Ionising Radiation Induces Promoter DNA Hypomethylation and Perturbs Transcriptional Activity of Genes Involved in Morphogenesis during Gastrulation in Zebrafish.

Embryonic development is particularly vulnerable to stress and DNA damage, as mutations can accumulate through cell proliferation in a wide number of cells and organs. However, the biological effects of chronic exposure to ionising radiation (IR) at low and moderate dose rates (< 6 mGy/h) remain largely controversial, raising concerns for environmental protection. The present study focuses on the molecular effects of IR (0.005 to 50 mGy/h) on zebrafish embryos at the gastrula stage (6 hpf), at both the transcriptomics and epigenetics levels. Our results show that exposure to IR modifies the expression of genes involved in mitochondrial activity from 0.5 to 50 mGy/h. In addition, important developmental pathways, namely, the Notch, retinoic acid, BMP and Wnt signalling pathways, were altered at 5 and 50 mGy/h. Transcriptional changes of genes involved in the morphogenesis of the ectoderm and mesoderm were detected at all dose rates, but were prominent from 0.5 to 50 mGy/h. At the epigenetic level, exposure to IR induced a hypomethylation of DNA in the promoter of genes that colocalised with both H3K27me3 and H3Kme4 histone marks and correlated with changes in transcriptional activity. Finally, pathway enrichment analysis demonstrated that the DNA methylation changes occurred in the promoter of important developmental genes, including morphogenesis of the ectoderm and mesoderm. Together, these results show that the transcriptional program regulating morphogenesis in gastrulating embryos was modified at dose rates greater than or equal to 0.5 mGy/h, which might predict potential neurogenesis and somitogenesis defects observed at similar dose rates later in development.

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.

Molecular markers for X-ray-insensitive differentiated cells in the Inner and outer regions of the mesenchymal space in planarian Dugesia japonica.

Planarian's strong regenerative ability is dependent on stem cells (called neoblasts) that are X-ray-sensitive and proliferative stem cells. In addition to neoblasts, another type of X-ray-sensitive cells was newly identified by recent research. Thus, planarian's X-ray-sensitive cells can be divided into at least two populations, Type 1 and Type 2, the latter corresponding to planarian's classically defined "neoblasts". Here, we show that Type 1 cells were distributed in the outer region (OR) immediately underneath the muscle layer at all axial levels from head to tail, while the Type 2 cells were distributed in a more internal region (IR) of the mesenchymal space at the axial levels from neck to tail. To elucidate the biological significance of these two regions, we searched for genes expressed in differentiated cells that were locate close to these X-ray-sensitive cell populations in the mesenchymal space, and identified six genes mainly expressed in the OR or IR, named OR1, OR2, OR3, IR1, IR2 and IR3. The predicted amino acid sequences of these genes suggested that differentiated cells expressing OR1, OR3, IR1, or IR2 provide Type 1 and Type 2 cells with specific extracellular matrix (ECM) environments.

Irradiation Decreases the Neuroendocrine Biomarker Pro-Opiomelanocortin in Small Cell Lung Cancer Cells In Vitro and In Vivo.

BACKGROUND: Small cell lung cancer (SCLC) is an extremely aggressive disease, commonly displaying therapy-resistant relapse. We have previously identified neuroendocrine and epithelial phenotypes in SCLC tumours and the neuroendocrine marker, pro-opiomelanocortin (POMC), correlated with worse overall survival in patients. However, the effect of treatment on these phenotypes is not understood. The current study aimed to determine the effect of repeated irradiation treatment on SCLC cell phenotype, focussing on the neuroendocrine marker, POMC. RESULTS: Human SCLC cells (DMS 79) were established as subcutaneous xenograft tumours in CBA nude mice and then exposed to repeated 2Gy irradiation. In untreated animals, POMC in the blood closely mirrored tumour growth; an ideal characteristic for a circulating biomarker. Following repeated localised irradiation in vivo, circulating POMC decreased (p< 0.01), in parallel with a decrease in tumour size, but remained low even when the tumours re-established. The excised tumours displayed reduced and distinctly heterogeneous expression of POMC compared to untreated tumours. There was no difference in the epithelial marker, cytokeratin. However, there were significantly more N-cadherin positive cells in the irradiated tumours. To investigate the tumour response to irradiation, DMS79 cells were repeatedly irradiated in vitro and the surviving cells selected. POMC expression was reduced, while mesenchymal markers N-cadherin, ß1-integrin, fibroblast-specific protein 1, ß-catenin and Zeb1 expression were amplified in the more irradiation-primed cells. There were no consistent changes in epithelial marker expression. Cell morphology changed dramatically with repeatedly irradiated cells displaying a more elongated shape, suggesting a switch to a more mesenchymal phenotype. CONCLUSIONS: In summary, POMC biomarker expression and secretion were reduced in SCLC tumours which regrew after irradiation and in repeatedly irradiation (irradiation-primed) cells. Therefore, POMC was no longer predictive of tumour burden. This highlights the importance of fully evaluating biomarkers during and after therapy to assess clinical utility. Furthermore, the gain in mesenchymal characteristics in irradiated cells could be indicative of a more invasive phenotype.

