A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy.

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

HSD17B4 methylation enhances glucose dependence of BT-474 breast cancer cells and increases lapatinib sensitivity.

PURPOSE: HER2-positive breast cancer has a high chance of achieving pathological complete response when HSD17B4, responsible for peroxisomal ß-oxidation of very long-chain fatty acids (VLCFA) and estradiol, is methylation-silenced. Here, we aimed to identify the underlying molecular mechanism. METHODS: Using a HER2-positive breast cancer cell line, BT-474, control and knock-out (KO) clones were obtained. Metabolic characteristics were analyzed using a Seahorse Flux analyzer. RESULTS: HSD17B4 KO suppressed cellular proliferation, and enhanced sensitivity to lapatinib approximately tenfold. The KO led to accumulation of VLCFA and a decrease of polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid. HSD17B4 KO increased Akt phosphorylation, possibly via decreased DHA, and genes involved in oxidative phosphorylation (OxPhos) and electron transport chain (ETC) were upregulated. Increased mitochondrial ATP production in the KO cells was confirmed by extracellular flux analyzer. Increased OxPhos led to severe dependence of the KO cells on pyruvate from glycolysis. Suppression of glycolysis by lapatinib led to severe delayed suppression of OxPhos in KO cells. CONCLUSION: HSD17B4 KO in BT-474 cells caused a decrease of PUFAs, increased Akt phosphorylation, enhanced glucose dependence of OxPhos, and increased sensitivity to inhibition of HER2, upstream of Akt. This mechanism may be applicable to other HER2-positive glucose-dependent breast cancer cells with HSD17B4 silencing.

Targeting aberrant DNA hypermethylation as a driver of ATL leukemogenesis by using the new oral demethylating agent OR-2100.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1-infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

Prediction of tissue origin of adenocarcinomas in the esophagogastric junction by DNA methylation.

BACKGROUND: Prediction of tissue origin of esophagogastric junction (EGJ) adenocarcinomas can be important for therapeutic decision, but no molecular marker is available. Here, we aimed to develop such a marker taking advantage of tissue-specific profiles of DNA methylation. METHODS: DNA methylation profiles of gastric adenocarcinomas (GACs) were obtained by an Infinium HumanMethylation450 BeadChip array, and those of esophageal adenocarcinoma (EACs) were obtained from the TCGA database. DNA from formalin-fixed paraffin-embedded (FFPE) samples was analyzed by bisulfite pyrosequencing. RESULTS: In the screening set, 51 of 145,841 CpG sites in CpG islands were methylated at significantly higher levels in 30 GACs compared to those in 30 EACs. Among them, SLC46A3 and cg09177106 were unmethylated in all the 30 EACs. Predictive powers of these two markers were successfully confirmed in an independent validation set (18 GACs and 18 EACs) (SLC46A3, sensitivity = 77.8%, specificity = 100%; cg09177106, sensitivity = 83.3%, specificity = 94.4%), and could be applied to FFPE samples (37 GACs and 18 EACs) (SLC46A3, P = 0.0001; cg09177106, P = 0.0028). On the other hand, EAC-specific markers informative in the FFPE samples could not be isolated. Using these GAC-specific markers, nine of 46 (19.6%) TCGA EGJ adenocarcinomas were predicted to be GACs. CONCLUSIONS: Two GAC-specific markers, SLC46A3 and cg09177106, had a high specificity for identifying the tissue origin of EGJ adenocarcinoma.

SAA1 is upregulated in gastric cancer-associated fibroblasts possibly by its enhancer activation.

