) explain what it means for a function to be θ(1).
Oncotarget. 2014 January; 5(ane): 211–223.
AID downregulation is a novel role of the DNMT inhibitor 5-aza-deoxycytidine
Chiou-Tsun Tsai
ane Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Pei-Ming Yang
i Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Ting-Rong Chern
1 Department of Pharmacology, College of Medicine, National Taiwan Academy, Taipei, Taiwan
Shu-Hui Chuang
1 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Jung-Hsin Lin
2 School of Chemist's, College of Medicine, National Taiwan University, Taipei, Taiwan
3 Research Heart for Practical Sciences, Academia Sinica, Taipei, Taiwan
4 Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan
Lars Klemm
five Section of Laboratory Medicine, University of California San Francisco, San Francisco, California
Markus Müschen
v Department of Laboratory Medicine, University of California San Francisco, San Francisco, California
Ching-Chow Chen
1 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Received 2013 Aug 23; Accepted 2013 Nov 23.
Abstract
Activation-induced cytidine deaminase (AID) was originally identified every bit an inducer of somatic hypermutation (SHM) and class switch recombination (CSR) in immunoglobulin genes. However, AID can also cause mutations in host genes and contribute to cancer progression and drug resistance. In this written report, molecular docking showed the interaction of free v-aza-CdR and Zebularine (Zeb) with Help. Yet, merely 5-aza-CdR-incorporated ssDNA bound to the active site of AID and inhibited AID expression through proteasomal degradation. 5-aza-CdR demonstrated cytotoxicity against Assist-positive and -negative hematopoietic cancer cells. In contrast, Zeb exhibited a cytotoxic outcome just in Help-negative cells due to its disability to inhibit Assist expression. This differential effect might be due to the DNMT1 stabilization induced past Assist, thus restricting the power of Zeb to deplete DNMT1 and induce tumor suppressor genes (TSGs), such equally p21, in AID-positive cells. Moreover, the in vivo anticancer effect of 5-aza-CdR but not Zeb in AID-positive hematopoietic cancer cells was demonstrated. The study not only displays the association of Assist and DNMT1 and identifies a novel biological function of Aid, only also provides novel information regarding the use of DNMT inhibitors to treat AID-positive hematopoietic cancers.
Keywords: AID, 5-aza-CdR, Zebularine, DNMT1
INTRODUCTION
Help, encoded by the AICDA gene, belongs to the apolipoprotein B-editing catalytic polypeptide (APOBEC) family and was originally described as a B cell–specifc factor unique to activated germinal center B cells. During CSR, AID is recruited to the switch region to deaminate the nucleoside cytidine and catechumen information technology to uridine, causing Dna indicate mutations and double strand breakage [ane]. This activity is essential for SHM and CSR, which generates immunoglobulin diversity after V(D)J recombination [2]. In dissimilarity to the favorable role of AID in the immune system, Help can cause chromosomal translocations and/ or mutations in proto-oncogenes, thus promoting tumor germination [3]. For example, AID induces double strand breaks in the c-myc factor, resulting in its translocation to the Ig loci and uncontrolled expression of c-Myc in Burkett's lymphoma [4, v].
Assist likewise plays an essential role in the progression of Philadelphia-positive (Ph+) leukemias, including chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) [6, 7]. The Ph chromosome originates from a translocation between the c-abl on chromosome 9 and the bcr gene on chromosome 22, leading to a BCR/ABL1 fusion protein. The forced expression of the Abelson tyrosine kinase ABL1 can phosphorylate a wide range of substrates that regulate jail cell proliferation, differentiation, migration, survival, and DNA repair and drive the pathogenesis of Ph+ leukemias [8]. Clinically, CML follows a triphasic pattern of chronic, accelerated, and blast crisis. The bulk of patients (85%) in the chronic stage will progress to the accelerated phase and blast crisis if untreated [ix]. AID is expressed in a subset of CML patients in lymphoid nail crisis, which promotes the genetic instability of tumor suppressors and DNA repair genes through point mutations and re-create number alterations. In addition, AID mutates BCR-ABL1, providing a rationale for the rapid evolution of imatinib resistance in blast crisis progression [half-dozen].
Aid is also expressed in Ph+ ALL patients, who prove an increased mutation frequency of oncogenes and TSGs, such as MYC, BCL6, and p16, which may be relevant to the unfavorable prognosis in this subset of ALL [7]. Bone marrow isolated from wild-type and Aid knockout mice were transduced with BCR-ABL1 to induce BCR-ABL1-driven ALL; then, ALL cells were transplanted into the mice. The mice engrafted with AID-/- ALL cells showed prolonged survival compared with those transplanted with Help+/+ ALL cells. Molecular analyses showed that Assist-/- ALL cells had lower frequencies of amplification, deletion and point mutation in non-Ig genes, such as Pax5 and Rhoh, and failed to repress TSGs including Rhon, p21 and Blnk. The results indicate that AID may be an oncoprotein that accelerates the evolution of ALL through aberrant hypermutation and TSG downregulation [10]. A study has also shown that AID can induce B-lymphoma/leukemia in a bone marrow transplantation mouse model, and its activity to induce CSR and SHM is essential for lymphomagenesis [xi].
The cytidine ribose nucleoside analogue 5-aza-CR (5-azacytidine) was initially identified equally a potential anticancer drug and was subsequently shown to be a Deoxyribonucleic acid methyltransferase (DNMT) inhibitor [12]. After incorporation into Dna, 5-aza-CR is recognized past DNMT1 to form stable covalent protein-DNA adducts. DNMT1 are trapped and degraded, leading to rapid protein diminish as early on as 24 hours treatment [13, 14]. Eventually, Dna demethylation occurs, and then TSGs are induced to inhibit cancer jail cell proliferation [15]. 5-aza-2-deoxycytidine (5-aza-CdR), the deoxyribose counterpart of 5-aza-CR, was subsequently developed [16]. Cytidine deaminase (CDA) is a key enzyme in the pyrimidine salvage pathway, catalyzing the deamination of cytidine and deoxycytidine into uridine and deoxyuridine. CDA besides deaminates cytidine analogues, including 5-aza-CR and five-aza-CdR, to reduce their stability [17, 18]. Another DNMT inhibitor, 1-(β-D-ribofuranosyl)-2(1H)-pyrimidinone (zebularine; Zeb), is a cytidine analogue that contains a ii-(1H)-pyrimidinone ring. Zeb was originally synthesized as a CDA inhibitor [19]. Because both CDA and AID can catalyze cytidine deamination, AID might also be targeted past cytidine analogues.
In this study, molecular docking assay showed the interaction of both complimentary 5-aza-CdR and gratuitous Zeb with Assist. All the same, only 5-aza-CdR-incorporated single-strand Deoxyribonucleic acid (ssDNA) bound to the active site of AID and inhibited AID expression through proteasomal degradation. The cytotoxicity of 5-aza-CdR was observed in both Aid-positive and -negative hematopoietic cancer cells. In dissimilarity, Zeb cytotoxicity was merely observed in Aid-negative cells due to its inability to inhibit AID expression. This differential upshot might exist due to the association and stabilization of DNMT1 by AID, thus restricting the ability of Zeb to deplete DNMT1 and induce TSGs, such as p21, in AID-positive cells. Moreover, the anticancer result of 5-aza-CdR simply not Zeb on AID-positive hematopoietic cancers was also demonstrated in vivo. The study not only displays the association of AID and DNMT1 and identifies a novel biological function of Assist, but also provides a novel role of a DNMT inhibitor for treating AID-positive hematopoietic cancers.
