Department of Pediatrics Works

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Absence of Respiratory Burst in X-linked Chronic Granulomatous Disease Mice Leads to Abnormalities in Both Host Defense and Inflammatory Response to Aspergillus fumigatus
(Rockefeller University Press, 1997) Morgenstern, David E.; Gifford, Mary A. C.; Li, Ling Lin; Doerschuk, Claire M.; Dinauer, Mary C.; Pediatrics, School of Medicine
Mice with X-linked chronic granulomatous disease (CGD) generated by targeted disruption of the gp91phox subunit of the NADPH-oxidase complex (X-CGD mice) were examined for their response to respiratory challenge with Aspergillus fumigatus. This opportunistic fungal pathogen causes infection in CGD patients due to the deficient generation of neutrophil respiratory burst oxidants important for damaging A. fumigatus hyphae. Alveolar macrophages from X-CGD mice were found to kill A. fumigatus conidia in vitro as effectively as alveolar macrophages from wild-type mice. Pulmonary disease in X-CGD mice was observed after administration of doses ranging from 10(5) to 48 spores, none of which produced disease in wild-type mice. Higher doses produced a rapidly fatal bronchopneumonia in X-CGD mice, whereas progression of disease was slower at lower doses, with development of chronic inflammatory lesions. Marked differences were also observed in the response of X-CGD mice to the administration of sterilized Aspergillus hyphae into the lung. Within 24 hours of administration, X-CGD mice had significantly higher numbers of alveolar neutrophils and increased expression of the proinflammatory cytokines IL-1 beta and TNF-alpha relative to the responses seen in wild-type mice. By one week after administration, pulmonary inflammation was resolving in wild-type mice, whereas X-CGD mice developed chronic granulomatous lesions that persisted for at least six weeks. This is the first experimental evidence that chronic inflammation in CGD does not always result from persistent infection, and suggests that the clinical manifestations of this disorder reflect both impaired microbial killing as well as other abnormalities in the inflammatory response in the absence of a respiratory burst.
The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity
(1997-10) Sandigursky, Margarita; Yacoub, Adly; Kelley, Mark R.; Xu, Yi; Franklin, William A.; Deutsch, Walter A.
The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine). Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3. Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5′ apurinic/apyrimidinic (AP) sites via a β-elimination reaction. Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3′ incised AP site by a Mg2+-dependent hydrolytic mechanism. In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities. A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5′ AP sites. Activity was also detected for the Mg2+-dependent removal of trans-4-hydroxy-2-pentenal-5-phosphate at 3′ incised AP sites and from intact AP sites. Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair. However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans-4-hydroxy-2-pentenal-5-phosphate left at a 3′ terminus created by a β-elimination catalyst.
Nf1 Regulates Hematopoietic Progenitor Cell Growth and Ras Signaling in Response to Multiple Cytokines
(Rockefeller University Press, 1998) Zhang, You-Yan; Vik, Terry A.; Ryder, John W.; Srour, Edward F.; Jacks, Tyler; Shannon, Kevin; Clapp, D. Wade; Pediatrics, School of Medicine
Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21(ras) (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1-/- hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1-/- progenitors. Nf1-/- fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1-/- progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.
Murine Cutaneous Mastocytosis and Epidermal Melanocytosis Induced by Keratinocyte Expression of Transgenic Stem Cell Factor
(Rockefeller University Press, 1998) Kunisada, Takahiro; Lu, Shu-Zhuang; Yoshida, Hisahiro; Nishikawa, Satomi; Nishikawa, Shin-ichi; Mizoguchi, Masako; Hayashi, Shin-ichi; Tyrrell, Lynda; Williams, David A.; Wang, Xiaomei; Longley, B. Jack; Pediatrics, School of Medicine
The growth and differentiation of mast cells and melanocytes require stem cell factor (SCF), the ligand for the kit receptor tyrosine kinase. SCF may exist as a membrane-bound or soluble molecule. Abnormalities of the SCF-kit signaling pathway, with increased local concentrations of soluble SCF, have been implicated in the pathogenesis of the human disease cutaneous mastocytosis, but have not yet been shown to play a causal role. To investigate both the potential of SCF to cause mastocytosis and its role in epidermal melanocyte homeostasis, we targeted the expression of SCF to epidermal keratinocytes in mice with two different transgenes controlled by the human keratin 14 promoter. The transgenes contained cDNAs that either produced SCF, which can exist in both membrane-bound and soluble forms, or SCF, which remains essentially membrane bound. Murine epidermal keratinocyte expression of membrane-bound/ soluble SCF reproduced the phenotype of human cutaneous mastocytosis, with dermal mast cell infiltrates and epidermal hyperpigmentation, and caused the maintenance of a population of melanocytes in the interadnexal epidermis, an area where melanocytes and melanin are found in human skin but where they are not typically found in murine skin. Expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. We conclude, first, that a phenotype matching that of human mastocytosis can be produced in mice by keratinocyte overproduction of soluble SCF, suggesting a potential cause of this disease. Second, we conclude that keratinocyte expression of membrane-bound SCF results in the postnatal maintenance of epidermal melanocytes in mice. Since the resulting animals have skin that more closely approximates human skin than do normal mice, their study may be more relevant to human melanocyte biology than the study of skin of normal mice.
