Genome dynamics are intimately linked to the regulation of gene expression, the most fundamental mechanism in biology, yet we still do not know whether the very process of transcription drives spatial organization at specific gene loci. Here, we have optimized the ANCHOR/ParB DNA-labeling system for real-time imaging of a single-copy, estrogen-inducible transgene in human cells. Motion of an ANCHOR3-tagged DNA locus was recorded in the same cell before and during the appearance of nascent MS2-labeled mRNA. We found that transcription initiation by RNA polymerase 2 resulted in confinement of the mRNA-producing gene domain within minutes. Transcription-induced confinement occurred in each single cell independently of initial, highly heterogeneous mobility. Constrained mobility was maintained even when inhibiting polymerase elongation. Chromatin motion at constant step size within a largely confined area hence leads to increased collisions that are compatible with the formation of gene-specific chromatin domains, and reflect the assembly of functional protein hubs and DNA processing during the rate-limiting steps of transcription.

Photodynamic therapy (PDT) is an anticancer approach utilizing a light-absorbing molecule and visible light irradiation to generate, in the presence of O 2 , cytotoxic reactive oxygen species, which cause tumor ablation. Given that the photosensitizer hypericin is under consideration for PDT treatment of bladder cancer we used oligonucleotide microarrays in the T24 bladder cancer cell line to identify differentially expressed genes with therapeutic potential. This study reveals that the expression of several genes involved in various metabolic processes, stress-induced cell death, autophagy, proliferation, inflammation and carcinogenesis is strongly affected by PDT and pinpoints the coordinated induction of a cluster of genes involved in the unfolded protein response pathway after endoplasmic reticulum stress and in antioxidant response. Analysis of PDT-treated cells after p38 MAPK inhibition or silencing unraveled that the induction of an important subset of differentially expressed genes regulating growth and invasion, as well as adaptive mechanisms against oxidative stress, is governed by this stress-activated kinase. Moreover, p38 MAPK inhibition blocked autonomous regrowth and migration of cancer cells escaping PDT-induced cell death. This analysis identifies new molecular effectors of the cancer cell response to PDT opening attractive avenues to improve the therapeutic efficacy of hypericin-based PDT of bladder cancer.

Photodynamic therapy (PDT) is an established anticancer modality utilizing the photogeneration of reactive oxygen species (ROS) to kill the cancer cells and hypericin is a promising photosensitizer for the treatment of bladder tumors. In this paper we characterize the signaling pathways and the mechanisms leading to the up-regulation of the antioxidant enzyme heme oxygenase (HO-1) in PDT treated cancer cells. We show that PDT engages the p38(MAPK) and PI3K signaling cascades for HO-1 induction. p38(MAPK) inhibitors or small interfering RNA (siRNA) for p38(MAPK) suppress HO-1 induction after PDT and complete repression is attained when p38 and PI3K antagonists are combined. Blocking these signaling pathways increases additively the propensity of the cells to undergo PDT-induced apoptosis, mirroring the effect of HO-1 silencing. Conversely, increasing HO-1 protein level by hemin prior to irradiation is cytoprotective. HO-1 stimulation by PDT is dependent on transcription and de novo protein synthesis and it is preceded by the nuclear accumulation of the Nrf2 transcription factor, which is reduced by inhibitors of p38(MAPK) and PI3K. Altogether these results indicate that stimulation of HO-1 expression by hypericin-PDT is a cytoprotective mechanism governed by the p38(MAPK) and PI3K pathways, likely through the control of the nuclear availability of the Nrf2 pool.