The variant histone H2AX is phosphorylated in response to UV irradiation of primary human fibroblasts in a complex fashion that is radically different from that commonly reported after DNA double-strand breaks. H2AX phosphorylation after exposure to ionizing radiation produces foci, which are detectable by immunofluorescence microscopy and have been adopted as clear and consistent quantitative markers for DNA double-strand breaks. Here we show that in contrast to ionizing radiation, UV irradiation mainly induces H2AX phosphorylation as a diffuse, even, pannuclear staining. UV induced pan-nuclear phosphorylation of H2AX is present in all phases of the cell cycle and is highest in S phase. H2AX phosphorylation in G1 cells depends on nucleotide excision repair factors that may expose the S-139 site to kinase activity, is not due to DNA double-strand breaks, and plays a larger role in UV-induced signal transduction than previously realized.ultraviolet light ͉ xeroderma pigmentosum ͉ chromatin structure ͉ Wortmannin ͉ human fibroblasts H 2AX phosphorylation plays a major role in nuclear events during meiosis and DNA double-strand breaks (DSBs). DSBs cause phosphorylation of histone H2AX on large areas of chromatin flanking the breaks (1). Phosphorylation, coupled with the acetylation-dependent condensation of chromatin, permits microscopic visualization of discrete nuclear foci (2). Phosphorylated H2AX (␥H2AX) foci formation is a powerful tool used to study DSB formation and repair after genomic damage, chromosome dynamics, and signaling mechanisms (3-7). Analysis of knockout mice and cell lines implicate H2AX phosphorylation in genomic stability, tumor suppression, and spermatogenesis (8, 9).Although H2AX phosphorylation in response to ionizing radiation and other radiomimetic agents garners a great deal of attention, its role in the UV-induced DNA damage response is not well characterized. Reports have limited ␥H2AX formation to S phase after exposure to UV-B (10, 11). We have reported previously coincidence of ␥H2AX, MRE11, and PCNA in foci that represent replication fork breakage in the S phase of SV40-transformed cells (12,13). In contrast, a recent report finds that ␥H2AX is present in HeLa and HL60 cells at 1 h after UV-B irradiation in all phases of the cell cycle (14). These studies speculate that H2AX phosphorylation after UV irradiation is induced only by processing of UV-induced lesions into DNA DSBs, but we report a more complex response.Historically, direct DNA breakage from UV-C has been undetectable in mammalian cells (15). Calculation of the maximum frequency of DNA breaks during excision repair after UV light, from these experiments, yields an estimate of 3,000-4,000 single strand breaks per diploid cell associated with nucleotide excision repair (NER) at a maximum rate within 1 h of 10-20 J͞m 2 with a separation of Ͼ10 9 Da (4 ϫ 10 6 nt) between excision sites (16-18). The number of photoproducts per cell under these conditions is Ϸ2 ϫ 10 6 , and repair operates on only Ϸ0.1% of the initial photopr...
Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/ mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR 1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR 1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR 1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR 1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.O ncogenic mutations in codons 12, 13, or 61 of the rat sarcoma (RAS) family of small GTPases, Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma RAS viral oncogene homolog (NRAS) occur in approximately one-third of all human cancers with NRAS mutations found in about 15-20% of melanomas (1-7). Mutated RAS proteins activate signaling pathways that promote the cell division cycle and cell growth and suppress apoptosis. Small interfering RNA (siRNA)-mediated depletion of NRAS in melanoma cell lines inhibits proliferation and renders cells sensitive to chemotherapy, making mutant NRAS and its signaling effectors relevant targets for melanoma therapy (8, 9). Efforts at developing therapeutics that inhibit mutant RAS directly have so far not been successful. The high affinity of RAS for GTP and the high concentrations of GTP intracellularly has meant that the identification of small molecules, which selectively prevent accumulation of RAS-GTP, has not been possible (10). Targeting mutant NRAS with siRNA is still limited to preclinical models because of the significant challenge in delivering antisense oligonucleotides in vivo. The response of NRAS mutant melanoma and other melanomas to various chemotherapeutic regiments has been very scarce with only 6% of patients responding (11). Alternatively, farnesyltransferase inhibitors (FTIs) were thought to inhibit RAS activation by blocking farnesy...
UV irradiation induces histone variant H2AX phosphorylated on serine 139 (γH2AX) foci and high levels of pan-nuclear γH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of γH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of γH2AX response: a minority of γH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of γH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear γH2AX without foci of either γH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear γH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear γH2AX were further increased by loss of the bypass polymerase Pol η and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damagebinding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced γH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear γH2AX, a preapoptotic signal associated with ATM-and JNK-dependent apoptosis during replication. The formation of γH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.T o maintain the integrity of genomic information under threat of DNA damage, cells employ a range of responses including DNA repair, replication bypass, recombination, cell-cycle checkpoints, and senescence or apoptosis (1, 2). These responses are described by the all-embracing concept of the DNA damage response (DDR) (1). DNA double-strand breaks (DSBs) are important but not the only starting lesion for the DDR.In eukaryotic cells, the DDR is rapidly initiated by the PI3K kinases ataxia-telangiectasia and Rad3-related (ATR) and ataxiatelangiectasia mutated kinase (ATM) (3, 4). ATR is recruited to single-stranded DNA regions at stalled replication forks in response to replication stress (5-8); ATM is activated primarily in response to DSBs (3, 9-11). ATR and ATM phosphorylate serine threonine protein kinase and CHK2, respectively, which activate cell-cycle checkpoints and p53, leading to a p53-dependent apoptotic pathway (12). This kinase cascade phosphorylates serine 139 and tyrosine 142 of the histone variant H2AX. After ionizing radiation accumulates in foci, phosphorylated H2AX (γH2AX) serine threonine protein kinase colocalizes with other markers of DSBs (53BP1, pNBS1, MDC1, and Brca1) in immunofluorescent microscopy (IF) (13-16). γH2AX foci are frequently adopted as quantitative markers for DSBs in IF (17, 18). However, the exact correspondence between γH2AX and D...
The phosphorylation of H2Ax on its S139 site, γH2Ax, is important during DNA double-strand repair and is considered necessary for assembly of repair complexes, but its functional role after other kinds of DNA damage is less clear. We have measured the survival of isogenic mouse cell lines with the H2Ax gene knocked out, and replaced with wild-type or mutant (S139A) H2Ax genes, exposed to a range of agents with varied mechanisms of DNA damage. Knockout and mutant cells were sensitive to γ-rays, etoposide, temozolamide, and endogenously generated reactive oxygen species, each of which can include double-strand breaks among their spectra of DNA lesions. The absence or mutation of H2Ax had no influence on sensitivity to cisplatin or mitomycin C. Although UV light induced the highest levels of γH2Ax, mutation of S139 had no influence on UV sensitivity or the UV DNA damage response. Complete loss of H2Ax reduced the survival of cells exposed to UV light and reduced pChk1 induction, suggesting that sites other than S139 may impact the ATR-pChk1 pathway. The relative intensity of γH2Ax measured in Western blots in wild-type cells did not correlate with the functional importance of γH2Ax. The use of γH2Ax as a general biomarker of DNA damage is therefore potentially misleading because it is not an unambiguous indicator of double-strand breaks, and a significant fraction of DNA repair, especially involving nucleotide excision or crosslink repair, can occur without functional involvement of γH2Ax.alkylation | rotenone | caffeine | poly(ADP-ribose) polymerase | veliparib
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