No abstract

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.

The effects of estrogens, particularly 17␤-estradiol (E2), are mediated by estrogen receptor ␣ (ER␣) and ER␤. 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 ER␣ and ER␤ 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 ER␣ and/or ER␤ 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 ER␣ dimer. These results suggest that ER␣ is the functionally dominant partner in the ER␣/␤ heterodimer.Estrogen hormones, particularly 17␤-estradiol (E2), exert their effects through a complex array of convergent and divergent signaling pathways that mediate genomic and nongenomic events, resulting in target tissue-specific responses (11, 31). The E2 information is conveyed by the transcription factors, estrogen receptor ␣ (ER␣) and ER␤ (11, 31), which are encoded by distinct genes and are expressed in different tissues as well as in the same tissue at various levels (11,31).Upon binding to E2, ER dimerizes and interacts with permutations of a palindromic DNA sequence separated by three nonspecific nucleotides: 5Ј-GGTCAnnnTGACC-3Ј, the consensus estrogen responsive element (ERE) (11,18,31). The E2-ER-ERE complex subsequently recruits coactivators/regulators to promote local chromatin remodeling and to bridge with general transcription factors for the initiation of transcription (11, 31). This pathway is called ERE-dependent ER signaling. The E2-ER complex also regulates gene expression through functional tethering to a transcription factor bound to its cognate regulatory element on DNA. This is the DNAdependent and ERE-independent signaling pathway (22, 36). Furthermore, E2 elicits effects through the membrane and cytoplasmic ERs (24, 39).ER␣ and ER␤ share high amino acid identity (96%) in their DNA-binding domains (DBDs) (11, 31), which is reflected in...

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.

Estrogen receptors (ER) alpha and beta are members of a superfamily of nuclear receptors and mediate estrogen [17beta-estradiol (E2)] signaling. ERbeta has considerably less transcription potency than ERalpha in heterologous expression systems that use E2 response elements (ERE) in tandem as the trans-acting unit. We show here that despite similar intracellular characteristics, ERbeta, in contrast to ERalpha, fails to induce gene transcription synergistically in response to E2 from tandem EREs. Moreover, our results indicate that ERalpha-specific partial agonistic activity of antagonists occurs additively. Although synergy contributes, it is not sufficient for differences in the transcription potencies between the ER subtypes. We demonstrate here that differences in the abilities of ERs to integrate activation functions through functional interactions between amino and carboxyl termini are critical for the transcriptional strength of ER subtypes.