Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.

Feedback regulation of Gab2-dependent signaling by the Ras/MAPK pathway

La voie de signalisation des Récepteurs Tyrosine Kinase (RTK) occupe un rôle central dans la régulation de la croissance cellulaire, la prolifération, la différentiation et la motilité. Une régulation anormale des RTKs mène à plusieurs maladies humaines telles que le cancer du sein, la seconde cause de mortalité chez les femmes à cause de l’amplification et la mutation fréquente de la protéine tyrosine kinase HER2 (ERBB2). Grb2-associated binder (Gab) 2 est une protéine adaptatrice qui agit en aval de plusieurs RTKs, y compris HER2, pour réguler de multiples voies de signalisation. En réponse à la stimulation par de nombreux facteurs de croissances et cytokines, Gab2 est recruté à la membrane plasmique où il potentialise l’activation des voies de signalisation Ras/mitogen-activated protein kinase (MAPK) et PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B). En plus d’occuper un rôle essentiel durant le développement du système hématopoïétique, Gab2 est souvent amplifié dans les cancers, notamment le cancer du sein et les mélanomes. Cependant, les mécanismes moléculaires qui régulent le fonctionnement de Gab2 sont peu connus. Il est établi que lors de l’activation des RTKs, Gab2 est phosphorylé au niveau de plusieurs résidus Tyrosine, menant à l’association de différentes protéines comme p85 et Shp2. En plus de la phosphorylation en Tyrosine, notre laboratoire ainsi que d’autres groupes de recherche avons identifié que Gab2 est aussi phosphorylé au niveau de résidus Ser/Thr suite à l’activation de la voie de signalisation MAPK. Cependant, la régulation des fonctions de Gab2 par ces modifications post-traductionnelles est encore peu connue. Dans le but de comprendre comment Gab2 est régulé par la voie de signalisation MAPK, nous avons utilisé différentes approches. Dans la première partie de ma thèse, nous avons déterminé un nouveau mécanisme démontrant que la voie de signalisation Ras/MAPK, par le biais des protéines kinases RSK (p90 ribosomal S6 kinase), phosphoryle Gab2. Ce phénomène se produit à la fois in vivo et in vitro au niveau de trois résidus Ser/Thr conservés. Des mutations au niveau de ces sites de phosphorylation entrainent le recrutement de Shp2 menant à l’augmentation de la motilité cellulaire, ce qui suggère que les protéines RSK restreignent les fonctions dépendantes de Gab2. Ce phénomène est le résultat de la participation de RSK dans la boucle de rétroaction négative de la voie de signalisation MAPK. Dans la seconde partie de ma thèse, nous avons démontré que les protéines ERK1/2 phosphorylent Gab2 au niveau de plusieurs résidus pS/T-P à la fois in vitro et in vivo, entrainant l’inhibition du recrutement de p85. De plus, nous avons établi pour la première fois que Gab2 interagit physiquement avec ERK1/2 dans des cellules lors de l’activation de la voie de signalisation MAPK. Par ailleurs, nous avons montré un nouveau domaine d’attache du module ERK1/2 sur Gab2. Des mutations sur les résidus essentiels de ce domaine d’attache n’entrainent pas seulement la dissociation de ERK1/2 avec Gab2, mais diminuent également la phosphorylation dépendante de ERK1/2 sur Gab2. Ainsi, nos données montrent que la voie de signalisation MAPK régule les fonctions de la protéine Gab2 par le biais des kinases RSK et ERK1/2. Cette thèse élabore par ailleurs un schéma complet des fonctions de Gab2 dépendantes de la voie de signalisation MAPK dans le développement de nombreux cancers.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41Δ, sap190Δ, ppm1Δ, rrd1Δ) and U34 modification defects (elp3Δ, kti12Δ, urm1Δ, ncs2Δ) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3Δ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3Δ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3Δ, urm1Δ) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TORsensitive NCR branch via Gln3 misregulation.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Deregulated ephrin-B2 signaling in mammary epithelial cells alters the stem cell compartment and interferes with the epithelial differentiation pathway

