Analysis of an antioxidative defence system of hydrogen peroxide-treated pancreatic islet-derived 1.1B4 cells and siRNA targeting NR4A3-treated cells by microarray.

Pancreatic islet ß-cells weaken under oxidative stress. In this study, human pancreatic islet-derived 1.1B4 cells were exposed to H2O2 and analysed using a human microarray, which revealed that heme oxygenase 1 (HMOX1), glutamate-cysteine ligase, early growth response 1, nuclear receptor subfamily 4 group A member 3 (NR4A3) and jun B proto-oncogene were upregulated, whereas superoxide dismutase 1 and catalase were not. Expression of NR4A3 rapidly increased after H2O2 addition, and the 1.1B4 cells treated with siRNA targeting NR4A3 became sensitive to H2O2; further, HMOX1 expression was strongly inhibited, suggesting that NR4A3 is an oxidative stress-responsive transcription factor that functions through HMOX1 expression in pancreatic islet ß-cells. Expression of cyclin E1 and cyclin-dependent kinase 1 was also inhibited by siRNAs targeting NR4A3.

Time dependency and unique etiology of barotrauma in COVID-19: A retrospective cohort study with landmark analysis and pathological approach.

BACKGROUND: Barotrauma frequently occurs in coronavirus disease 2019. Previous studies have reported barotrauma to be a mortality-risk factor; however, its time-dependent nature and pathophysiology are not elucidated. To investigate the time-dependent characteristics and the etiology of coronavirus disease 2019-related-barotrauma. METHODS AND FINDINGS: We retrospectively reviewed intubated patients with coronavirus disease 2019 from March 2020 to May 2021. We compared the 90-day survival between the barotrauma and non-barotrauma groups and performed landmark analyses on days 7, 14, 21, and 28. Barotrauma within seven days before the landmark was defined as the exposure. Additionally, we evaluated surgically treated cases of coronavirus disease 2019-related pneumothorax. We included 192 patients. Barotrauma developed in 44 patients (22.9%). The barotrauma group's 90-day survival rate was significantly worse (47.7% vs. 82.4%, p < 0.001). In the 7-day landmark analysis, there was no significant difference (75.0% vs. 75.7%, p = 0.79). Contrastingly, in the 14-, 21-, and 28-day landmark analyses, the barotrauma group's survival rates were significantly worse (14-day: 41.7% vs. 69.1%, p = 0.044; 21-day: 16.7% vs. 62.5%, p = 0.014; 28-day: 20.0% vs. 66.7%, p = 0.018). Pathological examination revealed a subpleural hematoma and pulmonary cyst with heterogenous lung inflammation. CONCLUSIONS: Barotrauma was a poor prognostic factor for coronavirus disease 2019, especially in the late phase. Heterogenous inflammation may be a key finding in its mechanism. Barotrauma is a potentially important sign of lung destruction.

Commissioning and performance evaluation of commercially available mobile imager for image guided total body irradiation.

BACKGROUND: The setup of lung shield (LS) in total body irradiation (TBI) with the computed radiography (CR) system is a time-consuming task and has not been quantitatively evaluated. The TBI mobile imager (TBI-MI) can solve this problem through real-time monitoring. Therefore, this study aimed to perform commissioning and performance evaluation of TBI-MI to promote its use in clinical practice. METHODS: The source-axis distance in TBI treatment, TBI-MI (CNERGY TBI, Cablon Medical B.V.), and the LS position were set to 400, 450, and 358 cm, respectively. The evaluation items were as follows: accuracy of image scaling and measured displacement error of LS, image quality (linearity, signal-to-noise ratio, and modulation transfer function) using an EPID QC phantom, optimal thresholding to detect intra-fractional motion in the alert function, and the scatter radiation dose from TBI-MI. RESULTS: The accuracy of image scaling and the difference in measured displacement of the LS was <4 mm in any displacements and directions. The image quality of TBI imager was slightly inferior to the CR image but was visually acceptable in clinical practice. The signal-to-noise ratio was improved at high dose rate. The optimal thresholding value to detect a 10-mm body displacement was determined to be approximately 5.0%. The maximum fraction of scattering radiation to irradiated dose was 1.7% at patient surface. CONCLUSION: MI-TBI can quantitatively evaluate LS displacement with acceptable image quality. Furthermore, real-time monitoring with alert function to detect intrafraction patient displacement can contribute to safe TBI treatment.

