Transgenic Expression of Wild Type JAK2 Suppresses Myeloproliferative Disorder Phenotypes Induced by Mutant JAK2V617F in Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 180-180
Author(s):  
Shu Xing ◽  
Wanming Zhao ◽  
Wanting Tina Ho ◽  
Zhizhuang Joe Zhao
Keyword(s):  
Transgenic Mice ◽  
Myeloproliferative Disorders ◽  
Heterozygous Mutation ◽  
Homozygous Mutation ◽  
Mutant Form ◽  
Dependent Manner ◽  
Wild Type ◽  
Transgenic Expression ◽  
Cell Counts ◽  
Invaluable Tool

Abstract JAK2V617F, a mutant form of tyrosine kinase JAK2, is found in the majority of patients with myeloproliferative disorders (MPDs). It displays increased kinase activity and causes MPD phenotypes in transgenic mice in a transgence dosage-dependent manner. Following our initial generation and characterization of JAK2V617F transgenic mice, we further generated transgenic mice expressing wild type JAK2 by using the same vav promoter employed for JAK2V617F. Three lines of JAK2 transgenic mice were generated. Real time PCR analyses revealed transgene copy numbers of 38, 2, and 1. All these mice are viable and fertile, and they displayed normal blood cell counts. This proves that the V617F mutation but not gene overexpression per se caused MPD phenotypes in JAK2V617F transgenic mice. We then crossed the JAK2 and JAK2V617F transgenic mice to generate JAK2/JAK2V617F double transgenic hybrids. Interestingly, these hybrid mice developed no or mild MPD phenotypes with only a slight increase in blood counts in contrast to the striking elevation observed in JAK2V617F transgenic mice. Expression of wild type JAK2 also blocked the constitutive activation of signal transduction components caused by JAK2V617F. Our data indicates that over-expression of wild type JAK2 suppresses the pathogenic function of mutant JAK2V617F. Therefore, JAK2V617F is not a typical dominant oncogene. Homozygous mutation or in the case of heterozygous mutation, its amplification with concurrent deletion or suppression of wild type JAK2, is required to produce MPD phenotypes. Our transgenic mouse models will serve as an invaluable tool to study the interplay of JAK2 and JAK2V617F and the mechanism by which specific MPD phenotypes develop.

scholarly journals Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5109-5117 ◽  
Author(s):  
Shu Xing ◽  
Tina Ho Wanting ◽  
Wanming Zhao ◽  
Junfeng Ma ◽  
Shaofeng Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Gene Promoter ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Transgenic Expression ◽  
Cell Counts

Abstract The JAK2V617F mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2V617F, showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2V617F expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2V617F can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2V617F and to develop treatment for MPDs.

HH-002 Effectively Suppresses Myeloproliferative Neoplasm Phenotypes in JAK2V617F Transgenic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4095-4095
Author(s):  
Wan-Ting Ho ◽  
Wanke Zhao ◽  
Paul Hallenbeck ◽  
Frank Rong ◽  
Zhizhuang Joe Zhao
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Blood Cells ◽  
Hematopoietic Cells ◽  
Mutant Form ◽  
Dependent Manner ◽  
Combination Drug ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Abstract Ph-negative myeloproliferative neoplasms (MPNs) represent a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). A major molecular defect in MPNs is JAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2 found in the majority of MPN patients. In earlier studies, we generated JAK2V617F transgenic mice by using the vav promoter which drives gene expression in all hematopoietic cells. These mice develop MPN-like phenotypes in a transgene dose-dependent manner. The objective of this study is to use this animal MPN model to test the efficacy of HH-002, a derivative of natural plant alkaloid homoharringtonine. Treatment of JAK2V617F transgenic mice with a daily dose of 1 mg/kg HH-002 through subcutaneous injection reduced blood cell counts to the normal range or slightly below the normal level. HH-002 causes a preferential reduction of myeloid cells in JAK2V617F transgenic mice since percentages of lymphocytes (CD3e+ T cells and CD19+ B cells) were increased (despite somewhat decreases in absolute numbers) while the percentage of Gr-1+/CD11b+ granulocytes sharply declined. HH-002 also effectively reduced the spleen size of JAK2V617F transgenic mice and prevented development of myelofibrosis. With a JAK2V617F bone marrow transplant mouse model, HH-002 showed a similar effect. Although it did not increase the ratio of JAK2V617F-negative cells to JAK2V617F-positive, it stopped further expansion of JAK2V617F-containing malignant cells in JAK2V617F bone marrow recipient mice. In vitro experiments with primary hematopoietic cells demonstrated that HH-002 potently inhibited formation of erythroid and myeloid colonies with an IC50 value of 1-3 nM. However, it does not show a preferential inhibition of JAK2V617F-containing cells. This is not unexpected since homoharringtonine is known to inhibit protein synthesis. Taken together, HH-002 is a promising candidate for development of therapeutic drugs to treat MPNs and related diseases. Combination drug therapies with JAK2 inhibitors need to be explored. Disclosures Hallenbeck: Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Rong:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Zhao:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Research Funding.

