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Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

We previously reported the establishment of a rare xenograft derived from a recurrent oligodendroglioma with 1p/19q codeletion. Here, we analyzed in detail the exome sequencing datasets from the recurrent oligodendroglioma (WHO grade III, recurrent O2010) and the first-generation xenograft (xenograft1). Somatic SNVs and small InDels (n = 80) with potential effects at the protein level in recurrent O2010 included variants in IDH1 (NM_005896:c.395G>A; p. Arg132His), FUBP1 (NM_003902:c.1307_1310delTAGA; p.Ile436fs), and CIC (NM_015125:c.4421T>G; p.Val1474Gly). All but 2 of these 80 variants were also present in xenograft1, along with 7 new variants. Deep sequencing of the 87 SNVs and InDels in the original tumor (WHO grade III, primary O2005) and in a second-generation xenograft (xenograft2) revealed that only 11 variants, including IDH1 (NM_005896:c.395G>A; p. Arg132His), PSKH1 (NM_006742.2:c.650G>A; p.Arg217Gln), and SNX12 (NM_001256188:c.470G>A; p.Arg157His), along with a variant in the TERT promoter (C250T, NM_198253.2: c.-146G>A), were already present in primary O2005. Allele frequencies of the 11 variants were calculated to assess their potential as putative driver genes. A missense change in NDST4 (NM_022569:c.2392C>G; p.Leu798Val) on 4q exhibited an increasing allele frequency (~ 20%, primary O2005, 80%, recurrent O2010 and 100%, xenograft1), consistent with a selection event. Sequencing of NDST4 in a cohort of 15 oligodendrogliomas, however, revealed no additional cases with potential protein disrupting variants. Our analysis illuminated a tumor evolutionary series of events, which included 1p/19q codeletion, IDH1 R132H, and TERT C250T as early events, followed by loss of function of NDST4 and mutations in FUBP1 and CIC as late events.

Summary Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC−/−;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Background The evolution of primary glioblastoma (GBM) is poorly understood. Multifocal GBM (ie, multiple synchronous lesions in one patient) could elucidate GBM development. Methods We present the first comprehensive study of 12 GBM foci from 6 patients using array-CGH, spectral karyotyping, gene expression arrays, and next-generation sequencing. Results Multifocal GBMs genetically resemble primary GBMs. Comparing foci from the same patient proved their monoclonal origin. All tumors harbored alterations in the 3 GBM core pathways: RTK/PI3K, p53, and RB regulatory pathways with aberrations of EGFR and CDKN2A/B in all (100%) patients. This unexpected high frequency reflects a distinct genetic signature of multifocal GBMs and might account for their highly malignant and invasive phenotype. Surprisingly, the types of mutations in these genes/pathways were different in tumor foci from the same patients. For example, we found distinct mutations/aberrations in PTEN, TP53, EGFR, and CDKN2A/B, which therefore must have occurred independently and late during tumor development. We also identified chromothripsis as a late event and in tumors with wild-type TP53. Only 2 events were found to be early in all patients: single copy loss of PTEN and TERT promoter point mutations. Conclusions Multifocal GBMs develop through parallel genetic evolution. The high frequency of alterations in 3 main pathways suggests that these are essential steps in GBM evolution; however, their late occurrence indicates that they are not founder events but rather subclonal drivers. This might account for the marked genetic heterogeneity seen in primary GBM and therefore has important implications for GBM therapy.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol molecule from green tea and is known to exhibit antioxidative as well as tumor suppressing activity. In order to examine EGCG tumor invasion and suppressing activity against adult T-cell leukemia (ATL), two HTLV-1 positive leukemia cells (HuT-102 and C91- PL) were treated with non-cytotoxic concentrations of EGCG for 2 and 4 days. Proliferation was significantly inhibited by 100 μM at 4 days, with low cell lysis or cytotoxicity. HTLV-1 oncoprotein (Tax) expression in HuT- 102 and C91-PL cells was inhibited by 25 μM and 125 μM respectively. The same concentrations of EGCG inhibited NF-kB nuclearization and stimulation of matrix metalloproteinase-9 (MMP-9) expression in both cell lines. These results indicate that EGCG can inhibit proliferation and reduce the invasive potential of HTLV-1- positive leukemia cells. It apparently exerted its effects by suppressing Tax expression, manifested by inhibiting the activation of NF-kB pathway and induction of MMP-9 transcription in HTLV-1 positive cells.

