Functional characterization of all-trans retinoic acid-induced differentiation factor (ATRAID).

All-trans retinoic acid-induced differentiation (ATRAID) factor was first identified in HL60 cells. Several mRNA isoforms exist, but the respective proteins have not been fully characterized. In transfected cells expressing Myc-Flag-tagged ATRAID Isoform (Iso) A, B, and C, Iso C was found to be expressed at high levels, Iso A was found to be expressed at low levels due to rapid degradation, and the predicted protein expressed from Iso B was not detected. Iso C was present mainly in an N-glycosylated form. In subcellular fractionation experiments, Iso C localized to the membranous and nuclear fractions, while immunofluorescence analysis revealed that Iso C is located close to the plasma membrane, mainly in cytoplasmic vesicles and in the Golgi area. We confirm that Iso C colocalizes to some extent with endosomal/lysosomal markers LAMP1 and LAMP2. Furthermore, we show that ATRAID co-localizes with RAB11, a GTPase associated with recycling endosomes and implicated in regulating vesicular trafficking.

A Pellino-2 variant is associated with constitutive NLRP3 inflammasome activation in a family with ocular pterygium-digital keloid dysplasia.

Ocular pterygium-digital keloid dysplasia (OPDKD) is a rare hereditary disease characterized by corneal ingrowth of vascularized conjunctival tissue early in life. Later, patients develop keloids on fingers and toes but are otherwise healthy. In a recently described family with OPDKD, we report the presence of a de novo c.770C > T, p.(Thr257Ile) variant in PELI2 in the affected individual. PELI2 encodes for the E3 ubiquitin ligase Pellino-2. In transgenic U87MG cells overexpressing Pellino-2 with the p.(Thr257Ile) amino acid substitution, constitutive activation of the NLRP3 inflammasome was observed. However, the Thr257Ile variant did not affect Pellino-2 intracellular localization, its binding to known interaction partners, nor its stability. Our findings indicate that constitutive autoactivation of the NLRP3 inflammasome contributes to the development of PELI2-associated OPDKD.

Residual methylation of tumor suppressor gene promoters, RASSF6 and RASSF10, as novel biomarkers for minimal residual disease detection in adult acute lymphoblastic leukemia.

Aberrant promoter methylation of RASSF6 and RASSF10 occurs at a high frequency in acute lymphoblastic leukemia (ALL). Because of the complexity of the current minimal residual disease (MRD) detecting-methods, the DNA methylation status of the RASSF6 and RASSF10 genes could potentially become biomarkers for the assessment of MRD levels in ALL patients. The promoter methylation status of RASSF6 and RASSF10 was assessed by using methylation-specific PCR (MSP) in the DNA isolated from 280 peripheral blood (PB) samples taken at the time of diagnosis, day 14, 28, and from the subsequent 30-month follow-ups of 45 adult ALL patients. The relative methylation level obtained during the follow-ups by MSP was compared to the MRD results obtained by the amplification of IG/TCR clonal rearrangements using the allele-specific quantitative-PCR (ASO-PCR) assay. Frequently, RASSF6 was methylated in B-ALL, and RASSF10 was methylated in T-ALL. The applicability and accuracy of the assays were increased when these markers were combined (91.1% and 93.8%, respectively). When a cutoff was defined for the PCR-MRD level, results of the 30 months of MRD detection showed a significant correlation between the PCR and MSP techniques (r = 0.848; p < 0.001). Due to the high applicability, the non-invasiveness, and promising prospect of longitudinal assessment, the DNA methylation status of the RASSF6 and RASSF10 genes could be potential biomarkers for the assessment of residual disease in PB of patients with ALL.

Minimal residual disease (MRD) detection using rearrangement of immunoglobulin/T cell receptor genes in adult patients with acute lymphoblastic leukemia (ALL).

MRD detection with allele-specific oligonucleotide-quantitative polymerase chain reaction (ASO-qPCR) and using clone-specific immunoglobulin/T cell receptor rearrangements is considered as a powerful prognostic factor in acute lymphoblastic leukemia (ALL). In the present study, we evaluated an ASO-qPCR assay for MRD quantification in peripheral blood (PB) samples of adult patients with ALL. DNA was isolated from PB samples of patients with newly diagnosed ALL. They were first investigated by multiplex-PCR assay to identify V/J usage. An ASO-qPCR technique was then applied for 2.5-year monthly MRD quantification for detection of patient-specific Ig/TCR receptor rearrangements as a molecular target. From 98 patients who were diagnosed as ALL, 72 (73.5%) were enrolled in the present study for MRD detection. MRD was successfully quantified in patients with 1-month interval time. MRD level at the end of induction therapy up to day 88 was the only significant prognostic factor. Regarding MRD level, patients were categorized into two groups of low and high-risk. 2.5-year OS in all three time points (days 28, 58 and 88) were significantly lower in high-risk group (P < 0.008). The results of the 2.5-year MRD detection indicate that MRD level at the end of induction up to about 6 months after the first diagnosis was associated with clinical outcome. This study may highlight the usefulness of PB and the definitions of cut-off level for early prediction of relapse and for stratifying ALL patients. Short-interval time points and frequent PB sampling to monitor MRD level is suggested for early clinical relapse prediction and clinical management of the disease.

DNA hypermethylation of tumor suppressor genes RASSF6 and RASSF10 as independent prognostic factors in adult acute lymphoblastic leukemia.

