Russell Hilf

A fluorometric method for determination of oxidized and reduced glutathione in tissues

Abstract A method for measurement of both oxidized (GSSG) and reduced (GSH) glutathione has been developed, with use of o -phthalaldehyde (OPT) as a fluorescent reagent. The method takes advantage of the reaction of GSH with OPT at pH 8 and of GSSG with OPT at pH 12; GSH can be complexed to N -ethylmaleimide to prevent interference of GSH with measurement of GSSG. The method gave “recoveries” of 91 to 110% for both GSH and GSSG and was quite specific for glutathione; and none of the manipulations appeared to influence the amount of glutathione present in the tissue. Results for GSH levels agreed well with earlier reports but levels of GSSG estimated here were higher than earlier reported values. The reasons for the apparently higher levels of GSSG are discussed.

Oxygen consumption and diffusion effects in photodynamic therapy.

Effects of oxygen consumption in photodynamic therapy (PDT) are considered theoretically and experimentally. A mathematical model of the Type II mechanism of photooxidation is used to compute estimates of the rate of therapy-dependent in vivo oxygen depletion resulting from reactions of singlet oxygen (1O2) with intracellular substrate. Calculations indicate that PDT carried out at incident light intensities of 50 mW/cm2 may consume 3O2 at rates as high as 6-9 microM s-1. An approximate model of oxygen diffusion shows that these consumption rates are large enough to decrease the radius of oxygenated cells around an isolated capillary. Thus, during photoirradiation, cells sufficiently remote from the capillary wall may reside at oxygen tensions that are low enough to preclude or minimize 1O2-mediated damage. This effect is more pronounced at higher power densities and accounts for an enhanced therapeutic response in tumors treated with 360 J/cm2 delivered at 50 mW/cm2 compared to the same light dose delivered at 200 mW/cm2. The analysis further suggests that the oxygen depletion could be partially overcome by fractionating the light delivery. In a transplanted mammary tumor model, a regimen of 30-s exposures followed by 30-s dark periods produced significantly longer delays in tumor growth when compared to the continuous delivery of the same total fluence.

Sequence Requirements for Estrogen Receptor Binding to Estrogen Response Elements

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Single-Chain Estrogen Receptors (ERs) Reveal that the ERα/β Heterodimer Emulates Functions of the ERα Dimer in Genomic Estrogen Signaling Pathways

ABSTRACT The effects of estrogens, particularly 17β-estradiol (E2), are mediated by estrogen receptor α (ERα) and ERβ. Upon binding to E2, ERs homo- and heterodimerize when coexpressed. The ER dimer then regulates the transcription of target genes through estrogen responsive element (ERE)-dependent and -independent pathways that constitute genomic estrogen signaling. Although ERα and ERβ have similar ERE and E2 binding properties, they display different transregulatory capacities in both ERE-dependent and -independent signaling pathways. It is therefore likely that the heterodimerization provides novel functions to ERs by combining distinct properties of the contributing partners. The elucidation of the role of the ER heterodimer is critical for the understanding of physiology and pathophysiology of E2 signaling. However, differentially determining target gene responses during cosynthesis of ER subtypes is difficult, since dimers formed are a heterogeneous population of homo- and heterodimers. To circumvent the pivotal dimerization step in ER action and hence produce a homogeneous ER heterodimer population, we utilized a genetic fusion strategy. We joined the cDNAs of ERα and/or ERβ to produce single-chain ERs to simulate the ER homo- and heterodimers. The fusion ERs interacted with ERE and E2 in a manner similar to that observed with the ER dimers. The homofusion receptors mimicked the functions of the parent ER dimers in the ERE-dependent and -independent pathways in transfected mammalian cells, whereas heterofusion receptors emulated the transregulatory properties of the ERα dimer. These results suggest that ERα is the functionally dominant partner in the ERα/β heterodimer.