Clinical impact of p16 positivity in nasopharyngeal carcinoma.

Purpose: The clinical characteristics and prognosis of HPV-related nasopharyngeal cancer (NPC) remain controversial. The relationship between p16 status and outcome was retrospectively investigated in the NPC patients. Materials and Methods: Between May 2009 and May 2019, 81 NPC patients who received definitive radiation therapy, in a hospital in Japan, were identified and the prognosis was investigated. p16, p53, and Epstein-Barr virus (EBV) status were assessed. Also, circumferential tumor extent in the nasopharyngeal cavity was assessed on a 5-point scale. Results: Nine and 72 patients were p16-positive and p16-negative, respectively. Fewer patients were EBV-encoded RNA in situ hybridization (EBER-ISH)-positive in the p16-positive group than in the p16-negative group (p < .01). Seventy-five patients were nonkeratinizing NPCs, and six patients were keratinizing NPCs. There were two p16-positive patients among the keratinizing NPCs.The mean circumferential tumor extent scores of 16-positive and p16-negative NPCs were 4.2 and 3.2, respectively with a statistically significant difference (p = .02). Two-year progression-free survival (PFS) of p16-positive and p16-negative patients undergoing chemoradiation therapy were 100% and 69%, respectively (p = .13). Conclusion: In this study conducted in Japan, p16-positive NPC patients are minor but not very low, and the proportion of keratinizing NPCs was small. p16-positive NPCs were seen both in keratinizing and nonkeratinizing NPCs. P16-positive NPC had a tendency of better PFS than p16-negative NPC. This better prognosis might be due to the higher radiosensitivity of the p16-positive cell. Additionally, p16-positive NPCs seemed to spread more extensively in circumference along the nasopharyngeal mucosa than p16-negative NPCs.

Effect of Hyaluronate Acid Injection on Dose-Volume Parameters in Brachytherapy for Cervical Cancer.

Purpose: Hyaluronate gel has been injected as a spacer into the rectovaginal fossa and vesicouterine fossa during brachytherapy for patients with cervical cancer at our institution. The effect of hyaluronate gel injection (HGI) on dose-volume parameters was investigated in this study. Methods and Materials: Between July 2008 to January 2020, a total of 104 patients (non-HGI group: 52 patients; HGI group: 52 patients) who underwent curative radiation therapy for cervical cancer were selected. The total doses of external beam radiation therapy and brachytherapy for high-risk clinical target volume (CTVHR) D90, bladder D2cc, and rectal D2cc were converted to the equivalent dose in 2 Gy fractions (EQD2) and were analyzed for association with HGI. Results: Median CTVHR D90 (EQD2) in the non-HGI group was 76.0 Gy (63.7-99.5 Gy), and in the HGI group it was 79.4 Gy (52.6-97.5 Gy) (P = .017). The median bladder D2cc and rectal D2cc (EQD2) were 62.9 Gy and 56.0 Gy in the non-HGI group and 63.7 Gy and 54.8 Gy in the HGI group, which had no significant difference. Conclusions: In cases with HGI, a significant CTVHR D90 dose increase was obtained with sufficient bladder and rectal doses suppression.

[Giant cell arteritis diagnosed by a temporal artery biopsy without abnormal imaging and physical findings in an elderly patient presenting with fever].

