Amanda K. Ashley

Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.

More forks on the road to replication stress recovery

High-fidelity replication of DNA, and its accurate segregation to daughter cells, is critical for maintaining genome stability and suppressing cancer. DNA replication forks are stalled by many DNA lesions, activating checkpoint proteins that stabilize stalled forks. Stalled forks may eventually collapse, producing a broken DNA end. Fork restart is typically mediated by proteins initially identified by their roles in homologous recombination repair of DNA double-strand breaks (DSBs). In recent years, several proteins involved in DSB repair by non-homologous end joining (NHEJ) have been implicated in the replication stress response, including DNA-PKcs, Ku, DNA Ligase IV-XRCC4, Artemis, XLF and Metnase. It is currently unclear whether NHEJ proteins are involved in the replication stress response through indirect (signaling) roles, and/or direct roles involving DNA end joining. Additional complexity in the replication stress response centers around RPA, which undergoes significant post-translational modification after stress, and RAD52, a conserved HR protein whose role in DSB repair may have shifted to another protein in higher eukaryotes, such as BRCA2, but retained its role in fork restart. Most cancer therapeutic strategies create DNA replication stress. Thus, it is imperative to gain a better understanding of replication stress response proteins and pathways to improve cancer therapy.

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.

Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes.

DJ-1 mutation induces early-onset Parkinsons disease, and conversely over-expression of DJ-1 is associated with cancer in numerous tissues. A gene-trap screening library conducted in embryonic stem cells was utilized for generation of a DJ-1 mutant mouse. Real-time PCR and immunoblotting were utilized to confirm functional mutation of the DJ-1 gene. Normal DJ-1 protein expression in adult mouse tissue was characterized and demonstrates high expression in brain tissue with wide systemic distribution. Primary astrocytes isolated from DJ-1(-/-) mice reveal a decreased nuclear localization of DJ-1 protein in response to rotenone or LPS, with a concomitant increase in mitochondrial localization of DJ-1 found only in the rotenone exposure. Resting mitochondrial membrane potential was significantly lower in DJ-1(-/-) astrocytes, as compared to controls. Our DJ-1 knockout mouse provides an exciting tool for exploring the molecular and physiological roles of DJ-1 to further explicate its functions in neurodegeneration.

Activation of the CXCL12/CXCR4 signaling axis may drive vascularization of the ovine placenta

Early pregnancy, when most embryonic losses occur, is a critical period in which vital placental vascularization is established. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, and factors that regulate VEGF function, expression, or both may ultimately affect vascularization. Activation of the C-X-C chemokine receptor type 4 (CXCR4) by its cognate ligand, C-X-C chemokine ligand 12 (CXCL12), increases VEGF synthesis and secretion, which in turn stimulates CXCL12 and CXCR4 production and this synergistic regulation may influence placental vascularization. We hypothesized that expression of CXCL12, CXCR4, select angiogenic factors, and their receptors would increase in placental tissues during early pregnancy and that treatment of ovine trophectoderm cells with CXCL12 would increase production of angiogenic factors. To test this hypothesis, maternal caruncle (CAR) and fetal extraembryonic membrane (FM) tissues were collected on days 18, 20, 22, 25, 26, and 30 of pregnancy and on day 10 of the estrous cycle (control, NP) to determine relative mRNA or protein expression of CXCL12 and CXCR4 and selected angiogenic factors. In CAR, expression of mRNA for CXCR4 increased on day 18, 20, 22, and 25 and CXCL12 increased on day 18 and 20 compared with NP ewes. CXCL12 protein followed a similar pattern in CAR tissue, with greater levels on day 20 than in NP tissue. Greater levels of fibroblast growth factor 2 (FGF2) mRNA was observed in CAR on day 20 of gestation than on day 30. In FM, CXCL12, CXCR4, angiopoietin 1, VEGF, and VEGF receptor 1 were enhanced with advancing pregnancy, whereas FGF2 and kinase insert domain receptor (or VEGF receptor 2) peaked on day 25. An increase in protein levels occurred on day 25 compared with day 20 in FM for CXCL12 and CXCR4, as well as a similar tendency for FGF2 protein. Both CXCL12 and CXCR4 are specifically localized to trophoblast cells and to the uterine luminal and glandular epithelium. Treatment of ovine trophectoderm cells with CXCL12 increased mRNA expression for VEGF and FGF2. The relationship between VEGF, FGF2, and the CXCL12/CXCR4 signaling underscores the potential role for this chemokine axis in driving placentation.