scholarly journals Transgenic Expression of JAK2V617F Causes Myeloproliferative Disorders in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2527-2527
Author(s):  
Shu Xing ◽  
Zhizhuang Joe Zhao
Keyword(s):  
Transgenic Mice ◽  
Myeloproliferative Disorders ◽  
Extramedullary Hematopoiesis ◽  
Transgenic Expression ◽  
Cell Counts ◽  
Serum Erythropoietin Level ◽  
Hyper Sensitivity ◽  
Serum Erythropoietin

Abstract Recently, an acquired mutation of tyrosine kinase JAK2 was found in most patients with myeloproliferative disorders (MPDs) including polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis. We have generated transgenic mice expressing the mutation enzyme, JAK2V617F, in the hematopoietic system driven by the promoter of the vav gene. The mice are viable and fertile. One line of the transgenic mice expressed a lower level of JAK2V617F and displayed elevated blood cell counts, while the other line expressed a higher level of JAK2V617F and exhibited a marked increase in blood counts and developed phenotypes that closely resembled human ET and PV. The latter line of mice also developed marrow and spleen fibrosis as the animal aged. In general, the transgenic mice had megakaryocytic hyperplasia in the bone morrow and extramedullary hematopoiesis resulting in splenomegaly, and their serum erythropoietin level was also significantly reduced. In vitro colony assays demonstrated that transgenic mice possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow and that these cells displayed hyper-sensitivity to growth factors and cytokines. The data prove that JAK2V617F is a cause of MPDs. Our study thus provides a permanent mouse system for further study to define the pathological role of JAK2V617F and to develop treatment for MPDs.

scholarly journals the Prevalence of UGT1A1*93 and ABCC5 Polymorphisms in Cancer Patients Receiving Irinotecan-Based Chemotherapy at Al-Najaf Al-Ashraf

2019 ◽  
Vol 28 (2) ◽  
pp. 24-29
Author(s):  
Ahmed F. Al_talkani ◽  
Sarmed H. Kathem
Keyword(s):  
Cancer Patients ◽  
Topoisomerase I ◽  
Antineoplastic Agent ◽  
Limiting Factors ◽  
Cancer Center ◽  
Heterozygous Mutation ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Isolation And Purification ◽  
Wide Range