Microgravity and cosmic rays as found in space are difficult to recreate on earth. However, ground-based models exist to simulate space flight experiments. In the present study, an experimental model was utilized to monitor gene expression changes in fetal skin fibroblasts of murine origin. Cells were continuously subjected for 65 h to a low dose (55 mSv) of ionizing radiation (IR), comprising a mixture of high‑linear energy transfer (LET) neutrons and low-LET gamma-rays, and/or simulated microgravity using the random positioning machine (RPM), after which microarrays were performed. The data were analyzed both by gene set enrichment analysis (GSEA) and single gene analysis (SGA). Simulated microgravity affected fetal murine fibroblasts by inducing oxidative stress responsive genes. Three of these genes are targets of the nuclear factor‑erythroid 2 p45-related factor 2 (Nrf2), which may play a role in the cell response to simulated microgravity. In addition, simulated gravity decreased the expression of genes involved in cytoskeleton remodeling, which may have been caused by the downregulation of the serum response factor (SRF), possibly through the Rho signaling pathway. Similarly, chronic exposure to low-dose IR caused the downregulation of genes involved in cytoskeleton remodeling, as well as in cell cycle regulation and DNA damage response pathways. Many of the genes or gene sets that were altered in the individual treatments (RPM or IR) were not altered in the combined treatment (RPM and IR), indicating a complex interaction between RPM and IR.

IDH1 R132H mutations are considered to play a key role in the development of low grade gliomas and therefrom derived secondary glioblastomas (GBM). Wild type IDH1 converts isocitrate to α-ketoglutarate (a-KG) while reducing NADP+. IDH1R132H has a neomorphic enzymatic function using a-KG to generate high amounts of the oncometabolite D-2-hydroxyglutarate (D-2-HG). While the effects of D-2-HG have been subject to intense research, D-2-HG independent effects of IDH1R132H on energy homeostasis and redox status are not well studied. Here we demonstrate that IDH1R132H transduction but not D-2-HG alone leads to significantly decreased Krebs cycle metabolite concentrations and proliferation in U87 and the primary GBM cell line HT7606 as well as in immortalized astrocytes SVGp12. Furthermore, IDH1R132H mutation, but not D-2-HG treatment, resulted in a significant drop in NADPH levels in tumor cells (U87 and HT7606), whereas immortalized astrocytes retained normal NADPH levels. Since NAPDH levels can be restored via the reaction of the NAD-kinase, we analyzed NAD levels and enzymes involved in NAD-synthesis in our cell lines. Indeed we found a significant drop of NAD levels and in the activity of the NAD-dependent enzyme sirtuin in IDH1R132H mutant U87 and HT7606 but not in NADPH stable SVGp12-IDH1R132H. Interestingly, there were marked differences in expression of NAD-synthesis enzymes between the different cell-lines. In particular, NAMPT-levels were much higher in U87 and HT7606 then in astrocytes (SVGp12) and significantly decreased in U87-IDH1R132H and HT7606-IDH1R132H. Importantly, we also found decreased levels of NAMPT in primary tumor tissues and patient derived glioma cell lines with IDH1 R132H compared to wild type gliomas. Altogether our results for the first time show that the IDH1 mutation directly affects energy homeostasis and redox status in a cell-type dependent manner. We hypothesize that this leads to a drop in NADPH and NAD-levels during malignant progression, resulting in a disadvantage for proliferating tumor cells. This is in line with the favorable prognosis and good response to chemo- and radiation therapy clinically observed in IDH-mutated gliomas. Our findings suggest that the impaired metabolism in IDH1-mutant tumors might be a promising target for future therapies. Citation Format: Evelin Schrock, Julia Biedermann, Khalil Abou-El-Ardat, Matthias Lehmann, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Matthias Meinhardt, William P. J. Leenders, Christel Herold-Mende, Graeme Eisenhofer, Simone P. Niclou, Leoni Kunz-Schughart, Achim Temme, Barbara Klink. Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-308.

