Sequence-specific activation of TAK1-D by short double-stranded RNAs induces apoptosis in NCI-H460 cells.

Short double-stranded RNAs (dsRNA) are potent biological entities triggering a number of cellular effects. Most prominent among these is the post-transcriptional gene silencing of target genes by small interfering RNAs (siRNAs). In addition dsRNAs activate signal transduction processes through molecules like PKR or the Toll-like receptor important in viral defense and in explaining off target effects of siRNAs. Only a few of these dsRNA triggered pathways have been characterized yet. Here we show that the splicing variant D of the TAK1 gene is activated by short double-stranded RNAs in a sequence-specific manner. Activation of TAK1-D leads to the downstream activation of the p38 MAPK and of SAPK/JNK but not the NFkappaB pathway. In the human lung cancer cell line NCI-H460 the activation of these pathways leads to cell cycle arrest and apoptosis. Our results demonstrate that TAK1-D is activated by siRNAs of specific sequences, offering a new explanation for off target effects triggered by these molecules. In addition the dsRNA triggered activation of a cell death pathway in the human lung cancer cell line studied suggests that TAK1-D might be a new and promising therapeutic target for the treatment of nonsmall cell lung cancer.

2'-5'-Oligoadenylate synthetase is activated by a specific RNA sequence motif.

2'-5'-Oligoadenylate synthetase plays a central role in the cellular innate antiviral response. Although activation of 2'-5'-oligoadenylate synthetase by double stranded RNA was discovered more than 30 years ago it is still unclear which sequence features are required by an RNA to activate the enzyme. A pool of chemically synthesized short double stranded RNAs of specific sequence was used to probe 2'-5'-oligoadenylate synthetase activation. It was found that activating double stranded RNAs contain the following motif: NNWWNNNNNNNNNWGN. Verification of this sequence motif in a pool of 102 small double stranded RNAs demonstrated a false positive prediction rate of 8% and a false negative prediction rate of 12%. The sequence motif identified provides mechanistic insight into the mechanism of 2'-5'-oligoadenylate synthetase activation by double stranded RNA and allows theoretical predictions whether a given RNA molecule has the capability to activate 2'-5'-oligoadenylate synthetase.

Sustained metaphase arrest in response to ionizing radiation in a non-small cell lung cancer cell line.

Kodym, E., Kodym, R., Choy, H. and Saha, D. Sustained Metaphase Arrest in Response to Ionizing Radiation in a Non-small Cell Lung Cancer Cell Line. Radiat. Res. 169, 46-58 (2008). In solid tumors, non-apoptotic forms of tumor cell inactivation such as mitotic catastrophe appear to be predominant in the response to DNA-damaging agents. Despite its importance, the underlying molecular mechanisms of mitotic catastrophe have been only partially elucidated. We found that a large fraction of HCC2279 non-small cell lung cancer cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual gamma-H2AX foci. Although TP53 was nonfunctional, we detected a prompt radiation response on the level of checkpoint kinases. In contrast, CDC25A was the only checkpoint phosphatase that was responsive to radiation. CDC25B was not detectable, and CDC25C was constitutively phosphorylated at serine 216, leading to its cytoplasmic sequestration and functional inactivation. Therefore, radiation-induced mitotic catastrophe in HCC2279 cells appears to be induced by a combination of relative insufficiencies in the p53-mediated and checkpoint kinase-mediated pathways leading to premature entry into mitosis. Displaced chromosome fragments triggering an intra-M checkpoint in cells entering mitosis presumably result in a sustained metaphase arrest. The phenomenon found in these cells, which were derived directly from a human patient, might be responsible for therapy-induced genetic instability of tumors.

DNA-PKcs-dependent modulation of cellular radiosensitivity by a selective cyclooxygenase-2 inhibitor.

PURPOSE: Inhibition of cyclooxygenase-2 has been shown to increase radiosensitivity. Recently, the suppression of radiation-induced DNA-dependant protein kinase (DNA-PK) activity by the selective cyclooxygenase-2 inhibitor celecoxib was reported. Given the importance of DNA-PK for repair of radiation-induced DNA double-strand breaks by nonhomologous end-joining and the clinical use of the substance, we investigated the relevance of the DNA-PK catalytic subunit (DNA-PKcs) for the modulation of cellular radiosensitivity by celecoxib. METHODS AND MATERIALS: We used a syngeneic model of Chinese hamster ovarian cell lines: AA8, possessing a wild-type DNK-PKcs; V3, lacking a functional DNA-PKcs; and V3/WT11, V3 stably transfected with the DNA-PKcs. The cells were treated with celecoxib (50 muM) for 24 h before irradiation. The modulation of radiosensitivity was determined using the colony formation assay. RESULTS: Treatment with celecoxib increased the cellular radiosensitivity in the DNA-PKcs-deficient cell line V3 with a dose-enhancement ratio of 1.3 for a surviving fraction of 0.5. In contrast, clonogenic survival was increased in DNA-PKcs wild-type-expressing AA8 cells and in V3 cells transfected with DNA-PKcs (V3/WT11). The decrease in radiosensitivity was comparable to the radiosensitization in V3 cells, with a dose-enhancement ratio of 0.76 (AA8) and 0.80 (V3/WT11) for a survival of 0.5. CONCLUSIONS: We have demonstrated a DNA-PKcs-dependent differential modulation of cellular radiosensitivity by celecoxib. These effects might be attributed to alterations in signaling cascades downstream of DNA-PK toward cell survival. These findings offer an explanation for the poor outcomes in some recently published clinical trials.