Giant cell arteritis (GCA) is considered in the differential diagnosis of fever of unknown origin in the elderly. We describe the case of an 83-year-old man with GCA diagnosed by temporal artery biopsy (TBA), who did not exhibit abnormal physical and imaging findings. The patient had fever and elevated C-reactive protein (CRP), which had persisted for two months. He was examined and treated with antibiotics and antipyretic analgesics in a local clinic, but they had little effect. He was referred to us. He showed no abnormal physical findings. Image examinations, including ultrasonography, CT, MRI, and PET-CT, showed no abnormal findings. We performed TBA. The histological examination of the artery showed inflammatory cell invasion and rupture of the internal elastic membrane, indicating GCA. We initiated oral corticosteroid treatment. The patient's fever quickly disappeared and his CRP level returned to normal. TBA has been the gold standard for the diagnosis of GCA. However, TBA is an invasive procedure and the sensitivity depends on the operator's skill level. Recently, imaging examinations have frequently been used for the diagnosis of GCA. The sensitivity of imaging examinations is similar to that of TBA. However, our case did not show any abnormal imaging findings and was only diagnosed by TBA. This case suggested that TBA remains a useful examination for elderly patients with fever that persists for a long time.

Salvage image-guided freehand interstitial brachytherapy for pelvic sidewall recurrence after hysterectomy for uterine malignancies.

PURPOSE: Pelvic sidewall recurrence after hysterectomy for uterine malignances has a poor prognosis, and the salvage therapy for this type of recurrence is still challenging. The purpose of this study was to investigate the efficacy of freehand high-dose-rate interstitial brachytherapy (HDR-ISBT) through the perineum using transrectal ultrasonography for this disease. METHODS AND MATERIALS: We retrospectively evaluated 42 patients with pelvic sidewall recurrence after hysterectomy for uterine cervical and endometrial cancers. We investigated patients' characteristics, the 2-year local control and survival rates, and late adverse events of the rectum and bladder. RESULTS: The 2-year overall survival, local control, and progression-free survival rates were 73.7% (95% confidence interval [CI], 60.8-89.3%), 69.4% (95% CI, 55.4-80.1%), and 37.3% (95% CI, 24.6-56.5%), respectively. In Cox multivariate analysis, tumor size at recurrence (<45 mm vs. ≥45 mm) (p = 0.04) and disease-free periods after hysterectomy (<10 months vs. ≥10 months) (p < 0.01) were significant prognostic factors for overall survival. Lymph node metastasis at recurrence (p < 0.01) was also a significant prognostic factor for progression-free survival. Three patients experienced Grade 3-4 late proctitis (7%). CONCLUSIONS: Transperineal freehand salvage HDR-ISBT using transrectal ultrasonography was demonstrated to be a curative treatment option for patients with pelvic sidewall recurrence following hysterectomy. Based on the findings of this study, we emphasize the importance of HDR-ISBT for pelvic sidewall recurrence.

Non-Robustness of Ang's Risk Classification in Human Papillomavirus-Related Oropharyngeal Squamous Cell Carcinoma in Japanese Patients.

BACKGROUND: Validity of the risk classification by Ang for human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) remains to be studied in the patients treated by modalities other than chemoradiotherapy and in Japanese patients. MATERIALS AND METHODS: Between 2010 and 2018, 122 patients with HPV-related OPSCC in stages III and IV by the TNM classification 7th edition (TNM-7) were treated curatively at a single institution in Japan. The median age was 62.7 years. Over 50% of the patients underwent surgery with or without adjuvant therapy. The influence of multiple factors on survival was analyzed. RESULTS: The amount of smoking dichotomized at 10 pack-year, which was used in Ang's risk classification, was not predictive of prognosis, and Ang's risk classification was not significantly influential on prognosis in multivariate analysis. In the patients treated with definitive radiation therapy, Ang's risk classification was not predictive of the prognosis in univariate analysis. The impact of smoking was significant only in the patients undergoing the definitive operation. CONCLUSIONS: Ang's risk classification was not robust in predicting the prognosis of general Japanese HPV-related OPSCC patients. The amount of smoking might have different prognostic influences depending on the therapeutic method.