Human chorionic gonadotropin increases serum progesterone, number of corpora lutea and angiogenic factors in pregnant sheep

Early gestation is a critical period when implantation and placental vascularization are established, processes influenced by progesterone (P4). Although human chorionic gonadotropin (hCG) is not endogenously synthesized by livestock, it binds the LH receptor, stimulating P4 synthesis. We hypothesized treating pregnant ewes with hCG would increase serum P4, number of corpora lutea (CLs) and concepti, augment steroidogenic enzymes, and increase membrane P4 receptors (PAQRs) and angiogenic factors in reproductive tissues. The objective was to determine molecular alterations induced by hCG in pregnant sheep that may promote pregnancy. Ewes received either 600 IU of hCG or saline i.m. on day 4 post mating. Blood samples were collected daily from day 0 until tissue collection for serum P4 analysis. Reproductive tissues were collected on either day 13 or 25 of gestation and analyzed for PAQRs, CXCR4, proangiogenic factors and steroidogenic enzymes. Ewes receiving hCG had more CL and greater serum P4, which remained elevated. On day 25, StAR protein production decreased in CL from hCG-treated ewes while HSD3B1 was unchanged; further, expression of CXCR4 significantly increased and KDR tended to increase. PAQR7 and CXCR4 protein was increased in caruncle tissue from hCG-treated ewes. Maternal hCG exposure influenced fetal extraembryonic tissues, as VEGFA, VEGFB, FLT1, and ANGPT1 expression increased. Our results indicate hCG increases serum P4 due to augmented CL number per ewe. hCG treatment resulted in greater PAQR7 and CXCR4 in maternal endometrium and promoted expression of proangiogenic factors in fetal extraembryonic membranes. Supplementing livestock with hCG may boost P4 levels and improve reproductive efficiency.

Progestin-mediated activation of MAPK and AKT in nuclear progesterone receptor negative breast epithelial cells: The role of membrane progesterone receptors

Progesterone (P4), a steroid produced during estrous cycles and gestation for maintenance of pregnancy, also plays key roles in breast development to allow lactation post-parturition. Progestins (P4 and related steroids) are also implicated in breast cancer etiology. Hormone replacement therapy containing both estrogen and progestins increases breast cancer incidence while estrogen hormone therapy lowers breast cancer risk. P4 signaling via nuclear P4 receptors (PRs) has been extensively studied in breast cancer, however, progestin signaling via non-classical membrane bound progestin receptors (MPRs and PGRMC1) remains unclear. Moreover, P4 metabolites and synthetic progestins may bind membrane progestin receptors. We hypothesized that PR-negative breast epithelial cells express non-classical progestin receptors, which activate intracellular signaling pathways differently depending on nature of progestin. Therefore, our objectives for the current study were to determine expression of MPRs and PGRMC1 in two PR-negative non-tumorigenic breast epithelial cell lines, assess progestin-mediated signaling and biological functions. We determined five MPR isoforms and PGRMC1 were present in MCF10A cells and all progestin receptors but MPRβ in MCF12A cells. MCF10A and MCF12A cells were treated with P4, select P4 metabolites (5αP and 3αHP), medroxyprogesterone acetate (MPA), or a specific MPR-Agonist (MPR-Ag) and phosphorylation of ERK, p38, JNK, and AKT was characterized following treatment. To our knowledge this is the first report of ERK and JNK activation in MCF10A and MCF12A cells with P4, P4 metabolites, MPA, and MPR-Ag. Activation of ERK and JNK in cells treated with MPR-Ag implicates MPRs may serve as the receptors responsible for their activation. In contrast, p38 activation varied with cell type and with progestin treatment. P4 and MPA promoted AKT phosphorylation in the MCF12A cell line only whereas no activation was observed in MCF10A cells. Interestingly, cellular proliferation increased in MCF10A cells treated with MPA or 5αP, while MPR-Ag tended to slightly decrease proliferation. Collectively, our data highlights the importance of investigating the effects of synthetic progestins in breast cancer biology. Our results add to the understanding that various progestins have on breast epithelial cells and underscores the importance of considering both membrane bound receptors and progestin type in breast cancer development.

