Author Correction: Selective Priming of Tumor Blood Vessels by Radiation Therapy Enhances Nanodrug Delivery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Selective Priming of Tumor Blood Vessels by Radiation Therapy Enhances Nanodrug Delivery.

Effective drug delivery is restricted by pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, poorly-permeable blood vessels limit the intratumoral permeation and penetration of chemo or nanotherapeutic drugs. New and clinically viable strategies are urgently sought to breach the neoplastic barriers that prevent effective drug delivery. Here, we present an original idea to boost drug delivery by selectively knocking down the tumor vascular barrier in a human pancreatic cancer model. Clinical radiation activates the tumor endothelial-targeted gold nanoparticles to induce a physical vascular damage due to the high photoelectric interactions. Active modulation of these tumor neovessels lead to distinct changes in tumor vascular permeability. Noninvasive MRI and fluorescence studies, using a short-circulating nanocarrier with MR-sensitive gadolinium and a long-circulating nanocarrier with fluorescence-sensitive nearinfrared dye, demonstrate more than two-fold increase in nanodrug delivery, post tumor vascular modulation. Functional changes in altered tumor blood vessels and its downstream parameters, particularly, changes in Ktrans (permeability), Kep (flux rate), and Ve (extracellular interstitial volume), reflect changes that relate to augmented drug delivery. The proposed dual-targeted therapy effectively invades the tumor vascular barrier and improve nanodrug delivery in a human pancreatic tumor model and it may also be applied to other nonresectable, intransigent tumors that barely respond to standard drug therapies.

Nanoparticle-Mediated X-Ray Radiation Enhancement for Cancer Therapy.

Metallic nanoparticles with a high atomic number release Auger electrons in response to external beam X-ray radiation. When these nanoparticles are selectively delivered to tumors, they have the potential to locally enhance the effects of radiation therapy. Optimizing the therapeutic efficacy of these nanoparticles, however, remains a challenging and time-consuming task. Here we describe three different assays that can be used to experimentally quantify and optimize the in vitro therapeutic efficacy of nanoparticle-mediated X-ray radiation enhancement. These include an IC50 extended dose response curve, clonogenic cell survival assay, and immunoblotting. Collectively, these assays provide information about whether a given nanoparticle provides radiosensitization, the extent of the radiosensitization, and the potential mechanism of radiosensitization.

Ubiquitin-specific peptidase 20 regulates Rad17 stability, checkpoint kinase 1 phosphorylation and DNA repair by homologous recombination.

Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.

The role of the human psoralen 4 (hPso4) protein complex in replication stress and homologous recombination.

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G2/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.

The Role of PCNA Posttranslational Modifications in Translesion Synthesis.

Organisms are predisposed to different types in DNA damage. Multiple mechanisms have evolved to deal with the individual DNA lesions. Translesion synthesis is a special pathway that enables the replication fork to bypass blocking lesions. Proliferative Cell Nuclear Antigen (PCNA), which is an essential component of the fork, undergoes posttranslational modifications, particularly ubiquitylation and sumoylation that are critical for lesion bypass and for filling of DNA gaps which result from this bypass. A special ubiquitylation system, represented by the Rad6 group of ubiquitin conjugating and ligating enzymes, mediates PCNA mono- and polyubiquitylation in response to fork stalling. The E2 SUMO conjugating enzyme Ubc9 and the E3 SUMO ligase Siz1 are responsible for PCNA sumoylation during undisturbed S phase and in response to fork stalling as well. PCNA monoubiquitylation mediated by Rad6/Rad18 recruits special polymerases to bypass the lesion and fill in the DNA gaps. PCNA polyubiquitylation achieved by ubc13-mms2/Rad 5 in yeast mediates an error-free pathway of lesion bypass likely through template switch. PCNA sumoylation appears required for this error-free pathway, and it plays an antirecombinational role during normal replication by recruiting the helicase Srs2 to prevent sister chromatid exchange and hyper-recombination.

Gene expression profiling in the mammary gland of rats treated with 7,12-dimethylbenz[a]anthracene.

Identification of molecular markers of early-stage breast cancer development is important for the diagnosis and prevention of the disease. In the present study, we used microarray analysis to examine the differential expression of genes in the rat mammary gland soon after treatment with a known chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), and prior to tumor development. Six weeks after DMBA, differential expression of multiple genes involved in cell growth, differentiation and microtubule dynamics were observed. Gene expression changes were further validated by a combination of techniques, including real-time PCR, RT-PCR, Western blotting and immunohistochemistry. An inhibition of differentiation in this early stage was suggested by the lower expression of beta-casein and transferrin and higher expression of hsp27 in glands from DMBA-treated rats. Possible cell cycle deregulation was indicated by an increased expression of cyclin D1 and hsp86, a heat shock protein associated with cyclin D1. Prior to tumor development, DMBA increased cellular proliferation as detected by Ki-67 and stathmin immunostaining in histologically normal mammary gland. Genes regulating microtubule function, including stathmin, Ran, alpha-tubulin and hsp27, were all overexpressed in the mammary gland of DMBA-treated rats, raising the possibility that disruption of microtubule dynamics and abnormal mitosis may be critical events preceding breast cancer development. Several of the altered proteins, including hsp27, hsp86 and stathmin, may ultimately serve as markers of early breast cancer development.

Rhesus monkey simian immunodeficiency virus infection as a model for assessing the role of selenium in AIDS.

The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of macaques could be used as a model system to assess the role of selenium in AIDS. Plasma and serum selenium levels were determined by standard assays in monkeys before and after inoculation of SIV. SIV-infected cells or cells expressing the HIV Tat protein were labeled with 75Se, and protein extracts were prepared and electrophoresed to analyze selenoprotein expression. Total tRNA was isolated from CEMx174 cells infected with SIV or from KK1 cells infected with HIV, and selenocysteine tRNA isoforms were characterized by reverse phase chromatography. SIV-infected monkeys show a decrease in blood selenium levels similar to that observed in AIDS with development of SAIDS. Cells infected with SIV in vitro exhibit reduced selenoprotein levels and an accumulation of small molecular weight selenium compounds relative to uninfected cells. Examination of the selenocysteine tRNA isoforms in HIV-infected KK1 cells or SIV-infected CEMx174 cells reveals an isoform distribution characteristic of selenium-deficient cells. Furthermore, transfection of Jurkat E6 cells with the Tat gene selectively altered selenoprotein synthesis, with GPX4 and Sep15 being the most inhibited and TR1 the most enhanced. Taken together, the data show that monkeys infected with SIV in vivo and cells infected with SIV in vitro will provide appropriate models for investigating the mechanism(s) responsible for reduced selenium levels that accompany the progression of AIDS in HIV disease.