Elevated expression of Rad18 regulates melanoma cell proliferation.

The E3 ligase Rad18 is a key regulator for the lesion bypass pathway, which plays an important role in genomic stability. However, the status of Rad18 expression in melanoma is not known. Using melanoma tissue microarray (TMA), we showed that nuclear Rad18 expression was upregulated in primary and metastatic melanoma compared to dysplastic nevi. Rad18 expression was significantly reduced in sun-exposed sites compared to the sun-protected sites. Strong Rad18 expression correlated with worse 5-year patient survival and was an independent prognostic factor for melanoma found in the sun-protected sites. Furthermore, we showed that melanoma cell proliferation and the expression of pAkt and cyclin D1 were reduced upon Rad18 knockdown. We, for the first time, showed that Rad18 is significantly increased in melanoma and predicts the poor outcome for melanoma in the sun-protected sites. Rad18 is involved in the regulation of melanoma cell proliferation, which can be exploited in designing new strategy for melanoma treatment.

Tumour suppressor ING1b maintains genomic stability upon replication stress.

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.

The ING family tumor suppressors: from structure to function.

The Inhibitor of Growth (ING) proteins belong to a well-conserved family which presents in diverse organisms with several structural and functional domains for each protein. The ING family members are found in association with many cellular processes. Thus, the ING family proteins are involved in regulation of gene transcription, DNA repair, tumorigenesis, apoptosis, cellular senescence and cell cycle arrest. The ING proteins have multiple domains that are potentially capable of binding to many partners. It is conceivable, therefore, that such proteins could function similarly within protein complexes. In this case, within this family, each function could be attributed to a specific domain. However, the role of ING domains is not definitively clear. In this review, we summarize recent advances in structure-function relationships in ING proteins. For each domain, we describe the known biological functions and the approaches utilized to identify the functions associated with ING proteins.

Radically rethinking agriculture for the 21st century.

Population growth, arable land and fresh water limits, and climate change have profound implications for the ability of agriculture to meet this century's demands for food, feed, fiber, and fuel while reducing the environmental impact of their production. Success depends on the acceptance and use of contemporary molecular techniques, as well as the increasing development of farming systems that use saline water and integrate nutrient flows.

Loss of SNF5 expression correlates with poor patient survival in melanoma.

PURPOSE: Aberrant expression of SWI/SNF chromatin remodeling complex is involved in cancer development. The tumor suppressor SNF5, the core subunit of SWI/SNF complex, has been shown to regulate cell differentiation, cell cycle control, and apoptosis. To investigate the role of SNF5 in the development of melanoma, we examined the expression of SNF5 in melanocytic lesions at different stages and analyzed the correlation between SNF5 expression and clinicopathologic variables and patient survival. EXPERIMENTAL DESIGN: Using tissue microarray and immunohistochemistry, we evaluated SNF5 staining in 51 dysplastic nevi, 88 primary melanomas, and 48 metastatic melanomas. We studied chemosensitivity of melanoma cells with reduced SNF5 expression by siRNA using cell survival and apoptosis assays. RESULTS: SNF5 expression was reduced in metastatic melanoma compared with dysplastic nevi (P = 0.005), in advanced primary melanoma (Clark's level V) compared with low risk Clark's level II melanoma (P = 0.019), and in melanoma at sun-exposed sites compared with sun-protected sites (P = 0.044). Furthermore, we showed a strong correlation between negative SNF5 expression and a worse 5-year survival in melanoma patients (P = 0.016). Multivariate Cox regression analysis revealed that negative SNF5 expression is an independent prognostic factor to predict patient outcome in primary melanomas (P = 0.031). Finally, we showed that knockdown of SNF5 in melanoma cell lines resulted in significant chemoresistance. CONCLUSIONS: Our data indicate that SNF5 may be an important marker for human melanoma progression and prognosis as well as a potential therapeutic target.

The role of ING tumor suppressors in UV stress response and melanoma progression.

The INhibitor of Growth (ING) genes were discovered during the past decade and identified as type II tumor suppressor genes. Previous studies demonstrated that ING family members participate in various cellular stress responses and thus play important roles in the pathogenesis of various types of cancers, including melanoma. Epidemiological studies showed that UV radiation is the primary etiological factor in melanoma development. Here we review the studies on the role of ING proteins in cellular responses to UV irradiation, melanoma cell motility, and melanoma progression.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33(ING1b).