Irinotecan (CPT-11) is a semisynthetic derivative of the antineoplastic agent camptothecin used in a wide range as an anti-cancer agent in many solid tumors because of its cytotoxic effect through the interaction with the topoisomerase I enzyme. The major limiting factors for irinotecan treatment are its association with potentially life-threatening toxicities including neutropenia and acute or delayed-type diarrhea, results from distinct interindividual and interethnic variability due to gene polymorphism. This is a cross sectional pharmacogentics study was conducted on 25 cancer patients to estimate the prevalence of UGT1A1*93 and ABCC5 allele single nucleotide polymorphism (SNP) in Iraqi cancer patients treated with irinotecan-based therapy at Middle Euphrates Cancer Center. Four drops of venous blood was drawn for each patient and was applied onto the FTA classic card to perform a genotyping assay for the 2 SNPs. After DNA isolation and purification, real time PCR was performed to detect the SNPs of each gene. Results of this study showed the prevalence of one allele variant (heterozygous mutation) of UGT1A1*93 was 64% compared to 36% of patients were wild type to this SNP. No patient (0%) could be detected with homozygous polymorphism of the UGT1A1*93. For the ABCC5 polymorphism, results revealed that 32% of patients have one polymorphic allele (heterozygous), while 28% of them have two polymorphic alleles (homozygous mutation). Wild type ABCC5 gene constitutes 40% of patients.   As a conclusion, high prevalence of UGT1A1*93 and ABCC5 polymorphic alleles were detected in patients at Middle Euphrates Cancer Center which may explain the high toxicity features associated with irinotecan therapy. 

Analysis of the JAK2 Gene cDNA Full-Length In One Chinese Familial Myeloproliferative Disorders

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5056-5056
Author(s):  
Ru Feng ◽  
Lixia Hao ◽  
Yongmin Zhang ◽  
Yongqiang Wei ◽  
Fen Huang ◽  
...  
Keyword(s):  
Family Members ◽  
Gene Mutations ◽  
Myeloproliferative Disorders ◽  
Full Length ◽  
The Other ◽  
Jak2v617f Mutation ◽  
Chinese Family ◽  
Heterozygous Mutation ◽  
Homozygous Mutation ◽  
The Family

Abstract Abstract 5056 Introduction: JAK2V617F point mutation have been confirmed to be one of the major molecular mechanism of BCR/ABL negative myeloproliferative disorders(MPD). Besides, some other gene mutations such as JAK2 exon12, MPL W515L/K, c-mpl and EPOR have extended the scope of the research in this field. Most of the MPD patients are sporadic and there are seldom reports in Chinese familial MPD. 2008 ASH metting we have reported in a Chinese family of MPD's findings, the two brothers in our hospital diagnosis for MPD (one is a PV, another is ET), then we investigated the 15 members of the family. We discovered that there were three male members carried the JAK2V617F mutation in this family, including the two MPD patients and their father, which affected in two generations. All the family members were confirmed as BCR/ABL, MPL W515L/K, c-mpl, and EPOR negative. Subsequently, in order to understand the existence of family members in addition to the gene JAK2 V617F mutation, the existence of JAK2 gene mutations in other parts of the? if other mutations in existence and the high incidence of family members of MPD? We focus on the cDNA full-length of JAK2 gene to provide some theory basis on the pathogenesis in MPD. Methods: A total of 15 family members were enrolled in our study, including 2 brothers of MPD patients (the older one was thrombocythemia (ET), and another is polycythemia vera (PV)) and the other members in the same family. The mRNA of mononuclear cells from peripheral blood sample was extracted according to the manufacturer's instruction (TAKARA). RT-PCR and DNA sequencing have been used to analyze the cDNA full-length of the JAK2 gene. Results: All of the samples can be analyzed for JAK2 cDNA full-length. 3 members carried the JAK2V617F mutation (1849G®T) in this family, including the two MPD patients and their father. And the older brother was homozygous mutation and the other two were heterozygous mutation. All of the 15 samples were JAK2 exon12 gene mutation negative. 2 persons who were the male ET patient's children had a heterozygous mutation (380G®A) in JAK2 exon 3, caused a glycine-to-asparticacid substitution at position 127. Besides, 13 persons had 489C®T mutation in exon 4 and 14 persons had 2490G→A mutation in exon 17 in this family, But they were both same-sense mutation. Conclusion: It is necessary to do routine analysis of blood and other related inspection for MPD patient's family members, so as to make diagnosis earlier. However, we are not sure that the sequencing results are unique to all the familial MPD and need to be confirmed by more cases. We still do not determine the current discovery point mutations have biological significance, still need to be further explored. Disclosures: No relevant conflicts of interest to declare.