RESULTS
Molecular docking of DNMT inhibitors to Help
A previous study showed that the DNMT inhibitor Zeb, a cytidine analogue, is a competitive inhibitor of CDA due to its lack of a 4-amino group on the cytosine (Fig. 1A) [20]. Because Aid also catalyzes cytidine deamination, we hypothesized that cytidine analogues, such as five-aza-CR, 5-aza-CdR, and Zeb, might inhibit Aid. Molecular modeling simulation was performed to test this hypothesis. To our noesis, the crystal construction of AID has not yet been solved. Because the sequences of AID and the APOBEC2 fragment shared 51.0% similarity and 33% identity (suppl. Fig. S1A), the AID structure was predicted based on the crystal structure of APOBEC2 [21] (suppl. Fig. S1B). The proposed binding modes of 5-aza-CR, five-aza-CdR, and Zeb are illustrated in Figure 1B. Free 5-aza-CR, 5-aza-CdR, and Zeb spring to the active site of AID and interacted with the zinc ion that is crucial for the enzymatic reaction by the ii',3' hydroxyl group and the 2' carbonyl group (Fig. 1B and suppl. S1C). Based on CDOCKER interaction energy, the binding capacity of 5-aza-CR (-37.7) and 5-aza-CdR (-35.95) was higher than that of Zeb (-27.312). These cytidine analogues are incorporated into nucleic acids after entering the cell membrane [13]. A previous study has shown that AID targets the immunoglobulin H (IgH) switch region, which contains 5'-AGCT-3' repeats in its core [22]. Therefore, v'-AGCT-3' ssDNA, in which the cytidine base was substituted by the respective azacytidine (5'-AG-azaC-T-3') or Zeb (five'-AG-ZebC-T-3'), was analyzed by molecular docking. The ten predicted binding modes are illustrated in Figure 1C. Compared with 5'-AG-ZebC-T-3' (Fig. 1C, right panel), 5'-AG-azaC-T-3' was closer to the catalytic site of Assist, and both of them inserted into the active site (Fig. 1C, left panel). The best insertion model showed that the substituted 5-aza-cytosine inserted exactly into the minor active site (Fig. 1D), while the remainder of the ssDNA interacted with the polar surface (as indicated by the orange color) of Assistance (Fig. 1D, right console). These findings propose that only azacytidine-incorporated ssDNA could interact with Help.
Molecular docking of DNMT inhibitors to AID
(A) Molecular structures of cytidine and its analogs. (B) The docking sites of DNMT inhibitors were analyzed using CDOCKER, and the best structures were selected based on the lowest CDOCKER_ INTERACTION_ENERGY for each ligand. (C) 5-aza-CdR and Zeb were incorporated into the DNA sequence (v'-AGCT-3') built with "Build and Edit Nucleic Acrid" tools in Discovery Studio 2.55, and the bounden modes were further analyzed. The top 10 binding structures are shown. (D) The best insertion model is presented past a ribbon (left panel) and surface model (right console).
v-aza-CdR but not Zeb destabilizes AID through ubiquitin-proteasomal degradation
Once incorporated into Dna, azacytidine traps DNMT and triggers its degradation [xiv]. Because azacytidine- but not Zeb-substituted ssDNA interacted with Help (Fig. 1C), we proposed that 5-aza-CdR and 5-aza-CR would also trigger AID deposition. Therefore, the inhibitory consequence of DNMT inhibitors on AID expression was examined in Burkitt'due south lymphoma Raji and Ph+ ALL SUP-B15 cells, which possess AID. 5-aza-CdR, 5-aza-CR, and Zeb all downregulated DNMT1 expression [13]; however, simply 5-aza-CdR and five-aza-CR inhibited Aid expression (Fig. 2A, 2B and suppl. Fig. S2A). However, AICDA mRNA was not significantly affected by five-aza-CdR (Fig. 2C and 2D), indicating that 5-aza-CdR might inhibit AID expression through post-transcriptional regulation.
5-aza-CdR downregulated Aid
Raji cells and SUP-B15 were treated with 5-aza-CdR (1-10 μM), Zeb (50-200 μM), or TSA (1 μM) for 4 days (A) or 5-aza-CdR (5 μM) for 24, 48, and 72 hrs (B). The poly peptide expression levels of AID, DNMT1 and actin were analyzed through immunoblotting. (C) Raji cells were treated with 5-aza-CdR (1-10 μM) or Zeb (50-200 μM) for four days (left panel) or 5-aza-CdR (5 μM) for 24, 48, and 72 hrs (right panel). The mRNA levels of AICDA and actin were analyzed through RT-PCR. (D) Raji cells were treated with v-aza-CdR (5-ten μM) or Zeb (100 μM) for 4 day. The relative mRNA levels of AICDA were analyzed through QRT-PCR
Assistance stability has been reported to be regulated through the proteasome degradation pathway [23]. To investigate how five-aza-CdR downregulates Assistance, the cells were treated with 5-aza-CdR in the presence of the proteasome inhibitor MG132. Restoration of AID expression was observed (Fig. 3A, upper console), suggesting the involvement of proteasomal degradation in this consequence. To further ostend this ascertainment, AID protein stability was examined in the presence of cycloheximide. Every bit shown in Figure 3A, lower panel, 5-aza-CdR reduced Assist poly peptide stability, which was reversed by MG132. Because proteasome degradation is usually triggered by polyubiquitination [23], nuclear AID ubiquitination was analyzed using an immunoprecipitation assay. The smear blotting was more intense after co-treatment with five-aza-CdR and MG132 (Fig. 3B, left panel), indicating that five-aza-CdR enhanced Assistance polyubiquitination. Assist degradation has been reported to occur in the nucleus [23]; therefore, nuclear Assist expression was examined. AID was substantially downregulated in the nucleus by 5-aza-CdR (Fig. 3B, right panel). To confirm this finding, Assistance-negative CML K562 cells were transfected with flag-AID through electroporation, and stable clones were established. Nuclear flag-AID was downregulated by 5-aza-CdR simply not Zeb, although total flag-AID was increased by 5-aza-CdR and Zeb (Fig. 3C, left console). Immunofluorescence (IFA) also indicated the degradation of nuclear AID by five-aza-CdR (Fig. 3C, right panel). Because AID is normally restricted in cytoplasm to prevent off-target deamination [24], a Crm1 inhibitor, leptomycin B (LMB), which accumulates Help in the nucleus [25], was further utilized to demonstrate the occurrence of this consequence in the nucleus (Fig. 3D).
