Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells.

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair.

Severe PATCHED1 Deficiency in Cancer-Prone Gorlin Patient Cells Results in Intrinsic Radiosensitivity.

PURPOSE: Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome. METHODS AND MATERIALS: The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5 Gy) and low (50-250 mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods. RESULTS: Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity. CONCLUSIONS: In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations.

Biochemical alterations in human cells irradiated with alpha particles delivered by macro- or microbeams.

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.

Transcriptional response of wild-type and ataxia telangiectasia lymphoblasts following exposure to equitoxic doses of ionizing radiation.

Experiments were designed to compare the transcriptional response to ionizing radiation (IR) of wild-type (WT) and ataxia telangiectasia (AT) cells. mRNA levels were assessed 2, 4 and 24 h after exposure to equitoxic doses using cDNA microarrays. Data reveal distinct patterns of gene expression between AT and WT cells since IR-responsive genes were mostly cell-type specific, this group representing 87 and 94% of the responding genes in WT and AT cells, respectively. In both cell lines, transcriptional alterations of genes associated with proliferation correlated with the observed cell cycle and growth data. Deregulated genes involved in apoptosis suggest that wild-type cells were more prone to cell death by apoptosis than AT cells. Furthermore, genes associated with the response to oxidative stress were particularly deregulated in wild-type cells whereas alterations of genes related to unexpected pathways including RNA processing, protein synthesis and lipid metabolism were specifically found in irradiated AT cells. These data suggest that under radiation conditions leading to a similar survival of WT and AT cells, the mechanisms triggered after radiation were mainly dependent on ATM status and thus on the intrinsic radiosensitivity.

Infrared microspectroscopy study of gamma-irradiated and H2O2-treated human cells.

PURPOSE: Fourier transform infrared microspectroscopy (FT-IRM), which allows simultaneous detection of biochemical changes in the various cellular compartments, was used as a new analytical tool to study early radiation- and oxidation-induced cellular damage at the molecular level in single human cells. MATERIALS AND METHODS: HaCaT keratinocytes were given a single dose of 6 Gy (137Cs) or 650 microM H2O2, neither of which is cytotoxic (neutral red assay) but both of which result in less than 10% clonogenic survival, and deposited on zinc sulphur (ZnS) windows for infra-red (IR) spectra acquisition, immediately and 2 h after treatment. DNA damage was assessed by comet assays in alkaline conditions. RESULTS: Comet assays showed that the yield of DNA damage was higher after H2O2 treatment than after gamma-irradiation. The comparison between spectra of irradiated and H2O2-treated cells showed common changes, but H2O2 treatment presented a broader spectrum of cellular oxidation than ionizing radiation. The bands characteristic of deoxyribose/ribose in nucleic acids centered at 966 and 997 cm(-1), the bands characteristic of nucleic acid bases centered at 1572, 1599, and 1691 cm(-1), as well as the bands characteristic of ordered secondary structure of DNA centered at 1713-1716 cm(-1), were changed in absorbance, sometimes accompanied by a shift. The bands characteristic of proteins centered at 1515, 1530, 1544 and 1640 cm(-1) were changed in absorbance indicating a decrease in secondary structure of proteins. Moreover, the absorbance of the bands at 1515 and 1630 cm(-1) was correlated the yield of reactive oxygen species. Two hours after both treatments most changes were persistent, suggesting either irreversible or not easily repaired damage or persistent oxidative stress. CONCLUSION: As we previously demonstrated in radiation-induced apoptosis studies, these results show that FT-IRM, in correlation with other cellular biology techniques, might be useful for assessing immediate radiation- and oxidative-induced damage to nucleic acids and proteins in single human cells.

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation.

PURPOSE: Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. MATERIALS AND METHODS: The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). RESULTS: SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. CONCLUSIONS: FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2.

Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males.

BACKGROUND: The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both in vitro and in vivo, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes. METHODS: Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent in situ labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test. RESULTS: Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, RELB was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells. CONCLUSION: The molecular defects in the silencing of RELB involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic RELB silencing as a new marker of the progressive disease in males.