A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Apurinic/Apyrimidinic Endonuclease 1 Restricts the Internalization of Bacteria Into Human Intestinal Epithelial Cells Through the Inhibition of Rac1.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.

Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X.

Nanomaterials with elongated architectures have been shown to possess differential tumor homing properties compared to their spherical counterparts. Here, we investigate whether this phenomenon is mirrored by plant viral nanoparticles that are filamentous (Potato virus X) or spherical (Cowpea mosaic virus). Our studies demonstrate that Potato virus X (PVX) and Cowpea mosaic virus (CPMV) show distinct biodistribution profiles and differ in their tumor homing and penetration efficiency. Analogous to what is seen with inorganic nanomaterials, PVX shows enhanced tumor homing and tissue penetration. Human tumor xenografts exhibit higher uptake of PEGylated filamentous PVX compared to CPMV, particularly in the core of the tumor. This is supported by immunohistochemical analysis of the tumor sections, which indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity make it a potentially superior platform for applications in cancer drug delivery and imaging applications.

Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors.

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [ß-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies.

Synthesis of bombesin-functionalized iron oxide nanoparticles and their specific uptake in prostate cancer cells.

The imaging of molecular markers associated with disease offers the possibility for earlier detection and improved treatment monitoring. Receptors for gastrin-releasing peptide are overexpressed on prostate cancer cells offering a promising imaging target, and analogs of bombesin, an amphibian tetradecapeptide have been previously demonstrated to target these receptors. Therefore, the pan-bombesin analog [ß-Ala11, Phe13, Nle14]bombesin-(7-14) was conjugated through a linker to dye-functionalized superparamagnetic iron oxide nanoparticles for the development of a new potential magnetic resonance imaging probe. The peptide was conjugated via click chemistry, demonstrating a complementary alternative methodology to conventional peptide-nanoparticle conjugation strategies. The peptide-functionalized nanoparticles were then demonstrated to be selectively taken up by PC-3 prostate cancer cells relative to unfunctionalized nanoparticles and this uptake was inhibited by the presence of free peptide, confirming the specificity of the interaction. This study suggests that these nanoparticles have the potential to serve as magnetic resonance imaging probes for the detection of prostate cancer.