Showing papers in "Cell Research in 2008"

Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

Abstract: Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

Regulation of DNA double-strand break repair pathway choice

Abstract: DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

Mechanisms and functions of DNA mismatch repair

Abstract: DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSalpha and MutLalpha, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.

MicroRNA-21 targets tumor suppressor genes in invasion and metastasis

Abstract: MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level. Our previous studies suggest that mir-21 functions as an oncogene and has a role in tumorigenesis, in part through regulation of the tumor suppressor gene tropomyosin 1 (TPM1). Given that TPM1 has been implicated in cell migration, in this study we further investigated the role of mir-21 in cell invasion and tumor metastasis. We found that suppression of mir-21 in metastatic breast cancer MDA-MB-231 cells significantly reduced invasion and lung metastasis. Consistent with this, ectopic expression of TPM1 remarkably reduced cell invasion. Furthermore, we identified two additional direct mir-21 targets, programmed cell death 4 (PDCD4) and maspin, both of which have been implicated in invasion and metastasis. Like TPM1, PDCD4 and maspin also reduced invasiveness of MDA-MB-231 cells. Finally, the expression of PDCD4 and maspin inversely correlated with mir-21 expression in human breast tumor specimens, indicating the potential regulation of PDCD4 and maspin by mir-21 in these tumors. Taken together, the results suggest that, as an oncogenic miRNA, mir-21 has a role not only in tumor growth but also in invasion and tumor metastasis by targeting multiple tumor/metastasis suppressor genes. Therefore, suppression of mir-21 may provide a novel approach for the treatment of advanced cancers.

Homologous recombination in DNA repair and DNA damage tolerance

Abstract: Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

Wnt signaling and stem cell control

Abstract: Wnt signaling has been implicated in the control over various types of stem cells and may act as a niche factor to maintain stem cells in a self-renewing state. As currently understood, Wnt proteins bind to receptors of the Frizzled and LRP families on the cell surface. Through several cytoplasmic relay components, the signal is transduced to s-catenin, which then enters the nucleus and forms a complex with TCF to activate transcription of Wnt target genes. Wnts can also signal through tyrosine kinase receptors, in particular the ROR and RYK receptors, leading to alternative modes of Wnt signaling. During the growth of tissues, these ligands and receptors are dynamically expressed, often transcriptionally controlled by Wnt signals themselves, to ensure the right balance between proliferation and differentiation. Isolated Wnt proteins are active on a variety of stem cells, including neural, mammary and embryonic stem cells. In general, Wnt proteins act to maintain the undifferentiated state of stem cells, while other growth factors instruct the cells to proliferate. These other factors include FGF and EGF, signaling through tyrosine kinase pathways.

SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

Abstract: While severe acute respiratory syndrome coronavirus (SARS-CoV) was initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that virus entry may also involve endocytosis. We have found that SARS-CoV enters cells via pH- and receptor-dependent endocytosis. Treatment of cells with either SARS-CoV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic pathway inhibitors and dominant-negative Eps15 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells.

Abstract: Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3' OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3' phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight.

Abstract: Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci (QTLs). We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases.

Abstract: In their seminal publication describing the structure of the DNA double helix, Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis.

Abstract: Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase (FRO2) and ferrous transporter (IRT1) promoters. Overexpression of FIT with either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

Abstract: The encounter of elongating RNA polymerase II (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA- E3-ubiquitin ligase complex to the stalled RNAPIIo. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcription-blocking lesions, but are also likely to contribute to DNA damage signalling events.

MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth

Abstract: A microRNA expression screen was performed analyzing 157 different microRNAs in laser-microdissected tissues from benign melanocytic nevi (n = 10) and primary malignant melanomas (n = 10), using quantitative real-time PCR. Differential expression was found for 72 microRNAs. Members of the let-7 family of microRNAs were significantly downregulated in primary melanomas as compared with benign nevi, suggestive for a possible role of these molecules as tumor suppressors in malignant melanoma. Interestingly, similar findings had been described for lung and colon cancer. Overexpression of let-7b in melanoma cells in vitro downregulated the expression of cyclins D1, D3, and A, and cyclin-dependent kinase (Cdk) 4, all of which had been described to play a role in melanoma development. The effect of let-7b on protein expression was due to targeting of 3'-untranslated regions (3'UTRs) of individual mRNAs, as exemplified by reporter gene analyses for cyclin D1. In line with its downmodulating effects on cell cycle regulators, let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells. Taken together, these findings not only point to new regulatory mechanisms of early melanoma development, but also may open avenues for future targeted therapies of this tumor.

The endless tale of non-homologous end-joining.

Abstract: DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

STAT3 as a target for inducing apoptosis in solid and hematological tumors.

Abstract: Studies in the past few years have provided compelling evidence for the critical role of aberrant Signal Transducer and Activator of Transcription 3 (STAT3) in malignant transformation and tumorigenesis. Thus, it is now generally accepted that STAT3 is one of the critical players in human cancer formation and represents a valid target for novel anticancer drug design. This review focuses on aberrant STAT3 and its role in promoting tumor cell survival and supporting the malignant phenotype. A brief evaluation of the current strategies targeting STAT3 for the development of novel anticancer agents against human tumors harboring constitutively active STAT3 will also be presented.

MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Abstract: Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.

Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model.

Abstract: Human mesenchymal stem cells (hMSCs) can home to tumor sites and inhibit the growth of tumor cells. Little is known about the underlying molecular mechanisms that link hMSCs to the targeted inhibition of tumor cells. In this study, we investigated the effects of hMSCs on two human hepatoma cell lines (H7402 and HepG2) using an animal transplantation model, a co-culture system and conditioned media from hMSCs. Animal transplantation studies showed that the latent time for tumor formation was prolonged and that the tumor size was smaller when SCID mice were injected with H7402 cells and an equal number of Z3 hMSCs. When co-cultured with Z3 cells, H7402 cell proliferation decreased, apoptosis increased, and the expression of Bcl-2, c-Myc, proliferating cell nuclear antigen (PCNA) and survivin was downregulated. After treatment with conditioned media derived from Z3 hMSC cultures, H4702 cells showed decreased colony-forming ability and decreased proliferation. Immunoblot analysis showed that beta-catenin, Bcl-2, c-Myc, PCNA and survivin expression was downregulated in H7402 and HepG2 cells. Taken together, our findings demonstrate that hMSCs inhibit the malignant phenotypes of the H7402 and HepG2 human liver cancer cell lines, which include proliferation, colony-forming ability and oncogene expression both in vitro and in vivo. Furthermore, our studies provide evidence that the Wnt signaling pathway may have a role in hMSC-mediated targeting and tumor cell inhibition.

A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1.

Abstract: Bone-marrow-derived mesenchymal stem cells (MSCs) have been shown to possess immunosuppressive properties, e.g., by inhibiting T cell proliferation. Activated T cells can also enhance the immunosuppression ability of MSCs. The precise mechanisms underlying MSC-mediated immunosuppression remain largely undefined, although both cell-cell contact and soluble factors have been implicated; nor is it clear how the immunosuppressive property of MSCs is modulated by T cells. Using MSCs isolated from mouse bone marrow, we show here that interferon gamma (IFNgamma), a well-known proinflammatory cytokine produced by activated T cells, plays an important role in priming the immunosuppressive property of MSCs. Mechanistically, IFNgamma acts directly on MSCs and leads to up-regulation of B7-H1, an inhibitory surface molecule in these stem cells. MSCs primed by activated T cells derived from IFNgamma-/- mouse exhibited dramatically reduced ability to suppress T cell proliferation, a defect that can be rescued by supplying exogenous IFNgamma. Moreover, siRNA-mediated knockdown of B7-H1 in MSCs abolished immunosuppression by these cells. Taken together, our results suggest that IFNgamma plays a critical role in triggering the immunosuppresion by MSCs through up-regulating B7-H1 in these cells, and provide evidence supporting the cell-cell contact mechanism in MSC-mediated immunosuppression.

Feedback regulation of cholesterol synthesis: sterol-accelerated ubiquitination and degradation of HMG CoA reductase

Abstract: 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).

Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses

Abstract: Jasmonic acid (JA) is an important phytohormone that regulates plant defense responses against herbivore attack, pathogen infection and mechanical wounding. In this report, we provided biochemical and genetic evidence to show that the Arabidopsis thaliana NAC family proteins ANAC019 and ANAC055 might function as transcription activators to regulate JA-induced expression of defense genes. The role of the two NAC genes in JA signaling was examined with the anac019 anac055 double mutant and with transgenic plants overexpressing ANAC019 or ANAC055. The anac019 anac055 double mutant plants showed attenuated JA-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) and LIPOXYGENASE2 (LOX2) expression, whereas transgenic plants overexpressing the two NAC genes showed enhanced JA-induced VSP1 and LOX2 expression. That the JA-induced expression of the two NAC genes depends on the function of COI1 and AtMYC2, together with the finding that overexpression of ANAC019 partially rescued the JA-related phenotype of the atmyc2-2 mutant, has led us to a hypothesis that the two NAC proteins act downstream of AtMYC2 to regulate JA-signaled defense responses. Further evidence to substantiate this idea comes from the observation that the response of the anac019 anac055 double mutant to a necrotrophic fungus showed high similarity to that of the atmyc2-2 mutant.

Roles of unphosphorylated STATs in signaling.

Abstract: The seven members of the signal transducer and activator of transcription (STAT) family of transcription factors are activated in response to many different cytokines and growth factors by phosphorylation of specific tyrosine residues. The STAT1 and STAT3 genes are specific targets of activated STATs 1 and 3, respectively, resulting in large increases in the levels of these unphosphorylated STATs (U-STATs) in response to the interferons (STAT1) or ligands that active gp130, such as IL-6 (STAT3). U-STATs drive gene expression by novel mechanisms distinct from those used by phosphorylated STAT (P-STAT) dimers. In this review, we discuss the roles of U-STATs in transcription and regulation of gene expression.

FXR: a metabolic regulator and cell protector.