The tumour suppressor p33(ING1b) ((ING1b) for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33(ING1b) phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33(ING1b) with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33(ING1b). We found that p33(ING1b) is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33(ING1b). Furthermore, ultraviolet irradiation induces p33(ING1b) phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.

Myeloid leukemia-1 expression in benign and malignant melanocytic lesions.

Myeloid leukemia-1 (Mcl-1) is an anti-apoptotic protein implicated in tumor progression. Its expression was found to be elevated in many types of human cancers and is correlated with tumor progression. The expression of Mcl-1 in melanoma is not fully understood. We investigated the expression of Mcl-1 in normal nevi, dysplastic nevi, primary melanoma and melanoma metastases by tissue microarray and immunohistochemistry. We found that Mcl-1 expression was significantly increased in dysplastic nevi, primary melanoma and melanoma metastases when compared to normal nevi, though the expression of Mcl-1 was decreased in metastatic melanoma when compared to dysplastic nevi. We did not find any correlation between Mcl-1 expression and melanoma patient survival. Our data suggest that Mcl-1 may play a critical role in the initiation of melanoma development.

The role of integrin-linked kinase in melanoma cell migration, invasion, and tumor growth.

Melanoma is a life-threatening disease with a high mortality rate due to rapid metastasis. Currently, there is no effective treatment for metastatic melanoma. Integrin-linked kinase (ILK) is a serine/threonine kinase and has its role implicated in connecting cell-extracellular matrix interaction and growth factor signaling to cell survival, cell migration, invasion, anchorage-independent growth, angiogenesis, and epithelial-mesenchymal transition. However, the functional role of ILK in melanoma progression is not completely understood. We have previously shown that strong ILK expression was significantly associated with melanoma thickness. In this study, we further elucidate the role of ILK in melanoma cell migration, invasion, anchorage-independent growth, and tumor growth in vivo by specific ILK knockdown using small interfering RNA and short hairpin RNA. We found that ILK knockdown impeded melanoma cell migration, which was associated with reduced stress fiber formation, cell spreading, and cell adhesion. Furthermore, ILK knockdown decreased the invasion ability of melanoma cells and the formation of anchorage-independent colonies in soft agar. Moreover, ILK knockdown significantly impaired the growth of melanoma xenografts in severe combined immunodeficient mice. This study highlights the importance of ILK in melanoma progression and provides an attractive target for the treatment of melanoma.

The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA.

ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.

Characterization of four rice mutants with alterations in the defence response pathway.

SUMMARY A fast-neutron mutagenized population of rice seedlings was screened with Magnaporthe grisea, the causal agent of rice blast disease, to identify mutants with alterations in the defence response. Three mutant lines, ebr1, ebr2 and ebr3 (enhanced blast resistance) were identified that display enhanced resistance to M. grisea. ebr1 and ebr3 also confer enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). ebr3 develops a lesion mimic (LM) phenotype upon inoculation with M. grisea, and the phenotype is also induced by a shift in environmental conditions. The fourth mutant line, ncr1 (necrosis in rice), has an LM phenotype under all conditions tested and lacks enhanced resistance to either M. grisea or Xoo. Complementation testing using the mutant lines ebr3 and ncr1 indicates that the ebr3 and ncr1 loci are nonallelic and recessive. ebr1 and ebr2 display no alterations in expression of the rice pathogenesis-related (PR) genes PBZ1 and PR1, compared to wild-type CO39. ebr3 has an elevated expression of PBZ1 and PR1 only in tissue displaying the LM phenotype. ncr1 strongly expresses PBZ1 in tissue displaying the LM phenotype, whereas PR1 expression in this tissue is similar to wild-type CO39.

Use of Pi5(t) markers in marker-assisted selection to screen for cultivars with resistance to Magnaporthe grisea.