Background: In the thyroid, iodine deficiency (ID) induces angiogenesis via a tightly controlled reactive oxygen species (ROS)-hypoxia inducible factor-1 (HIF-1)-vascular endothelial growth factor (VEGF) dependent pathway (ROS-HIF-VEGF). Deficient iodine intake may be associated with increased thyroid cancer incidence. The hypothesis of this work is to test whether ID affects the angiogenic processes in thyroid malignant cells by altering the ROS-HIF-VEGF pathway. Methods: Goiters were obtained in RET/PTC3 transgenic and wild-type (wt) mice and ID was induced in three thyroid carcinoma cell lines (TPC-1, 8305c, and R082-w1). Thyroid blood flow, VEGF mRNA and protein, and HIF-1α protein expression were measured. The role of HIF-1 and of ROS was assessed using echinomycin and N-acetylcysteine (NAC), respectively. Results: The goitrogen treatment increased the thyroid blood flow in wt and RET/PTC3 mice. Compared with wt mice, basal VEGF expression was higher in RET/PTC3 mice and increased with goitrogen treatment. In the three cell lines, ID induced marked increases in VEGF mRNA, and moderate increases in HIF-1α protein expression that were not transient as in normal cells. ID-induced VEGF mRNA expression was fully (8305c), partially (TPC-1), or not (R082-w1) blocked by echinomycin. NAC had no effect on ID-induced VEGF mRNA and HIF-1α protein expression in the three cell lines. Conclusions: ID induces a long lasting angiogenic phenotype in thyroid cancer cells that occurs through VEGF induction via a pathway partially mediated by HIF-1, but not by ROS. These results suggest that, in contrast with normal cells, ID-induced angiogenesis in cancer cells occurs via alternative and likely less controlled routes, thereby leading to uncontrolled growth.

The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4 Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4 Gy in both RET/PTC-positive systems but no common genes at 62.5 mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.

The link between high doses of radiation and thyroid cancer has been well established in various studies, as opposed to the effects of low doses. In this study, we investi-gated the effects of low-dose X-ray irradiation in a papillary thyroid carcinoma model with wild-type and mutated p53. A low dose of 62.5 mGy was enough to cause an upregulation of p16 and a decrease in the number of TPC-1 cells in the S phase, but not in the number of BCPAP p53-mutant cells. At a dose of 0.5 Gy, visible signs of senescence appeared only in the TPC-1 cells. We conclude that low doses of X-rays are enough to cause a change in cell cycle distribution, possibly p53-dependent p16 activation, but no significant apoptosis. Senescence requires higher doses of X-irradiation via a mechanism involving both p16 and p21.