BACKGROUND: The Hypermethylation of Ras association domain family (RASSF) often plays a key role in malignant progression of solid tumors; however, their impact on the prognosis and survival of adult ALL patients remain elusive. METHODS: The frequency of the promoter methylation pattern of RASSF6 and RASSF10 were analyzed in the peripheral blood (PB) samples taken at the time of diagnosis of 45 ALL patients. The methylation-specific PCR (MSP) assay was used to detect the DNA methylation patterns. RESULTS: RASSF6 was frequently hypermethylated in patients diagnosed with pre-B-ALL (90.9%) and B-ALL (87.5%), followed by T-ALL (66.7%); whereas, RASSF10 methylation was more confined to T-ALL (80%) as compared to B-ALL (25%) and pre-B ALL (9.1%) patients. Moreover, hypermethylation of RASSF6 was significantly associated with a poor prognosis and shorter overall survival (OS) in patients with pre-B-ALL (log-rank test; P=0.041). CONCLUSION: RASSF6 and RASSF10 were frequently hypermethylated in the samples at the time of diagnosis of adult ALL patients. Our study represents the first report of methylation of RASSF6 at a high frequency in patients with pre-B ALL. Furthermore, hypermethylation of RASSF6 was significantly associated with inferior overall survival in pre-B ALL patients. It may suggest that the frequent epigenetic inactivation of RASSF6 plays an important role in the pathogenesis and progression of pre-B-ALL.

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs.

BACKGROUND: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. METHODS: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. RESULTS: Maximum CB-CD34+ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34+ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34+ and CD34- cells in the cytokine co-culture system was observed. CONCLUSION: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

Gene Expression and Methylation Pattern in HRK Apoptotic Gene in Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDSs) are a clonal bone marrow (BM) disease characterized by ineffective hematopoiesis, dysplastic maturation and progression to acute myeloid leukemia (AML). Methylation silencing of HRK has been found in several human malignancies. In this study, we explored the association of HRK methylation status with its expression, clinical parameters and MDS subtypes in MDS patients. To study the methylation status of HRK gene, we applied Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) in MDS patients, as well as healthy controls and EpiTect®PCR Control DNA. Real time RT-PCR was used for gene expression analysis. Methylation frequency in promoter region of HRK in patient samples was 20.37%. Methylation of HRK was significantly related to transcriptional downregulation (P=0.023). The difference in frequency of hypermethylated HRK gene was significant between good (10%) and poor (71.42%) cytogenetic risk groups (P= 0.001), advanced stage MDS patients (66.66%) in comparison with early stage MDS patients (2.56%) (P= 0.00), higher- risk MDS group (61.53%) and lower- risk MDS group (7.31%) (P= 0.00). HRK hypermethylation was associated with advanced- stage MDS and downregulation of HRK gene may play a role in the progression of MDS.

Apoptosis, DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells.

Expansion of umbilical cord blood-derived CD34(+) cells can potentially provide them in numbers sufficient for clinical applications in adult humans. In this study apoptosis rate of expanded cells, mRNA expression and promoter methylation status of DAPK1 were evaluated during cord blood hematopoietic stem cell (CB-HSC) ex vivo expansion using cytokines and a co-culture system with mesenchymal stromal cells (MSCs). Ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs feeder layer without cytokine. Total number of cells, CD34(+) cells and colony forming unit assay were performed during expansion. Flow cytometric analysis by propidium iodide was performed to detection of apoptosis rate in expanded cells. Methylation status of the DAPK1 gene promoter was analyzed using methylation specific PCR, and DAPK1 mRNA expression was evaluated by real time-PCR. Maximum CB-CD34(+) cells expansion was observed in day 10 of expansion. The highest apoptosis rate was observed in cytokine culture without feeder layer that showed significant difference with co-culture condition. The data showed that ex vivo expansion of CD34(+) cells in all three culture conditions after 10 days resulted in, significant increased expression of DAPK1, also a significant difference between co-culture without cytokine and two other cytokine culture was observed (p < 0.01). DAPK1gene promoter of expanded CD34(+) cells at days 5, 10 and 14 of culture remained in unmethylated form similar to fresh CD34(+) cells. Expression of DAPK1 in hematopoietic cells was increased during 10 days expansion of CD34(+) cells. Also no methylation of DAPK1 promoter was observed; otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Human fibroblast switches to anaerobic metabolic pathway in response to serum starvation: a mimic of warburg effect.

Fibroblasts could be considered as connective tissue cells that are morphologically heterogeneous with diverse functions depending on their location and activity. These cells play critical role in health and disease such as cancer and wound by Production of collagen, fibronectin, cytokines and growth factors. Absence of insulin and other growth factors in serum deprivation condition and similarity of this condition to the environment of tumor cells and ulcer made us to investigate anaerobic glycolysis in these cells. To this end, we cultured fibroblasts isolated from fresh human newborn foreskin in serum free medium for 16, 24, 48 and 72 hrs and measured glucose consumption, lactate secretion and intracellular LDH in these cells. The results showed despite the lack of insulin, the 16hr serum starved fibroblasts consumed glucose similar to non-starved fibroblasts control. Moreover, in this condition these cells secreted higher levels of lactate and exhibited higher levels of intracellular LDH in comparison to non-starved fibroblasts control. Thus it could be concluded that in serum starvation condition, the newborn human dermal fibroblasts may change the metabolic strategy to Warburg effect. This finding opens a new perspective to further understanding the basic mechanisms involved in communication between tumor cells and fibroblasts.

Mesenchymal stem cells as a feeder layer can prevent apoptosis of expanded hematopoietic stem cells derived from cord blood.

Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34(+) cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) ( ) cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34(+) cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.