222 Effective donor selection before superovulation treatment for Japanese Black beef cattle

2020 ◽  
Vol 32 (2) ◽  
pp. 239
Author(s):  
N. Muraguchi ◽  
H. Uemura ◽  
C. Kubota
Keyword(s):  
Beef Cattle ◽  
T Test ◽  
Donor Selection ◽  
Heterozygous Mutation ◽  
Corpora Lutea ◽  
Polymorphism Analysis ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Embryo Yield ◽  
Study Results

Japanese Black beef cattle (JBBC) are a leading Wagyu breed in Japan. Embryo transfers are being used to improve JBBC, a sturdy, fast-maturing breed prized for its marbled beef; however, relevant donor selection techniques have not been fully established. Accordingly, in this study, in order to produce embryos efficiently, we aimed to investigate donor selection techniques based on follicle counts at different time points (Experiment 1) and an associated genetic marker (Experiment 2). Statistical differences were evaluated with Student's t-test or Welch's t-test, regarding P<0.05 as significant. In Experiment 1, we initially targeted 57 JBBCs for evaluation. Their follicle counts were determined by ultrasound 3 days before the start of superovulation (pre), close to the time of follicular wave emergence, and they were grouped according to number of follicles (0-19, 20-29, 30-39, and ≥40). Each cow was administered a tapered dose of FSH-R (20 AU) for superovulation, and then subjected to AI with cryopreserved semen from bulls of proven fecundity. Embryos were collected 7 days after AI by non-surgical intrauterine reflux. Post-AI follicle counts were done before ovulation and did not differ significantly from pre-superovulation follicle count in any group (range: −1.09 to 0.61). We then targeted a further 12 JBBC undergoing superovulation and AI with the same procedures, for counting of follicles at 30 days before (Day −30) embryo recovery (ER), pre-superovulation, and AI, and counting of corpora lutea and large follicles at ER, to investigate correlations between counts. We used decision coefficient (R2), regarding R2 ≥ 0.5 as significant. We found positive correlations (R2) at Day −30 with pre-superovulation (0.91), AI (0.63), and ER (0.63), and at pre-superovulation with AI (0.69) and ER (0.74). In Experiment 2, we targeted 69 JBBCs for detection of mutations in the GRIA1 gene, which encodes inotropic glutamate receptor AMPA1 with a known association with ovulation rate, and embryo yield measurements. DNA obtained from blood was subject to microsatellite polymorphism analysis, for mutations at base 917 of GRIA1 exon 7. We identified 33 cows (48%) as bearers of the wild-type allele (GG), 29 cows (42%) as bearers of the heterozygous mutation (GA), and seven cows (10%) as bearers of the homozygous mutation (AA). Average embryo yield was highest in wild-type cows, followed by heterozygous mutants and then homozygous mutants, and was significantly lower for homozygous mutants than for the other two types. Our study results demonstrated that follicle count before superovulation and GRIA1 gene analysis have utility for selecting donors for embryo transfer in JBBC. Furthermore, follicle counts at the early time point of 30 days before ER can be a useful indicator for this selection.

Transgene Dose- and Age-Dependent Development of Myeloproliferative Neoplasm Phenotypes In JAK2V617F Transgene Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1980-1980
Author(s):  
Wanting Ho ◽  
Wanming Zhao ◽  
Zhizhuang Joe Zhao
Keyword(s):  
Transgenic Mice ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Gene Copy ◽  
Cell Transplant ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Age Dependent

