Figure 5. Histological review of salivary glands 30 days following Roscovitine pretreatment in irradiated tissues. A) Untreated parotid gland showing structure of acini and ducts. B) Parotid gland treated with a single dose of 5Gy radiation C) Parotid gland treated with Roscovitine. D) Parotid gland pretreated with Roscovitine thirty days following a single dose of 5Gy radiation. Abbreviations represent untreated (UT), irradiated with vehicle pre-treatment (IR+V), Roscovitine alone (Rosco), and Roscovitine prior to irradiation (IR+Rosco). growth in vivo [18,19,22]. In contrast to these studies, this work focused on the in vivo effects of Roscovitine in combination with radiation on normal salivary glands. We found that Roscovitine administered prior to radiation induced a significant increase in the accumulation of cells in the G2/M phase of the cell cycle. This induction of cell cycle arrest leads to a decrease in the level of apoptosis in irradiated tissues pretreated with Roscovitine. These results are similar to the peptide inhibitor studies where cellviability was unaltered by treatment of non-transformed normal cells [20,21]. We hypothesize that one mechanism of salivary gland sensitivity to radiation involves inefficient cell cycle arrest leading to poor DNA repair and elevated levels of apoptosis [10]. The current study supports the notion that transient activation of cell cycle arrest in normal tissues can have a beneficial effect in preserving cell viability and function.
Radiation causes DNA damage, which activates p53 leading to cell death or cell cycle arrest and DNA repair. It has previously been documented that radiation-induced apoptosis of acinar cells is regulated by p53 [23]. In addition, IGF-1 mediated cell cycle arrest following radiation is p53-dependent. Therefore, protection of normal tissues from radiation-damage may involve directing p53 activity toward cell cycle arrest rather than apoptosis. Our previous work has demonstrated that the p53 ubiquitin ligase, MDM2, is required for Akt-mediated suppression of apoptosis [24] and treatment with IGF-1 prior to radiation enhances p53 binding to the p21 promoter leading to sustained expression [10]. In the current study, we observed increased p21 expression and elevated phosphorylation of Akt and MDM2 in salivary gland tissues treated with Roscovitine prior to radiation. Interestingly, Mendoza et al reported that Roscovitine reduced cellular viability in cells with inactivated Rb and p53 function when compared to parental cells with functional Rb and p53 [21]. This suggests that Roscovitine-mediated protection of normal tissues may be dependent on wildtype p53 activity. This is an important distinction between normal tissues and cancer cells, since a number of tumors have mutated or altered p53 activity [6]. Important for the translation of the findings of the current study into the clinic, R-roscovitine (seliciclib) is currently in clinical trials as a small molecule therapeutic for treatment of advanced solid tumors and non-small cell lung carcinomas [25]. In this study, Roscovitine preserved the physiological function of normal salivary glands at acute and chronic time points by arresting the cell cycle at the G2/M phase and suppressing apoptosis. This is clinically significant because salivary glands have demonstrated little capacity for regeneration and therefore tissues exposed to higher cumulative doses of radiation (.30 Gy) often have permanent loss of function [26]. In addition, this is potentially broadly applicable to other highly differentiated tissues in close proximity to tumor margins. Based on the new knowledge from the current study, Roscovitine has a potential additional use as a small molecule therapeutic in preventing the damage of incidental radiation to surrounding normal tissues like salivary glands. This would be a significant benefit to the quality of life of patients undergoing radiation treatment for head and neck cancer.
Abstract
Prion diseases, including sheep scrapie, are neurodegenerative diseases with the fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC) to disease-associated prion protein (PrPSc). Chemical inhibition of prion accumulation is widely investigated, often using rodent-adapted prion cell culture models. Using a PrPSc-specific ELISA we discovered a monocationic phenyl-furan-benzimidazole (DB772), which has previously demonstrated anti-pestiviral activity and represents a chemical category previously untested for anti-prion activity, that inhibited PrPSc accumulation and prion infectivity in primary sheep microglial cell cultures (PRNP 136VV/154RR/171QQ) and Rov9 cultures (VRQ-ovinized RK13 cells). We investigated potential mechanisms of this anti-prion activity by evaluating PrPC expression with quantitative RT-PCR and PrP ELISA, comparing the concentration-dependent anti-prion and anti-pestiviral effects of DB772, and determining the selectivity index. Results demonstrate at least an approximate two-log inhibition of PrPSc accumulation in the two cell systems and confirmed that the inhibition of PrPSc accumulation correlates with inhibition of prion infectivity. PRNP transcripts and total PrP protein concentrations within cell lysates were not decreased; thus, decreased PrPC expression is not the mechanism of PrPSc inhibition. PrPSc accumulation was multiple logs more resistant than pestivirus to DB772, suggesting that the anti-PrPSc activity was independent of anti-pestivirus activity. The anti-PrPSc selectivity index in cell culture was approximately 4.6 in microglia and 5.5 in Rov9 cells. The results describe a new chemical category that inhibits ovine PrPSc accumulation in primary sheep microglia and Rov9 cells, and can be used for future studies into the treatment and mechanism of prion diseases.
Citation: Stanton JB, Schneider DA, Dinkel KD, Balmer BF, Baszler TV, et al. (2012) Discovery of a Novel, Monocationic, Small-Molecule Inhibitor of Scrapie Prion Accumulation in Cultured Sheep Microglia and Rov Cells. PLoS ONE 7(11): e51173. doi:10.1371/journal.pone.0051173 Editor: Roberto Chiesa, Dulbecco Telethon Institute and Mario Negri Institute for Pharmacological Research, Italy Received June 14, 2012; Accepted October 30, 2012; Published November 30, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by United States Department of Agriculture/Agricultural Research Service Specific Cooperative Agreement No. 58-5348-7-577, the Washington State University New Faculty Seed Grant, and National Institutes of Health AI064200. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
