The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent

<p>Abstract</p> <p>Background</p> <p>Cigarette smoke (CS) is the main cause in the development of chronic obstructive pulmonary disease (COPD), the pathogenesis of which is related to an extended inflammatory response. In this study, we investigated the effect of low and high doses of gas phase cigarette smoke (GPS) on cultured lymphocyte progenitor cells, using techniques to assess cell viability and to elucidate whether cells die of apoptosis or necrosis upon exposure to different doses of GPS.</p> <p>Methods</p> <p>In our approach we utilised a newly-established system of exposure of cells to GPS that is highly controlled, accurately reproducible and simulates CS dosage and kinetics that take place in the smokers' lung. This system was used to study the mode of cell death upon exposure to GPS in conjunction with a range of techniques widely used for cell death studies such as Annexin V staining, activation of caspase -3, cytoplasmic release of cytochrome C, loss of mitochondrial membrane potential and DNA fragmentation.</p> <p>Results</p> <p>Low doses of GPS induced specific apoptotic indexes in CCRF-CEM cells. Specifically, cytochrome C release and cleaved caspase-3 were detected by immunofluorescence, upon treatment with 1-3 puffs GPS. At 4 h post-exposure, caspase-3 activation was observed in western blot analysis, showing a decreasing pattern as GPS doses increased. Concomitant with this behaviour, a dose-dependent change in Δψ_mdepolarization was monitored by flow cytometry 2 h post-exposure, while at 4 h Δψ_mcollapse was observed at the higher doses, indicative of a shift to a necrotic demise. A reduction in DNA fragmentation events produced by 5 puffs GPS as compared to those provoked by 3 puffs GPS, also pointed towards a necrotic response at the higher dose of GPS.</p> <p>Conclusion</p> <p>Collectively, our results support that at low doses gas phase cigarette smoke induces apoptosis in cultured T-lymphocytes, whereas at high doses GPS leads to necrotic death, by-passing the characteristic stage of caspase-3 activation and, thus, the apoptotic route.</p

The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity.We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity.In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs

Dissociation of CAK from Core TFIIH Reveals a Functional Link between XP-G/CS and the TFIIH Disassembly State

Transcription factor II H (TFIIH) is comprised of core TFIIH and Cdk-activating kinase (CAK) complexes. Here, we investigated the molecular and cellular manifestation of the TFIIH compositional changes by XPG truncation mutations. We showed that both core TFIIH and CAK are rapidly recruited to damage sites in repair-proficient cells. Chromatin immunoprecipitation against TFIIH and CAK components revealed a physical engagement of CAK in nucleotide excision repair (NER). While XPD recruitment to DNA damage was normal, CAK was not recruited in severe XP-G and XP-G/CS cells, indicating that the associations of CAK and XPD to core TFIIH are differentially affected. A CAK inhibition approach showed that CAK activity is not required for assembling pre-incision machinery in vivo or for removing genomic photolesions. Instead, CAK is involved in Ser5-phosphorylation and UV-induced degradation of RNA polymerase II. The CAK inhibition impaired transcription from undamaged and UV-damaged reporter, and partially decreased transcription of p53-dependent genes. The overall results demonstrated that a) XP-G/CS mutations affect the disassembly state of TFIIH resulting in the dissociation of CAK, but not XPD from core TFIIH, and b) CAK activity is not essential for global genomic repair but involved in general transcription and damage-induced RNA polymerase II degradation

Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries.

It is widely believed that translation occurs only in the cytoplasm of eukaryotes, but recent results suggest some takes place in nuclei, coupled to transcription. Support for this heterodoxy comes from studies of the nonsense-mediated decay (NMD) pathway; this pathway probably uses ribosomes to proofread messenger RNAs. We find components of the machineries involved in transcription, translation and NMD colocalise, interact and copurify, and that interactions between them are probably mediated by the C-terminal domain of the catalytic subunit of RNA polymerase II. These results are simply explained if the NMD machinery uses nuclear ribosomes to translate - and so proofread - newly made transcripts; then, faulty transcripts and any truncated peptides produced by nuclear translation would be degraded

Influence of irofulven, a transcription-coupled repair-specific antitumor agent, on RNA polymerase activity, stability and dynamics in living mammalian cells.

Transcription-coupled repair (TCR) plays a key role in the repair of DNA lesions induced by bulky adducts and is initiated when the elongating RNA polymerase II (Pol II) stalls at DNA lesions. This is accompanied by alterations in Pol II activity and stability. We have previously shown that the monofunctional adducts formed by irofulven (6-hydroxymethylacylfulvene) are exclusively recognized by TCR, without involvement of global genome repair (GGR), making irofulven a unique tool to characterize TCR-associated processes in vivo. Here, we characterize the influence of irofulven on Pol II activity, stability and mobility in living mammalian cells. Our results demonstrate that irofulven induces specific inhibition of nucleoplasmic RNA synthesis, an important decrease of Pol II mobility, coupled to the accumulation of initiating polymerase and a time-dependent loss of the engaged enzyme, associated with its polyubiquitylation. Both proteasome-mediated degradation of the stalled polymerase and new protein synthesis are necessary to allow Pol II recycling into preinitiating complexes. Together, our findings provide novel insights into the subsequent fate of the stalled RNA polymerase II and demonstrate the essential role of the recycling process for transcriptional reinitiation and viability of mammalian cells

Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells

CK2 denotes a ubiquitous and pleiotropic protein kinase whose holoenzyme is composed of two catalytic (α and/or α') and two regulatory β subunits. The CK2 consensus sequence, S/T-x-x-D/E/pS/pT is present in numerous phosphosites, but it is not clear how many of these are really generated by CK2. To gain information about this issue, advantage has been taken of C2C12 cells entirely deprived of both CK2 catalytic subunits by the CRISPR/Cas9 methodology. A comparative SILAC phosphoproteomics analysis reveals that, although about 30% of the quantified phosphosites do conform to the CK2 consensus, only one-third of these are substantially reduced in the CK2α/α'(-/-) cells, consistent with their generation by CK2. A parallel study with C2C12 cells deprived of the regulatory β subunit discloses a role of this subunit in determining CK2 targeting. We also find that phosphosites notoriously generated by CK2 are not fully abrogated in CK2α/α'(-/-) cells, while some phosphosites unrelated to CK2 are significantly altered. Collectively taken our data allow to conclude that the phosphoproteome generated by CK2 is not as ample and rigidly pre-determined as it was believed before. They also show that the lack of CK2 promotes phosphoproteomics perturbations attributable to kinases other than CK2

Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining

DNA double-strand break (DSB) repair occurs within chromatin and can be modulated by chromatin modifying enzymes. Here we identify the related human histone deacetylases HDAC1 and HDAC2 as two participants in the DNA-damage response (DDR). We show that acetylation of histone H3 lysine 56 (H3K56) is regulated by HDAC1/2, and that HDAC1/2 are rapidly recruited to DNA-damage sites to promote H3K56 hypo-acetylation. Furthermore, we establish that HDAC1/2-depleted cells are hypersensitive to DNA-damaging agents and exhibit sustained DNA-damage signaling, phenotypes that reflect defective DSB repair, particularly by the pathway of non-homologous end-joining (NHEJ). Collectively, these results demonstrate that HDAC1 and HDAC2 function in the DDR by promoting DSB repair and thus provide important insights into the radio-sensitizing effects of HDAC inhibitors that are being developed as cancer therapies