Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.
DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C3W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. Although a strong correlation between promoter methylation and gene silencing has been extensively demonstrated (5,24,35), the molecular mechanisms of this methylation-modulated gene inactivation remains unclear. Two hypotheses have been proposed to explain transcriptional inactivation from promoter methylation. One of them is based on the finding that methyl-CpG-binding proteins (MBPs), such as MeCP2, specifically bind to symmetrically methylated DNA through a methyl-CpG-binding domain (11,41). MBPs then recruit transcriptional repressors such as Sin3, NuRD, and histone deacetylases (HDACs) through its transcriptional-repression domain (25,32,54). Since Sin3 and HDACs are known transcriptional repressors (2, 50), methylated DNA may repress gene expression indirectly through MeCP2 and other MBPs. In addition, deacetylation of histones results in a net increase in positively charged lysines and arginines at the N-terminal tail of the histones (18, 21), thus inducing a tighter noncovalent linkage between the positively charged histones and the negatively charged DNA (3, 47). Consequently, transcription factors have difficulty accessing their DNA-binding sites (4, 29, 47), with a reduction or silencing of gene transcription. This hypothesis, based on the interaction between DNA methylation and histone acetylation status, has been extensively supported by accumulated experimental evidence (7,16,37,40). For example, trichostatin A (TSA), an inhibitor of HDAC, induces a robust reexpression of silenced genes when used with minimal doses of the demethylating agent, 5-aza-2Ј-deoxycytidine (5-azaCdR), although TSA or 5-aza-CdR alone do not lead to gene reexpression (7). Our previous data also show a link between histone acetylation status and DNA methylation, such that 5-aza-CdR significantly enhances acetylation of histones H3 and H4 induced by a HDAC inhibitor, depsipeptide. Related to this, depsipeptide-induced apoptosis is dramatically increased in cells pretreated with 5-aza-CdR (56). In addition, p19 INK4D expression is greatly enhanced when human lung cancer cells are treated with depsipeptide and 5-aza-CdR together compared to treatment with each agent alone (55). These studies support the notion that methylation and histone acetylation work cooperatively to influence gene expression and other biological processes.A...
In addition to its demethylating function, 5-aza-2-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 M) induces inhibition of cell proliferation as well as increased p53/p21Waf1/Cip1 expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21 As demethylating agents, 5-aza-cytidine and 5-aza-2Ј-deoxycytidine (5-aza-CdR) 1 have been extensively used for epigenetic research (1-4). Both demethylating agents are incorporated into DNA where they bind DNA methyltransferase (DNMT) in an irreversible, covalent manner, thus sequestering the enzyme and preventing maintenance of the methylation state (5-7). Consequently, silenced genes induced by hypermethylation are re-expressed after treatment with these demethylating agents.Originally, 5-aza-cytidine and 5-aza-CdR were developed as anticancer agents (5, 8) and have been shown to have significant cytotoxic and antineoplastic activities in many experimental tumors (9 -12). 5-Aza-CdR is reported to be noncarcinogenic and incorporates into DNA but not RNA or protein (13,14). 5-Aza-CdR has been found empirically to have more potent therapeutic effects than 5-aza-cytidine in cell culture and animal models of human cancers. However, 5-aza-CdR-induced cytotoxicity may not be linked to its demethylating function (3,(15)(16)(17). In addition, the therapeutic effects of 5-aza-CdR in the treatment of different human cancer cells are conflicting. 5-Aza-CdR appears to be beneficial in the treatment of human leukemias (9,18,19), myelodysplastic syndromes (20, 21), and hemoglobinopathies (22, 23). On the other hand, there has been less positive experience in the effectiveness of 5-aza-CdR for the treatment of human solid tumors (10, 24). Therefore, it is possible that one or more critical factors may be involved in regulating the cellular response to 5-aza-CdR treatment that vary in different cell types.p53 is a very important tumor suppressor gene and is reported to be abnormal in more than 50% of human cancers (25). Chemotherapeutic agents frequently act through the mechanism of DNA damage, and p53 plays an important role in the induction of cell cycle arrest and apoptosis in response to DNA damage (26). 5-Aza-CdR has also shown anticancer activity that may be related to its ability to induce DNA damage (15,27). Based on the scenario mentioned above, it is hypothesized that 5-aza-CdR may induce DNA damage, thereby activating p53, which in turn increases p21Waf1/Cip1 expression, leading to the inhibition of cell proliferation.To confirm the role of p53 in the 5-aza-CdR-induced inhibition of cell proliferation, human lung cancer cells with different p53 status were selected as the targets for this study. As an important downstream target of p53 activation, p21Waf1/Cip1 plays a critical role in inhibit...
Maintenance of p53 function is important for normal cell growth and development, and loss of p53 function contributes directly to malignant tumor development. The recently discovered Pirh2 protein is an ubiquitin-protein ligase that negatively regulates p53 through activity by targeting it for degradation. To determine how Pirh2 may mediate lung tumorigenesis, we evaluated Pirh2 expression in human and mouse lung tumor samples and paired normal lung tissues using immunoblot analysis and immunohistochemistry. Pirh2 protein expression was higher in 27 (84%) of 32 human lung neoplasms than in matched normal lung tissue and in 14 of 15 mouse lung tumors evaluated. In addition, p53 protein was more ubiquitinated in the mouse lung tumors than in normal tissue, and overall p53 expression was lower than that in normal tissue. These results are consistent with the hypothesis that increased Pirh2 expression affects lung tumorigenesis by reducing p53 activity. To our knowledge, this is the first description of altered Pirh2 expression in human and mouse tumors.
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