Identification of the PCR markers tightly linked to genes that encode important agronomic traits is useful for marker-assisted selection (MAS). The rice Pi5(t) locus confers broad-spectrum resistance to Magnaporthe grisea, the causal agent of rice blast disease. It has been hypothesized that the Pi5(t) locus carries the same gene as that encoded by the Pi3(t) and Pii(t) loci. We developed three PCR-based dominant markers (JJ80-T3, JJ81-T3, and JJ113-T3) from three previously identified BIBAC clones-JJ80, JJ81, and JJ113-that are linked to the Pi5(t) locus. PCR analysis of 24 monogenic lines revealed that these markers are present only in lines that carry Pi5(t), Pi3(t), and Pii(t). PCR and DNA gel-blot analysis of candidate resistance lines using JJ80-T3, JJ81-T3, and JJ113-T3 indicated that Tetep is the likely donor of Pi5(t). Of the 184 rice varieties tested, 34 carried the JJ80-T3-, JJ81-T3-, and JJ113-T3-specific bands. Disease evaluation of those 34 varieties revealed that all conferred resistance to PO6-6. The genomic structure of three of these resistant varieties (i.e., IR72, Taebaeg, Jahyangdo) is most similar to that of Pi5(t). Our results demonstrate the usefulness of the JJ80-T3, JJ81-T3, and JJ113-T3 markers for MAS for M. grisea resistance.

Development of Co-Dominant Amplified Polymorphic Sequence Markers in Rice that Flank the Magnaporthe grisea Resistance Gene Pi7(t) in Recombinant Inbred Line 29.

ABSTRACT Pi7(t), a dominant blast resistance gene derived from the rice cultivar Moroberekan, confers complete resistance against the fungal pathogen Magnaporthe grisea. Pi7(t) previously was positioned on chromosome 11 by restriction fragment length polymorphism (RFLP) mapping of a recombinant inbred line population. One derivative of this population, recombinant inbred line (RIL)29, was designated as the representative line for Pi7(t). A segregating F2 population was created from RIL29 in order to determine the location of Pi7(t). The new mapping data indicate a position for Pi7(t) 30 centimorgans distal to the original location. Pi7(t) shares a common position with the previously mapped Pi1 M. grisea resistance gene. RIL29 carries DNA not derived from either parent used to create the RIL population at the newly assigned Pi7(t) locus. RFLP analysis has identified a possible donor source.

Isolation and characterization of rice mutants compromised in Xa21-mediated resistance to X. oryzae pv. oryzae.

The rice gene, Xa21, confers resistance to diverse races of Xanthomonas oryzae pv. oryzae (Xoo) and encodes a receptor-like kinase with leucine-rich repeats in the extra-cellular domain. To identify genes essential for the function of the Xa21 gene, 4,500 IRBB21 ( Xa21 isogenic line in IR24 background) mutants, induced by diepoxybutane and fast neutrons, were screened against Philippine race six (PR6) Xoo for a change from resistance to susceptibility. From two greenhouse screens, 23 mutants were identified that had changed from resistant to fully (6) or partially (17) susceptible to PR6. All fully susceptible mutants carried rearrangements at the Xa21 locus as detected by PCR and Southern hybridization. For the partially susceptible mutants, no changes were detected at the Xa21 locus based on Southern and PCR analyses. However, two of these mutants were confirmed via genetic analysis to have mutations at the Xa21 locus. Partially susceptible mutants exhibited variation in level of susceptibility to different Xoo strains, suggesting that they may carry different mutations required for the Xa21-mediated resistance. The mutants identified in this study provide useful materials for dissecting the Xa21-mediated resistance pathway in rice.

Genetic and physical mapping of Pi5(t), a locus associated with broad-spectrum resistance to rice blast.