Lower grade gliomas can be histologically classified into astrocytomas, oligoastrocytomas, or oligodendrogliomas. The latter have come into focus because of their interesting characteristics: they have a more favorable outcome and better response to chemo/radiotherapy. This is correlated with a unique unbalanced translocation der(1;19)(q10;p10) causing a codeletion of the short arm of chromosome 1 and the long arm of chromosome 19. Like the other lower grade gliomas, a large percentage of oligodendrogliomas carry the IDH1 R132 mutation and this is associated with the presence of the codeletion. We identified a high number of mutations in the remaining alleles of the Capicua homolog gene (CIC) and in the far upstream element binding protein (FUBP1) in 1p/19q codeleted oligodendrogliomas (Eisenreich et al. 2013, doi:10.1371/journal.pone.0076623). Both genes are suspected to act as tumor suppressor genes. We then analyzed the expression profiles of 13 oligodendrogliomas and oligoastrocytomas both with the 1p/19q codeletion (nine samples) and without (four samples) using a combination of expression microarrays and transcriptome sequencing (RNA-Seq). Our results showed that all nine samples with the 1p/19q codeletion carried the heterozygous IDH1 R132H mutation while only one tumor without the codeletion carried said mutation. There was a high concordance between the results of RNA-Seq and the microarray experiments and both showed a distinctive expression signature between samples with the 1p/19q codeletion and those without. Using MSigDB, we found that the genes whose expression was altered in in tumors with the 1p/19q codeletion showed significant overlap with those involved in stemness and differentiation and genes that had CpG-rich promoters. Furthermore, there was an overlap with genes with altered expression in chronic myelogenous leukemia (CML). When we compared the expression of genes located on 1p and 19q between the tumors with the 1p/19q codeletion and those without, we found that only 82% of genes on 1p and 19q were downregulated significantly. When we ran the upregulated genes in PathScan, we found enrichment in GA binding protein alpha (GABPA)binding sites. GABPA expression levels were increased in our glioma samples with and without the 1p/19q codeletion. Investigating the expression levels of GABPA in the Cancer Genome Atlas (TCGA) GBM dataset also revealed an upregulation of this transcription factor. An increase in GABPA-mediated transcription was recently reported in CML but has not been yet reported in gliomas. We therefore propose that GABPA is an important transcription factor in gliomas. We are currently evaluating the function of GABPA in glioma what will also be presented. Acknowledgements: This project was partially supported by a MeDDrive grant of the Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden. Citation Format: Barbara Klink, Karol Szafranski, Jaime Campos-Valenzuela, Sophie Eisenreich, Dietmar Krex, Eva-Maria Gerlach, Karl Hackmann, Andreas Rump, Gabriele Schackert, Matthias Platzer, Lars Kaderali, Evelin Schröck, Khalil Abou-El-Ardat. Upregulation of gene expression in oligodendrogliomas is linked to an increase in GA binding protein alpha transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2014-3387

Glioblastoma (GBM) is the most common and malignant type of brain tumor in adults with an average survival time of less than a year. A hallmark of GBM is its infiltrating growth hampering successful surgical treatment and precluding curative therapy. Multifocal GBMs - multiple synchronous lesions in the same patient - represent approximately 8-10% of all gliomas and generally have a poor prognosis. There are two theories as to the nature of these multifocal GBMs: they could either have originated from one main GBM or they can be independent tumors. Genetic studies of multifocal GBMs are very rare and such studies could elucidate the nature of these tumors. We have analyzed eleven tumor foci of six patients with multifocal GBMs using array comparative genome hybridization (aCGH), spectral karyotyping (SKY), and Sanger sequencing of PTEN, TP53, and IDH1/2. Array CGH analysis of ten of the tumors revealed multiple genetic aberrations. The most frequent were gain of chromosome 7 (9/10), amplification of EGFR (10/10), loss of chromosome 10 (10/10), and partial loss of chromosome 9 (7/10). SKY analysis identified multiple translocations including complex rearrangements. None of the tumors carried a mutation in IDH1/2 and they were genetically similar to primary GBMs although they exhibited unique characteristics; e.g. all carried an EGFR amplification, indicating that it is important for the multifocal phenotype. Tumor foci derived from the same patient always shared identical aberrations, proving monoclonal origin of these tumors but also exhibited additional aberrations, which were specific for each focus and therefore must have occurred later in tumor development. Moreover, our results show that events important for gliomagenesis could occur at different time points in tumor evolution: for example, the region involved in the EGFR amplification was different in both foci from three patients and identical in two foci from one patient. The same can be said of point mutations in TP53 and PTEN; TP53 mutations were identical in the different foci from one patient and different in another patient while PTEN mutations were different in the foci from two patients and similar in those of one. Interestingly, in each patient one focus always contained more aberrations than the other, highlighting the possibility that one derived from the other; this would further support the hypothesis that multifocal GBMs are of monoclonal origin and then develop independently of each other by clonal evolution. Taken together, multifocal GBM provide an excellent model for investigating tumor progression and invasion and might help to distinguish between driver and passenger alterations in GBM. Acknowledgements: This work was partially supported by a grant (MeDDrive program) of the Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden. Citation Format: Khalil Abou-El-Ardat, Dietmar Krex, Michael Seifert, Kerstin Becker, Morten Hillmer, Sophie Eisenreich, Arleta Käßner-Frensel, Petra Freitag, Eva-Maria Gerlach, Karl Hackmann, Andreas Rump, Gabriele Schackert, Evelin Schröck, Barbara Klink. Genetic analysis of multifocal glioblastoma multiforme foci points to their monoclonal evolution and highlights early and late events in their development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3415. doi:10.1158/1538-7445.AM2014-3415