Cancer-associated fibroblasts (CAFs) tend to have tumor-promoting capacity, and can provide therapeutic targets. Even without cancer cells, CAF phenotypes are stably maintained, and DNA methylation and H3K27me3 changes have been shown to be involved. Here, we searched for a potential therapeutic target in primary CAFs from gastric cancer and a mechanism for its dysregulation. Expression microarray using eight CAFs and seven non-CAFs (NCAFs) revealed that serum amyloid A1 (SAA1), which encodes an acute phase secreted protein, was second most upregulated in CAFs, following IGF2. Conditioned medium (CM) derived from SAA1-overexpressing NCAFs was shown to increase migration of gastric cancer cells compared with that from control NCAFs, and its tumor-promoting effect was comparable to that of CM from CAFs. In addition, increased migration of cancer cells by CM from CAFs was mostly canceled with CM from CAFs with SAA1 knockdown. Chromatin immunoprecipitation (ChIP)-quantitative PCR showed that CAFs had higher levels of H3K27ac, an active enhancer mark, in the promoter and the two far upstream regions of SAA1 than NCAFs. Also, BET bromodomain inhibitors, JQ1 and mivebresib, decreased SAA1 expression and tumor-promoting effects in CAFs, suggesting SAA1 upregulation by enhancer activation in CAFs. Our present data showed that SAA1 is a candidate therapeutic target from gastric CAFs and indicated that increased enhancer acetylation is important for its overexpression.

Combination of a synthetic retinoid and a DNA demethylating agent induced differentiation of neuroblastoma through retinoic acid signal reprogramming.

BACKGROUND: The CpG island methylator phenotype of neuroblastoma (NBL) is strongly associated with poor prognosis and can be targeted by 5-aza-2'-deoxycytidine (5-aza-dC). Differentiation therapy is a standard maintenance therapy for high-risk NBLs. However, the in vivo effect of tamibarotene, a synthetic retinoic acid, and the efficacy of its combination with 5-aza-dC have not been studied. Here, we conducted a preclinical study to assess the in vivo tamibarotene effect and the combination. METHODS: Treatment effects were analysed by in vitro cell growth and differentiation state and by in vivo xenograft suppression. Demethylated genes were analysed by DNA methylation microarrays and geneset enrichment. RESULTS: Tamibarotene monotherapy induced neural extension and upregulation of differentiation markers of NBL cells in vitro, and tumour regression without severe side effects in vivo. 5-Aza-dC monotherapy suppressed tumour growth both in vitro and in vivo, and induced demethylation of genes related to nervous system development and function. Pre-treatment with 5-aza-dC in vitro enhanced upregulation of differentiation markers and genes involved in retinoic acid signaling. Pre-treatment with 5-aza-dC in vivo significantly suppressed tumour growth and reduced the variation in tumour sizes. CONCLUSIONS: Epigenetic drug-based differentiation therapy using 5-aza-dC and TBT is a promising strategy for refractory NBLs.

Genetic and epigenetic alterations in normal tissues have differential impacts on cancer risk among tissues.

Genetic and epigenetic alterations are both involved in carcinogenesis, and their low-level accumulation in normal tissues constitutes cancer risk. However, their relative importance has never been examined, as measurement of low-level mutations has been difficult. Here, we measured low-level accumulations of genetic and epigenetic alterations in normal tissues with low, intermediate, and high cancer risk and analyzed their relative effects on cancer risk in the esophagus and stomach. Accumulation of genetic alterations, estimated as a frequency of rare base substitution mutations, significantly increased according to cancer risk in esophageal mucosae, but not in gastric mucosae. The mutation patterns reflected the exposure to lifestyle risk factors. In contrast, the accumulation of epigenetic alterations, measured as DNA methylation levels of marker genes, significantly increased according to cancer risk in both tissues. Patients with cancer (high-risk individuals) were precisely discriminated from healthy individuals with exposure to risk factors (intermediate-risk individuals) by a combination of alterations in the esophagus (odds ratio, 18.2; 95% confidence interval, 3.69-89.9) and by only epigenetic alterations in the stomach (odds ratio, 7.67; 95% confidence interval, 2.52-23.3). The relative importance of epigenetic alterations upon genetic alterations was 1.04 in the esophagus and 2.31 in the stomach. The differential impacts among tissues will be critically important for effective cancer prevention and precision cancer risk diagnosis.

Cancer cell niche factors secreted from cancer-associated fibroblast by loss of H3K27me3.