Abstract Abstract 1980 Myeloproliferative neoplasms (MPNs) are heterogeneous hematologic disorders represented by three main phenotypes: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The major molecular lesion in these diseases is JAK2V617F, which occurs in over 95% patients with PV and in over 50% of patients with ET or PMF. The pathogenic effects of JAK2V617F have been demonstrated by retrovirus-mediated gene transfer, transgenic, and knock-in mouse models, but the precise mode of JAK2V617F action is not clear. Interestingly, in the knock-in model, expression of JAK2V617F causes severe PV-like disease but not ET-like phenotype as seen in patients. To verify the pathogenic role of JAK2V617F, we further characterized the phenotypes of three lines of JAK2V617F transgenic mice generated by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. These mice developed MPN-like phenotypes in a transgene dose- and age-dependent manner. Line A mice have a JAK2V617F gene copy number of 13; they develop MPN phenotype with marked increases in blood counts and enlarged spleens as early as 4–6 weeks after birth. In contrast, lines B and D mice have a transgene copy number of 2 and 1, respectively, and it takes nearly 70 weeks for these mice to show MPN-like phenotypes. The phenotype of line A mice is particularly noteworthy. Essentially all the hemizygous line A mice displayed an ET-like phenotype with marked elevations in platelet counts (usually over 4000×109/L by the age of 15 weeks), but only a slight increase in red cell and white cell counts. In contrast, all the homozygous mice exhibited a clear PV-like phenotype with elevations in all three types of blood cells, although their platelets hardly ever went over 4000×109/L. The hemizygous mice developed myelofibrosis after 30 weeks while the homozygous mice showed the symptom within only 10 weeks. As expected, the increased blood cell counts and formation of myelofibrosis are associated with mobilization of hematopoietic stem/progenitor cells to peripheral hematopoietic tissues (blood, spleen, and liver). By conducting stem cell transplant experiments, we further proved that JAK2V617F-induced ET and PV-like phenotypes are transplantable. Our study demonstrates that transgenic expression of JAK2V617F is capable of producing all three phenotypes of MPNs in a transgense dose- and age-dependent manner. Our transgenic mice thus represent an excellent model system to study MPNs. Disclosures: No relevant conflicts of interest to declare.

The Role Of p53 In JAK2V617F-Induced Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4103-4103 ◽  
Author(s):  
Wanke Zhao ◽  
Yanhong Du ◽  
Yun Chen ◽  
Wanting Ho ◽  
Zhizhuang Joe Zhao
Keyword(s):  
Transgenic Mice ◽  
Effective Treatment ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Leukemia Cell ◽  
Dependent Manner ◽  
Myeloid Metaplasia ◽  
Cell Counts ◽  
Jak2 Inhibitors

Abstract Ph- myeloproliferative neoplasms (MPNs) are hematopoietic malignancies in which one or more myeloid lineages are abnormally amplified. These diseases represent a group of chronic conditions including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. MPNs mainly affect older people and have an average onset age of 55 years. Complications associated with MPNs include development of acute leukemia as well as thrombosis, hemorrhage, and myeloid metaplasia. JAK2V617F is found in the majority of patients with MPNs, and an overwhelming number of studies have demonstrated its pathogenicity. However, the precise action of JAK2V617F is not well defined, and other molecular defects involved in MPNs remain elusive. In earlier studies, we have generated transgenic mice expressing JAK2V617F in the hematopoietic system and demonstrated that JAK2V617F can cause MPN-like phenotypes in an age- and transgene dose-dependent manner. In this study, we address the involvement of tumor suppressor p53 in the progression of JAK2V617F-induced MPN phenotypes. We crossed our JAK2V617F transgenic mice with p53 knockout mice. Under the p53+/+ background, our JAK2V617F transgenic mice developed a mild MPN-like phenotypes in 15 weeks. However, under the heterozygous knockout p53-/+ condition, the mice developed a strong MPN-like phenotype in 8 weeks, manifested in higher blood cell counts, more severe splenomegaly, and earlier onset of myelofibrosis. This suggests that p53 affects the progression of MPNs, and reduced levels of p53 activity may be responsible for the heterogeneous phenotypes observed in MPN patients. Furthermore, when p53 was deleted from both chromosomes, JAK2V617F mice developed acute erythroleukemia, megakaryoblastic leukemia, or myeloid leukemia and died in 20 weeks with greatly enlarged spleen (>10 times the normal size) and liver (twice the normal size) infiltrated with leukemic cells. This indicates that loss of p53 in addition to JAK2V617F causes leukemic transformation. This is consistent with the earlier findings that p53 mutations exist in JAK2V617F-positive leukemia cell lines and JAK2V617F-positive MPNs patients who developed acute myeloid leukemia. Our study also suggests that targeting JAK2V617F and p53 simultaneously may provide effective treatment for MPNs. We employed nutlin-3 that disrupts the MDM2-p53 interaction thereby reducing degradation of p53. In vitro cell culture studies demonstrated that JAK2 inhibitors and nutlin-3 synergistically inhibited the growth of hematopoietic progenitor cells from JAK2V617F transgenic mice and MPN patients. Finally, from one of the p53-/- JAK2V617F transgenic mice, we derived an erythroleukemia cell line designated J53Z1. J53Z1 cells are CD71high and Ter119low and are dependent on erythropoietin for survival. They were effectively inhibited by known JAK2 inhibitors and should be useful in cell-based assays for screening of JAK2 inhibitors. Altogether, our study demonstrates that the level of p53 dictates the progression of JAK2V617F-induced MPNs and targeting p53 and JAK2V617F simultaneously may provide effective treatment for JAK2V617F-positive MNPs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Chk2 Activation Dependence on Nbs1 after DNA Damage