Cell dynamics in Hertwig's epithelial root sheath and surrounding mesenchyme in mice irradiated to the head.

OBJECTIVE: To investigate the mechanisms that cause damage to root formation as a result of irradiation to the mouse head, morphological changes in molar dental roots and cell dynamics in Hertwig's epithelial root sheath (HERS), and surrounding mesenchymal tissue were examined. MATERIALS AND METHODS: To perform the experiments, 5-day-old C57BL/6 mice were randomly divided into three groups: the control group (0 Gy) and irradiated groups (10 and 20 Gy). Micro-CT analysis, HE staining, immunohistochemistry analysis, and TUNEL assay were then performed. RESULTS: Roots in irradiated mice were dose-dependently shorter than those of control mice. Cells located outside the root dentin, with abnormal morphology in irradiated mice, were positive for an odontoblast marker. HERS fragmentation occurred earlier in irradiated mice than in control mice, and HERS was trapped by the calcified apical tissue. A dose-dependent reduction in the number of proliferating cells within the apical dental pulp and periapical periodontal ligament surrounding HERS was observed in irradiated mice. Apoptotic cells in the dental pulp and periodontal ligament surrounding HERS were hardly seen. CONCLUSIONS: These results indicate that the early disappearance of HERS and the proliferative suppression of the surrounding mesenchymal cells, which was induced by irradiation, caused dental root malformation.

Hair follicle disruption facilitates pathogenesis to UVB-induced cutaneous inflammation and basal cell carcinoma development in Ptch(+/-) mice.

Hairless mice carrying homozygous mutations in hairless gene manifest rudimentary hair follicles (HFs), epidermal cysts, hairless phenotype, and enhanced susceptibility to squamous cell carcinomas. However, their susceptibility to basal cell carcinomas (BCCs), a neoplasm considered originated from HF-localized stem cells, is unknown. To demonstrate the role of HFs in BCC development, we bred Ptch(+/-)/C57BL6 with SKH-1 hairless mice, followed by brother-sister cross to get F2 homozygous mutant (hairless) or wild-type (haired) mice. UVB-induced inflammation was less pronounced in shaved haired than in hairless mice. In hairless mice, inflammatory infiltrate was found around the rudimentary HFs and epidermal cysts. Expression of epidermal IL1f6, S100a8, vitamin D receptor, repetin, and major histocompatibility complex II, biomarkers depicting susceptibility to cutaneous inflammation, was also higher. In these animals, HF disruption altered susceptibility to UVB-induced BCCs. Tumor onset in hairless mice was 10 weeks earlier than in haired littermates. The incidence of BCCs was significantly higher in hairless than in haired animals; however, the magnitude of sonic hedgehog signaling did not differ significantly. Overall, 100% of hairless mice developed >12 tumors per mouse after 32 weeks of UVB therapy, whereas haired mice developed fewer than three tumors per mouse after 44 weeks of long-term UVB irradiation. Tumors in hairless mice were more aggressive than in haired littermates and manifested decreased E-cadherin and enhanced mesenchymal proteins. These data provide novel evidence that disruption of HFs in Ptch(+/-) mice enhances cutaneous susceptibility to inflammation and BCCs.

The effect of oxidized low-density lipoprotein (ox-LDL) on radiation-induced endothelial-to-mesenchymal transition.