5-aza-CdR reduced the protein stability of nuclear Assistance
(A) Upper-left panel: Raji cells were treated with five-aza-CdR (one-10 μM) for 40 hrs, and MG132 (10 μM) was added for another 8 hrs. Upper-right panel: Raji cells were co-treated with 5-aza-CdR (5 μM) and MG132 (10 μM) for eight hrs. The protein expression levels of AID and actin were analyzed through immunoblotting. Lower panel: Raji cells were pretreated with 5-aza-CdR (5 μM) for xvi hrs. Then, the cells were exposed to cycloheximide (twenty μM) or cycloheximide/ MG132 (10 μM) for 4, 8, 10, and 12 hrs. The poly peptide expression levels of Help and actin were analyzed through immunoblotting. (B) Left console: Raji cells were pretreated with v-aza-CdR (10 μM) for 19 hrs; so, the cells were exposed to MG132 (10 μM) for 5 hrs. AID was immunoprecipitated, and AID ubiquitination was examined through immunoblotting. Correct panel: Raji cells were treated with 5-aza-CdR (5-10 μM) for 48 hrs, and the nuclear and cytosolic extracts were harvested. The protein expression levels of AID, tubulin, and lamin A/C were analyzed through immunoblotting. (C) Upper-left panel: K562AID4 cells were treated with 5-aza-CdR (10-50 μM) and Zeb (l μM) for 48 hrs. Lower-left panel: K562AID4 cells were treated with 5-aza-CdR (10 μM) and Zeb (ten μM) for 48 hrs, and the nuclear and cytosolic extracts were harvested. The poly peptide expression levels of flag-Assist and actin were analyzed through immunoblotting. Right panel: K562AID4 cells were treated with 10 μM DNMT inhibitors for 4 days. The cells were fixed and stained with an anti-fag antibody and DAPI and subjected to confocal microscopy analysis. (D) K562AID4 cells were treated with 5-aza-CdR (x μM), LMB (5 ng/ml), or both for 48 hrs. The cells were stock-still and stained with an anti-fag antibiotic (xanthous) and DAPI (blue) and subjected to confocal microscopy analysis. The images are shown at 1X and 3X magnification.
The function of Help expression in the cytotoxicity of DNMT inhibitors
Because 5-aza-CdR but non Zeb was plant to induce AID degradation, the cytotoxicity of each agent was examined in hematopoietic cells expressing different Aid levels. The cell viability of K562 (Help negative), Raji (loftier AID) and SUP-B15 (low AID) cells was assessed. Both 5-aza-CdR and Zeb inhibited the cell viability of K562 cells, while Raji and SupB15 cells were sensitive to 5-aza-CdR but resistant to Zeb (Fig. 4A and 4B). This differential effect indicated that AID might play an interfering role in the anticancer effect of DNMT inhibitors. To confirm this, the growth inhibitory furnishings of five-aza-CdR and Zeb were examined in AID-knockdown Raji or Assistance-overexpressing K562 cells. The inhibition of jail cell viability by Zeb was enhanced in AID-knockdown Raji cells only attenuated in AID-overexpressing K562 cells (suppl. Fig. S3, Fig. 4C and 4D), while the inhibitory ability of five-aza-CdR was not influenced by Assistance expression (Fig. 4C). These results suggest that five-aza-CdR targets both AID-positive and -negative cancer cells due to its ability to deplete Assist. To exclude the possibility that Assist-mediated genetic mutations alter Zeb cytotoxicity, K562 cells were transiently transfected with Assist. Consistently, the cytotoxicity of Zeb was attenuated by AID overexpression (Fig. 4D, right console).
Assistance interferes with the cytotoxic issue of DNMT inhibitor in vitro
(A) K562, Raji, and SUP-B15 cells were treated with 5-aza-CdR (1-10 μM) or Zeb (l-200 μM) for 4 days, and jail cell viability was analyzed with the Alamar blue assay. Fault bars indicate the mean ± SD of three independent experiments. (B) K562 and Raji cells were treated with 5-aza-CdR (5-10 μM) and Zeb (200 μM) for 0-half dozen days, and cell viability was analyzed with the trypan bluish assay. (C) Left panel: Raji-shluc and Raji-shAIDH1 cells were treated with 5-aza-CdR (v-10 μM) or Zeb (l μM) for 4 days, and jail cell viability was analyzed with the Alamar bluish analysis. **, p<0.01. Right panel: K562 and pooled K562AID cells were treated with 5-aza-CdR (10 μM) or Zeb (ten μM) for iv days. Then, cell viability was analyzed with the Alamar bluish analysis. *, p<0.05. (D) Left panel: K562 and pooled K562AID cells were treated with Zeb (ane-10 μM) for four days. Then, jail cell viability was analyzed with the Alamar blue assay. *, p<0.01. Correct console: K562 cells were transiently transfected with pCMV or pCMV-AID plasmids and exposed to Zeb (5 μM) for 3 days. The protein expression levels of flag-Aid and the jail cell viability were examined through immunoblotting and the Alamar blueish assay, respectively.
AID interferes with the issue of DNMT1 inhibitor through stabilizing DNMT1
DNMT inhibitors induce growth arrest by downregulating DNMT1 to promote the expression of tumor suppressor genes [xv]. Because AID expression differentially influenced the cytotoxic issue of v-aza-CdR and Zeb, its function in DNMT1 inhibition was further examined. Both 5-aza-CdR and Zeb depleted DNMT1 in AID-deficient K562 cells, whereas the consequence of Zeb was apparently attenuated in AID-positive Raji cells (Fig. 5A, left console). Consistently, Zeb finer downregulated DNMT1 in Assistance-knockdown Raji cells but not in AID-overexpressing K562 cells (Fig. 5A, right panel and 5B, left console), indicating that the inhibitory effect of Zeb on DNMT1 might be attenuated by endogenous Aid levels due to its inability to deplete Aid. Similarly, the induction of the tumor suppressor gene p21 by Zeb but not five-aza-CdR was diminished in Assistance-overexpressing K562 cells (Fig. 5B, right panel).
AID blocks DNMT inhibitor-induced degradation of DNMT1 and expression of TSG.s
(A) Left panel: K562 and Raji cells were treated with v-aza-CdR (v-ten μM) or Zeb (200 μM) for 24 hrs. Correct console: Raji cells transduced with the shluc or shAID plasmid were treated with 5-aza-CdR (5-ten μM) or Zeb (50 μM) for 24 hrs. (B) Left panel: K562 and pooled K562AID cells were treated with v-aza-CdR (5-10 μM) or Zeb (ten μM) for 24 hrs. Right panel: K562 and pooled K562AID cells were treated with 5-aza-CdR (10 μM) or Zeb (10 μM) for 96 hrs. The protein expression levels of DNMTs, p21, flag-Assist, and actin were examined through immunoblotting. (C) Left panel: Raji-shluc, Raji-shAID, K562 and K562AID cells were treated with cycloheximide (xx μM) for 4, viii, and 12 hrs. Right panel: K562 and K562AID cells were pretreated with Zeb (x μM) for xviii hrs and so exposed to cycloheximide (20 μM) for ane, 3, and five hrs. The protein expression levels of DNMT1, flag-AID, and actin were analyzed through immunoblotting. (D) Upper panel: Assist and DNMT1 were co-immunoprecipitated from the total lysate of K562AID4 cells. Flag-Assistance was precipitated using the anti-fag antibody. Normal mouse IgG (mIgG) was used every bit the IP control. The poly peptide expression levels of DNMT1 and AID were analyzed through immunoblotting. Lower panel: Raji cells were fixed and stained with anti-Help antibody (light-green), anti-DNMT1 antibody (red) and DAPI (bluish) and subjected to confocal microscopy analysis. (Due east) Proteins were harvested from Raji-shluc and Raji-shAIDH1 cells, and poly peptide expression levels of DNMT1, p21, Rassf1a, p53, AID, and tubulin were examined through immunoblotting.
