Environmental exposure to mercury is suggested to contribute to human immune dysfunction. To shed light on the mechanism, we identified changes in the phosphoproteomic profile of the WEHI-231 B cell ...line after intoxication with Hg2+. These changes were compared to changes in the phosphoproteome that were induced by pervanadate or okadaic acid exposure. Both 250 μM HgCl2 and pervanadate, a known phosphotyrosine phosphatase inhibitor, caused an increase in the number of proteins identified after TiO2 affinity selection and LC-MS/MS analysis. Pervanadate treatment had a larger effect than Hg2+ on the number of Scansite motifs that were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling pathways activated, 4, with score >5.0. However, Hg2+ had a more focused effect, primarily causing tyrosine-phosphorylation in src homology 2 domains in proteins that are in the B cell receptor signaling pathway. The finding that many of the changes induced by Hg2+ overlap with those of pervanadate, indicates that at high concentrations Hg2+ inhibits protein tyrosine phosphatases.
Maspin is an epithelial-specific tumor suppressor gene. Previous data suggest that maspin expression may redirect poorly differentiated tumor cells to better differentiated phenotypes. Further, ...maspin is the first and only endogenous polypeptide inhibitor of histone deacetylase 1 (HDAC1) identified thus far. In the current study, to address what central program of tumor cell redifferentiation is regulated by maspin and how tumor microenvironments further define the effects of maspin, we conducted a systematic and extensive comparison of prostate tumor cells grown in 2-dimensional culture, in 3-dimensional collagen I culture, and as in vivo bone tumors. We showed that maspin was sufficient to drive prostate tumor cells through a spectrum of temporally and spatially polarized cellular processes of redifferentiation, a reversal of epithelial-to-mesenchymal transition (EMT). Genes commonly regulated by maspin were a small subset of HDAC target genes that are closely associated with epithelial differentiation and TGFβ signaling. These results suggest that a specific endogenous HDAC inhibitor may regulate one functionally related subset of HDAC target genes, although additional maspin-induced changes of gene expression may result from tumor interaction with its specific microenvironments. Currently, EMT is recognized as a critical step in tumor progression. To this end, our current study uncovered a link between maspin and a specific mechanism of prostate epithelial differentiation that can reverse EMT.
Abstract▪2101▪This icon denotes a clinically relevant abstract
Arnaud et ala recently reported on two children with an as yet unclassified CDA, characterized by hemolytic anemia with high fetal ...hemoglobin (HbF), presence of embryonic hemoglobin Portland (HbPort) and absence on red blood cells (RBC) of CD44, an adhesion molecule, aquaporin 1 (AQP1) and the linked In(Lu) and Colton blood groups. The underlying molecular defect involves the erythroid transcription factor KLF1 with a mutation in zinc finger 2 at position 973 of Guanine to Adenine. The G973A mutation results in substitution of glutamate to lysine at position 325 in the transcribed protein. The CDA results from a dominant negative effect of KLF1 E325K on the gamma-globin repressor BCL11A, leading to the persistence of fetal erythropoiesis, high Hb F and HbPort. The absence of CD44, AQP1 and deficiency of certain cytoskeletal proteins in the RBC indicate that KLF1 is necessary for the terminal differentiation of RBC. The presence of CD44 on the leucocytes in these cases would appear to suggest an erythroid restrictive effect of the mutation. Of note, the child in whom the E325K mutation was first identified, a 46XY male, was described to have micropenis and hypospadias; the hemolytic anemia was severe and required splenectomy. The original case was that of phenotypic female with 46XX and the clinical course was much milder.
We have identified a new case (a caucasian child) with the KLF1 E325K mutation. Comparison with the clinical findings in the two published cases indicates that KLF1 E325K mutation may cause profound developmental disturbances in organs beyond erythroid development, particularly of the gonads. Our patient, now 5 yrs old, was followed with an undiagnosed hemolytic anemia, which started with fetal hydrops at 23 weeks gestation, requiring intrauterine transfusions. Because of the presence of spherocytes on the blood smear and history of renal stones, a mutant band 3 was suspected; however direct nucleotide sequence analysis of the promoter and coding region exons of the band 3 gene from amplified genomic DNA of the proband was normal. Splenectomy was done at 4-years-of-age and the emerging picture was one of extreme normoblastemia, with the presence of both spherocytes and massively enlarged erythrocytes. Osmotic gradient ektacytometry showed reduced erythrocyte deformability with a population of swollen erythrocytes. Hemoglobin analyses revealed a HbF of31% and, HgPort (isoelectric focusing done by James Hoyer at Mayo Laboratories). Flow cytometry demonstrated that CD44 was not expressed on patient's erythrocytes but was expressed on lymphocytes. AQP1 was absent on western blots of RBC ghosts. Nucleotide sequence analysis of the KLF1 gene identified the KLF1 E325K mutation in lymphocyte DNA from the patient, but not in her parents. In addition to the severe anemia, the child who is 46XY, has female external genitalia. Ultrasonography demonstrated a vagina and uterus. Surgically excised streak gonads showed no cellular elements. Array comparative genomic hybridization studies did not reveal any copy number variations in male sex determining genes, but identified polymorphic deletion of estrogen metabolizing UGT2B17.