Oligodendroglial tumors form a distinct subgroup of gliomas, characterized by a better response to treatment and prolonged overall survival. Most oligodendrogliomas and also some oligoastrocytomas are characterized by a unique and typical unbalanced translocation, der(1,19), resulting in a 1p/19q co-deletion. Candidate tumor suppressor genes targeted by these losses, CIC on 19q13.2 and FUBP1 on 1p31.1, were only recently discovered. We analyzed 17 oligodendrogliomas and oligoastrocytomas by applying a comprehensive approach consisting of RNA expression analysis, DNA sequencing of CIC, FUBP1, IDH1/2, and array CGH. We confirmed three different genetic subtypes in our samples: i) the "oligodendroglial" subtype with 1p/19q co-deletion in twelve out of 17 tumors; ii) the "astrocytic" subtype in three tumors; iii) the "other" subtype in two tumors. All twelve tumors with the 1p/19q co-deletion carried the most common IDH1 R132H mutation. In seven of these tumors, we found protein-disrupting point mutations in the remaining allele of CIC, four of which are novel. One of these tumors also had a deleterious mutation in FUBP1. Only by integrating RNA expression and array CGH data, were we able to discover an exon-spanning homozygous microdeletion within the remaining allele of CIC in an additional tumor with 1p/19q co-deletion. Therefore we propose that the mutation rate might be underestimated when looking at sequence variants alone. In conclusion, the high frequency and the spectrum of CIC mutations in our 1p/19q-codeleted tumor cohort support the hypothesis that CIC acts as a tumor suppressor in these tumors, whereas FUBP1 might play only a minor role.

Several lines of evidence have linked limb teratogenesis to radiation-induced apoptosis and to the p53 status in murine fetuses. In previous reports, we studied the occurrence of various malformations after intrauterine irradiation and showed that these malformations were modulated by p53-deficiency as well as by the developmental stage at which embryos were irradiated. In this new study, we focused onto one particular phenotype namely forelimb defects to further unravel the cellular and molecular mechanisms underlying this malformation. We measured various parameters expected to be directly or indirectly influenced by irradiation damage. The mouse fetuses were irradiated at day 12 p.c. (post conception) and examined for forelimb defects on gestational days 15, 16, 17 and 19 of development. The release of inflammatory cytokines was determined in the amniotic fluid on day 16 p.c. and the mean telomere lengths assessed at days 12, 13 and 19 p.c. Differential gene expression within the forelimb bud tissues was determined using Real Time quantitative PCR (RTqPCR) 24 h following irradiation. Apoptosis was investigated in the normal and malformed fetuses using the TUNEL assay and RTqPCR. First, we found that irradiated fetuses with forelimb defects displayed excessive apoptosis in the predigital regions. Besides, overexpression of the pro-apoptotic Bax gene indicates a mitochondrial-mediated cell death. Secondly, our results showed overexpression of MKK3 and MKK7 (members of the stress-activated MAP kinase family) within the malformed fetuses. The latter could be involved in radiation-induced apoptosis through activation of the p38 and JNK pathways. Thirdly, we found that irradiated fetuses exhibiting forelimb defects showed a marked telomere shortening. Interestingly, telomere shortening was observed as the malformations became apparent. Fourthly, we measured cytokine levels in the amniotic fluid and detected a considerable inflammatory reaction among the irradiated fetuses as evidenced by the increase in pro-inflammatory cytokine levels. Altogether, our data suggest that transcriptional modulations of apoptotic, inflammation, stress, and DNA damage players are early events in radiation-induced forelimb defects. These changes resulted in harsh developmental conditions as indicated by a marked increase in cytokine levels in the amniotic fluid and telomere shortening, two features concomitant with the onset of the forelimb defect phenotype in our study.