2001 ◽  
Vol 21 (15) ◽  
pp. 5214-5222 ◽  
Author(s):  
Giacomo Buscemi ◽  
Camilla Savio ◽  
Laura Zannini ◽  
Francesca Miccichè ◽  
Debora Masnada ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Nijmegen Breakage Syndrome ◽  
Mutant Form ◽  
Dependent Manner ◽  
Wild Type ◽  
Normal Cells ◽  
Chromosome Fragility ◽  
Carboxy Terminal

ABSTRACT The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G1 arrest. Here we show that the ATM-dependent activation of Chk2 by γ- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.

scholarly journals Autoimmune Oophoritis with Multiple Molecular Targets Mitigated by Transgenic Expression of Mater

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2465-2473 ◽  
Author(s):  
Noriyuki Otsuka ◽  
Zhi-Bin Tong ◽  
Konstantina Vanevski ◽  
Wei Tu ◽  
Mickie H. Cheng ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Autoimmune Response ◽  
Dependent Manner ◽  
Wild Type ◽  
Transgenic Expression ◽  
Autoimmune Polyglandular Syndrome ◽  
Autoimmune Polyglandular Syndrome Type ◽  
Specific Tolerance ◽  
Autoimmune Oophoritis

Primary ovarian insufficiency (POI) resulting from ovarian autoimmunity is a poorly understood clinical condition lacking in effective treatments. Understanding the targets of the autoimmune response and induction of ovarian-specific tolerance would allow development of focused therapies to preserve fertility in an at-risk population. MATER (maternal antigen that embryos require) is a known ovarian autoantigen targeted in autoimmune syndromes of POI. We attempt to induce ovarian-specific tolerance via transgenic expression of the MATER antigen on potentially tolerogenic antigen-presenting cells (APC), which typically present antigen via the major histocompatibility complex (MHC) class II molecule. We hypothesize that expression of MATER in a MHC class II-dependent manner on APC can mediate induction of ovarian tolerance. We utilized a well-characterized murine model of ovarian autoimmunity, whereby oophoritis develops after d 3 neonatal thymectomy (NTx). Wild-type and transgenic mice, carrying an MHC Class II-driven Mater gene (IE-Mater), were subjected to NTx and assessed for evidence of autoimmune oophoritis. After disease induction by NTx, female mice carrying the IE-Mater transgene had significant reductions in histological oophoritis (56%) and circulating ovarian autoantibodies (28%) compared with wild-type females (94% and 82%, respectively). Incidence of other autoimmunity was unaffected as assessed by antinuclear autoantibodies. Transgenic expression of MATER in APC can induce antigen-specific tolerance with a significant reduction in ovarian autoimmunity. Lack of complete disease protection suggests that other antigens may also play a role in autoimmune oophoritis. As a known autoantigen in the human APS1 (autoimmune polyglandular syndrome type 1), which is associated with POI, MATER may represent a relevant target for future diagnostic and therapeutic clinical interventions.

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