Human adipose-derived mesenchymal stem cells prevent type 1 diabetes induced by immune checkpoint blockade.

AIMS/HYPOTHESIS: Immunomodulators blocking cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) have improved the treatment of a broad spectrum of cancers. These immune checkpoint inhibitors (ICIs) reactivate the immune system against tumour cells but can also trigger autoimmune side effects, including type 1 diabetes. Mesenchymal stem cell (MSC) therapy is the most prevalent cell therapy, with tissue-regenerating, anti-fibrosis and immunomodulatory functions provided by the secretome of the cells. Here, we examined whether systemic MSC treatment could prevent the development of type 1 diabetes in a NOD mouse model. METHODS: The purified PD-L1 monoclonal antibody was administered to induce diabetes in male NOD mice which normally do not develop diabetes. Human adipose-derived MSCs were administered by tail vein injections. T cells, macrophages and monocyte-derived macrophages expressing C-X-C motif chemokine ligand 9 (CXCL9) in pancreatic sections of NOD mice and a cancer patient who developed diabetes following the ICI treatments were analysed by immunofluorescence. Tissue localisation of the injected MSCs, plasma exosome levels and plasma cytokine profiles were also investigated. RESULTS: PD-1/PD-L1 blockade induced diabetes in 16 of 25 (64%) NOD mice which received anti-PD-L1 mAb without hMSCs [MSC(-)], whereas MSC administration decreased the incidence to four of 21 (19%) NOD mice which received anti-PD-L1 mAb and hMSCs [MSC(+)]. The PD-1/PD-L1 blockade significantly increased the area of CD3-positive T cells (6.2-fold) and macrophage-2 (Mac-2) antigen (2.5-fold)- and CXCL9 (40.3-fold)-positive macrophages in the islets. MSCs significantly reduced T cell (45%) and CXCL9-positive macrophage (67%) accumulation in the islets and the occurrence of diabetes. The insulin content (1.9-fold) and islet beta cell area (2.7-fold) were also improved by MSCs. T cells and CXCL9-positive macrophages infiltrated into the intricate gaps between the beta cells in the islets by PD-1/PD-L1 blockade. Such immune cell infiltration was largely prevented by MSCs. The most striking difference was observed in the CXCL9-positive macrophages, which normally did not reside in the beta cell region in the islets but abundantly accumulated in this area after PD-1/PD-L1 blockade and were prevented by MSCs. The CXCL9-positive macrophages were also observed in the islets of a cancer patient who developed diabetes following the administration of ICIs but few CXCL9-positive macrophages were observed in a control patient. Mechanistically, the injected MSCs accumulated in the lung but not in the pancreas and strongly increased plasma exosome levels and changed plasma cytokine profiles. CONCLUSIONS/INTERPRETATION: Our results suggest that MSCs can prevent the incidence of diabetes associated with immune checkpoint cancer therapy and may be worth further consideration for new adjuvant cell therapy.

Physiological background parenchymal uptake of 18F-FDG in normal breast tissues using dedicated breast PET: correlation with mammographic breast composition, menopausal status, and menstrual cycle.