A positive correlation betwixt the protein but non mRNA expression levels of AID and DNMT1 was observed in several stable clones of Assist-knockdown Raji cells and AID-overexpressing K562 cells (suppl. Fig. S3A-S3C). AID overexpression also increased ectopic DNMT1 expression, indicating the positive regulation of these 2 enzymes at the protein level (suppl. Fig. S3D). Furthermore, DNMT1 stability was decreased in AID-knockdown Raji cells merely increased in AID-overexpressing K562 cells (Fig. 5C, left panel), and the inhibition past Zeb was attenuated by AID overexpression (Fig. 5C, right panel). These results indicate that AID may stabilize DNMT1 to prevent its deposition by DNMT inhibitors. Flag-Assistance ectopically expressed in K562 cells and endogenous AID in Raji cells were co-immunoprecipitated with DNMT1 (Fig. 5D, upper pamel), and the co-localization of Help and DNMT1 in Raji cells was demonstrated (Fig. 5D, lower panel). These results suggest that the clan of AID and DNMT1 might stabilize DNMT1. The silencing of AID by shAID caused DNMT1 depletion and induction of TSGs, such as p21 and Rassf1a (Fig. 5E).
v-aza-CdR inhibits cell growth in a mouse model
Because Help accelerates CML progression by causing imatinib resistance [6], the event of DNMT inhibitors on KCL22+Assistance cells was investigated. Consistent with the results from K562 cells, Zeb- but not 5-aza-CdR-induced cytotoxicity and DNMT1 downregulation were partially attenuated by AID overexpression, and 5-aza-CdR also downregulated nuclear Assistance levels (Fig. 6A and 6B). KCL22 cells harboring AID fused to a luciferase reporter were intra-tibially injected into NOD/SCID mice to examine the in vivo anticancer effect of 5-aza-CdR and Zeb. Later transplantation, the cells grew in the os marrow and spread throughout the trunk; However, KCL22+Aid cells displayed higher growth rate (83%) than KCL22 cells (42%) (suppl. Fig. S4). The tumor-bearing mice were so selected and treated with Zeb (500 mg/kg) or 5-aza-CdR (0.five-5 mg/kg). Zeb inhibited the tumor growth of KCL22-engrafted but non KCL22+Assist-engrafted mice (suppl. Fig. S5A and Fig. 6C, left panel). On the contrary, 5-aza-CdR effectively inhibited tumor growth of both KCL22 and KCL22+AID grafts (suppl. Fig. S5B and Fig. 6C, right panel), and extended the mouse lifespan (Fig. 6D). These data indicate that five-aza-CdR but non Zeb could care for CML-LBC and Help-positive hematopoietic malignancies.
The anti-cancer effect of DNMT inhibitors on tumor-engrafted mice
(A) KCL22 and KCL22+AID cells were treated with five-aza-CdR (0.ane-x μM, left panel) and Zeb (x-100 μM, right console) for four days, and cell viability was analyzed with the Alamar blue assay. The mistake confined represent the mean ± SD. **, p<0.01. (B) Left panel: KCL22 and KCL22+Aid cells were treated with v-aza-CdR (five-10 μM, left console) and Zeb (10-25 μM, right panel) for 48 hrs. The poly peptide expression levels of AID, DNMT1, and actin were analyzed through immunoblotting. Right panel: KCL22+AID cells were treated with v-aza-CdR (5 and 10 μM) and Zeb (fifty μM) for 4 days, and the nuclear extracts were harvested. The protein expression levels of AID and lamin A/C were examined through immunoblotting. (C) Left panel: Firefly luciferase-labeled KCL22+AID cells were i.t. injected into NOD/SCID recipient mice. Later 21 days, tumor-bearing mice were selected and treated with PBS (northward=5) or Zeb (500 mg/kg, n=v). Prison cell growth was examined using an in vivo imaging system (IVIS) at mean solar day 21, 37, and 43. Correct panel: Firefly luciferase-labeled KCL22+Assistance cells were i.t. injected into NOD/SCID mice, and after 21 days, the mice were then treated with PBS (n=five) or five-aza-CdR (0.5 mg/kg, n=five) for 21 days. Prison cell growth was examined using IVIS at twenty-four hours 21, 28, and 35. (D) Then the overall survival was depicted with Kaplan-Meier assay.
DISCUSSION
For the by decade, targeted therapy has developed and go dominant for cancer treatment. All the same, cancers are notwithstanding a cureless disease because of drug resistance, which has never been resolved. Recently, substantial evidences accept indicated that AID non only contributes to tumor formation and progression merely likewise causes drug resistance by mutating tumor suppressor genes and oncoproteins [6, 10]. In addition, AID correlates with poor prognosis [26, 27]. Therefore, AID might be a potential target for preventing cancer progression and drug resistance. In this report, we establish that Assistance interacted with and may stabilize DNMT1. This association interfered with the ability of a DNMT inhibitor to deplete DNMT1 and induce p21. 5-aza-CdR inhibited nuclear AID expression through ubiquitin-proteasome degradation, thus exerting a cytotoxic issue in AID-positive hematopoietic cancers. In contrast, Zeb furnishings were limited due to its disability to inhibit AID expression.
Our data have indicated that Assistance might interact with DNMT1 and stabilize its expression (Fig. 5 and suppl. Fig. S3). Still, how Aid and DNMT1 interact with each other is currently unclear. DNMT1 transfers methyl groups into the CpG islands presently subsequently replication to maintain Deoxyribonucleic acid methylation patterns in the newly synthesized single strand. Directly binding of DNMT1 to proliferating cell nuclear antigen (PCNA) in DNA replication sites has been reported[28, 29]. We constitute the association of AID with PCNA (suppl. Fig. S6). Therefore, PCNA might human action as a bridge for AID and DNMT1 interaction. In add-on, H3K9 trimethylation past G9a creates a binding platform for HP1, which recruits DNMT1 and increases Deoxyribonucleic acid methylation [30]. Information technology has been reported that AID forms a complex with KRAB domain-associated poly peptide 1 (KAP1) and HP1 during CSR, leading to Assist recruitment to switch regions [31]. Therefore, HP1 might also human activity equally a bridge or scaffold for Help and DNMT1 interaction. Information technology has been also shown that post-translational modifications of DNMT1 modulate its stability. SET7/nine, a histone methyltransferase, mediates DNMT1 methylation at Lys142 and Lys1094 to induce its proteasomal deposition [32, 33]. In contrast, DNMT1 tin can exist stabilized by Akt1 phosphorylation at Ser143 which inhibits Lys142 methylation [33, 34]. PKA phosphorylation of Assist at Ser38 is crucial for its action in CSR [35]. Whether PKA phosphorylation of DNMT1 at Ser143 occurs through AID/DNMT1 complex remains to be investigated.
