Bernard W Futscher

PMID: 2097077;Abstract:

Urine samples were collected at 3-hr intervals over a single 24-hr period from each of seven clinically healthy men who ranged in age from 21-25 years. Urines at each collection time were subsequently pooled using 20% of each volume and serially dialyzed against ammonium-barbituric acid buffer (pH 7.35 +/- 0.02), using a cellulose membrane permeable to compounds of less than 12,000-14,000 molecular weight (mw). When the dialyzed portions were then analyzed for total proteins, the sum of proteins in eight pools amounted to 74 mg. A 1 ml aliquot of each pool, representing approximately 50 micrograms of proteins, was concentrated and reconstituted. Approximately 20 micrograms of reconstituted proteins were then subjected to polyacrylamide gel electrophoresis. The stained gel was then scanned by laser densitometry and planimetry. Each aliquot revealed eight segments as identified by Coomassie and silver staining. Their molecular weights, estimated by extrapolation from concurrently run protein standards, and their total protein amounts were: 116,000 mw (9.44 mg), 91,000 mw (3.3 mg), 68,000 mw (11.58 mg), 53,000 mw (2.58 mg), 43,000 mw (9.12 mg), 32,000 mw (7.13 mg), 24,000 mw (4.52 mg) and 20,000 mw (5.27 mg). A statistically significant rhythm (P = 0.022 from ANOVA and 0.011 from Single Cosinor) was found for the excretion of total proteins, with an acrophase in the afternoon (1537) for these diurnally-active subjects.

PMID: 16636674;Abstract:

Activating enhancer-binding protein 2α (AP-2α) and activating enhancer-binding protein 2γ (AP-2γ) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2α and AP-2γ interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2γ as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2γ but also AP-2α mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2α and AP-2γ promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2α or γ inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation. © 2006 Nature Publishing Group. All rights reserved.

PMID: 24355420;Abstract:

The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Exposure to 1% O2 prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity. © 2013 Elsevier Inc.

p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2'-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.

PMID: 9268689;Abstract:

Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxorubicin in the absence of MDR1 overexpression or topo II mutations. Consistent with its drug resistant phenotype, a 2- to 3-fold decrease in topo II expression was identified. To investigate the molecular basis for decreased topo II expression in Dox1V, a semi-quantitative analysis of Topo II activity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level. Southern blot and sequencing experiments revealed wild-type sequence of the topo II promoter in the drug resistant cells. Transient gene expression assays demonstrated that topo II is transcriptionally down-regulated in Dox1V independent of the promoter sequence of the endogenous alleles. Instead, the activity of a ubiquitous transcription factor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with the decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regulation resulted from a reduced CP-1/NF-Y activity is responsible for decreased topo II expression in Dox1V cells.