OBJECTIVE: This study aimed to quantitatively evaluate the effects of age, mammographic density, menopausal status, and menstrual cycle on background parenchymal uptake (BPU) using ring-shaped dedicated breast positron emission tomography (dbPET). METHODS: This study included 186 adult women who underwent mammography and dbPET on the same day and had no abnormalities classified as Breast Imaging Reporting and Data System (BI-RADS) category 1 on both examinations. The volume of interest (VOI) was placed in the glandular tissue of both breasts, and the maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), and metabolic breast volume (MBV) were measured as indicators of BPU. We analyzed the correlation between BPU and age, mammographic density, menopausal status, and menstrual cycle. RESULTS: The SUVmax and SUVmean for normal breast tissue were inversely correlated with age (both p < 0.001). The SUVmax, SUVmean, and MBV of mammographically dense breast tissues were significantly higher than those of non-dense breast tissues (all p < 0.001). The SUVmax, SUVmean, and MBV of normal breast tissue in premenopausal women were significantly higher than those in postmenopausal women (p < 0.001, p < 0.001, p = 0.002, respectively). In the study, 59 premenopausal women, the SUVmax of normal breast tissue in the menstrual-follicular phase was significantly lower than that in the periovulatory-luteal phase (p = 0.02). When we sorted the premenopausal women by mammographic breast composition, the SUVmax and SUVmean of normal breast tissues in the menstrual-follicular phase were significantly lower than those in the periovulatory-luteal phase in the 44 premenopausal women with dense breasts (p = 0.007, and p = 0.038, respectively), whereas no statistically significant difference was found between the menstrual-follicular phase and the periovulatory-luteal phase in the 15 premenopausal women with non-dense breasts. CONCLUSIONS: BPU in normal breast tissues assessed using ring-shaped dbPET was associated with mammographic density, menopausal status, and women's menstrual cycle. The menstrual cycle was significantly associated with BPU in premenopausal women with dense breasts but not in women with non-dense breasts.

[Biopsy-proven autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy with characteristic image findings: a case report].

A 53-year-old woman was admitted to the hospital because she developed headache and malaise 3 months prior to her arrival, followed by gait disturbance, abnormal behavior, and hallucinations. On admission, she was stupor and showed left hemispatial neglect, and brain MRI showed extensive FLAIR high-signal lesions with contrast enhancement in the bilateral periventricular white matter, and CSF examination showed pleocytosis and elevated protein. A stereotactic brain biopsy was performed from the right temporal lobe lesion, and pathological findings demonstrated a perivascular inflammatory cell infiltrate. After the administration of intravenous methylprednisolone followed by oral prednisolone, she recovered almost completely within three months and the abnormal MRI findings disappeared. Anti-glial fibrillary acidic protein (GFAP) antibody in the cerebrospinal fluid turned out to be positive, then the diagnosis of autoimmune GFAP astrocytopathy was made. Reports of this disease are still rare, and we report this case because of its slowly progressive course and pathological evaluation by brain biopsy.

High-Dose-Rate Interstitial Brachytherapy for Deeply Situated Gynecologic Tumors Guided by Combination of Transrectal and Transabdominal Ultrasonography: A Technical Note.

BACKGROUND AND PURPOSE: High-dose-rate interstitial brachytherapy (HDR-ISBT) is recommended to obtain a better local tumor control for uterine cancer patients in specific situations such as bulky lesions, an extension to the lateral parametrium, or tumors with irregular shapes. Our group uses real-time transrectal ultrasonography (TRUS) to guide freehand interstitial needle insertion. Occasionally, target tumors locate deeper beyond the rectum and cannot be visualized by TRUS. CT can guide needles to deeply located tumors, but in such cases, repeated image obtainment is required to achieve ideal needle localization. In this report, we present nine cases of patients who underwent HDR-ISBT for deeply situated tumors guided by a combination of transrectal and transabdominal ultrasonography (TR/TA-US). MATERIAL AND METHODS: Nine uterine cancer patients whose tumors were located deeper than the reach of TRUS and underwent HDR-ISBT guided by TR/TA-US were presented. All nine cases had no distal organ metastasis and underwent external beam radiation therapy (EBRT) to the pelvic region for 45-50.4 Gy in 25-28 fractions followed by boost HDR-ISBT for deeply situated tumors guided by TR/TA-US. RESULTS: There were seven cervical cancer and two endometrial cancer patients: six with extensive uterine corpus invasion, one cervical cancer with massive pelvic lymph node metastasis, one cervical cancer with postoperative pelvic recurrence, and one with left ovarian direct tumor invasion. The median follow-up period was 15 months (range 3-28 months). The average clinical target volume at the time of first HDR-ISBT was 131 ml (range 44-335 ml). The linear distance from the vaginal entrance to the deepest part of the tumor at first time brachytherapy of nine cases was 14.0 (9.0-17.0) cm. HDR-ISBT dose fractionation was 24-30 Gy in four or five fractions. Seven out of nine cases had no local recurrence in the follow-up period. One had local in-field recurrence 25 months after HDR-ISBT. Another case with carcinosarcoma could not obtain local control and underwent salvage hysterectomy for a residual uterine tumor 11 months after HDR-ISBT. Four cases had extra-field recurrence in lymph nodes or distant organs. CONCLUSIONS: In brachytherapy for gynecologic malignancies, deeply situated tumors located out of reach of TRUS may obtain favorable local control by HDR-ISBT guided with TR/TA-US.

