Understanding the molecular circuitry of the radiation survival response might strongly assist optimization of tumor therapy, particularly radiotherapy, and issues related to radioprotection. Owing to a great lack of knowledge in this area of research, we examined the radiation survival response of cells under adhesion versus suspension conditions in this study. In recent years, observations from our group and others pinpointed the importance of FA signaling for the survival of cells exposed to X-rays and chemotherapeutics. The multiprotein complex characteristic of FA suggests more molecules inevitable involved in such stress reactions than integrins and growth factor receptors. Concluding from previous findings that PINCH1 confers radioresistance 
, the present study elucidated whether PINCH1 also mediates its prosurvival effects under suspension conditions in different mouse and human cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion is independent from adhesion and can also be observed under suspension conditions. Despite a reduced DSB repair ability in one of the two tested tumor cell lines, other possible determinants of clonogenic data analysis such as proliferation as well as adhesion and apoptosis were taken into account or could be excluded, respectively. Our findings can also not be explained by modifications of adhesion and growth factor associated signaling involving FAK, Paxillin, p130CAS
, AKT1 and MAPK under suspension and serum withdrawal relative to adhesion plus serum conditions. The presented data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adherent and suspension conditions.
In various human normal and tumor cell line models, adhesion to ECM confers resistance against ionizing radiation (X-rays) and cytotoxic agents 
. These phenomena, called cell adhesion mediated radioresistance (CAM-RR) and cell adhesion mediated drug resistance (CAM-DR), arise from interactions of cells with components of their microenvironment through binding of integrins to ECM or through cytokine binding to their cognate receptors with subsequent channeling of prosurvival biochemical cues 
. However, the tested immortalized PINCH1fl/fl
MEF showed insensitivity to ECM adhesion with regard to radiation survival. With remaining PINCH1-dependent differences in radiosensitivity, these cells similarly survived under suspension as compared to adhesion conditions after exposure to increasing single doses of X-rays, a finding confirmed by PINCH1 reconstitution in PINCH1−/−
MEF. From our analysis on the influence of growth factors/FCS on radiation survival, PINCH1fl/fl
MEF seem to become more sensitive to serum withdrawal over time as compared to PINCH1−/−
MEF, whose radiation survival remained unchanged.
The comparison of protein expression and phosphorylation in this MEF system suggests a PINCH1-related modification of protein tyrosine phosphorylation rather than protein expression. From further analysis, we identified moderate to strong dephosphorylation of FAK and the associated proteins Paxillin and p130CAS
as well as AKT, GSK3β and ERK1/2. Despite these clear differences resulting from PINCH1 depletion, which have been partly pursued as recently published 
and provide a molecular basis for untangling the radiosensitization by PINCH1 knockout, the unsuspected similarity between radiation survival of adhesion and suspension cultures cannot be explained and requires further evaluation. Certainly, as this is only a snapshot analysis of a very limited number of signaling molecules, additional signal transduction events and changes in other important cell processes such as gene expression and post-translational protein modification have also to be taken into consideration.
As next step, we decided to examine the generality of this phenomenon in human cancer cell lines from different origin, i.e. head and neck and cervix. Subsequent to observing the heterogenic expression of the complex proteins PINCH1, ILK and α-Parvin among human cancer cell lines in contrast to a well known and clear pattern in both PINCH1 MEF and ILK mouse kidney fibroblasts 
, we performed a siRNA-mediated knockdown of PINCH1 in HTB43 and HTB35 cells. The resulting radiosensitization in these cell lines confirmed our previously reported data in other cell line models 
. Intriguingly, it is shown that the absence/strong downregulation of PINCH1 does not result in any adhesion defects, which might account for the differences in clonogenic survival, an observation also supported by the presented similarity in plating efficiencies of PINCH1 knockdown HTB43 and HTB35 cell cultures relative to siRNA controls. Moreover, in line with unaltered cell adhesion, changes in cell morphology under reduced PINCH1 expression were undetectable.
The role of PINCH1 on tumor cell apoptosis has been recently documented by Chen and colleagues 
. They showed a regulation of Bim translocation to mitochondria by PINCH1 in a HT-1080 fibrosarcoma cell model that seems relevant for antiapoptotic effects in tumor cells. In our hands, a PINCH1 knockdown failed to induce apoptosis in the two tumor cell lines used. A second highly relevant parameter in the context of radiobiology is the repair of DSBs as most life-threatening DNA lesions upon exposure to ionizing radiation 
. With the presented inconsistencies in these two human tumor cell lines, it is impossible to assess the impact of PINCH1 on DSB repair. With one report on PINCH1's nuclear import and export sequence and its nuclear localization 
, PINCH1 might influence such kind of processes via its function as adapter protein and/or via its putative function as transcription factor.
Of further importance for the presented study is the fact that the degree of radiosensitization was maintained when cells were irradiated in suspension. However, the dose-effect curves of adherent PINCH1 knockdown cultures and suspension siRNA controls were superimposable, thus, indicating that the tested human tumor cell lines receive prosurvival/radioresistance signals through ECM adhesion, which are cut-off under detachment conditions.
In comparison with the analysis of MEFs, HTB43 but not HTB35 cells showed alterations of protein phosphorylations upon PINCH1 silencing. In addition to ILK and α-Parvin repression, PINCH1 depleted HTB43 cells revealed reduced levels of FAK Tyr397 and Tyr576/577 and Paxillin Tyr31. Under suspension, however, broad dephosphorylation occurred in both HTB43 and HTB35 cells with the exception of Src, whose Tyr416 phosphorylation was induced. Similar to MEFs, the analysis of signaling molecules involved in adhesion and growth factor receptor signaling provided no definite and molecular explanation for the radiosensitization through PINCH1 knockdown.
In conclusion, our data generated in MEF and human tumor cells suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells independent from cell adhesion. For clarification of a function of PINCH1 aside from FAs and whether other adapter proteins are key to the cellular radiation response remains to be solved in future examinations.