An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation
(2000-03) Tell, Gianluca; Zecca, Alessandro; Pellizzari, Lucia; Spessotto, Paola; Colombatti, Alfonso; Kelley, Mark R.; Damante, Giuseppe; Pucillo, Carlo
The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H2O2 induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse–chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.
Enhanced mtDNA repair and cellular survival following oxidative stress by targeting the hOGG repair enzyme to mitochondria.
(2000-12) Dobson, Allison W.; Xu, Yi; Kelley, Mark R.; LeDoux, Susan P.; Wilson, Glenn L.
Oxidative damage to mtDNA has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared to cells transfected with vector only, while nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of tetrazolium compound to formazan, Trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.
A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase
(2001-04) Kreklau, Emiko L.; Limp-Foster, Melissa; Liu, Naili; Xu, Yi; Kelley, Mark R.; Erickson, Leonard C.
DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O6‐methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.
Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization
(2001-05) Yang, Feng-Chun; Atkinson, Simon J; Gu, Ying; Borneo, Jovencio B; Roberts, Andrew W; Zheng, Yi; Pennington, Janice; Williams, David A
Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.
Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation
(2001-05) Hsieh, Marlene M.; Hegde, Vijay; Kelley, Mark R.; Deutsch, Walter A.
Reactive oxygen species (ROS) arise through normal cellular aerobic respiration, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, can have a profound negative effect on cellular macromolecules such as DNA that may lead to a number of human pathological disorders including accelerated aging and cancer. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidinic (AP) sites, which without removal are known to halt mRNA and DNA synthesis, or act as non-coding lesions resulting in the increased generation of DNA mutations. In human cells, the major enzyme in correcting the deleterious effects of AP sites in DNA is through the participation of AP endonuclease (APE), which initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5′ and adjacent to an AP site. Interestingly, APE also possesses an activity (Ref-1) that controls the redox status of a number of transcription factors including Fos and Jun. The means by which APE/Ref-1 is directed to carry out such disparate roles are unknown. The presence of a number of phosphorylation sites scattered throughout both functional domains of APE/Ref-1 however offered one possible mechanism that we reasoned could play a role in dictating how this protein responds to different stimuli. Here we show that the in vitro redox activity of APE/Ref-1 is stimulated by PKC phosphorylation. Furthermore, when human cells were exposed to the PKC activator phorbol 12-myristate 13-acetate, an increase in redox activity was observed that corresponded to an increase in the phosphorylation status of APE/Ref-1. Importantly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by APE/Ref-1 that also involved an increase in PKC activity and a corresponding increase in the phosphorylation of APE/Ref-1. These results suggest that the ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in response to DNA damaging agents.
Conversion of the Bifunctional 8-oxoguanine/β-δ AP DNA Repair Activities of Drosophila Ribosomal Protein S3 into the Human S3 Monofunctional β- elimination Catalyst Through a Single Amino Acid Change
(2001-07) Hegde, Vijay; Kelley, Mark R.; Xu, Yi; Mian, Saira; Deutsch, Walter A.
The Drosophila S3 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that S3 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila S3 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a β-elimination reaction that cleaves phosphodiester bonds 3′ and adjacent to an AP lesion in DNA. In certain situations, this is followed by a δ-elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5′- and 3′-phosphate groups. The human S3 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a β-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a δ-elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human S3 were suggested by the absence of a conserved glutamine residue in human S3 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the δ-elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human S3 protein. In support of this were experiments executed in vivo that showed that human S3 and the Drosophila site-directed glutamine-changed S3 performed poorly when compared with Drosophilawild-type S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.

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