Abstract: Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. As a metabolic regulator, FXR plays key roles in bile acid, cholesterol, lipid, and glucose metabolism. Therefore, FXR is a potential drug target for a number of metabolic disorders, especially those related to the metabolic syndrome. More recently, our group and others have extended the functions of FXR to more than metabolic regulation, which include anti-bacterial growth in intestine, liver regeneration, and hepatocarcinogenesis. These new findings suggest that FXR has much broader roles than previously thought, and also highlight FXR as a drug target for multiple diseases. This review summarizes the basic information of FXR but focuses on its new functions.

Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology

Abstract: Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER, while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

The IkappaB kinase - a bridge between inflammation and cancer.

Abstract: A potential link between inflammation and cancer has been suspected for over a century, but the exact molecular mechanisms connecting the two remained nebulous. We proposed that NF-kappaB transcription factors regulated via the IkappaB kinase (IKK) complex play a critical role in coupling inflammation and cancer and have set out to test this hypothesis in mouse models of cancer. Using mice bearing mutations in the genes coding for the IKKbeta and IKKalpha catalytic subunits we obtained evidence supporting a critical role for IKKbeta in tumor promotion and more recently identified the involvement of IKKalpha in metastatogenesis. Whereas the major pro-tumorigenic function of IKKbeta is mediated via NF-kappaB, the pro-metastatic function of IKKalpha is NF-kappaB-independent. In addition to illustrating the critical roles of the two IKK molecules in linking inflammation and cancer and providing an explanation for increased cancer risk in response to persistent infections and inflammation, these results also identify new targets for development of novel anti-cancer therapies and preventive strategies. Instead of targeting the cancer cell itself, as done by conventional anti-cancer drugs, the new therapeutics will target processes that occur within inflammatory cells that are essential for cancer development and progression. Unlike cancer cells, inflammatory cells retain a normal and stable genome and therefore are unlikely to become genetically resistant to therapeutic intervention.

Actions of human telomerase beyond telomeres.

Abstract: Telomerase has fundamental roles in bypassing cellular aging and in cancer progression by maintaining telomere homeostasis and integrity. However, recent studies have led some investigators to suggest novel biochemical properties of telomerase in several essential cell signaling pathways without apparent involvement of its well established function in telomere maintenance. These observations may further enhance our understanding of the molecular actions of telomerase in aging and cancer. This review will provide an update on the extracurricular activities of telomerase in apoptosis, DNA repair, stem cell function, and in the regulation of gene expression.

The roles of MAPKs in disease

Abstract: MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA.

Abstract: In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.

The DNA damage response pathways: at the crossroad of protein modifications.

Abstract: Post-translational modifications play a crucial role in coordinating cellular response to DNA damage. Recent evidence suggests an interplay between multiple protein modifications, including phosphorylation, ubiquitylation, acetylation and sumoylation, that combine to propagate the DNA damage signal to elicit cell cycle arrest, DNA repair, apoptosis and senescence. Utility of specific post-translational modifiers allows temporal and spatial control over protein relocalization and interactions, and may represent a means for trans-regulatory activation of protein activities. The ability to recognize these specific modifiers also underscores the capacity for signal amplification, a crucial step for the maintenance of genomic stability and tumor prevention. Here we have summarized recent findings that highlight the complexity of post-translational modifications in coordinating the DNA damage response, with emphasis on the DNA damage signaling cascade.

Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.

Abstract: MYB-type transcription factors contain the conserved MYB DNA-binding domain of approximately 50 amino acids and are involved in the regulation of many aspects of plant growth, development, metabolism and stress responses. From soybean plants, we identified 156 GmMYB genes using our previously obtained 206 MYB unigenes, and 48 were found to have full-length open-reading frames. Expressions of all these identified genes were examined, and we found that expressions of 43 genes were changed upon treatment with ABA, salt, drought and/or cold stress. Three GmMYB genes, GmMYB76, GmMYB92 and GmMYB177, were chosen for further analysis. Using the yeast assay system, GmMYB76 and GmMYB92 were found to have transactivation activity and can form homodimers. GmMYB177 did not appear to have transactivation activity but can form heterodimers with GmMYB76. Yeast one-hybrid assay revealed that all the three GmMYBs could bind to cis-elements TAT AAC GGT TTT TT and CCG GAA AAA AGG AT, but with different affinity, and GmMYB92 could also bind to TCT CAC CTA CC. The transgenic Arabidopsis plants overexpressing GmMYB76 or GmMYB177 showed better performance than the GmMYB92-transgenic plants in salt and freezing tolerance. However, these transgenic plants exhibited reduced sensitivity to ABA treatment at germination stage in comparison with the wild-type plants. The three GmMYB genes differentially affected a subset of stress-responsive genes in addition to their regulation of a common subset of stress-responsive genes. These results indicate that the three GmMYB genes may play differential roles in stress tolerance, possibly through regulation of stress-responsive genes.

XRCC1 and DNA polymerase beta in cellular protection against cytotoxic DNA single-strand breaks.

Abstract: Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1(-/-) mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase beta (pol beta) is specific to this pathway, whereas pol beta is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1(-/-), and to a lesser extent in pol beta(-/-) cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol beta(-/-) cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1(-/-) cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.