Together these 3 cases suggest that KLF1 E325K may cause more severe anemia and gonadal dysgenesis in 46XY fetuses while the disease is considerably milder in 46XX individuals. Both of the 46 XY cases also had fetal hydrops and were salvaged with transfusions in utero, suggesting that KLF1 mutations may cause early loss of male fetuses. Given that KFL1 interacts with more than 1000 proteins,b,c it is likely that KLF1 is important in the developmental regulation of non erythroid genes including male sex determining genes.
No relevant conflicts of interest to declare.
MicroRNAs (miRNAs) constitute short non-coding RNAs that can post-transcriptionally modulate the expression of many oncogenes and tumor suppressor genes engaged in key cellular processes. Deregulated ...serum miRNA signatures have been detected in various solid cancers including prostate cancer, suggesting that circulating miRNAs could function as non-invasive biomarkers of tumor emergence and progression. To determine whether serum miRNA expression levels are different between patients with aggressive and non-aggressive prostate cancer, we analyzed a panel of miRNAs from the blood of African American (AA) prostate cancer patients using a new recursive partitioning method that allows hypothesis testing of each split. We observed that both extrema of circulating miR-17, i.e. upregulation and downregulation, are associated with aggressive prostate cancer. A similar effect was observed in tumor samples from a separate dataset representing a different population of prostate cancer patients and in AA prostate cancer samples from the TCGA. The dual effect is consistent with the contradictory findings on the role of miR-17 in prostate cancer progression, whereby it controls important oncogenic and tumor-suppressive genes.
XPC is an important DNA damage recognition protein involved in DNA nucleotide excision repair. We have studied the role of the XPC protein in cisplatin treatment‐mediated cell cycle regulation. ...Through the comparison of microarray data obtained from human normal fibroblasts and two individual XPC‐defective cell lines, 486 genes were identified as XPC‐responsive genes in the cisplatin treatment (with a minimal 1.5‐fold change) and 297 of these genes were further mapped to biological pathways and gene ontologies. The cell cycle and cell proliferation‐related genes were the most affected genes by the XPC defect in the cisplatin treatment. Many other cellular function genes were also affected by the XPC defect in the treatment. Western blot hybridization results revealed that the XPC defect reduced the p53 responses to the cisplatin treatment. The ability to activate caspase‐3 was also attenuated in the XPC cells with the treatment. These results suggest that the XPC protein plays a critical role in initiating the cisplatin DNA damaging treatment‐mediated signal transduction process, resulting in activation of the p53 pathway and cell cycle arrest that allow DNA repair and apoptosis to take place. These results reveal an important role of the XPC protein in the cancer prevention.
Abstract 2039
Poster Board II-16
Acute megakaryocytic leukemia (AMkL; M7) is a biologically heterogeneous form of AML, representing ∼10% of pediatric and 1-2% of adult AML cases. AMkL is the most ...common AML subtype of children with Down syndrome (DS). DS children with AMkL have an excellent prognosis with EFS rates of 80-100% when treated with ara-C/anthracycline-based protocols, in contrast to the <30% EFS rates of non-DS children with AMkL. This also contrasts to the ∼50% EFS rates of non-DS children with AML overall, indicating that AMkL is an extremely poor risk group amongst non-DS children with AML despite the use of intensive chemotherapy-based protocols. These clinical data make a compelling argument that new therapies are essential to improve the treatment outcome of this aggressive disease. Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected uniformly in nearly all DS AMkL cases, but not in non-DS AML and non-AMkL DS leukemia cases. The net effect of GATA1 mutations is an introduction of early stop codons and synthesis of a shorter GATA1 protein (designated GATA1s) that has altered transactivation activity, potentially contributing to the uncontrolled proliferation of immature megakaryocytes. It is conceivable that the altered GATA1 function between DS and non-DS AMkL may account for differential expression of GATA1 target genes in these two groups of patients. On the other hand, overexpression of GATA1 in megakaryoblasts from non-DS children with AMkL compared to myeloblasts from non-DS children with other subtypes of AML may contribute to differences in chemotherapy sensitivity via regulation of GATA1 target genes. We previously reported that GATA1 mutations in DS AMkL are associated with decreased expression of cytidine deaminase (encodes an enzyme which can convert ara-C to ara-U, the inactive form of the drug), thus contributing to the enhanced ara-C sensitivity of DS AMkL blasts. Further, when GATA1 was ectopically expressed in a DS AMkL cell line, CMK, it caused significantly increased