Oral Contraceptive Disturbed the Recovery of the Adrenal Function after Adrenalectomy in Cushing Syndrome.

Estrogen is known to increase exogenous corticosteroid levels. In this case, a 27-year-old Japanese woman was referred to our hospital for examination of an adrenal tumor and was diagnosed with Cushing syndrome. Resection of the tumor resulted in secondary adrenal insufficiency. She also developed microcytic anemia due to hypermenorrhea, which was masked by Cushing syndrome. An oral contraceptive was administered for the treatment of hypermenorrhea, but this led to a marked increase in serum cortisol and the reduction of plasma adenocorticotropic hormone, disturbing the recovery of the adrenal function. Attention is required when oral contraceptives are used to treat hypermenorrhea masked by Cushing syndrome.

Mucinous Syringometaplasia: Surrounding Acanthosis With Hyperkeratosis Tends to Conceal the Change.

A 57-year-old man had a 2-year history of a painful nodule on the right sole. Physical examination revealed an 8 × 8 mm hyperkeratotic plaque with a central fissure. Excisional biopsy disclosed epithelial invagination surrounded by the acanthotic epidermis with parakeratotic hyperkeratosis and focal hypergranulosis. The invaginated epithelium lacked a cornified layer and was composed of a mixture of small basaloid squamous cells and goblet cells showing tubular structures. The patient was diagnosed with mucinous syringometaplasia. Our literature review established that surrounding acanthosis with hyperkeratosis typically tends to conceal mucinous syringometaplastic changes. Because mucinous syringometaplasia often presents as an asymptomatic papule/nodule with no distinct ulcer, fissure, or depressed area, cases may be overlooked.

Adiponectin Stimulates Exosome Release to Enhance Mesenchymal Stem-Cell-Driven Therapy of Heart Failure in Mice.

Mesenchymal stem/stromal cells (MSCs) are cultured adult stem cells that originally reside in virtually all tissues, and the gain of MSCs by transplantation has become the leading form of cell therapy in various diseases. However, there is limited knowledge on the alteration of its efficacy by factors in recipients. Here, we report that the cardioprotective properties of intravenously injected MSCs in a mouse model of pressure-overload heart failure largely depend on circulating adiponectin, an adipocyte-secreted factor. The injected MSCs exert their function through exosomes, extracellular vesicles of endosome origin. Adiponectin stimulated exosome biogenesis and secretion through binding to T-cadherin, a unique glycosylphosphatidylinositol-anchored cadherin, on MSCs. A pharmacological or adenovirus-mediated genetic increase in plasma adiponectin enhanced the therapeutic efficacy of MSCs. Our findings provide novel insights into the importance of adiponectin in mesenchymal-progenitor-mediated organ protections.

A disintegrin and metalloproteinase 12 prevents heart failure by regulating cardiac hypertrophy and fibrosis.

