Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing.

Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.

Potent Estrogen Receptor ß Agonists with Inhibitory Activity In Vitro , Fail to Suppress Xenografts of Endocrine-Resistant Cyclin-dependent Kinase 4/6 inhibitor-Resistant Breast Cancer Cells.

Objective: Seventy percent of newly diagnosed breast cancers are estrogen receptor-α positive and HER2/neu negative [1]. First-line treatments incorporate endocrine therapy and cyclin-dependent kinase 4/6 inhibitors [2]. However, therapy resistance occurs in most patients [3-5]. Hence, there is an urgent need for effective second-line treatments. We previously showed that the potent estrogen receptor-ß agonists, OSU-ERb-12 and LY500307, synergized with the selective estrogen receptor modulator, tamoxifen, in vitro. Furthermore, we showed that these compounds inhibited endocrine-resistant and cyclin-dependent kinase 4/6-inhibitor-resistant estrogen receptor α-positive cell lines in vitro [6]. Here, we used fulvestrant- and abemaciclib-resistant T47D-derived cell line xenografts to determine the efficacy of the combination of OSU-ERb-12 and LY500307 with tamoxifen in vivo. Results: Despite efficacy in vitro, treatments failed to reduce xenograft tumor volumes. Hence, we conclude that this treatment strategy lacks direct cancer cell-intrinsic cytotoxic efficacy in vivo.

AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia.

We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19(+) spleen B cells from Eµ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.

Structure-activity relationships for 4-anilinoquinoline derivatives as inhibitors of the DNA methyltransferase enzyme DNMT1.

A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing side chains of varying pK(a), were prepared by acid-catalysed coupling of the pre-formed side chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were effective, whereas less strongly basic ((diaminomethylene)hydrazono)ethyl or 3-methylpyrimidine-2,4-diamine side chains allowed both NHCO- and CONH-linked compounds to show activity. In contrast, the pK(a) of the quinoline unit had little apparent influence on activity.

Lipid-based nanoparticle delivery of Pre-miR-107 inhibits the tumorigenicity of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. Our laboratory showed that miR-107 expression is reduced and functions as a tumor suppressor gene in HNSCC suggesting the potential application of miR-107 as a novel anticancer therapeutic. In this study, we determined the efficiency and efficacy of cationic lipid nanoparticles to deliver pre-miR-107 (NP/pre-miR-107) in HNSCC cells in vitro and in vivo. NP/pre-miR-107 increased delivery of miR-107 into HNSCC cells by greater than 80,000-fold compared to free pre-miR-107. Levels of known miR-107 targets, protein kinase Cε (PKCε), cyclin-dependent kinase 6 (CDK6), and hypoxia-inducible factor 1-ß (HIF1-ß), decreased following NP/pre-miR-107 treatment. Clonogenic survival, cell invasion, and cell migration of HNSCC cells was inhibited with NP/pre-miR-107. Moreover, NP/pre-miR-107 reduced the cancer-initiating cell (CIC) population and dampened the expression of the core embryonic stem cell transcription factors, Nanog, Oct3/4, and Sox2. In a preclinical mouse model of HNSCC, systemic administration of NP/pre-miR-107 significantly retarded tumor growth by 45.2% compared to NP/pre-miR-control (P < 0.005, n = 7). Kaplan-Meier analysis showed a survival advantage for the NP/pre-miR-107 treatment group (P = 0.017). Our results demonstrate that cationic lipid nanoparticles are an effective carrier approach to deliver therapeutic miRs to HNSCC.

Augmentation of Extracellular ATP Synergizes With Chemotherapy in Triple Negative Breast Cancer.

Introduction: Breast cancer affects two million patients worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP is degraded by eATPases. Given that eATP is toxic to cancer cells, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy. Methods: TNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPases or specific antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively. Results: In the present study, we observed dose-dependent declines of cell viability and increases in eATP of paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases, but not of the MCF-10A cell line. These effects were reversed by specific antagonists of P2RXs. Similar results, as those observed with eATPase inhibitors, were seen with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels. Conclusion: These results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines, which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.

Activity of Estrogen Receptor ß Agonists in Therapy-Resistant Estrogen Receptor-Positive Breast Cancer.

