Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks

<p>Abstract</p> <p>Background</p> <p>An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of <it>CD133 </it>gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express <it>CD133 </it>gene.</p> <p>Results</p> <p>A reporter analysis revealed that P5 promoter, located far upstream in a human <it>CD133 </it>gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of <it>CD133 </it>mRNA expression.</p> <p>Conclusions</p> <p>In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.</p

Clinical significance of RAS pathway alterations in pediatric acute myeloid leukemia

RAS pathway alterations have been implicated in the pathogenesis of various hematological malignancies. However, their clinical relevance in pediatric acute myeloid leukemia (AML) is not well characterized. We analyzed the frequency, clinical significance, and prognostic relevance of RAS pathway alterations in 328 pediatric patients with de novo AML. RAS pathway alterations were detected in 80 (24.4%) of 328 patients: NF1 (n=7, 2.1%), PTPN11 (n=15, 4.6%), CBL (n=6, 1.8%), NRAS (n=44, 13.4%), KRAS (n=12, 3.7%). Most of these alterations in the RAS pathway were mutually exclusive also together with other aberrations of signal transduction pathways such as FLT3-ITD (P=0.001) and KIT mutation (P=0.004). NF1 alterations were frequently detected in patients with complex karyotype (P=0.031) and were found to be independent predictors of poor overall survival (OS) in multivariate analysis (P=0.007). At least four of seven patients with NF1 alterations had biallelic inactivation. NRAS mutations were frequently observed in patients with CBFB-MYH11 and were independent predictors of favorable outcomes in multivariate analysis (OS, P=0.023; event-free survival [EFS], P=0.037). Patients with PTPN11 mutations more frequently received stem cell transplantation (P=0.035) and showed poor EFS than patients without PTPN11 mutations (P=0.013). Detailed analysis of RAS pathway alterations may enable a more accurate prognostic stratification of pediatric AML and may provide novel therapeutic molecular targets related to this signal transduction pathway

A proposed new rotating reference axis for the tibial component after proximal tibial resection in total knee arthroplasty.

PURPOSE:During total knee arthroplasty, few rotating reference axes can be reliably used after tibial resection. We speculated that a line that passes through the lateral edge of the posterior cruciate ligament (PCL) at its tibial attachment after resection and the most prominent point of the tibial tubercle [after-tibial resection (ATR) line] will provide a good reference axis. In this study, we aimed to evaluate the association between ATR and Akagi's lines. MATERIALS AND METHODS:In this case-control simulation study, we retrospectively evaluated 38 patients with varus knee and 28 patients with valgus knee. We defined the reference cutting plane as 10 mm distal from the lateral articular surface of the tibia in varus group and as 7 mm distal from the medial articular surface in the valgus group. We measured angles between Akagi's line and the ATR line (ATR line angle) as well as between Akagi's line and 1/3 Akagi's line (1/3 Akagi's line angle), which passes through the midpoint of PCL and the medial third of the patellar tendon. We used paired t-tests to determine the significance of differences between these angles, with p < 0.05 indicating statistical significance. Intra- and interclass correlation coefficients for the reproducibility of 1/3 Akagi's line angle and ATR line angle were analyzed by two surgeons. RESULTS:We found that 1/3 Akagi's line angle was 10.2° ± 1.3° in the varus group and 10.9° ± 1.3° in the valgus group (p = 0.017). The ATR line was positioned externally compared with Akagi's line in all patients. Mean ATR line angles at 0°, 3° and 7° posterior slopes were 6.1° ± 1.9°, 5.8° ± 2.0° and 6.0° ± 1.7° in the varus group and 6.3° ± 2.3°, 6.2° ± 2.3° and 5.4° ± 2.1° in the valgus group, respectively. There were no significant differences in the ATR line angle between the varus and valgus groups. (p = 0.34-0.67) Intra- and interclass correlation coefficients for the reproducibility of 1/3 Akagi's line angle were 0.936 and 0.986 and those for the reproducibility of ATR line angle were 0.811 and 0.839. CONCLUSIONS:The ATR line was positioned between Akagi's line and 1/3 Akagi's line in all patients and was a valid option for evaluating rotational tibial alignment after tibial resection