OBJECTIVE: Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not fully understood. We aimed to show the critical role of epigenetic changes in CAFs in maintaining their tumour-promoting capacity and to show the validity of the epigenomic approach in identifying therapeutic targets from CAFs to starve cancer cells. DESIGN: Twelve pairs of primary gastric CAFs and their corresponding non-CAFs (NCAFs) were established from surgical specimens. Genome-wide DNA methylation and H3K27me3 analyses were conducted by BeadArray 450K and ChIP-on-Chip, respectively. Functions of potential a therapeutic target were analysed by inhibiting it, and prognostic impact was assessed in a database. RESULTS: CAFs had diverse and distinct DNA methylation and H3K27me3 patterns compared with NCAFs. Loss of H3K27me3, but not DNA methylation, in CAFs was enriched for genes involved in stem cell niche, cell growth, tissue development and stromal-epithelial interactions, such as WNT5A, GREM1, NOG and IGF2. Among these, we revealed that WNT5A, which had been considered to be derived from cancer cells, was highly expressed in cancer stromal fibroblasts, and was associated with poor prognosis. Inhibition of secreted WNT5A from CAFs suppressed cancer cell growth and migration. CONCLUSIONS: H3K27me3 plays a crucial role in defining tumour-promoting capacities of CAFs, and multiple stem cell niche factors were secreted from CAFs due to loss of H3K27me3. The validity of the epigenetic approach to uncover therapeutic targets for cancer-starving therapy was demonstrated.

Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease.

End-stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of 2 major histopathological types: clear-cell RCCs (ESRD-ccRCCs) and acquired cystic disease (ACD)-associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. Here, we investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD-ccRCCs and 7 ACD-associated RCCs to identify their molecular alterations and cellular origins. Targeted sequencing of 409 cancer-related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD-ccRCCs harbored frequent VHL mutations, while ACD-associated RCCs did not. CNA analysis showed that ESRD-ccRCCs had a frequent loss of chromosome 3p while ACD-associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD-ccRCCs had methylation profiles similar to those of sporadic ccRCCs, while ACD-associated RCCs had profiles similar to those of papillary RCCs. Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD-ccRCCs and ACD-associated RCCs had high expression of proximal tubule cell marker genes, while chromophobe RCCs had high expression of distal tubule cell/collecting duct cell marker genes. In conclusion, ESRD-ccRCCs and ACD-associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these 2 histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron.

Epigenetic priming sensitizes gastric cancer cells to irinotecan and cisplatin by restoring multiple pathways.

BACKGROUND: Gastric cancer is heavily influenced by aberrant DNA methylation that alters multiple cancer-related pathways, and may respond to DNA demethylating agents, such as 5-aza-2'-deoxycytidine (5-aza-dC). Here, we aimed to analyze whether 5-aza-dC can sensitize gastric cancer cells to clinically used cytotoxic drugs. METHODS: Ten gastric cancer cell lines were treated with 5-aza-dC for 72 h and their growth was analyzed by conducting WST assay. In vivo effect of the drugs was analyzed using xenografts of OCUM-2 M/SN38 cells. Genome-wide expression and DNA methylation analyses were conducted using microarrays, and biological functions were identified through ingenuity pathway analysis. RESULTS: The cell lines most resistant to SN38 (an active metabolite of irinotecan), CDDP, PTX, and 5-FU, were identified. 5-Aza-dC pre-treatment of the resistant cell lines decreased the IC50 values for SN38 (TMK1, 226.4 nM to 32.91 nM; 44As3, 128.2 nM to 19.32 nM; OCUM2 M/SN38, 74.43 nM to 16.47 nM) and CDDP (TMK1, 5.05 µM to 2.26 µM; OCUM2 M, 10.79 µM to 2.77 µM), but not PTX and 5-FU. The reactivation of apoptosis-related genes, such as RUNX3, PYCARD, TNF, FAS, and FASLG, was induced by pre-treatment with 5-aza-dC, and the DNA demethylation of promoter CpG islands of RUNX3 and PYCARD was confirmed. In a xenograft model with OCUM2 M/SN38, treatment with 5-aza-dC before irinotecan showed markedly enhanced tumor suppression. CONCLUSION: Epigenetic priming with 5-aza-dC can improve the sensitivity of gastric cancer cells to SN38 and CDDP.