A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based on the finding that a small-molecule ADAM12 inhibitor, KB-R7785, ameliorated cardiac function in a transverse aortic constriction (TAC) model by inhibiting the proteolytic activation of heparin-binding-EGF signaling. However, this compound has poor selectivity for ADAM12, and the role of ADAM12 in cardiac dysfunction has not yet been investigated using genetic loss-of-function mice. We revealed that ADAM12 knockout mice showed significantly more advanced cardiac hypertrophy and higher mortality rates than wild-type mice 4 wk after TAC surgery. An ADAM12 deficiency resulted in significantly more expanded cardiac fibrosis accompanied by increased collagen-related gene expression in failing hearts. The results of a genome-wide transcriptional analysis suggested a strongly enhanced focal adhesion- and fibrosis-related signaling pathway in ADAM12 knockout hearts. The loss of ADAM12 increased the abundance of the integrinß1 subunit and transforming growth factor (TGF)-ß receptor types I and III, and this was followed by the phosphorylation of focal adhesion kinase, Akt, mammalian target of rapamycin, ERK, and Smad2/3 in the heart, which resulted in cardiac dysfunction. The present results revealed that the loss of ADAM12 enhanced focal adhesion and canonical TGF-ß signaling by regulating the abundance of the integrinß1 and TGF-ß receptors.NEW & NOTEWORTHY In contrast to a long-believed cardio-damaging role of a disintegrin and metalloproteinase (ADAM)12, cardiac hypertrophy was more severe, cardiac function was lower, and mortality was higher in ADAM12 knockout mice than in wild-type mice after transverse aortic constriction surgery. The loss of ADAM12 enhanced focal adhesion- and fibrosis-related signaling pathways in the heart, which may compromise cardiac function. These results provide insights for the development of novel therapeutics that target ADAM12 to treat heart failure.

RUNX1 induces DNA replication independent active DNA demethylation at SPI1 regulatory regions.

BACKGROUND: SPI1 is an essential transcription factor (TF) for the hematopoietic lineage, in which its expression is tightly controlled through a -17-kb upstream regulatory region and a promoter region. Both regulatory regions are demethylated during hematopoietic development, although how the change of DNA methylation status is performed is still unknown. RESULTS: We found that the ectopic overexpression of RUNX1 (another key TF in hematopoiesis) in HEK-293T cells induces almost complete DNA demethylation at the -17-kb upstream regulatory region and partial but significant DNA demethylation at the proximal promoter region. This DNA demethylation occurred in mitomycin-C-treated nonproliferating cells at both regulatory regions, suggesting active DNA demethylation. Furthermore, ectopic RUNX1 expression induced significant endogenous SPI1 expression, although its expression level was much lower than that of natively SPI1-expressing monocyte cells. CONCLUSIONS: These results suggest the novel role of RUNX1 as an inducer of DNA demethylation at the SPI1 regulatory regions, although the mechanism of RUNX1-induced DNA demethylation remains to be explored.

RUNX1 regulates site specificity of DNA demethylation by recruitment of DNA demethylation machineries in hematopoietic cells.

RUNX1 is an essential master transcription factor in hematopoietic development and plays important roles in immune functions. Although the gene regulatory mechanism of RUNX1 has been characterized extensively, the epigenetic role of RUNX1 remains unclear. Here, we demonstrate that RUNX1 contributes DNA demethylation in a binding site-directed manner in human hematopoietic cells. Overexpression analysis of RUNX1 showed the RUNX1-binding site-directed DNA demethylation. The RUNX1-mediated DNA demethylation was also observed in DNA replication-arrested cells, suggesting an involvement of active demethylation mechanism. Coimmunoprecipitation in hematopoietic cells showed physical interactions between RUNX1 and DNA demethylation machinery enzymes TET2, TET3, TDG, and GADD45. Further chromatin immunoprecipitation sequencing revealed colocalization of RUNX1 and TET2 in the same genomic regions, indicating recruitment of DNA demethylation machinery by RUNX1. Finally, methylome analysis revealed significant overrepresentation of RUNX1-binding sites at demethylated regions during hematopoietic development. Collectively, the present data provide evidence that RUNX1 contributes site specificity of DNA demethylation by recruitment of TET and other demethylation-related enzymes to its binding sites in hematopoietic cells.