PURPOSE: Radiation-induced cardiovascular disease is a potentially severe side-effect of thoracic radiotherapy treatment. Clinically, this delayed side-effect presents as a form of accelerated atherosclerosis several years after irradiation. As general endothelial dysfunction is known to be an initiating event in radiation-induced vascular damage, we examined the effects of radiation on endothelial cells in radiation-induced atherosclerosis. MATERIALS AND METHODS: The effects of radiation on human aortic endothelial cells (HAoEC) were assessed by immunoblotting and immunofluorescence assays. Radiation-induced phenotypic changes of endothelial cells (ECs) were examined using atherosclerotic tissues of irradiated apoprotein E null (ApoE(-/-)) mice. RESULTS: Radiation induced the HAoEC to undergo phenotypic conversion to form fibroblast-like cells, called the endothelial-to-mesenchymal transition (EndMT), which leads to the upregulation of mesenchymal cell markers such as alpha-smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1), and vimentin, and downregulation of endothelial cell-specific markers such as CD31 and vascular endothelial (VE)-cadherin. Furthermore, compared with low-density lipoprotein (LDL), oxidized low-density lipoprotein (ox-LDL) significantly augmented radiation-induced EndMT in HAoEC. These fibrotic phenotypes of ECs were found in atherosclerotic tissues of irradiated ApoE(-/-) mice with increased levels of ox-LDL. CONCLUSIONS: Taken together, these observations suggest that ox-LDL accelerates radiation-induced EndMT and subsequently contributes to radiation-induced atherosclerosis, providing a novel target for the prevention of radiation-induced atherosclerosis.

[Pharmacological modulation of late radio-induced side effects]. / Modulation pharmacologique des effets tardifs de l'irradiation.

After normal tissue exposure to radiation therapy, late side effects can occur and may reduce patients' quality of life due to their progressive nature. Late toxicities occurrence is the main limiting factor of radiotherapy. Various biological disorders related to irradiation are involved in the development of late toxicities including fibrosis. The present review will focus on the recent physiopathological and molecular mechanisms described to be involved in the development of late radio-induced toxicities, that provide therapeutic perspective for pharmacomodulation.

X-ray irradiation and Rho-kinase inhibitor additively induce invasiveness of the cells of the pancreatic cancer line, MIAPaCa-2, which exhibits mesenchymal and amoeboid motility.

Tumor cells can migrate and invade tissue by two modes of motility: mesenchymal and amoeboid. X-ray or γ-ray irradiation increases the invasiveness of tumor cells with mesenchymal motility through the induction of matrix metalloproteinases (MMP), and this increase is suppressed by MMP inhibitors (MMPI). However, the effects of X-ray or γ-ray irradiation on the invasiveness of tumor cells with amoeboid motility remain unclear. We investigated the effect of irradiation on amoeboid motility by using cells of the human pancreatic cancer line, MIAPaCa-2, which exhibits both modes of motility. The X-ray-induced invasiveness of MIAPaCa-2 cells was associated with the upregulation of MMP2 at both the RNA and protein levels and was inhibited by MMPI treatment. Amoeboid-mesenchymal transition was slightly induced after irradiation. The MMPI treatment caused mesenchymal-amoeboid transition without significant increase in invasiveness, while the ROCK inhibitor (ROCKI) stimulated amoeboid-mesenchymal transition and enhanced invasiveness under both non-irradiated and irradiated conditions. This ROCKI-induced transition was accompanied by the upregulation of MMP2 mRNA and protein. Exposure to both irradiation and ROCKI further enhanced MMP2 expression and had an additive effect on the invasiveness of MIAPaCa-2 cells. Additionally, exposure to MMPI led to significant suppression of both radiation-induced and the basal invasiveness of MIAPaCa-2 cells. This suggests that ROCKI treatment, especially with concomitant X-ray irradiation, can induce invasion of cancer cells and should be used only for certain types of cancer cells. Simultaneous use of inhibitors, ROCKI and MMPI may be effective in suppressing invasiveness under both X-ray-irradiated and non-irradiated conditions.

Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow.

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFRalpha+Sca-1+CD45-TER119-) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.

Effects of white light on beta-catenin signaling pathway in retinal pigment epithelium.

This study investigated the effect of visible light exposure on retinal pigment epithelium (RPE). The activation of Wnt/beta-catenin pathway was investigated by immunofluorescence and Western blot analysis using human retinal pigment epithelial (ARPE-19) cells, which demonstrated that the exposure of white light induced the activation of the Wnt/beta-catenin pathway. Real time RT-PCR demonstrated that the mRNA of alpha-smooth muscle actin (alpha-SMA), and vimentin increased 2.5-4-fold and that of zona occludens 1 (ZO-1) decreased approximately 0.8-fold after white light exposure. The up-regulation of vimentin expression and the down-regulation of ZO-1 were evident by Western blot analysis and immunohistochemistry. Moreover, the ability of phagocytosis of ARPE-19 cells decreased 0.6-fold after light exposure. Together, white light exposure was supposed to induce the activation of Wnt/beta-catenin pathway, the changes in the expression markers of epithelial and mesenchymal cells in RPE cells, and the concomitant impairment of the ability of phagocytosis.

Tumor irradiation increases the recruitment of circulating mesenchymal stem cells into the tumor microenvironment.