Correction to: Epigenetic priming sensitizes gastric cancer cells to irinotecan and cisplatin by restoring multiple pathways.

Recently published the priming.

Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model.

Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation-related genes were observed in the colonic epithelial cells of the AOM/DSS-treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P < .01) and area of occupancy from 50.1 ± 57.4 to 0.5 ± 1.4 mm2 (P < .01). Aberrant DNA methylation of three marker CpG islands (Cbln4, Fosb, and Msx1) was induced by AOM/DSS treatment in colonic mucosae, but this increase was suppressed by 50%-92% (P < .05) with antibiotic treatment. Microbiome analysis showed that this change was associated with a decrease of the Clostridium leptum subgroup. These data indicate that antibiotics suppressed tumorigenesis through inhibition of aberrant DNA methylation induced by chronic inflammation.

Establishment of a DNA methylation marker to evaluate cancer cell fraction in gastric cancer.

BACKGROUND: Tumor samples are unavoidably contaminated with coexisting normal cells. Here, we aimed to establish a DNA methylation marker to estimate the fraction of gastric cancer (GC) cells in any DNA sample by isolating genomic regions specifically methylated in GC cells. METHODS: Genome-wide and gene-specific methylation analyses were conducted with an Infinium HumanMethylation450 BeadChip array and by quantitative methylation-specific PCR, respectively. Purified cancer and noncancer cells were prepared by laser-capture microdissection. TP53 mutation data were obtained from our previous study using next-generation target sequencing. RESULTS: Genome-wide DNA methylation analysis of 12 GC cell lines, 30 GCs, six normal gastric mucosae, one sample of peripheral leukocytes, and four noncancerous gastric mucosae identified OSR2, PPFIA3, and VAV3 as barely methylated in normal cells and highly methylated in cancer cells. Quantitative methylation-specific PCR using 26 independent GCs validated that one or more of them was highly methylated in all of the GCs. Using four pairs of purified cells, we confirmed the three genes were highly methylated (85 % or more) in cancer cells and barely methylated (5 % or less) in noncancer cells. The cancer cell fraction assessed by the panel of the three genes showed good correlation with that assessed by the TP53 mutant allele frequency in 13 GCs (r = 0.77). After correction of the GC cell fraction, unsupervised clustering analysis of the genome-wide DNA methylation profiles yielded clearer clustering. CONCLUSIONS: A DNA methylation marker-namely, the panel of the three genes-is useful to estimate the cancer cell fraction in GCs.

Identification of coexistence of DNA methylation and H3K27me3 specifically in cancer cells as a promising target for epigenetic therapy.

Alterations of epigenetic modifications are promising targets for cancer therapy, and several epigenetic drugs are now being clinically utilized. At the same time, individual epigenetic modifications have physiological functions in normal cells, and cancer cell specificity is considered difficult to achieve using a drug against a single epigenetic modification. To overcome this limitation, a combination of epigenetic modifications specifically or preferentially present in cancer cells is a candidate target. In this study, we aimed to demonstrate (i) the presence of a cancer cell-specific combination of epigenetic modifications by focusing on DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3) and (ii) the therapeutic efficacy of a combination of DNA demethylation and EZH2 inhibition. Analyses of DNA methylation and H3K27me3 in human colon, breast and prostate cancer cell lines revealed that 24.7±4.1% of DNA methylated genes had both DNA methylation and H3K27me3 (dual modification) in cancer cells, while it was 11.8±7.1% in normal cells. Combined treatment with a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC) and an EZH2 inhibitor, GSK126, induced marked re-expression of genes with the dual modification, including known tumor-suppressor genes such as IGFBP7 and SFRP1, and showed an additive inhibitory effect on growth of cancer cells in vitro. Finally, an in vivo combined treatment with 5-aza-dC and GSK126 inhibited growth of xenograft tumors more efficiently than a single treatment with 5-aza-dC. These results showed that the dual modification exists specifically in cancer cells and is a promising target for cancer cell-specific epigenetic therapy.

Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells.

Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.

Compendium of aberrant DNA methylation and histone modifications in cancer.

Epigenetics now refers to the study or research field related to DNA methylation and histone modifications. Historically, global DNA hypomethylation was first revealed in 1983, and, after a decade, silencing of a tumor suppressor gene by regional DNA hypermethylation was reported. After the proposal of the histone code in the 2000s, alterations of histone methylation were also identified in cancers. Now, it is established that aberrant epigenetic alterations are involved in cancer development and progression, along with mutations and chromosomal losses. Recent cancer genome analyses have revealed a large number of mutations of epigenetic modifiers, supporting their important roles in cancer pathogenesis. Taking advantage of the reversibility of epigenetic alterations, drugs targeting epigenetic regulators and readers have been developed for restoration of normal pattern of the epigenome, and some have already demonstrated clinical benefits. In addition, DNA methylation of specific marker genes can be used as a biomarker for cancer diagnosis, including risk diagnosis, detection of cancers, and pathophysiological diagnosis. In this paper, we will summarize the major concepts of cancer epigenetics, placing emphasis on history.

Corrigendum to "Cigarette smoke, but not novel tobacco vapor products, causes epigenetic disruption and cell apoptosis" [Biochem. Biophys. Rep. 24 (2020) 100865].

[This corrects the article DOI: 10.1016/j.bbrep.2020.100865.].

Intrinsic signaling pathways modulate targeted protein degradation.

Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.

DNA Methylation Analysis.

DNA methylation of promoter CpG islands silences their downstream genes, and enhancer methylation can be associated with decreased or increased gene expression. DNA methylation alterations in normal and diseased cells provide rich information, such as tissue origin, disease risk, patient response, and prognosis. DNA methylation status is detected by bisulfite conversion, which converts unmethylated cytosines into uracils but methylated cytosines very inefficiently. A genome-wide DNA methylation analysis is conducted by a BeadChip microarray or next-generation sequencing (NGS) of bisulfite-treated DNA. A region-specific DNA methylation analysis can be conducted by various methods, such as methylation-specific PCR (MSP), quantitative MSP, and bisulfite sequencing. This chapter provides protocols for bisulfite-mediated conversion, a BeadChip array-based method (Infinium), quantitative MSP, and bisulfite sequencing.

Partial endothelial-to-mesenchymal transition mediated by HIF-induced CD45 in neointima formation upon carotid artery ligation.

AIMS: Endothelial-to-mesenchymal transition (EndMT) is a fundamental process in vascular remodelling. However, the precise regulatory mechanism of vascular remodelling during neointima formation and the source of neointima cells are not entirely understood. METHODS AND RESULTS: To investigate the origin of neointima cells and their relevance to vascular wall remodelling, we used an endothelial cell (EC)-specific lineage tracing system [VE-Cadherin (Cdh5)-BAC-CreERT2 mice] and carotid artery ligation model and showed evidence that resident ECs transdifferentiate into neointima cells with the expression of CD45. During the early stages of neointima formation, ECs transiently expressed CD45, a haematopoietic marker, accompanied by a host of EndMT markers, and CD31 and αSMA were prominently expressed in developing neointima. In vitro, CD45-positive EndMT was induced by stabilization of HIF1α with cobalt chloride or with a VHL inhibitor in human primary ECs, which mimicked the hypoxic condition of the ligated artery, and promoted the formation of an integrin α11-shank-associated RH domain-interacting protein (SHARPIN) complex. Notably, a CD45 phosphatase inhibitor disrupted this integrin α11-SHARPIN complex, thereby destabilizing cell-cell junctions. Deletion of Hif1α in ECs suppressed expression of CD45 and EndMT markers and ameliorated neointima formation. CONCLUSION: These results suggest that the HIF-induced CD45 expression is normally required for the retention of an EC fate and cell-cell junctions, CD45-positive EndMT (termed as 'partial EndMT') contributes to neointima formation and vascular wall remodelling.