resistance to ara-C. In the present study, we confirmed overexpression of GATA1 in non-DS AMkL blasts compared to non-DS AML blasts by real-time RT-PCR quantitation of GATA1 transcripts in our cohort of patient samples. shRNA knockdown of GATA1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivities to ara-C and daunorubicin, the two main drugs used for AML treatment, and significantly increased basal level apoptosis. This was accompanied by significantly decreased Bcl-xL transcript and protein levels in the GATA1 shRNA knockdown clones compared to a shRNA negative control. Binding of GATA1 to the two GATA elements in Bcl-x promoter and transactivation of Bcl-x promoter activity by GATA1 was demonstrated by ChIP assays and luciferase reporter assays, respectively, in Meg-01 cells. In our cohort of non-DS AMkL and AML patient samples, significant overexpression of Bcl-xL in non-DS AMkL compared to non-DS AML cases and a significant correlation between Bcl-xL and GATA1 transcripts were detected. Besides Bcl-xL, additional GATA1 targets (e.g. TNF) related to apoptosis were also identified by gene expression and ChIP-on-ChIP microarray analyses. Interestingly, our microarray data also suggest that GATA1 may have an impact on PI3-kinase/Akt pathway through modulating directly or indirectly a group of genes within the pathway. Western blotting revealed increased phosphorylation of Akt in the GATA1 knockdown clones compared to the negative control cells. Previous studies reported that histone deacetylase inhibitors (HDACIs) treatment causes hyperacetylation and subsequent degradation of GATA1, suggesting that these agents may be effective in targeting GATA1 in AMkL. Treatment of Meg-01 cells with an HDACI, valproic acid (VPA), resulted in decreased protein levels for GATA1 and Bcl-xL and increased phosphorylation of Akt. Co-treatment of Meg-01 cells with VPA and ara-C resulted in synergistic induction of apoptosis and activation of caspase-3. This drug synergy was amplified when a non-toxic dose of the PI3-kinase inhibitor LY294002 was added. Our results demonstrate that GATA1 causes resistance to chemotherapy in non-DS AMkL by promoting AMkL blast survival through regulating its target genes. Treatment of AMkL may be improved by integrating HDACI and PI3-kinase or Akt inhibitors into the chemotherapy of this disease.
No relevant conflicts of interest to declare.
Aging and DNA polymerase b deficiency (b-pol super(+/-)) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency ...(FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in b-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in b-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in b-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD b-pol haploinsufficient mice.
Hepatocyte nuclear factor (HNF)-4alpha is a liver-enriched transcription factor that regulates numerous liver-expressed genes including several sex-specific cytochrome P450 genes. Presently, a ...liver-specific HNF4alpha-deficient mouse model was used to characterize the impact of liver HNF4alpha deficiency on a global scale using 41,174 feature microarrays. A total of 4994 HNF4alpha-dependent genes were identified, of which about 1000 fewer genes responded to the loss of HNF4alpha in female liver as compared with male liver. Sex differences in the impact of liver HNF4alpha deficiency were even more dramatic when genes showing sex-specific expression were examined. Thus, 372 of the 646 sex-specific genes characterized by a dependence on HNF4alpha responded to the loss of HNF4alpha in males only, as compared with only 61 genes that responded in females only. Moreover, in male liver, 78% of 508 male-specific genes were down-regulated and 42% of 356 female-specific genes were up-regulated in response to the loss of HNF4alpha, with sex specificity lost for 90% of sex-specific genes. This response to HNF4alpha deficiency is similar to the response of male mice deficient in the GH-activated transcription factor signal transducer and activator of transcription 5b (STAT5b), where 90% of male-specific genes were down-regulated and 61% of female-specific genes were up-regulated, suggesting these two factors cooperatively regulate liver sex specificity by mechanisms that are primarily active in males. Finally, 203 of 648 genes previously shown to bind HNF4alpha near the transcription start site in mouse hepatocytes were affected by HNF4alpha deficiency in mouse liver, with the HNF4alpha-bound gene set showing a 5-fold enrichment for genes positively regulated by HNF4alpha. Thus, a substantial fraction of the HNF4alpha-dependent genes reported here are likely to be direct targets of HNF4alpha.
It has been previously shown that acute myeloid leukemia (AML) patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS) patients with acute ...megakaryocytic leukemia (AMKL), whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C) and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP) and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts) is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.
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