Mesenchymal stem cells (MSC) migrate to and proliferate within sites of inflammation and tumors as part of the tissue remodeling process. Radiation increases the expression of inflammatory mediators that could enhance the recruitment of MSC into the tumor microenvironment. To investigate this, bilateral murine 4T1 breast carcinomas (expressing renilla luciferase) were irradiated unilaterally (1 or 2 Gy). Twenty-four hours later, 2 x 10(5) MSC-expressing firefly luciferase were injected i.v. Mice were then monitored with bioluminescent imaging for expression of both renilla (tumor) and firefly (MSC) luciferase. Forty-eight hours postirradiation, levels of MSC engraftment were 34% higher in tumors receiving 2 Gy (P = 0.004) than in the contralateral unirradiated limb. Immunohistochemical staining of tumor sections from mice treated unilaterally with 2 Gy revealed higher levels of MSC in the parenchyma of radiated tumors, whereas a higher proportion of MSC remained vasculature-associated in unirradiated tumors. To discern the potential mediators involved in MSC attraction, in vitro migration assays showed a 50% to 80% increase in MSC migration towards conditioned media from 1 to 5 Gy-irradiated 4T1 cells compared with unirradiated 4T1 cells. Irradiated 4T1 cells had increased expression of the cytokines, transforming growth factor-beta1, vascular endothelial growth factor, and platelet-derived growth factor-BB, and this up-regulation was confirmed by immunohistochemistry in tumors irradiated in vivo. Interestingly, the chemokine receptor CCR2 was found to be up-regulated in MSC exposed to irradiated tumor cells and inhibition of CCR2 led to a marked decrease of MSC migration in vitro. In conclusion, clinically relevant low doses of irradiation increase the tropism for and engraftment of MSC in the tumor microenvironment.

Irradiation-induced epithelial-mesenchymal transition (EMT) related to invasive potential in endometrial carcinoma cells.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In this study, we hypothesized that ionizing irradiation would cause endometrial carcinoma cells (HEC1A) to undergo an increase of motility related to EMT. METHODS: We investigated the effect of ionizing irradiation on HEC1A cell migration. Furthermore, we examined whether this enhanced invasiveness was associated with epithelial-mesenchymal transition (EMT) and Twist siRNA transfections effects in ionizing irradiation-induced HEC1A cell migratory capacity. RESULTS: Ionizing irradiation leads to HEC1A cell phenotypic changes with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. Ionizing irradiation leads to a 2-fold increase in HEC1A cell migration. In immunofluorescence staining of HEC1A cell, the expression of Twist, an organizer of EMT, increased by ionizing irradiation. Additionally, the irradiation-induced HEC1A cell invasion was inhibited by Twist siRNA transfections. CONCLUSIONS: This report suggested that the inhibitory effect of cell invasion through targeting Twist may represent a new approach for improving the therapeutic strategy.

Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

The role of the Spemann organizer in anterior-posterior patterning of the trunk.

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.

Maintenance of radiation-induced intestinal fibrosis: cellular and molecular features.

Recent advances in cell and molecular radiobiology clearly showed that tissue response to radiation injury cannot be restricted to a simple cell-killing process, but depends upon continuous and integrated pathogenic processes, involving cell differentiation and crosstalk between the various cellular components of the tissue within the extracellular matrix. Thus, the prior concept of primary cell target in which a single-cell type (whatever it's epithelial or endothelial cells) dictates the whole tissue response to radiation injury has to be replaced by the occurrence of coordinated multicellular response that may either lead to tissue recovery or to sequel development. In this context, the present review will focus on the maintenance of the radiation-induced wound healing and fibrogenic signals triggered by and through the microenvironment toward the mesenchymal cell compartment, and will highlight how sequential and sustained modifications in cell phenotypes will in cascade modify cell-to-cell interactions and tissue composition.

Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells.

Low-intensity pulsed ultrasound (LIPUS) is known to accelerate bone regeneration, but the precise cellular mechanism is still unclear. The purpose of this study was to determine the effect of LIPUS on the differentiation of pluripotent mesenchymal cell line C2C12. The cells were cultured in differentiation medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm2 for 20 min for all cultures. To verify the cell lineage after LIPUS stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARgamma) was studied using real-time polymerase chain reaction analysis. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were performed using Western blotting. The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the LIPUS stimulation, whereas the expression of MyoD, C/EBP, and PPARgamma was drastically decreased. In the Western blot analysis, LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. Our study demonstrated that LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK.

Radiation-induced suppression of the Bmp2 signal transduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for prevention of heterotopic ossification by radiotherapy.

Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.