AID-/- ALL testify markedly distinct gene expression patterns, with no downregulation of TSGs, such every bit Rhon, p21, Blnk and TP53 in AID-/- ALL, suggesting that AID may alter gene expression patterns to increment leukemia malignancy [10]. Indeed, the ectopic expression of Help in CML cells increased cell survival in vitro and in vivo (suppl. Fig. S4 and S7). Its effect on gene instability could not fully explicate this event. Our results showed that silencing AID depleted DNMT1 with the concomitant induction of hypermethylated TSGs, such as p21 and Rassf1a, suggesting that differential gene expression in Aid+/+ and AID-/- cells may be attributed to DNMT1 levels in cells, in which the methylation status of tumor suppressive genes was altered. In addition, most of B cell lymphoma t(14;18) translocations occur in the CpG-rich sites where both Deoxyribonucleic acid methylation and Assistance are required for creating the breakpoints for Dna recombination [36]. The positive association of AID and DNMT1 may be also crucial for t(14;18) translocations.
AID expression is regulated at the transcriptional and post-transcriptional levels [37]. Our results showed that AID mRNA levels were not significantly afflicted by 5-aza-CdR in hematopoietic cancers. Instead, 5-aza-CdR reduced the protein stability of AID past promoting its degradation in the nucleus, suggesting that five-aza-CdR downregulated AID expression at the post-transcriptional level. Nuclear Assist has been shown to be polyubiquitinated and degraded by proteasomes through an unclear mechanism [23]. 5-aza-CdR induces the degradation of DNMT1 through APC/CCdh1-mediated polyubiquitination [14]. Whether APC/CCdh1 E3 ligase is likewise involved in the AID polyubiquitination and degradation induced by v-aza-CdR requires investigation. Recently, the E3 ligase RING finger protein 126 (RFN126) has been plant for the ubiquitination of Help [38]. Whether 5-aza-CdR could upregulate RFN126 to degrade AID will also be investigated. Alternatively, AID tin undergo ubiquitin-contained protein degradation through the REGγ proteasome pathway [39]. Although 5-aza-CdR-induced polyubiquitination of AID has been demonstrated, an ubiquitin-independent pathway cannot be excluded.
The structures and metabolisms of 5-aza-CdR, v-aza-CR and Zeb are different. 5-aza-CdR is phosphorylated by deoxycytidine kinase and other kinases into triphosphate, which tin be incorporated into newly synthesized Dna [40]. In dissimilarity, 5-aza-CR and Zeb are primarily phosphorylated by uridine-cytidine kinase and other kinases into triphosphates, which are ultimately incorporated into RNA [41]. However, the diphosphate forms of 5-aza-CR and Zeb can as well be reduced past ribonucleoside reductase into deoxy-diphosphates, which can be incorporated into Deoxyribonucleic acid [42, 43]. Degradation of DNMT1 by 5-aza-CdR and Zeb demonstrated their incorporation into Deoxyribonucleic acid [13, 44]. However, only 5-aza-CdR could trigger AID degradation. Molecular docking analyses showed that azacytidine but not Zeb-substituted ssDNA could insert into the catalytic site of AID, which might explain the differential effect of five-aza-CdR and Zeb on Aid expression.
5-aza-CR and v-aza-CdR have been used for the treatment of MDS [45]. They accept likewise been considered powerful candidates for acute myeloid leukemia (AML), CML and ALL [46]. Phase II studies have shown that low doses of 5-aza-CdR exhibit clinical activity confronting CML, including imatinib-resistant cases [47, 48] . Furthermore, the combination of 5-aza-CdR and imatinib is well tolerated and active for CML patients in the accelerated or myeloid blastic phase [49]. five-aza-CdR is currently in a stage I clinical trial for refractory and relapsed ALL (ClinicalTrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT00349596","term_id":"NCT00349596"}}NCT00349596). Considering AID contributes to tumorigenesis, imatinib resistance, clonal evolution, and immune evasion in various hematopoietic malignancies [vi, 10, 50], our results provide a novel molecular basis for a new indication of 5-aza-CdR in treating AID-positive hematopoietic cancers.
Taken together, we propose a model for our hypothesis (Fig. vii). The incorporation of DNMT inhibitors into Deoxyribonucleic acid triggers DNMT1 deposition through the proteasomal pathway, resulting in Dna demethylation, TSG induction, and growth arrest. In Assist-overexpressing hematopoietic cancer cells, nonetheless, Help interacts with and stabilizes DNMT1, which blocks the anticancer consequence of Zeb due to its inability to downregulate Assistance and hinders DNMT1 deposition. In contrast, Deoxyribonucleic acid-incorporated 5-aza-CdR can trigger AID degradation through an ubiquitin-proteasome pathway. Thus, 5-aza-CdR can finer dethrone DNMT1 to exert its anti-cancer consequence against both AID-positive and AID-negative cells. Our results provide a novel role for the clinical utility of 5-aza-CdR to treat Assist-expressing cancers and indicate the crucial concern for the selection of DNMT inhibitors. Help downregulation past DNMT inhibitors, such as 5-aza-CdR, may be benign for the treatment of AID-expressing cancers.
A hypothetical model for the role of Aid in the anticancer upshot of DNMT inhibitors
(A) AID may co-localize with DNMT1 on Deoxyribonucleic acid and raise its stability in B-lymphoid malignancies. 5-aza-CdR and Zeb contain into Deoxyribonucleic acid. (B) 5-aza-CdR targets the active site of AID and DNMT1 and then destabilizes AID and DNMT1 through the proteasome degradation pathway. Eventually, DNA is demethylated, and TSGs are induced. (C) Considering Zeb is unable to bind to the agile site of AID, DNMT1 is stabilized and avoids degradation by Zeb. Thus, the anticancer effect of Zeb is inhibited
MATERIALS AND METHODS
Jail cell civilisation and construction of stable clones
AID-overexpressing CML KCL22+Aid cells (received from Dr. Markus Müschen, University of California San Francisco) and ALL SUP-B15 cells (obtained from Dr. Hu, Med. Biotech, National Taiwan University) were cultured in IMDM. The Burkett's lymphoma Raji cells (received from Dr. Doong, Section of Microbiology, National Taiwan University) and the CML K562 cells were maintained in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum (Gibco), 1% L-glutamine (Gibco), and 1% antibiotic:antimycotic solution (Gemini Bio Products), and the cells were incubated at 37°C in a humidified incubator containing five% CO2. For the generation of stable clones, the pLKO.1-shAID plasmid (purchased from the National RNAiCore Facility, Academia Sinica, Taipei, Taiwan) was transduced into Raji cells through lentivirus infection, and Raji-shAID cells were selected using 100 ng/ml puromycin. The pCMV-3XFLAG-Aid plasmid (received from Dr. Riccardo Dalla-Favera, Department of Microbiology, Columbia University) was transfected into K562 cells through electroporation (Nero Transfection System, Invitrogen), and K562AID cells were selected with ane mg/ml G418 (Gibco). 5-azaCR (Sigma Aldrich), 5-aza-CdR (Biovision) and zebularine (Sigma Aldrich) for in vitro experiments were prepared in DMSO at appropriate doses. Zebularine (NSC 309132) for animate being studies was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute.
