The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.Human embryonic stem cells (hESCs) 3 are capable of essentially unlimited self-renewal and retain the developmental potential to differentiate into almost any cell type. These characteristics of hESCs make them attractive for tissue and cellbased therapies (1, 2). Previously, basic fibroblast growth factor and activin A were identified as self-renewal factors (3-6). However, for reasons that are not clear, hESCs often display high rates of spontaneous apoptosis and differentiation in culture, thus making the process of expanding these cells highly inefficient (3, 7-10). For example, Dravid et al. (8) reported that, under routine culture conditions, Ͼ30% of hESCs undergo spontaneous apoptosis. Furthermore, Ezashi et al. (12) showed that nearly 40% of hESCs undergo spontaneous differentiation after 12 days of culture in normoxic conditions. Finally, Maitra et al. (13) reported that multiple passages of hESCs can cause genomic alterations, which may limit the therapeutic application of hESCs. In contrast to hESCs, mouse embryonic stem cells (mESCs) undergo lower rates of spontaneous apoptosis and differentiation (14). Moreover, they maintain their pluripotency and genomic stability longer than hESCs (15). The reason for these different species-specific phenotypes in embryonic stem cells is currently unknown.The p53 tumor suppressor gene is a strong candidate for playing a role in the observed phenotypes of hESCs, because it regulates various cellular processes, including apoptosis, differentiation, and genomic integrity (16). In many cell types p53 plays a crucial role in controlling apoptosis and cell cycle arrest when these cells are exposed to stress-inducing conditions (17). In response to stress, p53 accumulates and transactivates downstream target genes such as mdm2 (responsible for the feedback degradation circuitry of p53), p21 (responsible for cell cycle control), bax, noxa, and puma (responsible for DNA damage-induced ...

Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission (CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However, As2O3 treatment resulted in hepatic toxicity in 7 cases including 2 deaths, in contrast to the mild liver dysfunction in one third of the relapsed patients. Forty of forty-seven (85.1%) relapsed patients achieved CR. Two of three nonresponders showed clonal evolution at relapse, with disappearance of t(15;17) and PML-RAR fusion gene in 1 and shift to a dominant AML-1-ETO population in another, suggesting a correlation between PML-RAR expression and therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS was 17 months. Patients with white blood cell (WBC) count below 10 × 109/L at relapse had better survival than those with WBC count over 10 × 109/L (P = .038). The duration of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in patients treated with As2O3 combined with chemotherapy, compared with 12 of 18 relapses with As2O3 alone (P = .01). Reverse transcription polymerase chain reaction (RT-PCR) analysis in both newly diagnosed and relapsed groups showed long-term use of As2O3 could lead to a molecular remission in some patients. We thus recommend that ATRA be used as first choice for remission induction in newly diagnosed APL cases, whereas As2O3 can be either used as a rescue for relapsed cases or included into multidrug consolidation/maintenance clinical trials.

A central question in Wnt signaling is the regulation of ␤-catenin phosphorylation and degradation. Multiple kinases, including CKI␣ and GSK3, are involved in ␤-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of ␤-catenin. However, which phosphatase dephosphorylates ␤-catenin in vivo and how the specificity of ␤-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates ␤-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/␤-catenin signaling in Drosophila. Moreover, we have identified PR55␣ as the regulatory subunit of PP2A that controls ␤-catenin phosphorylation and degradation. PR55␣, but not the catalytic subunit, PP2Ac, directly interacts with ␤-catenin. RNA interference knockdown of PR55␣ elevates ␤-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55␣ enhances Wnt signaling. Taken together, our results suggest that PR55␣ specifically regulates PP2A-mediated ␤-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/␤-catenin signaling plays essential roles in development and tumorigenesis (1-3). Our previous work found that ␤-catenin is sequentially phosphorylated by CKI␣ 4 and GSK3 (4), which creates a binding site for ␤-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in ␤-catenin or APC genes that prevent ␤-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/ ␤-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (8 -10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Toward the goal of understanding the mechanism of ␤-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates ␤-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate ␤-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/BЈ, PR/BЉ, or PR/Bٞ). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55␣, a regulatory subunit of PP2A, specifically regulates ␤-catenin phosphorylation and degradation. Mechanistically, we found that PR55␣ directly interacts with ␤-catenin and regulates PP2A-mediated ␤-catenin dephosphorylation in Wnt signaling. EXPERIMENTAL PROCEDURESPlasmids-Myc-tagged PR55␣ was cloned into CS2ϩMT vector. FLAG-tagged PP2Ac was cloned into pRK5 vector. ␤-Catenin and Axin constructs have been described previously (4). PR55␣ shRNA constructs were purch...

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Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC is initiated by mutations of the tumor suppressor gene, adenomatous polyposis coli (APC) or β-catenin gene. These mutations stabilize β-catenin and constitutively activate Wnt/β-catenin target genes, such as c-Myc and Cyclin D1, ultimately leading to cancer. Naturally occurring stilbene derivatives, resveratrol and pterostilbene, inhibit Wnt signaling and repress CRC cell proliferation but are ineffective at concentrations lower than 10 µM. To understand the structure/activity relationship within these stilbene derivatives and to develop more efficacious Wnt inhibitors than these natural products, we synthesized and evaluated a panel of fluorinated N,N-dialkylaminostilbenes. Among this panel, (E)-4-(2,6-difluorostyryl)-N,N-dimethylaniline (4r) inhibits Wnt signaling at nanomolar levels and inhibits the growth of human CRC cell xenografts in athymic nude mice at a dosage of 20mg/kg. These fluorinated N,N-dialkylaminostilbenes appear to inhibit Wnt signaling downstream of β-catenin, probably at the transcriptional level.

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Background: Bmi1 is a potential marker for the intestinal stem cells. Results: Wnt regulates Bmi1 indirectly, while KLF4 directly inhibits Bmi1, as well as Bmi1-mediated histone ubiquitination in colon cancer cells. Conclusion: Bmi1 is required for colon cancer cell proliferation, and it is up-regulated in colon cancer tissues. Significance: Study of the mechanisms of Bmi1 regulation suggests potential targets for cancer therapeutics.

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