No abstract

Recent studies have revealed that fungi possess a mechanism similar to bacterial two-component systems to respond to extracellular changes in osmolarity. In Saccharomyces cerevisiae, Slnl p contains both histidine kinase and receiver (response regulator) domains and acts as an osmosensor protein that regulates the downstream HOG1 MAP kinase cascade. SLNI of Candida albicans was functionally cloned using an S. cerevisiae strain in which SLNI expression was conditionally suppressed. Deletion analysis of the cloned gene demonstrated that the receiver domain of C. albicans Slnlp was not necessary to rescue SLNI-deficient S. cerevisiae strains. Unlike 5. cerevisiae, a null mutation of C. albicans SLNI was viable under regular and high osmotic conditions, but it caused a slight growth retardation at high osmolarity. Southern blotting with C. albicans SLNI revealed the presence of related genes, one of which is highly homologous to the NIKl gene of Neurospora crassa. Thus, C. albicans harbours both SLNI-and NIKI-type histidine kinases.

The gene expression profiles of 33 renal cell carcinomas (RCCs) and nine normal kidney samples were examined using high-density oligonucleotide microarrays in an attempt to identify biomolecular markers for the diagnosis of tumour subtypes and also for prediction of prognosis. Hierarchical clustering demonstrated that clear-cell RCC, chromophobe RCC, and normal kidney tissue showed distinctive gene expression profiles. The mean expression levels of 149 of 12 500 genes were more than three times higher in clear-cell RCC than in chromophobe RCC and normal kidney tissue. Among the genes whose expression was upregulated in clear-cell RCC, adipose differentiation-related protein (ADFP) and nicotinamide N-methyltransferase (NNMT) were selected for further analysis. Consistent with the results of the microarray, increased levels of ADFP and NNMT mRNA were found more frequently in clear-cell RCCs than in other non-clear-cell tumour subtypes using real-time quantitative PCR. Immunohistochemistry for ADFP showed strong and unique tumour cell staining patterns in the majority of clear-cell RCCs. More importantly, patients bearing tumours with higher AFDP mRNA levels showed significantly better survival in both univariate and multivariate analyses. ADFP is a lipid storage droplet-associated protein and its transcription is considered to be regulated by the von Hippel-Lindau/hypoxia-inducible factor pathway. It is known that clear-cell RCC contains abundant lipids and cholesterols. Thus it is likely that sustained upregulation of ADFP following VHL inactivation is involved in the morphological appearance of clear-cell RCC. Moreover ADFP expression status may provide useful prognostic information as a biomolecular marker in patients with clear-cell RCC.

The cancer stem cell (CSC) concept has been proposed as an attractive theory to explain cancer development, and CSCs themselves have been considered as targets for the development of diagnostics and therapeutics. However, many unanswered questions concerning the existence of slow cycling/quiescent, drug-resistant CSCs remain. Here we report the establishment of colon cancer CSC lines, interconversion of the CSCs between a proliferating and a drug-resistant state, and reconstitution of tumor hierarchy from the CSCs. Stable cell lines having CSC properties were established from human colon cancer after serial passages in NOD/Shi-scid, IL-2Rc null (NOG) mice and subsequent adherent cell culture of these tumors. By generating specific antibodies against LGR5, we demonstrated that these cells expressed LGR5 and underwent self-renewal using symmetrical divisions. Upon exposure to irinotecan, the LGR5 1 cells transitioned into an LGR5 2 drug-resistant state. The LGR5 2 cells converted to an LGR5 1 state in the absence of the drug. DNA microarray analysis and immunohistochemistry demonstrated that HLA-DMA was specifically expressed in drug-resistant LGR5 2 cells, and epiregulin was expressed in both LGR5 1 and drug-resistant LGR5 2 cells. Both cells sustained tumor initiating activity in NOG mice, giving rise to a tumor tissue hierarchy. In addition, anti-epiregulin antibody was found to be efficacious in a metastatic model. Both LGR5 1 and LGR5 2 cells were detected in the tumor tissues of colon cancer patients. The results provide new biological insights into drug resistance of CSCs and new therapeutic options for cancer treatment.