Chicken Ovalbumin Upstream Promoter-Transcription Factor Interacts with Estrogen Receptor, Binds to Estrogen Response Elements and Half-Sites, and Inhibits Estrogen-induced Gene Expression

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purified with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response element (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE half-site with high affinity,K d = 1.24 nm. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyzed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor, peroxisome proliferator-activated receptor, or vitamin D receptor, were constituents of the COUP-TF·DNA binding complex detected in gel mobility shift assays. Direct interaction of COUP-TF with ER was indicated by GST “pull-down” and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half-site interaction decreased. Conversely, COUP-TF transcribed and translated in vitroenhanced the ERE binding of purified estradiol (E2)-liganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half-sites, to interact with ER, and to inhibit E2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding competition and through protein-protein interactions.

The relative importance of estrogen receptor analysis as a prognostic factor for recurrence or response to chemotherapy in women with breast cancer

The value of estrogen receptor (ER) analysis in primary breast cancer samples as a potential prognostic factor was examined in three clinical situations: time to recurrence in patients with no therapy after mastectomy, failure of patients receiving adjuvant therapy, and response of advanced disease patients to cytotoxic chemotherapy. Other prognostic factors analyzed were menopausal and nodal status. In none of these clinical settings were we able to demonstrate the usefulness of ER status as a prognosticator of the disease course or its response to therapy.

Interdependence of fluence, drug dose and oxygen on hematoporphyrin derivative induced photosensitization of tumor mitochondria.

Abstract— Studies were conducted to assess the interdependence of three discrete parameters known to influence hematoporphyrin derivative (Hpd)‐induced photosensitization. The effects of fiuence, drug dose and oxygen environment were examined for their role in causing an inhibition of the activity of mitochondrial cytochrome c oxidase. Experiments were performed on R3230AC mammary tumor mitochondria in vitro and on mitochondria isolated from tumors of animals pre‐treated with Hpd in vivo. Inhibition of cytochrome c oxidase activity was observed to be directly proportional to total energy density. Photosensitization was dependent on oxygen concentration, with total energy density dependent photosensitization being diminished in environments containing less than 5% oxygen. At 1% oxygen environments, photosensitization was significantly impaired and demonstrated no drug‐dose relationship. These results suggest that tissue oxygen concentration may represent a critical factor for the therapeutic usefulness of Hpd photodynamic therapy in treatment of cancer.

A regulatory role for porphobilinogen deaminase (PBGD) in delta-aminolaevulinic acid (delta-ALA)-induced photosensitization?

As an initial approach to optimize delta-aminolaevulinic acid (delta-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: delta-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time- and dose-related increase in tumours after intravenous administration of 300 mg kg(-1) delta-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mM delta-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after delta-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a rate-limiting determinant for the efficacy of delta-ALA-induced photosensitization when used in photodynamic therapy.

Photodynamic inactivation of selected intracellular enzymes by hematoporphyrin derivative and their relationship to tumor cell viability in vitro

The photosensitivity of freshly dissociated R3230AC mammary adenocarcinoma cells was examined by measurement of the activities of selected intracellular enzymes after treatment with hematoporphyrin derivative (Hpd) and exposure to light in vitro. Enzymes selected as representative of the cytosolic cell compartment showed no loss in activity, whereas malate dehydrogenase, located in the mitochondrial matrix, displayed a modest decrease (approximately 15%) in activity. In contrast, cytochrome c oxidase and succinate dehydrogenase, enzymes associated with the mitochondrial membrane, demonstrated a very rapid and more marked inhibition of activities, approximately 45% and 25%, respectively. The time-course of inhibition of these mitochondrial membrane enzymes preceded the loss of cell viability, which displayed slower kinetics as seen by a more gradual and progressive pattern of loss in viability. These data suggest that the mitochondria are an early-affected and important intracellular site for Hpd photosensitization.

Correlations between certain biochemical properties of breast cancer and response to therapy: A preliminary report

Biochemical analyses were carried out on tissues from 130 women with mammary carcinoma and 104 patients with benign breast lesions. The tissues were assayed for specific estrogen‐binding proteins (EBP) and for several enzymes involved in carbohydrate and lipid metabolism. Normal breast, fibrocystic disease, and fibroadenomas usually exhibited lower enzyme levels than malignant tissues, and rarely contained specific EBP. Intraductal carcinoma appears to contain certain biochemical characteristics unique to this tissue when compared with benign proliferative breast diseases; these differences may be of value in prediction of response to various types of therapy.