibitor p21WAF1/CIP1. Failure to inhibit E2F-1 activation of genes through its phosphorylation by the cyclin cdk2 kinase has been shown to result in S-phase arrest and apoptosis in a number of cell types. Although exposure of the normal mammary cells to CD437 does not result in modulation of cyclin A or cdk2 levels, an increase in E2F-1 levels and a marked inhibition of cyclin A/cdk2 kinase activity are observed. Exposure to CD437 results in enhanced E2F-1 binding to its DNA consensus sequences and transcriptional activity during S phase. We hypothesize that this enhanced E2F-1 transcriptional activity results in S-phase arrest and subsequent apoptosis that has been observed in other systems.

Monoclonal antibodies to 2,4-dichlorophenol hydroxylase as probes for the 2,4-D-degradative phenotype.

Two different monoclonal antibodies (MAb) were raised against 2,4-dichlorophenol hydroxylase (DCP-hydroxylase) of Ralstonia eutropha JMP134 (pJP4), the second enzyme in the 2,4-D-degradative pathway of this bacterium. The utility of these antibodies in detecting and characterizing 2,4-D-degrading soil bacteria was investigated. One MAb (F6) reacted with DCP-hydroxylase from 27 out of 36 strains tested, while the other (MAb C3) reacted with only 17 isolates. When used with the colony blot technique, MAb F6 was useful for detecting cross-reacting strains on plates of pure cultures or of mixtures containing nondegraders even when 2,4-D degraders were outnumbered 60 to 1. 2,4-D-degrading strains could also be detected from plates spread with enrichment cultures but not from primary isolation plates spread from soil dilutions, presumably because the ratio of degraders to nondegraders was too low. Colonies of some strains that were very distantly related genetically, but produced functionally similar DCP-hydroxlase enzymes, were detected by MAb F6. This result suggests that MAbs could be useful for detecting functionally similar proteins expressed from tfdB analogs, even in the absence of detectable DNA homology between the genes encoding them.

The 2,4-dichlorophenol hydroxylase of Alcaligenes eutrophus JMP134 is a homotetramer.

2,4-Dichlorophenol hydroxylase (DCP-hydroxylase) is a key enzyme in the pathway for degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in many bacteria. In Alcaligenes eutrophus JMP134, DCP-hydroxylase was reported to consist of two dissimilar types of subunit of 66 and 45 kDa, a structure which is different from that in other bacteria. Using a different procedure involving affinity purification and ion-exchange chromatography, we have purified active enzyme from JMP134 and show that it has a native molecular mass of approximately 245 kDa and consists of a single type of subunit of 66 kDa, similar to all other flavoprotein monooxygenase enzymes. A 45-kDa polypeptide, found in partially purified enzyme preparations, was not required for enzyme activity but had some serologic and N-terminal amino acid sequence similarity to the 66-kDa enzyme subunit.