Human glypican 3 (GPC3) is preferentially expressed in the tumor tissues of liver cancer patients. In this study, we obtained a monoclonal antibody (mAb) against the COOHterminal part of GPC3, which induced antibody-dependent cellular cytotoxicity (ADCC). The mAb, designated GC33, exhibited marked tumor growth inhibition of s.c. transplanted Hep G2 and HuH-7 xenografts that expressed GPC3 but did not inhibit growth of the SK-HEP-1 that was negative for GPC3. GC33 was efficacious even in an orthotopic model; it markedly reduced the blood A-fetoprotein levels of mice intrahepatically transplanted with Hep G2 cells. Humanized GC33 (hGC33) was as efficacious as GC33 against the Hep G2 xenograft, but hGC33 lacking carbohydrate moieties caused neither ADCC nor tumor growth inhibition. Depletion of CD56 + cells from human peripheral blood mononuclear cells markedly abrogated the ADCC caused by hGC33. The results show that the antitumor activity of hGC33 is mainly attributable to ADCC, and in human, natural killer cellmediated ADCC is one possible mechanism of the antitumor effects by GC33. hGC33 will provide a novel treatment option for liver cancer patients with GPC3-positive tumors. [Cancer Res 2008;68(23):9832-8]

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2⌬ and homozygous chs3⌬ null mutants.Like Saccharomyces cerevisiae, Candida albicans harbors three chitin synthase genes, designated CHS1, CHS2, and CHS3 (2, 6, 13). In S. cerevisiae, it was demonstrated by gene disruption experiments that chitin synthase 1 (Chs1p) is involved in the repair of damaged chitin, Chs2p is required for primary septum formation, and Chs3p is responsible for all other chitin syntheses (5,12,14). More recently, Kollar et al. reported that CHS3 also contributes to the formation of linkage between chitin and ␤-1,3-glucan in S. cerevisiae (10). In order to gain more insights into the physiological roles of the chitin synthases of C. albicans, we have disrupted both CHS2 and CHS3 in C. albicans by means of the URA blaster protocol (1).The homozygous chs2⌬ null mutant and the homozygous chs3⌬ null mutant strains of C. albicans were obtained by transforming CAI-4 cells (ura3⌬::imm34/ura3⌬::imm34) with DNA fragments containing either CHS2 in which the hisG-URA3-hisG cassette was inserted at the unique XhoI site or CHS3 in which the 0.8-kb NcoI-ClaI region was replaced by the hisG-URA3-hisG cassette by the lithium acetate method (9). These DNA fragments were successfully integrated into one of the diploid CHS2 or CHS3 alleles, respectively, and the URA3 gene was efficiently eliminated by 5-fluoroorotic acid (5-FOA) selection (11) (Fig. 1). Then these DNA fragments were again transfected into cells in which one of the diploid CHS2 or CHS3 alleles was already flanked by the hisG sequence. Although the second allele of the CHS2 locus was efficiently targeted by the same DNA fragment used to disrupt the first allele, the remaining CHS3 allele was not easily disrupted by transfection of the same DNA fragment. Therefore, we constructed another plasmid in which the hisG-URA3-hisG cassette was inserted at the NcoI site of CHS3. We assumed that use of this DNA for the second round of transfection would increase the efficiency of homologous recombination between the transfected DNA and the remaining intact CHS3 allele because the 0.8-kb NcoI-ClaI region of CHS3 was missing in the already targeted CHS3 locus. As expected, in 3 of 24 uracil auxotrophs, both of the CHS3 alleles were found to be flanked by the hisG sequence after 5-FOA selection, resulting in the homozygous chs3⌬ null mutation (Fig. 1).Cells lacking functional CHS3 grew in a rich medium such as YPD (1% peptone, 2% yeast extract, and 2% dextrose), but their growth was somewhat slower than that of cells missing CHS2 or the parental strain CAI-4 (the doubling times for CAI-4, the homozygous chs2⌬ null mutant, and the homozygous chs3⌬ null mutant were about 70, 72, and 90 min, respectively)...

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of ␤-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeastphase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall ␤-glucan levels by about 50%. The purified C. albicans ␤-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified ␤-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate ␤-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of ␤-1,3-glucan synthase.