Flipping of alkylated DNA damage bridges base and nucleotide excision repair.

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O(6)-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O(6)-methylguanine or cigarette-smoke-derived O(6)-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: enzyme activity, promoter methylation and immunohistochemistry.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O(6)-methylguanine. It also repairs larger adducts on the O(6)-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O(6)-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O(6)-alkylating drugs are still lacking.

O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy.

INTRODUCTION: Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities. SOURCES OF DATA: This review focuses on the mechanism of action of the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature. AREAS OF AGREEMENT: MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials. AREAS OF CONTROVERSY: MGMT inhibitors enhance the myelotoxic effect of O(6)-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development. GROWING POINTS: The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types. AREAS TIMELY FOR DEVELOPING RESEARCH: Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.

An atlas of differential gene expression during early Xenopus embryogenesis.

We have carried out a large-scale, semi-automated whole-mount in situ hybridization screen of 8369 cDNA clones in Xenopus laevis embryos. We confirm that differential gene expression is prevalent during embryogenesis since 24% of the clones are expressed non-ubiquitously and 8% are organ or cell type specific marker genes. Sequence analysis and clustering yielded 723 unique genes displaying a differential expression pattern. Of these, 18% were already described in Xenopus, 47% have homologs and 35% are lacking significant sequence similarity in databases. Many of them encode known developmental regulators. We classified 363 of the 723 genes for which a Gene Ontology annotation for molecular function could be attributed and found 'DNA binding' and 'enzyme' the most represented terms. The most common protein domains encoded in these embryonic, differentially expressed genes are the homeobox and RNA Recognition Motif (RRM). Fifty-nine putative orthologs of human disease genes, and 254 organ or cell specific marker genes were identified. Markers were found for nasal placode and archenteron roof, organs for which a specific marker was previously unavailable. Markers were also found for novel subdomains of various other organs. The tissues for which most markers were found are muscle and epidermis. Expression of cell cycle regulators fell in two classes, containing proliferation-promoting and anti-proliferative genes, respectively. We identified 66 new members of the BMP4, chromatin, endoplasmic reticulum, and karyopherin synexpression groups, thus providing a first glimpse of their probable cellular roles. Cluster analysis of tissues to measure tissue relatedness yielded some unorthodox affinities besides expectable lineage relationships. In conclusion, this study represents an atlas of gene expression patterns, which reveals embryonic regionalization, provides novel marker genes, and makes predictions about the functional role of unknown genes.

Combined endostatin/sFlt-1 antiangiogenic gene therapy is highly effective in a rat model of HCC.

Hepatocellular carcinoma (HCC) is regarded as a suitable target for antiangiogenic strategies. However, antiangiogenic agents aimed at single targets can be neutralized by upregulation of other proangiogenic factors. Therefore, combined approaches addressing at least two angiogenic targets should be more effective. Employing an appropriate rat hepatoma model, we examined the effects of sFlt-1 (soluble vascular endothelial growth factor [VEGF] receptor 1 as an indirect inhibitor of angiogenesis) and endostatin (a direct inhibitor of angiogenesis) in both single-agent as well as combined approaches under in vitro and in vivo conditions. Similar to human HCC, rat Morris hepatoma (MH) cells secreted high levels of VEGF, but no endogenous sFlt-1. Parental MH or MHES(r) cells, stably expressing rat endostatin, were adenovirally transduced either with AdsFlt-1 (encoding sFlt-1) or control vector Adnull (containing no transgene), followed by subcutaneous inoculation into syngeneic ACI rats. Compared with MH/Adnull cells, expressing no antiangiogenic factors at all, tumor weights were reduced fourfold in the MHES(r)/Adnull group, 19-fold in the MH/AdsFlt-1-group, and 77-fold in the MHES(r)/AdsFlt-1 combination therapy group. Analysis of variance did not show a significant interaction between the effects of the two factors ES(r) and sFlt-1; their effects multiplied. In conclusion, combined expression of sFlt-1 and endostatin effectively suppresses HCC growth under in vivo conditions. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).