Homology modeling
The sequence alignment between Aid and APOBEC-2 was performed with the ClustalW module using the BLOSUM multiple alignment scoring matrix in Discovery Studio 2.55. The sequence identity and similarity betwixt Assistance and APOBEC-two were 31.viii% and 51.0%, respectively. The homology model structure was generated and optimized using MODELLER in Discovery Studio 2.55. The APOBEC-two structure was obtained from the Poly peptide Information Bank (PDB ID: 2NYT). The construction with the everyman free energy score was selected as the final model.
Molecular docking
Docking simulations of single-stranded Deoxyribonucleic acid were performed using PLANTS ane.ii software. The structure of single-stranded DNA (5'-A-T-azaC-G-three') was built with the "Build and Edit Nucleic Acid" tool in Discovery Studio 2.55. The bounden site was set equally a sphere with a xl Å radius centered from the zinc cantlet to ensure that the entire poly peptide structure was included. All parameters were set to the default settings. Docking simulations of the pocket-sized molecules Zeb and 5-aza-CdR were performed using the CDOCKER programme in Discovery Studio 2.55 with the default parameters. Ligand structures were constructed using the ChemDraw Ultra 12 plan and further processed using the Set Ligands protocol in Discovery Studio 2.55 to convert to a 3D structure and assign the ionization land. All parameters were set to the default settings.
Immunofuorescence (IFA) and immunoblotting
For IFA, the slides with cells were immersed in ii% paraformaldehyde for twenty min. Afterwards fixation, TBST with i% goat serum was used to cake the slides. The cells were outset incubated with the primary antibody anti-fag (Sigma Aldrich) or co-incubated with anti-AID (Prison cell Signaling) and anti-DNMT1 at iv°C overnight; so, the secondary antibodies Alexa (488)-labeled anti-mouse and Alexa (647)-labeled anti-goat (Invitrogen) were added and incubated at 25°C for 30 min. The signal was observed using a confocal microscope (Leica SP5). For immunoblotting, the cells were lysed in sample buffer (50 mM Tris, ane mM EGTA, fifty mM NaF, 150 mM NaCl, 1 mM Na3VO4, 1 mM β-glycerophosphate, eight.5 mM sodium pyrophosphate, and 0.5% Triton X-100). lysates were resolved on a vii.5% or 13% sodium dodecyl sulfate-polyacrylamide gel followed by electrotransference to a nitrocellulose membrane (Hybon-C). Then, the membranes were incubated in TBST containing 5% milk for 1 hr. Ultimately, the membranes were incubated with the following principal antibodies: anti-DNMT1, anti-DNMT3a, anti-ubiquitin, anti-p21, anti-lamin A/C (all from Santa Cruz Biotechnology), anti-actin (Millipore), anti-tubulin (Sigma-Aldrich) or anti-Help (Cell Signaling).
Immunoprecipitation
Total cell lysates or nuclear extracts were diluted to 1 μg/μl, and 200-500 μl lysate was incubated with 2-five μl anti-AID (Jail cell Signaling) or anti-fag antibody (Sigma Aldrich) overnight at iv°C. The mixtures containing the lysates and antibody were added to 50% protein-A beads at a i:20 ratio (book) for i hr at iv°C; and so, the beads were precipitated at 2000 rpm for five min. And so the beads were done with PBS 3 times to clear nonspecific proteins. A total of 20 μl PBS and four μl 6X sample dye were mixed with beads to prepare samples for immunoblotting.
In vivo imaging system (IVIS)
KCL22+Assistance-luc cells (5x105) were intra-tibially (i.t.) injected into NOD/SCID mice. After the xenograft, the mice were imaged at unlike time points using IVIS (Xenogen, Caliper). The successfully transplanted mice were selected to examine the anticancer effect of the DNMT inhibitors. The treatments were administered weekly on 5 sequent days equally 300 μl i.p. injections of PBS, 5-aza-CdR (0.5 mg/kg) or Zeb (500 mg/kg). D-luciferin (Promega) dissolved in PBS was injected into mice at a dose of 2.v mg/mice, and the lite emission was measured one min afterwards. For anesthesia, 2.5% isoflurane was administered to the mice via a nose cone. All brute procedures were performed under protocols approved by the Institutional Animal Care and Utilise Commission of the College of Medicine, National Taiwan University.
RT-PCR
Total RNA was extracted from cells using TRIZOL Reagent (Invitrogen). RNA (2 μg) was reverse transcribed into 20 μl cDNA by Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Promega) at 420c for 1 hr, and then the PCR was performed using Taq polymerase (Geneaid). PCR products were resolved in 1% agarose gel and visualized past Gel Doc 200 (Bio Rad). The oligonucleotide primers for PCR amplifcation were as followed: Help, 5'-AGGCAAGAAGACACTCTGGACACC-3' (frontwards), 5'-GTGACATTCCTGGAAGTTGC-3' (reverse), β-actin, five'-TGACGGGGGTCACCCACTGTGCCCATCTA-3' (forrard), 5'-CTAGAAGCATTTGCGGGGGACGATGGAGGG-3' (opposite). For quantitative PCR, the cDNA mixed with SYBR Green Master (Roche) was amplifed and detected by AVI 7900 (AVI). The oligonucleotide primers for quantitative PCR were equally followed: AID, 5'-GGACTTTGGTTATCTTCGCAA-iii' (frontward), 5'-GTCGGGCACAGTCGTAGC-3' (opposite), β-actin, 5'-CCAACCGCGAGAAGATGA-three' (frontward), 5'-TCCATCACGATGCCAGTG-3' (contrary).
Cell viability analysis
Raji (five × 104 cells/ml), K562 (2 × 104 cells/ml) and SUP-B15 (v × tenfive cells/ml) were seeding in 12 well plate, and then treated with DNMT inhibitors at indicated doses for 96 hrs. Cells (100 μl) were transferred to 96 well and x μl alarma blueish (Invitrogen) was added to each well. Later on ii hrs, cell viability was analyzed by detecting fluorescence. The wavelength at 525-535 nm was used to excite fluorescence. Reference wavelength 535-590 nm was measured on a multiwell plate reader.
Statistical analysis
Data were analyzed using Pupil'due south t test. P values < 0.05 were considered significant.
Supplementary Figures
Acknowledgments
This piece of work was supported by research grants from NSC (to C-C. C.) and the NIH through R01CA139032, R01CA137060, and R01CA157644 (to M.M.).