Cancer most probably originates from stem/progenitor cells and exhibits a similar cell hierarchy as normal tissues. Moreover, there is growing evidence that only the stem cells are capable of metastasis formation. We have previously shown that overexpression of a dominant negative ephrin-B2 mutant interferes with mammary gland differentiation and confers a metastatic phenotype to NeuT-induced mammary tumors with an increase in cells with stem/progenitor characteristics. To investigate the role of ephrin-B2 in the control of the mammary stem cell niche, we analyzed the mammary stem and progenitor cell populations in transgenic mice overexpressing the mutant ephrin-B2. Quantification by FACS analysis revealed a significant increase of cells in the basal/alveolar cell-, the bi-potent progenitor- and the stem cell-enriched fractions. Moreover, the supposed precursors of estrogen receptor-positive cells were elevated in the stem cell-enriched fraction. In contrast, the epithelium from transgenic mice overexpressing the native ephrin-B2 gene showed an augmentation of the luminal cell- and the bi-potent progenitor-enriched fractions. Repopulation assays revealed that the epithelial cells of truncated ephrin-B2 transgenic epithelial cells have a higher regeneration capacity than those of controls and of native ephrin-B2 transgenic mice, confirming the augmentation of stem cells. Morphologically, these outgrowths exhibited impaired basal/luminal compartmentalization and epithelial polarization. These results demonstrate that deregulated ephrin-B2 expression interferes with the regulation of the stem cell niche and leads to a shift of the differentiation pathway and may thereby contribute to the acquisition of the metastatic phenotype long before carcinogenic growth becomes apparent.

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.

Reverse genetic analysis of Caenorhabditis elegans presenilins reveals redundant but unequal roles for sel-12 and hop-1 in Notch-pathway signaling

Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer’s disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, we used a reverse genetic strategy to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions displayed new phenotypes not observed in animals bearing either single deletion. These new phenotypes—germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects—resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling

Objective— Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. Methods and Results— sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. Conclusions— These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN

The tumor suppressor PTEN antagonizes phosphatidylinositol 3-kinase (PI3K), which contributes to tumorigenesis in many cancer types. While PTEN mutations occur in some melanomas, their precise mechanistic consequences have yet to be elucidated. We sought to identify novel downstream effectors of PI3K using a combination of genomic and functional tests. Microarray analysis of 53 melanoma cell lines identified 610 genes differentially expressed (P<0.05) between wild-type lines and those with PTEN aberrations. Many of these genes are known to be involved in the PI3K pathway and other signaling pathways influenced by PTEN. Validation of differential gene expression by qRT-PCR was performed in the original 53 cell lines and an independent set of 18 melanoma lines with known PTEN status. Osteopontin (OPN), a secreted glycophosphoprotein that contributes to tumor progression, was more abundant at both the mRNA and protein level in PTEN mutants. The inverse correlation between OPN and PTEN expression was validated (P<0.02) by immunohistochemistry using melanoma tissue microarrays. Finally, treatment of cell lines with the PI3K inhibitor LY294002 caused a reduction in expression of OPN. These data indicate that OPN acts downstream of PI3K in melanoma and provides insight into how PTEN loss contributes to melanoma development.

Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans

Skeletal muscle from strength- and endurance-trained individuals represents diverse adaptive states. In this regard, AMPK-PGC-1α signaling mediates several adaptations to endurance training, while up-regulation of the Akt-TSC2-mTOR pathway may underlie increased protein synthesis after resistance exercise. We determined the effect of prior training history on signaling responses in seven strength-trained and six endurance-trained males who undertook 1 h cycling at 70% VO2peak or eight sets of five maximal repetitions of isokinetic leg extensions. Muscle biopsies were taken at rest, immediately and 3 h postexercise. AMPK phosphorylation increased after cycling in strength-trained (54%; P<0.05) but not endurance-trained subjects. Conversely, AMPK was elevated after resistance exercise in endurance- (114%; P<0.05), but not strengthtrained subjects. Akt phosphorylation increased in endurance- (50%; P<0.05), but not strengthtrained subjects after cycling but was unchanged in either group after resistance exercise. TSC2 phosphorylation was decreased (47%; P<0.05) in endurance-trained subjects following resistance exercise, but cycling had little effect on the phosphorylation state of this protein in either group. p70S6K phosphorylation increased in endurance- (118%; P<0.05), but not strength-trained subjects after resistance exercise, but was similar to rest in both groups after cycling. Similarly, phosphorylation of S6 protein, a substrate for p70 S6K, was increased immediately following resistance exercise in endurance- (129%; P<0.05), but not strength-trained subjects. In conclusion, a degree of “response plasticity” is conserved at opposite ends of the endurancehypertrophic adaptation continuum. Moreover, prior training attenuates the exercise specific signaling responses involved in single mode adaptations to training.

Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.