Background: Among women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERß plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERß agonists without ERα activity has limited the exploration of ERß activation as a strategy for ERα+ breast cancer. Methods: We measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERß agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERß agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERß agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2-regulated genes. Results: In this study, we demonstrate the efficacy of highly selective ERß agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERß agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERß agonists and ERα antagonists suggested that the efficacy of ERß agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERß gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples. Conclusion: Our results demonstrate that highly selective ERß agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.

MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib.

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.

Lyn kinase and ZAP70 are substrates of PTPROt in B-cells: Lyn inactivation by PTPROt sensitizes leukemia cells to VEGF-R inhibitor pazopanib.

We have recently shown that the gene encoding the truncated form of protein tyrosine phosphatase receptor-type O (PTPROt) expressed predominantly in hematopoietic cells is epigenetically silenced in human primary chronic lymphocytic leukemia (B-CLL). To determine whether increased phosphorylation of the PTPROt substrates following PTPROt suppression alters signal transduction pathway(s) that impart a growth advantage to the leukemic lymphocytes, it is critical to discern the key substrates of PTPROt. Here, we used substrate-trapping assay to identify two novel substrates of PTPROt, the tyrosine kinases Lyn and ZAP70. Both Lyn and ZAP70 were dephosphorylated by wild-type PTPROt, but not by its catalytic site (CS) mutant. A critical phosphorylation site in Lyn, Y397, essential for its activity was dephosphorylated by PTPROt. Consequently, the activity of Lyn kinase was compromised when co-expressed with PTPROt-WT compared to vector control or upon co-expression with PTPROt-CS. Ectopic expression of PTPROt in Raji cells reduced phosphorylation of Lyn in the absence of any change in its protein levels. These results have revealed the physiological importance of PTPROt in regulating B-cell receptor signaling at Lyn kinase. Further, ectopic expression of PTPROt also sensitized the cells to the VEGF-R inhibitor Pazopanib.

Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription.

In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity.

Estrogen-mediated suppression of the gene encoding protein tyrosine phosphatase PTPRO in human breast cancer: mechanism and role in tamoxifen sensitivity.

We have previously demonstrated the tumor suppressor characteristics of protein tyrosine phosphatase receptor-type O (PTPRO) in leukemia and lung cancer, including its suppression by promoter methylation. Here, we show tumor-specific methylation of the PTPRO CpG island in primary human breast cancer. PTPRO expression was significantly reduced in established breast cancer cell lines MCF-7 and MDA-MB-231 due to promoter methylation compared with its expression in normal human mammary epithelial cells (48R and 184). Further, the silenced gene could be demethylated and reactivated in MCF-7 and MDA-MB-231 cells upon treatment with 5-Azacytidine, a DNA hypomethylating agent. Because PTPRO promoter harbors estrogen-responsive elements and 17beta-estradiol (E2) plays a role in breast carcinogenesis, we examined the effect of E2 and its antagonist tamoxifen on PTPRO expression in human mammary epithelial cells and PTPRO-expressing breast cancer cell line Hs578t. Treatment with E2 significantly curtailed PTPRO expression in 48R and Hs578t cells, which was facilitated by ectopic expression of estrogen receptor (ER)beta but not ERalpha. On the contrary, treatment with tamoxifen increased PTPRO expression. Further, knockdown of ERbeta by small interfering RNA abolished these effects of E2 and tamoxifen. Chromatin immunoprecipitation assay showed association of c-Fos and c-Jun with PTPRO promoter in untreated cells, which was augmented by tamoxifen-mediated recruitment of ERbeta to the promoter. Estradiol treatment resulted in dissociation of c-Fos and c-Jun from the promoter. Ectopic expression of PTPRO in the nonexpressing MCF-7 cells sensitized them to growth-suppressive effects of tamoxifen. These data suggest that estrogen-mediated suppression of PTPRO is probably one of the early events in estrogen-induced tumorigenesis and that expression of PTPRO could facilitate endocrine therapy of breast cancer.

Estrogen Receptor Beta (ERß): A Ligand Activated Tumor Suppressor.

Estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) belong to a superfamily of nuclear receptors called steroid hormone receptors, which, upon binding ligand, dimerize and translocate to the nucleus where they activate or repress the transcription of a large number of genes, thus modulating critical physiologic processes. ERß has multiple isoforms that show differing association with prognosis. Expression levels of the full length ERß1 isoform are often lower in aggressive cancers as compared to normal tissue. High ERß1 expression is associated with improved overall survival in women with breast cancer. The promise of ERß activation, as a potential targeted therapy, is based on concurrent activation of multiple tumor suppressor pathways with few side effects compared to chemotherapy. Thus, ERß is a nuclear receptor with broad-spectrum tumor suppressor activity, which could serve as a potential treatment target in a variety of human cancers including breast cancer. Further development of highly selective agonists that lack ERα agonist activity, will be necessary to fully harness the potential of ERß.

Metallothionein expression is suppressed in primary human hepatocellular carcinomas and is mediated through inactivation of CCAAT/enhancer binding protein alpha by phosphatidylinositol 3-kinase signaling cascade.

Reactive oxygen species (ROS) resulting from chronic inflammation cause liver injury leading to transformation of regenerating hepatocytes. Metallothioneins (MT), induced at high levels by oxidative stress, are potent scavengers of ROS. Here, we report that the levels of MT-1 and MT-2A are drastically reduced in primary human hepatocellular carcinomas (HCCs) and in diethylnitrosamine-induced liver tumors in mice, which is primarily due to transcriptional repression. Expression of the transcription factor, MTF-1, essential for MT expression, and its target gene Zn-T1 that encodes the zinc transporter-1 was not significantly altered in HCCs. Inhibitors of both phosphatidylinositol 3-kinase (PI3K) and its downstream target AKT increased expression of MT genes in HCC cells but not in liver epithelial cells. Suppression of MT-1 and MT-2A by ectopic expression of the constitutively active PI3K or AKT and their up-regulation by dominant-negative PI3K or AKT mutant confirmed negative regulation of MT expression by PI3K/AKT signaling pathway. Further, treatment of cells with a specific inhibitor of glycogen synthase kinase-3 (GSK-3), a downstream effector of PI3K/AKT, inhibited MT expression specifically in HCC cells. Short interfering RNA-mediated depletion of CCAAT/enhancer binding protein alpha (C/EBPalpha), a target of GSK-3, impeded MT expression, which could not be reversed by PI3K inhibitors. DNA binding activity of C/EBPalpha and its phosphorylation at T222 and T226 by GSK-3 are required for MT expression. MTF-1 and C/EBPalpha act in concert to increase MT-2A expression, which probably explains the high level of MT expression in the liver. This study shows the role of PI3K/AKT signaling pathway and C/EBPalpha in regulation of MT expression in hepatocarcinogenesis.

DNA methyltransferase 3b regulates nerve growth factor-induced differentiation of PC12 cells by recruiting histone deacetylase 2.

To elucidate the role of epigenetic reprogramming in cell- or tissue-specific differentiation, we explored the role of DNA methyltransferases (Dnmts) in the nerve growth factor (NGF)-induced differentiation of PC12 (pheochromocytoma) cells into neuronal cells. The mRNA and protein levels of de novo methyltransferase Dnmt3b increased, whereas those of Dnmt3a and Dnmt1 decreased, during NGF-induced neurite outgrowth. Dnmt3b localized in the nucleus, as well as in the growing neurites. When the expression of Dnmt3b was inhibited by antisense or small interfering RNA, PC12 cells continued to proliferate and failed to generate neurites. Cells depleted of Dnmt3b were unable to exit the cell cycle even after 6 days of NGF treatment. Furthermore, this failure in differentiation correlated with significant attenuation in tyrosine phosphorylation of TrkA (a marker for NGF-induced differentiation) and reduced the expression of neuronal markers, Hu antigen, and MAP2. The methyl-CpG content of the PC12 genome or the methylation status of repetitive elements was not significantly altered after differentiation and was not affected by Dnmt3b depletion. This was consistent with the ability of the catalytic-site mutant of Dnmt3b to induce differentiation in Dnmt3b-depleted cells after NGF treatment. The Dnmt3b-mediated differentiation was attributed to its N-terminal domain, which recruits histone deacetylase 2 (Hdac2), as demonstrated by (i) impeding of differentiation by the Hdac inhibitors, (ii) facilitation of the differentiation process by overexpression of the N-terminal domain of Dnmt3b, (iii) higher Hdac activity associated with Dnmt3b after NGF treatment, and (iv) coimmunoprecipitation and cosedimentation of Dnmt3b specifically with Hdac2 in a glycerol density gradient. These data indicate a novel role of Dnmt3b in neuronal differentiation.

5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal.

5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.