To gain an understanding of the molecular basis for resistance to rice blast (Magnaporthe grisea), we have initiated a project to clone Pi5(t), a locus associated with broad-spectrum resistance to diverse blast isolates. AFLP-derived markers linked to Pi5(t)-mediated resistance were isolated using bulked segregant analysis of F(2) populations generated by crossing three recombinant inbred lines (RILs), RIL125, RIL249, and RIL260 with the susceptible line CO39. The most tightly linked AFLP marker, S04G03, was positioned on chromosome 9 of the fingerprint-based physical map of Nipponbare, a well-characterized rice genotype. Flanking BAC-based Nipponbare markers were generated for saturation mapping using four populations, the three initial RILs and an additional one derived from a cross between M202 and RIL260. A BIBAC (binary BAC) library was constructed from RIL260. Using these resources Pi5(t) was mapped to a 170-kb interval, and a contiguous set of BIBAC clones spanning this region was constructed. It had previously been suggested that Pi3(t) and Pi5(t) might be allelic, due to their identical resistance spectrum and tight linkage. We therefore compared genomic regions for lines containing Pi3(t) using the Pi5(t)-linked markers. DNA gel-blot analyses indicated that the region around Pi3(t) is identical to that of Pi5(t), suggesting that Pi3(t) and Pi5(t) are the same resistance gene.

Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis.

The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Over-expression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade-sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1-rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequence-specifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.

Isolation and characterization of disease resistance gene homologues from rice cultivar IR64.

We initiated a search for disease resistance (R) gene homologues in rice cultivar IR64, one of the most agronomically important rice varieties in the world, with the assumption that some of these homologues would correspond to previously identified disease resistance loci. A family of rice R gene homologues was identified using the Arabidopsis NBS-LRR disease resistance gene RPS2 as a hybridization probe. Because member genes of this rice R gene family exhibit features characteristic of the NBS-LRR class of resistance genes, the family was given the name NRH (for NBS-LRR resistance gene homologues). Three members of the NRH family, NRH1, NRH2, and NRH3, were cloned and studied in detail. In IR64, NRH1 and NRH2 appear to encode full-length polypeptides, whereas NRH3 is prematurely truncated with a stop codon generated by a frameshift. NRH1 maps on chromosome 5, and NRH2 and NRH3 are less than 48kb apart on chromosome 11. Although NRH1, NRH2, and NRH3 map to regions of the rice genome where disease resistance loci to Xanthomonas oryzae pv. oryzae (Xoo) have been identified, susceptible rice varieties transformed with either NRH1 or NRH2 failed to exhibit increased resistance to a set of well-characterized Xoo strains.

The rice Rim2 transcript accumulates in response to Magnaporthe grisea and its predicted protein product shares similarity with TNP2-like proteins encoded by CACTA transposons.

A rice transcript, Rim2, was identified that accumulated in both incompatible and compatible interactions between rice and Magnaporthe grisea. The Rim2 transcript also accumulated in response to treatment with a cell wall elicitor derived from M. grisea. A 3.3-kb RIM2 cDNA clone was isolated and is predicted to encode a protein of 653 amino acids, which shares 32 55% identity with TNP2-like proteins encoded by CACTA transposons of other plants. A 1.05-kb segment of the Rim2 sequence shows 82% nucleotide sequence identity with sequences flanking the A1 and C members of the rice Xa21 disease resistance gene family. The 5'-upstream region of Rim2 was cloned and the transcriptional start sites were identified. The 5' and 3' noncoding termini of Rim2 are AT-rich. A cis-element showing similarity to a sequence that mediates defense-associated transcriptional activation of the tobacco retrotransposon Tnt1, and four motifs that fit the consensus sequence of the elicitor-responsive elements in the promoters of the parsley PR-1 genes were found in the 5'-upstream region. Four imperfect tandem repeats were identified in the 3' noncoding terminus. Southern analysis with genomic DNA from different rice species indicated that Rim2 is present in 3-4 copies per genome. These results suggest that Rim2 may be one component of a large CACTA-like element, whose transcript accumulates in response to attack by pathogens.

A high-resolution linkage map of the vicinity of the rice submergence tolerance locus Sub1.

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F3 families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F2 plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region.

The evolution of disease resistance genes.

Several common themes have shaped the evolution of plant disease resistance genes. These include duplication events of progenitor resistance genes and further expansion to create clustered gene families. Variation can arise from both intragenic and intergenic recombination and gene conversion. Recombination has also been implicated in the generation of novel resistance specificities. Resistance gene clusters appear to evolve more rapidly than other regions of the genome. In addition, domains believed to be involved in recognitional specificity, such as the leucine-rich repeat (LRR), are subject to adaptive selection. Transposable elements have been associated with some resistance gene clusters, and may generate further variation at these complexes.