Multiple isoforms of CDC25 oppose ATM activity to maintain cell proliferation during vertebrate development.

The early development of vertebrate embryos is characterized by rapid cell proliferation necessary to support the embryo's growth. During this period, the embryo must maintain a balance between ongoing cell proliferation and mechanisms that arrest or delay the cell cycle to repair oxidative damage and other genotoxic stresses. The ataxia-telangiectasia mutated (ATM) kinase is a critical regulator of the response to DNA damage, acting through downstream effectors, such as p53 and checkpoint kinases (CHK) to mediate cell-cycle checkpoints in the presence of DNA damage. Mice and humans with inactivating mutations in ATM are viable but have increased susceptibility to cancers. The possible role of ATM in limiting cell proliferation in early embryos has not been fully defined. One target of ATM and CHKs is the Cdc25 phosphatase, which facilitates cell-cycle progression by removing inhibitory phosphates from cyclin-dependent kinases (CDK). We have identified a zebrafish mutant, standstill, with an inactivating mutation in cdc25a. Loss of cdc25a in the zebrafish leads to accumulation of cells in late G(2) phase. We find that the novel family member cdc25d is essential for early development in the absence of cdc25a, establishing for the first time that cdc25d is active in vivo in zebrafish. Surprisingly, we find that cell-cycle progression in cdc25a mutants can be rescued by chemical or genetic inhibition of ATM. Checkpoint activation in cdc25a mutants occurs despite the absence of increased DNA damage, highlighting the role of Cdc25 proteins to balance constitutive ATM activity during early embryonic development.

Short double-stranded RNAs of specific sequence activate ribosomal TAK1-D and induce a global inhibition of translation.

We have previously shown that short double-stranded RNAs of specific sequence induce phosphorylation in the activation loop of splicing variant D of the transforming growth factor beta-activated protein kinase 1 (TAK1-D). Here, we further characterize this novel function of TAK1-D and the mechanisms of this dsRNA-triggered phenomenon. Using a dominant negative TAK1-D mutant we demonstrate that TAK1-D activation is functionally required to trigger the activation of p38 MAP kinase and c-JUN terminal kinase and to induce cell death in NCI-H460 cells. While total TAK1-D protein was found in the cytoplasm as well as in the ribosomal fraction, activated TAK1-D phosphorylated on T184 and T187 in the activation loop was found to be exclusively associated with the 80S ribosome. The association of TAK1-D with the ribosome suggests an involvement in translation-dependent signaling and we demonstrate here that dsRNA-mediated activation of TAK1-D leads to a downregulation of mRNA translation. In addition, we show that TAK1-D is also phosphorylated after the induction of ribotoxic stress. Our data indicate that TAK1-D plays a role in the signaling events triggered by selected types of ribotoxic stress.

The small-molecule CDK inhibitor, SNS-032, enhances cellular radiosensitivity in quiescent and hypoxic non-small cell lung cancer cells.

In solid tumors, including non-small cell lung carcinomas (NSCLC) the existence of radioresistant subpopulations, such as quiescent or hypoxic tumor cells, is well established, thus posing a critical therapeutic problem. Although small-molecule inhibitors targeting cyclin-dependent kinases (CDK) were demonstrated to enhance cellular radiosensitivity preferentially in proliferating tumor cells, cell cycle-independent activities of these substances were recently suggested. In this study, the potential of a newer generation small-molecule CDK inhibitor, SNS-032, to sensitize radioresistant tumor cells to ionizing radiation was tested in vitro using two NSCLC cell lines (NCI-H460 and A549). Exposure of quiescent and hypoxic lung tumor cells to SNS-032 at a clinically achievable concentration (500 nM) prior to irradiation resulted in a significant increase in cellular radiosensitivity indicating cell cycle-unrelated mechanisms. The effect of SNS-032 on non-cycling cells was not attributed to an enhanced toxicity of the drug. A SNS-032 mediated delay in the resolution of radiation-induced gammaH2AX foci a surrogate for DNA double-strand breaks was determined in non-cycling cells, suggesting a modulation of DNA double-strand break repair. These results indicate a modulation of DNA double-strand break repair to be partially attributed to the radiosensitization effects of SNS-032 observed in hypoxic and quiescent lung tumor cells. Considering the importance of therapy resistance for the radiocurability of solid tumors, our findings may provide the basis for an improvement of the well-established treatment regimens in clinical oncology.

Frequency of radiation recall dermatitis in adult cancer patients.

BACKGROUND: The re-occurrence of an inflammatory skin reaction which is limited to previously irradiated areas after the administration of a promoting agent is called radiation recall dermatitis. Despite a number of case reports no systematic analysis about the frequency of the phenomenon exists. Therefore we performed an observation study in order to obtain information about this phenomenon in clinical practice. PATIENTS AND METHODS: 142 patients who underwent palliative radiotherapy were included in the study. The skin reaction at the day of completion of radiotherapy was documented and the patients were followed until cytotoxic chemotherapy was administered or at least 6 months. RESULTS: 91 patients received different types of chemotherapy. In 8 patients a radiation recall dermatitis was observed, corresponding to a frequency of 8.8%. The radiation skin reactions occurred within 6 weeks after the completion of radiotherapy which corresponded to the time-period most of the chemotherapies were applied in. The grade of observed radiation recall dermatitis ranged from a mild erythema to a severe exfoliative dermatitis. CONCLUSION: The radiation recall dermatitis is a reaction of moderate frequency among adult cancer patients. Its possible severity as well as consecutive therapeutic consequences argue for its consideration in clinical settings.