Proteomic analysis of diaminochlorotriazine (DACT) adducts in three brain regions of Wistar Rats

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is detected frequently in drinking water at significant levels. Following oral exposure, metabolism of ATRA generates diaminochlorotriazine (DACT), an electrophilic molecule capable of forming covalent protein adducts. At high doses, both ATRA and DACT can disrupt the preovulatory luteinizing hormone (LH) surge in rats, thereby altering normal reproductive function. This research was designed to identify DACT protein adducts formed in three distinct brain regions of ATRA-exposed rats, including the preoptic area (POA), medial basal hypothalamus (MBH), and cortex (CTX). Proteins with DACT adducts were identified following 2-dimensional electrophoresis (2-DE), immunodetection, and MALDI-TOF mass spectrometry analysis. Western blots from exposed animals revealed over 30 DACT-modified spots that were absent in controls. Protein spots were matched to concurrently run 2-DE gels stained with Sypro Ruby, excised, and in-gel digested with trypsin.

Quantitative analysis of liver GST-P foci promoted by a chemical mixture of hexachlorobenzene and PCB 126: implication of size-dependent cellular growth kinetics

The objectives of this study were twofold: (1) evaluating the carcinogenic potential of the mixture of two persistent environmental pollutants, hexachlorobenzene (HCB) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), in an initiation-promotion bioassay involving the development of π glutathione S-transferase (GST-P) liver foci, and (2) analyzing the GST-P foci data using a biologically-based computer model (i.e., clonal growth model) with an emphasis on the effect of focal size on the growth kinetics of initiated cells. The 8-week bioassay involved a series of treatments of initiator, two-thirds partial hepatectomy, and daily oral gavage of the mixture of two doses in male F344 rats. The mixture treatment significantly increased liver GST-P foci development, indicating carcinogenic potential of this mixture. Our clonal growth model was developed to simulate the appearance and development of initiated GST-P cells in the liver over time. In the model, the initiated cells were partitioned into two subpopulations with the same division rate but different death rates. Each subpopulation was further categorized into single cells, mini- (2–11 cells), medium- (12–399 cells), and large-foci (>399 cells) with different growth kinetics. Our modeling suggested that the growth of GST-P foci is size-dependent; in general, the larger the foci, the higher the rate constants of division and death. In addition, the modeling implied that the two doses promoted foci development in different manners even though the experimental foci data appeared to be similar between the two doses. This study further illustrated how clonal growth modeling may facilitate our understanding in chemical carcinogenic process.

DJ-1 mutation decreases astroglial release of inflammatory mediators

Mutations in DJ-1, reactive gliosis and concomitant inflammatory processes are implicated in the pathogenesis and progression of Parkinsons disease (PD). To study the physiological consequences of DJ-1 mutation in the context of neuroinflammatory insult, primary cortical astrocytes were isolated from DJ-1 knockout mice. Astrocytes were exposed to 1μg/mL lipopolysaccharide (LPS) for 24h following 2h pre-exposure to inhibitors of MEK (U0126), JNK (JNK inhibitor II) or p38 (SB203580). Real-time PCR was used to assess the LPS-induced expression of pro-inflammatory mediators cyclooxygenase 2 (COX2), inducible nitric oxide synthetase (NOS2), and tumor necrosis factor α (TNFα). LPS-induced expression of COX2 decreased similarly in DJ-1(+/+) and DJ-1(-/-) astrocytes in response to inhibition of p38, but was unaffected by inhibition of MEK or JNK. No significant alterations in NOS2 expression were observed in any inhibitor-treated cells. The inhibitors did not affect expression of TNFα; however, DJ-1(-/-) astrocytes had consistently lower expression compared to DJ-1(+/+) counterparts. Secretion of TNFα and prostaglandin E2 (PGE2) into the culture medium was significantly decreased in DJ-1(-/-) astrocytes, and inhibition of p38 decreased this secretion in both genotypes. In conclusion, DJ-1(-/-) astrocytes may provide decreased neuroprotection to surrounding neurons due to alterations in pro-inflammatory mediator expression.