Effects of amifostine on clonogenic mesenchymal progenitors and hematopoietic progenitors exposed to radiation.

PURPOSE: To determine the radiation sensitivities of mesenchymal progenitors and hematopoietic progenitors, and to determine the in vitro effects of amifostine on hematopoietic and mesenchymal progenitors exposed to radiation. METHODS: Radiosensitivity of mesenchymal progenitor cells was determined by exposing marrow low-density cells to radiation at doses of 100 to 800 cGy. Mesenchymal cell colonies were established by plating 2.5 x 10(5) marrow low-density cells in long-term marrow culture medium (LTCM). The size, frequency, and cellular composition of the mesenchymal progenitor cells were scored after 14 days of incubation. Mesenchymal progenitor cells were subdivided into progenitors forming fibroblast and adipocyte mixed colonies (CFU-FA), and pure fibroblast colonies (CFU-F). Hematopoietic progenitors were assessed by methylcellulose-based assay. RESULTS: Radiation at 100 cGy caused a mild decrease in CFU-F and CFU-FA derived colonies by 12% and 13%, respectively; 200 cGy decreased CFU-F by 36% and CFU-FA by 52%; 400 cGy decreased CFU-F by 50% and CFU-FA by 86%; and 600 cGy decreased CFU-F by 24%, with total absence of CFU-FA. Pretreatment with amifostine protected 100% of CFU-F at 100 and 200 cGy, 84% at 400 cGy, 46% at 600 cGy, and 14% at 800 cGy. With CFU-FA colonies amifostine pretreatment provided only minimal radioprotection. For hematopoietic progenitors radiation at 100 cGy reduced CFU-GM by 74% but had no significant effect on CFU-GEMM and BFU-E. Radiation at 200 cGy decreased CFU-GEMM by 72%, BFU-E by 54%, and CFU-GM by 84%; 400 cGy further decreased CFU-GEMM by 83%, BFU-E by 81%, and CFU-GM by 93%. Pretreatment with amifostine resulted in twofold stimulation of CFU-GEMM and BFU-E colonies. All BFU-E colonies were protected up to 200 cGy. For CFU-GEMM amifostine pretreatment resulting in 68% at 200 cGy and 31% at 400 cGy. For CFU-GM colonies it was 54% at 100 cGy, 32% at 200 cGy, and 12% at 400 cGy. CONCLUSIONS: Mesenchymal progenitor cell subpopulations are differentially sensitive to radiation. Amifostine protects both mesenchymal and hematopoietic progenitors against radiation injury, though the level of protection appears to be dependent upon the sensitivities of these progenitor cells to radiation. Amifostine is a potent stimulant of BFU-E and CFU-GEMM progenitor colonies.

Radiation-induced reduction of BMP-induced proteoglycan synthesis in an embryonal mesenchymal tissue equivalent using the chicken "limb bud" test.

PURPOSE: Heterotopic ossification (HO) is a common complication following total hip replacement. Clinical studies showed the effectiveness of irradiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to find a suitable cell system, which can reproduce in-vitro data resulting from clinical in-vivo studies. The establishment of such a cell model allows detailed analyses of the mechanism of radiotherapy. METHOD: The chicken limb bud test was used as an in-vitro model. The cells acquired by the limb bud test were irradiated with different doses (0 Gy, 3 Gy, 7 Gy, 10 Gy, 20 Gy). Irradiation was set either 1 hour before, or 1 or 3 days after BMP-2 incubation. The synthesis of proteoglycans (PGS) upon treatment with bone morphogenetic protein (BMP)-2 was measured in cells incubated with BMP-2 for 4 days followed by 35SO4(2-) labeling for 6 hours. Labeled proteoglycans were precipitated using Alcian blue and measured in a raytest radio-TLC analyzer. The incubation with BMP-2 was defined to correlate the in-vivo stimulus meaning the operation. RESULTS: The proteoglycan synthesis was significantly reduced by irradiation 1 hour before or 1 day after BMP-2 incubation, if the dosage was at least 7 Gy. Higher doses than 7 Gy did not lead to lower proteoglycan levels. There was only a trend for a reduction of proteoglycan synthesis by 3 Gy irradiation, but no significant difference compared to the non-irradiated control. An irradiation 3 days after BMP-2 incubation had no effect on proteoglycan. CONCLUSION: A dose and time dependent effect of radiation on BMP-2-induced proteoglycan synthesis was observed. Therefore the results of clinical in-vivo studies were reproduced exactly by the limb bud test. We established an in-vitro cell model to analyze the mechanism of the prevention of heterotopic ossification by radiotherapy on cellular or sub-cellular level.