REFERENCES
1. Perez-Duran P, de Yebenes VG, Ramiro AR. Oncogenic events triggered past AID the adverse outcome of antibiotic diversifcation. Carcinogenesis. 2007;28(12):2427–2433. [PubMed] [Google Scholar]
two. Honjo T, Muramatsu One thousand, Fagarasan S. Help: how does it help antibody diversity? Immunity. 2004;20(half dozen):659–668. [PubMed] [Google Scholar]
3. Kinoshita K, Nonaka T. The dark side of activation-induced cytidine deaminase: relationship with leukemia and beyond. International journal of hematology. 2006;83(3):201–207. [PubMed] [Google Scholar]
4. Ramiro AR, Jankovic M, Callen Due east, Diflippantonio South, Chen HT, McBride KM, Eisenreich TR, Chen J, Dickins RA, Lowe SW, Nussenzweig A, Nussenzweig MC. Role of genomic instability and p53 in Help-induced c-myc-Igh translocations. Nature. 2006;440(7080):105–109. [PMC free article] [PubMed] [Google Scholar]
5. Takizawa M, Tolarova H, Li Z, Dubois Due west, Lim S, Callen E, Franco S, Mosaico M, Feigenbaum L, Alt FW, Nussenzweig A, Potter M, Casellas R. AID expression levels determine the extent of cMyc oncogenic translocations and the incidence of B cell tumor development. The Periodical of experimental medicine. 2008;205(9):1949–1957. [PMC free article] [PubMed] [Google Scholar]
half dozen. Klemm L, Duy C, Iacobucci I, Kuchen Southward, von Levetzow Chiliad, Feldhahn N, Henke Northward, Li Z, Hoffmann TK, Kim YM, Hofmann WK, Jumaa H, Groffen J, Heisterkamp N, Martinelli G, Lieber MR, et al. The B cell mutator Assist promotes B lymphoid blast crunch and drug resistance in chronic myeloid leukemia. Cancer Cell. 2009;16(3):232–245. [PMC complimentary article] [PubMed] [Google Scholar]
7. Feldhahn N, Henke N, Melchior K, Duy C, Soh BN, Klein F, von Levetzow K, Giebel B, Li A, Hofmann WK, Jumaa H, Muschen One thousand. Activation-induced cytidine deaminase acts equally a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells. The Journal of experimental medicine. 2007;204(5):1157–1166. [PMC costless article] [PubMed] [Google Scholar]
8. Quintas-Cardama A, Cortes J. Molecular biological science of bcr-abl1-positive chronic myeloid leukemia. Blood. 2009;113(8):1619–1630. [PMC free article] [PubMed] [Google Scholar]
9. Garcia-Manero G, Faderl S, O'Brien S, Cortes J, Talpaz M, Kantarjian HM. Chronic myelogenous leukemia: a review and update of therapeutic strategies. Cancer. 2003;98(three):437–457. [PubMed] [Google Scholar]
10. Gruber TA, Chang MS, Sposto R, Muschen Thou. Activation-induced cytidine deaminase accelerates clonal evolution in BCR-ABL1-driven B-cell lineage acute lymphoblastic leukemia. Cancer Res. 2010;70(19):7411–7420. [PMC costless article] [PubMed] [Google Scholar]
eleven. Komeno Y, Kitaura J, Watanabe-Okochi N, Kato Due north, Oki T, Nakahara F, Harada Y, Harada H, Shinkura R, Nagaoka H, Hayashi Y, Honjo T, Kitamura T. Help-induced T-lymphoma or B-leukemia/lymphoma in a mouse BMT model. Leukemia. 2010;24(5):1018–1024. [PubMed] [Google Scholar]
12. Sorm F, Piskala A, Cihak A, Vesely J. v-Azacytidine, a new, highly effective cancerostatic. Experientia. 1964;20(4):202–203. [PubMed] [Google Scholar]
xiii. Santi DV, Norment A, Garrett CE. Covalent bond germination between a Deoxyribonucleic acid-cytosine methyltransferase and Dna containing five-azacytosine. Proc Natl Acad Sci USA. 1984;81(22):6993–6997. [PMC gratis commodity] [PubMed] [Google Scholar]
14. Ghoshal Thousand, Datta J, Majumder S, Bai South, Kutay H, Motiwala T, Jacob ST. 5-Aza-deoxycytidine induces selective degradation of Dna methyltransferase ane past a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol. 2005;25(11):4727–4741. [PMC costless article] [PubMed] [Google Scholar]
15. Claus R, Lubbert M. Epigenetic targets in hematopoietic malignancies. Oncogene. 2003;22(42):6489–6496. [PubMed] [Google Scholar]
xvi. Momparler RL, Momparler LF, Samson J. Comparison of the antileukemic activity of 5-AZA-2'-deoxycytidine, 1-beta-D-arabinofuranosylcytosine and 5-azacytidine against L1210 leukemia. Leukemia enquiry. 1984;eight(6):1043–1049. [PubMed] [Google Scholar]
17. Laliberte J, Marquez VE, Momparler RL. Potent inhibitors for the deamination of cytosine arabinoside and five-aza-2'-deoxycytidine past homo cytidine deaminase. Cancer Chemother Pharmacol. 1992;30(ane):7–11. [PubMed] [Google Scholar]
18. Chabner BA, Drake JC, Johns DG. Deamination of v-azacytidine by a human leukemia cell cytidine deaminase. Biochem Pharmacol. 1973;22(21):2763–2765. [PubMed] [Google Scholar]
19. Kim CH, Marquez VE, Mao DT, Haines DR, McCormack JJ. Synthesis of pyrimidin-two-ane nucleosides as acid-stable inhibitors of cytidine deaminase. J Med Chem. 1986;29(viii):1374–1380. [PubMed] [Google Scholar]
20. Carlow D, Wolfenden R. Substrate connectivity effects in the transition state for cytidine deaminase. Biochemistry (Mosc) 1998;37(34):11873–11878. [PubMed] [Google Scholar]
21. Prochnow C, Bransteitter R, Klein MG, Goodman MF, Chen XS. The APOBEC-ii crystal structure and functional implications for the deaminase AID. Nature. 2007;445(7126):447–451. [PubMed] [Google Scholar]
22. Xu Z, Fulop Z, Wu K, Pone EJ, Zhang J, Mai T, Thomas LM, Al-Qahtani A, White CA, Park SR, Steinacker P, Li Z, Yates J, 3rd, Herron B, Otto M, Zan H, et al. 14-3-iii adaptor proteins recruit AID to 5'-AGCT-iii'-rich switch regions for grade switch recombination. Nat Struct Mol Biol. 2010;17(nine):1124–1135. [PMC free commodity] [PubMed] [Google Scholar]
23. Aoufouchi Due south, Faili A, Zober C, D'Orlando O, Weller Due south, Weill JC, Reynaud CA. Proteasomal degradation restricts the nuclear lifespan of Help. J Exp Med. 2008;205(6):1357–1368. [PMC free article] [PubMed] [Google Scholar]
24. Patenaude AM, Orthwein A, Hu Y, Campo VA, Kavli B, Buschiazzo A, Di Noia JM. Active nuclear import and cytoplasmic retention of activation-induced deaminase. Nature structural & molecular biology. 2009;xvi(5):517–527. [PubMed] [Google Scholar]
25. Ito S, Nagaoka H, Shinkura R, Begum N, Muramatsu M, Nakata M, Honjo T. Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide i. Proceedings of the National Academy of Sciences of the Usa of America. 2004;101(7):1975–1980. [PMC gratuitous commodity] [PubMed] [Google Scholar]
26. Leuenberger M, Frigerio Southward, Wild PJ, Noetzli F, Korol D, Zimmermann DR, Gengler C, Probst-Hensch NM, Moch H, Tinguely M. Assistance protein expression in chronic lymphocytic leukemia/pocket-sized lymphocytic lymphoma is associated with poor prognosis and complex genetic alterations. Mod Pathol. 2010;23(2):177–186. [PubMed] [Google Scholar]
