MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence.

Radiation-induced gastrointestinal (GI) syndrome as a function of age.

Previous studies show increased sensitivity of older mice (28-29 months) compared with young adult mice (3 months, possessing a mature immune system) to radiation-induced GI lethality. Age-dependent lethality was associated with higher levels of apoptotic stem cells in small intestinal crypts that correlated with sphingomyelinase activity, a source of pro-apoptotic ceramide. The objective of this study is to determine whether the cycling crypt base columnar cells (CBCs) in aging animals are specifically more sensitive to radiation effects than the CBCs in young adult mice, and to identify factors that contribute to increased radiosensitivity. Mortality induced by subtotal body radiation was assessed at different doses (13 Gy, 14 Gy, and 15 Gy) in young adult mice versus older mice. Each dose was evaluated for the occurrence of lethal GI syndrome. A higher death rate due to radiation-induced GI syndrome was observed in older mice as compared with young adult mice: 30 vs. 0% at 13 Gy, 90 vs. 40% at 14 Gy, and 100 vs. 60% at 15 Gy. Radiation-induced damage to crypts was determined by measuring crypt regeneration (H&E staining, Ki67 expression), CBC biomarkers (lgr5 and ascl2), premature senescence (SA-ß-gal activity), and apoptosis of CBCs. At all three doses, crypt microcolony survival assays showed that the older mice had fewer regenerating crypts at 3.5 days post-radiation treatment. Furthermore, in the older animals, baseline CBCs numbers per circumference were significantly decreased, correlating with an elevated apoptotic index. Analysis of tissue damage showed an increased number of senescent CBCs per crypt circumference in older mice relative to younger mice, where the latter was not significantly affected by radiation treatment. It is concluded that enhanced sensitivity to radiation-induced GI syndrome and higher mortality in older mice can be attributed to a decreased capacity to regenerate crypts, presumably due to increased apoptosis and senescence of CBCs.

Spatial Heterogeneity Analysis in Evaluation of Cell Viability and Apoptosis for Colorectal Cancer Cells.

In evaluation of cell viability and apoptosis, spatial heterogeneity is quantified for cancerous cells cultured in 3-D in vitro cell-based assays under the impact of anti-cancer agents. In 48-h experiments using human colorectal cancer cell lines of HCT-116, SW-620, and SW-480, incubated cells are divided into control and drug administered groups, to be grown in matrigel and FOLFOX solution, respectively. Our 3-D cell tracking and data acquisition system guiding an inverted microscope with a digital camera is utilized to capture bright field and fluorescent images of colorectal cancer cells at multiple time points. Identifying the locations of live and dead cells in captured images, spatial point process and Voronoi tessellation methods are applied to extract morphological features of in vitro cell-based assays. For the former method, spatial heterogeneity is quantified with the second-order functions of Poisson point process, whereas the deviation in the area of Voronoi polygons is computed for the latter. With both techniques, the results indicate that the spatial heterogeneity of live cell locations increases as the viability of in in vitro cell cultures decreases. On the other hand, a decrease is observed for the heterogeneity of dead cell locations with the decrease in cell viability. This relationship between morphological features of in vitro cell-based assays and cell viability can be used for drug efficacy measurements and utilized as a biomarker for 3-D in vitro microenvironment assays.