ATM/ATR checkpoint activation downregulates CDC25C to prevent mitotic entry with uncapped telomeres.

Shelterin component TRF2 prevents ATM activation, while POT1 represses ATR signalling at telomeres. Here, we investigate the mechanism of G2/M arrest triggered by telomeres uncapped through TRF2 or POT1 inhibition in human cells. We find that telomere damage-activated ATR and ATM phosphorylate p53, as well as CHK1 and CHK2, thus activating two independent pathways to prevent progression into mitosis with uncapped telomeres. Surprisingly, telomere damage targets the CDC25C phosphatase for proteasome degradation in G2/M. CHK1/CHK2-dependent phosphorylation of CDC25C at Ser 216 is required for CDC25C nuclear export and destruction, which in turn acts to sustain the G2/M arrest elicited by TRF2- or POT1-depleted telomeres. In addition, CDC25C is transcriptionally downregulated by p53 in response to telomere damage. These mechanisms are distinct from the canonical DNA damage response to ionizing radiation, which triggers cell-cycle arrest through CDC25A destruction. Thus, dysfunctional telomeres promote ATM/ATR-dependent degradation of CDC25C phosphatase to block mitotic entry, thereby preventing telomere dysfunction-driven genomic instability.

RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis.

Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability.

The chemoselective one-step alkylation and isolation of thiophosphorylated cdk2 substrates in the presence of native cysteine.

The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N(6) -(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.

Dose-dependent changes in cyclin D1 in response to 4-nitroquinoline 1-oxide-induced DNA damage.

Cells respond to DNA damage by either repairing the damage or committing to a death or senescence pathway, dependent on the level of damage sustained. In this study, we show that the protein levels of cyclin D1 and the CDK inhibitor, p21(CIP1), respond in a dose-dependent manner to the DNA damaging agent, 4-nitroquinoline 1-oxide (4NQO). Cyclin D1 responses were independent of p53 and resulted in a partial loss of Retinoblastoma protein phosphorylation. The differential responses of cyclin D1 and p21(CIP1) were associated with distinct cellular responses: in low dose treatments the cells recovered after a lag period whilst at medium and high doses, the cells died through seemingly distinct mechanisms. Our data suggest that the balance between cyclin D1 and p21(CIP1) following exposure to DNA damage may play a key role in determining the subsequent cellular responses.

ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

ARF is a tumour suppressor activated by oncogenic stress, which stabilizes p53. Although p53 is a key component of the response to DNA damage, a similar function for ARF has not been ascribed. Here we show that primary mouse and human cells lacking the tumour suppressor BRCA2 accumulate DNA damage, which triggers checkpoint signalling and ARF activation. Furthermore, senescence induced by Brca2 deletion in primary mouse and human cells is reversed by the loss of ARF, a phenotype recapitulated in cells lacking RAD51. Surprisingly, ARF is not necessary for p53 accumulation per se but for altering the spectrum of genes activated by this transcription factor. Specifically, ARF enables p53 transcription of Dusp4 and Dusp7, which encode a pair of phosphatases known to inactivate the MAP kinases ERK1/2. Our results ascribe a previously unanticipated function to the ARF tumour suppressor in genome integrity, controlled by replicative stress and ATM/ATR-dependent checkpoint responses.

A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.

In vitro DNA scission activity of heterometallocenes.

A comparative DNA scission activity study of azaferrocene, N-methyl-azaferrocene iodide and 3,3',4,4'-tetramethyl-1,1'-diphosphaferrocene (featuring iron in a +2 oxidation state), along with ferrocene (iron +2) and ferrocenium (iron +3) cation is described. Experiments indicate a high cleavage activity of azaferrocene and its N-methyl derivative in DMSO. DNA cleavage activity can be slowed down by addition of a free radical scavenger (thiourea) or triggered by addition of a reductive agent (dithiothreitol, DTT). The X-ray crystal structure of the N-methyl-2,5-dimethylazaferrocene cation (iron +2) with hexafluorophosphate as counter anion is also reported.

Synthesis, structure and assessment of the cytotoxic properties of 2,5-dimethylazaferrocenyl phosphonates.

The reaction of lithiated 2,5-dimethylazaferrocene 1 with diethyl chlorophosphate proceeds to give lateral and ring phosphonate products. The products 2 and 3 were characterized by spectroscopic (1H, 31P{1H} NMR, MS, IR) methods and 3 was treated with W(CO)5(thf) to form a crystalline W(CO)5-complex 4 which was characterized by single-crystal X-ray analysis. The new 2,5-dimethylazaferrocenyl phosphonates were transformed into the corresponding N-methyl iodide salts 5 and 6 in quantitative yields. Both salts are water soluble and stable compounds and an analysis of their cytotoxic and anti-proliferative activity was carried out. Compound 6 possesses anti-metabolic activity which exhibited some preference towards the cancerous HeLa cell line over the non-cancerous NIH 3T3 cell line. These new compounds are the first examples of azaferrrocene (i.e. non-ferrocene) derivatives featuring biologically important phosphonate groups. The preliminary studies into cytotoxic activity indicates that as with ferrocene, azaferrocene can also be regarded as a potential source for organometallic anticancer agents, featuring the iron centre in the +2 oxidation state rather than the often utilized ferrocenium +3 species.