27. McCarthy H, Wierda WG, Barron LL, Cromwell CC, Wang J, Coombes KR, Rangel R, Elenitoba-Johnson KS, Keating MJ, Abruzzo LV. High expression of activation-induced cytidine deaminase (AID) and splice variants is a distinctive feature of poor-prognosis chronic lymphocytic leukemia. Claret. 2003;101(12):4903–4908. [PubMed] [Google Scholar]
28. Chuang LS, Ian Hullo, Koh TW, Ng HH, Xu G, Li BF. Human Dna-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science. 1997;277(5334):1996–2000. [PubMed] [Google Scholar]
29. Leonhardt H, Folio AW, Weier HU, Bestor TH. A targeting sequence directs DNA methyltransferase to sites of Dna replication in mammalian nuclei. Cell. 1992;71(5):865–873. [PubMed] [Google Scholar]
xxx. Smallwood A, Esteve PO, Pradhan S, Carey M. Functional cooperation between HP1 and DNMT1 mediates gene silencing. Genes Dev. 2007;21(10):1169–1178. [PMC free article] [PubMed] [Google Scholar]
31. Jeevan-Raj BP, Robert I, Heyer V, Page A, Wang JH, Cammas F, Alt FW, Losson R, Reina-San-Martin B. Epigenetic tethering of Aid to the donor switch region during immunoglobulin class switch recombination. J Exp Med. 2011;208(viii):1649–1660. [PMC costless article] [PubMed] [Google Scholar]
32. Esteve PO, Chin HG, Benner J, Feehery GR, Samaranayake M, Horwitz GA, Jacobsen SE, Pradhan S. Regulation of DNMT1 stability through SET7-mediated lysine methylation in mammalian cells. Proceedings of the National Academy of Sciences of the The states. 2009;106(thirteen):5076–5081. [PMC gratis article] [PubMed] [Google Scholar]
33. Kar South, Deb G, Sengupta D, Shilpi A, Parbin S, Torrisani J, Pradhan S, Patra Due south. An insight into the various regulatory mechanisms modulating human DNA methyltransferase 1 stability and part. Epigenetics. 2012;seven(9):994–1007. [PMC costless article] [PubMed] [Google Scholar]
34. Esteve PO, Chang Y, Samaranayake M, Upadhyay AK, Horton JR, Feehery GR, Cheng X, Pradhan S. A methylation and phosphorylation switch between an adjacent lysine and serine determines man DNMT1 stability. Nature structural & molecular biology. 2011;xviii(one):42–48. [PMC free article] [PubMed] [Google Scholar]
35. Basu U, Franklin A, Alt FW. Post-translational regulation of activation-induced cytidine deaminase. Philos Trans R Soc Lond B Biol Sci. 2009;364(1517):667–673. [PMC free article] [PubMed] [Google Scholar]
36. Cui Ten, Lu Z, Kurosawa A, Klemm 50, Bagshaw AT, Tsai AG, Gemmell Northward, Muschen M, Adachi N, Hsieh CL, Lieber MR. Both CpG methylation and activation-induced deaminase are required for the fragility of the human bcl-ii major breakpoint region: implications for the timing of the breaks in the t(14;18) translocation. Molecular and cellular biology. 2013;33(v):947–957. [PMC free article] [PubMed] [Google Scholar]
37. Nagaoka H, Tran Th, Kobayashi M, Aida M, Honjo T. Preventing Assist a physiological mutator, from deleterious activation: regulation of the genomic instability that is associated with antibody diversity. Int Immunol. 2010;22(4):227–235. [PubMed] [Google Scholar]
38. Delker RK, Zhou Y, Strikoudis A, Stebbins CE, Papavasiliou FN. Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase. Proceedings of the National Academy of Sciences of the The states of America. 2013;110(iii):1029–1034. [PMC costless article] [PubMed] [Google Scholar]
39. Uchimura Y, Barton LF, Rada C, Neuberger MS. REG-gamma associates with and modulates the abundance of nuclear activation-induced deaminase. J Exp Med. 2011;208(12):2385–2391. [PMC free article] [PubMed] [Google Scholar]
40. Momparler RL, Derse D. Kinetics of phosphorylation of v-aza-two'-deoxyycytidine by deoxycytidine kinase. Biochemical pharmacology. 1979;28(8):1443–1444. [PubMed] [Google Scholar]
41. Van Rompay AR, Norda A, Linden K, Johansson M, Karlsson A. Phosphorylation of uridine and cytidine nucleoside analogs by 2 human uridine-cytidine kinases. Molecular pharmacology. 2001;59(5):1181–1186. [PubMed] [Google Scholar]
42. Ben-Kasus T, Ben-Zvi Z, Marquez VE, Kelley JA, Agbaria R. Metabolic activation of zebularine, a novel DNA methylation inhibitor, in homo bladder carcinoma cells. Biochem Pharmacol. 2005;70(ane):121–133. [PubMed] [Google Scholar]
43. Lee T, Karon K, Momparler RL. Kinetic studies on phosphorylation of 5-azacytidine with the purifed uridine-cytidine kinase from calf thymus. Cancer Res. 1974;34(10):2482–2488. [PubMed] [Google Scholar]
44. Schermelleh 50, Spada F, Easwaran HP, Zolghadr Yard, Margot JB, Cardoso MC, Leonhardt H. Trapped in action: directly visualization of Deoxyribonucleic acid methyltransferase activity in living cells. Nat Methods. 2005;2(10):751–756. [PubMed] [Google Scholar]
45. Lubbert 1000. Deoxyribonucleic acid methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of activeness. Curr Top Microbiol Immunol. 2000;249:135–164. [PubMed] [Google Scholar]
46. Saba Hi. Decitabine in myeloid malignancies. Hematology Meeting Reports. 2008;two(5):1–8. [Google Scholar]
47. Issa JP, Gharibyan V, Cortes J, Jelinek J, Morris Chiliad, Verstovsek Due south, Talpaz M, Garcia-Manero Thousand, Kantarjian HM. Phase II study of depression-dose decitabine in patients with chronic myelogenous leukemia resistant to imatinib mesylate. J Clin Oncol. 2005;23(17):3948–3956. [PubMed] [Google Scholar]
48. Kantarjian HM, O'Brien S, Cortes J, Giles FJ, Faderl S, Issa JP, Garcia-Manero G, Rios MB, Shan J, Andreeff M, Keating M, Talpaz M. Results of decitabine (five-aza-2'deoxycytidine) therapy in 130 patients with chronic myelogenous leukemia. Cancer. 2003;98(3):522–528. [PubMed] [Google Scholar]
49. Oki Y, Kantarjian HM, Gharibyan V, Jones D, O'Brien S, Verstovsek S, Cortes J, Morris GM, Garcia-Manero G, Issa JP. Phase 2 report of low-dose decitabine in combination with imatinib mesylate in patients with accelerated or myeloid blastic phase of chronic myelogenous leukemia. Cancer. 2007;109(5):899–906. [PubMed] [Google Scholar]
50. Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A, Tezcan I, Ersoy F, Kayserili H, Ugazio AG, Brousse N, Muramatsu M, et al. Activation-induced cytidine deaminase (Help) deficiency causes the autosomal recessive form of the syndrome (HIGM2) Jail cell. 2000;102(five):565–575. [PubMed] [Google Scholar]
Articles from Oncotarget are provided here courtesy of Impact Journals, LLC
fostergavempurneth.blogspot.com
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960202/
0 Response to ") explain what it means for a function to be θ(1)."
Post a Comment