SLC7A11 (xCT), together with SLC3A2 (4F2hc), encodes the heterodimeric amino acid transport system x c À , which mediates cystine-glutamate exchange and thereby regulates intracellular glutathione levels. We used microarrays to analyze gene expression of transporters in 60 human cancer cell lines used by the National Cancer Institute for drug screening (NCI-60). The expression of SLC7A11 showed significant correlation with that of SLC3A2 (r = 0.66), which in turn correlated with SLC7A5 (r = 0.68), another known partner for SLC3A2, and with T1A-2 (r = 0.60; all P < 0.0001). Linking expression of SLC7A11 with potency of 1,400 candidate anticancer drugs identified 39 showing positive correlations, e.g., amino acid analogue, L-alanosine, and 296 with negative correlations, e.g., geldanamycin. However, no significant correlation was observed with the geldanamycin analogue 17-allylamino, 17-demethoxygeldanamycin (17-AAG). Inhibition of transport system x c À with glutamate or (S)-4-carboxyphenylglycine in lung A549 and HOP-62, and ovarian SK-OV-3 cells, reduced the potency of L-alanosine and lowered intracellular glutathione levels. This further resulted in increased potency of geldanamycin, with no effect on 17-AAG. Down-regulation of SLC7A11 by small interfering RNA affected drug potencies similarly to transport inhibitors. The inhibitor of ;-glutamylcysteine synthetase, buthionine sulfoximine, also decreased intracellular glutathione levels and enhanced potency of geldanamycin, but did not affect L-alanosine. These results indicate that SLC7A11 mediates cellular uptake of L-alanosine but confers resistance to geldanamycin by supplying cystine for glutathione maintenance. SLC7A11 expression could serve as a predictor of cellular response to L-alanosine and glutathione-mediated resistance to geldanamycin, yielding a potential target for increasing chemosensitivity to multiple drugs. (Cancer Res 2005; 65(16): 7446-54)

MicroRNAs are strongly implicated in such processes as development, carcinogenesis, cell survival, and apoptosis. It is likely, therefore, that they can also modulate sensitivity and resistance to anticancer drugs in substantial ways. To test this hypothesis, we studied the pharmacologic roles of three microRNAs previously implicated in cancer biology (let-7i, mir-16, and mir-21) and also used in silico methods to test pharmacologic microRNA effects more broadly. In the experimental system, we increased the expression of individual microRNAs by transfecting their precursors (which are active) or suppressed the expression by transfection of antisense oligomers. In three NCI-60 human cancer cell lines, a panel of 60 lines used for anticancer drug discovery, we assessed the growthinhibitory potencies of 14 structurally diverse compounds with known anticancer activities. Changing the cellular levels of let-7i, mir-16, and mir-21 affected the potencies of a number of the anticancer agents by up to 4-fold. The effect was most prominent with mir-21, with 10 of 28 cell-compound pairs showing significant shifts in growthinhibitory activity. Varying mir-21 levels changed potencies in opposite directions depending on compound class; indicating that different mechanisms determine toxic and protective effects. In silico comparison of drug potencies with microRNA expression profiles across the entire NCI-60 panel revealed that f30 microRNAs, including mir-21, show highly significant correlations with numerous anticancer agents. Ten of those microRNAs have already been implicated in cancer biology. Our results support a substantial role for microRNAs in anticancer drug response, suggesting novel potential approaches to the improvement of chemotherapy. [Mol Cancer Ther 2008;7(1):1 -9]

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...

Advances in the understanding of cancer cell biology and response to drug treatment have benefited from new molecular technologies and methods for integrating information from multiple sources. The NCI-60, a panel of 60 diverse human cancer cell lines, has been used by the National Cancer Institute to screen >100,000 chemical compounds and natural product extracts for anticancer activity. The NCI-60 has also been profiled for mRNA and protein expression, mutational status, chromosomal aberrations, and DNA copy number, generating an unparalleled public resource for integrated chemogenomic studies. Recently, microRNAs have been shown to target particular sets of mRNAs, thereby preventing translation or accelerating mRNA turnover. To complement the existing NCI-60 data sets, we have measured expression levels of microRNAs in the NCI-60 and incorporated the resulting data into the CellMiner program package for integrative analysis.

Summary Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4R334X, as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient’s cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.