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IntroductionExpression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.MethodsIR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated.ResultsThe average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked repair of DNA-DSB. Likewise, YB-1 siRNA increased radiation sensitivity.ConclusionsIR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-Ras or IR enhances repair of DNA-DSB and postirradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways.

In the two cell lines investigated the mean number of residual foci of gammaH2AX can be used to predict differences in the radiation dose response relationship in vitro.

Cancer stem cells (CSC) have the unique ability to cause tumor recurrences if they survive treatment. Radiotherapy has curative potential because it has been functionally shown to sufficiently inactivate CSCs. It is well known that CSCs mediate the radiation resistance of tumors by tumor-specific factors, such as the pretreatment number of CSCs and repopulation or reoxygenation during fractionated radiotherapy. CSCs appear to have a higher intrinsic radioresistance than non-CSCs, a factor that is especially important for the development of predictive biomarkers that, if this finding holds true, can only be successfully established if they are stem-cell specific. Recent clinical data imply that stem-cell-related surface markers may be directly used as predictors for the radiocurability of tumors with comparable risk factors, such as histology and size. Future studies need to address the question of which additional markers need to be considered if more heterogeneous patient collectives are investigated. With the goal of developing a direct targeting approach, investigators are currently evaluating several drugs that are intended to target CSCs by inhibiting stem-cellrelated signal transduction pathways. We need to preclinically test such drugs as combined-modality therapies in combination with radiotherapy to evaluate their curative potential, and optimize them by increasing their specificity to CSCs over normal tissue stem cells to avoid increased radiation toxicity.

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Antibodies raised against fragments of synthetic peptides of human 5 alpha-reductase isoenzymes 1 (h5 alpha r1) and 2 (h5 alpha r2) were applied to paraffin sections of human skin (scalp, eyelid, lip, breast, scrotum). Immunoreactive sites were differentially distributed, in that h5 alpha r1 immunoreactivity was present in the nuclei of cells in the stratum germinativum (basal and lower portion of the spinous layer) of the epidermis, subepithelial fibroblasts, adipocytes, smooth muscle cells of the scrotal tunica dartos, basal cells of sebaceous glands, excretory duct cells of sweat glands, cells of the dermal papilla and fibrous and outer epithelial sheath of hair roots, as well as endothelial cells of small vessels and Schwann cells of cutaneous myelinated nerves. In contrast, immunoreactivity for h5 alpha r2 was found in the cytoplasm of the cells of the spinous layer (and far less intensely in the basal layer) of the epidermis, subepidermal fibrocytes, and especially in subcutaneous adipocytes. Immunoreactivity was strongest in the non-keratinized portion of the inner epithelial sheath and the cuticle of hair follicles, whereas other portions of the hair root were negative. Sweat glands were stained, whereas sebaceous glands showed only weak diffuse immunoreactivity. In mucocutaneous zones, salivary glands and conjunctival epithelium showed immunoreactive cells. Vascular endothelium displayed immunoreactivity only in the genital region. We present experimental evidence for a differential distribution of 5 alpha-reductase isoenzymes in human skin. This might reflect a diversity in the response of different areas of the skin to androgenic challenge.

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