Properties of iron-sulfur protein isolated from Pseudomonas ovalis.

Mutants of $\text{Escherichia coli}$ C were selected for resistance towards a set of cell wall LPS core specific bacteriophages, including øX174. Increasingly deficient LPS's from wt and mutant $\text{E. coli}$ C were tested for inactivation of øX174, and the core oligosaccharides were subjected to structural analysis by methylation/g.l.c./m.s. Loss of the terminal galactose in the following basic structure of the $\text{E. coli}$ C wt core was found to lead to adsorption resistance towards øX174:

     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila

Resistance and resilience constitute the two complementary aspects of epithelial host defenses in Drosophila. Epithelial cell homeostasis is necessary for the recovery of damages caused by stress or infections. However, the genes responsible for gut epithelial homeostasis remain poorly understood. Here, we show that rgn^G4035 mutant flies have higher mortality than wild-type flies after ingestion of sodium dodecyl sulfate (SDS). Excessive melanization and increased necrotic cells in the gut contribute to the reduced survival of rgn^G4035 mutant flies following SDS ingestion. rgn mutant flies have a defect in the replenishment of intestinal stem cells (ISCs) following gut damage. The antimicrobial peptide (AMP) expression is affected in rgn^G4035 mutant fly guts. Together, our study provides evidence that rgn gene is essential for gut cell homeostasis following damage in Drosophila.     

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5 mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (?1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5 mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (?1% oxygen) and hypoxia mimetic CoCl₂. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5 mM glucose after 48 h of physical hypoxia. However, combining the latter with 100 μM CoCl₂ was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl₂ in 5 mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.     

Effect of rate-limiting elongation on bacteriophage MS2 RNA-directed protein synthesis in extracts of Escherichia coli

The consequences of limiting the rate of elongation of protein synthesis in vitro have been examined. The concentration of Trp-tRNA^Trp was manipulated by varying the amount of exogenously added tryptophan in extracts from an Escherichia coli mutant in which the tryptophanyl-tRNA-synthetase has a higher K_M for tryptophan. The evidence presented supports the hypothesis that variation of the rate of elongation can be a means of regulating gene expression, both directly, by slowing or accelerating the rate of protein synthesis and indirectly, by leading to varying three-dimensional structures of the messenger RNA when progress of the ribosomes is perturbed. The data can be described by assuming that if a specific transfer RNA is limiting, to a first approximation the overall rate of protein synthesis is determined by the relative rate of reading past an individual codon requiring that tRNA raised to the power of how many times that codon appears in the message. This could be explained by a model in which, with a significant probability, the ribosome stops protein synthesis prematurely at these codons, falls off the messenger RNA and is available for further rounds of protein synthesis. In agreement with other work, evidence is also presented that suggests that under the most drastic available limitation of the elongation rate, that is, starvation for a given amino acid, reading through the corresponding “hungry codon” occurs in vitro at a surprisingly high rate, possibly due to mistranslation.     

Cell Cycle Regulation Targets of MYCN Identified by Gene Expression Microarrays

Background: We have previously shown that MYCN knockdown causes a G1 arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21WAF1 and hypo RB in p53 wt cell lines. 1 Hypothesis: MYCN acts by inhibiting p21WAF1, and also by p21 independent mechanisms to override the G1 checkpoint in exponentially growing cells. Methods: Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR-32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT-PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N). Results: MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+ Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57cip2) and TP53INP1 also increased after MYCN knockdown. Conclusions: MYCN may override the G1 checkpoint through down-regulation of SKP2 and TP53INP1 resulting in reduced p21WAF1 expression in p53 wt cell lines, and in addition may act through the WNT signalling pathway in a p53 independent manner.     

Partial characterizatio and proposed mode of action of inhibitory heLa cell surface polypeptides

Polypeptides removed from the HeLa cell surface by mild pronase treatment rapidly inhibit protein synthesis when added to HeLa cells or cell-free translation system derived from HeLa cells. The inhibitory activity is heat stable. Protein and carbohydrate components of these polypeptides are required for inhibition of protein synthesis in vivo and in vitro. Two peaks of activity can be recovered from polyacrylamide gels, corresponding to polypeptides with molecular weights of approximately 29 000 and 41 000. Inhibition of protein synthesis in cell-free translation systems appears to be primarily an effect on elongation of polypeptide chains, whereas in the intact cell the primary target may be polypeptide chain initiation.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

Short and long-term tumor cell responses to Aurora kinase inhibitors

Aurora kinases are essential for mitosis and are candidate targets of novel chemotherapeutic agents. The inhibitors ZM447439, MK-0457 (VX-680) as well as Hesperadin have been used to dissect the roles of Aurora kinases in the cell cycle and have been tested clinically for the treatment of cancer. Here we have carried out a detailed kinetic analysis of two isogenic cell lines differing in p53 function and have compared the effects of ZM447439 and VE-465 (related to MK-0457). We find that p53 is needed for efficient cell cycle arrest when Aurora kinases are inhibited by either ZM447439 or VE-465. However, the p53-induced cell cycle block is neither immediate nor absolute. ZM447439 induced the localized accumulation of γH2A.X indicating that p53 induction by this drug occurs in response to DNA damage. Our analysis of the long-term effects of ZM447439 indicates that cells can evade killing by the drug, but not via a classical drug-resistance mechanism. Several mechanisms to explain how cells may evade killing by Aurora kinase inhibitors are described.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

Distinct functions for WRN and TP53 in a shared pathway of cellular response to 1-beta-D-arabinofuranosylcytosine and bleomycin

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-beta-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway.     

Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae

Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10. The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype. MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10. The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the alpha-1,6-polymerizing activity of the mannosyltransferase complex. These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium. The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion. KlMNN10 has EMBL accession no. AJ575132.     

Ionizing radiation enhances the expression of the nonsteroidal anti-inflammatory drug-activated gene (NAG1) by increasing the expression of TP53 in human colon cancer cells

The induction of apoptosis in cells of human colon cancer cell lines after gamma irradiation was investigated to determine whether apoptosis was mediated by TP53 and the subsequent expression of its downstream target, the NSAID-activated gene (NAG1). HCT116 (TP53(+/+)), HCT15 (TP53 mutant) and TP53 null HCT116 (TP53(-/-)) cells were irradiated with gamma rays, and apoptosis was measured at various times after irradiation. In HCT116 TP53(+/+) cells, apoptosis was increased after irradiation; the increase was dependent on the time after treatment and the dose of gamma rays. However, in HCT15 TP53 mutant cells and HCT116 TP53(-/-) cells, there were no remarkable changes in apoptosis. The expression of TP53 protein in HCT116 cells was increased after irradiation and was followed by an increase in the expression of NAG1 protein. In contrast, the expression of NAG1 protein in TP53 mutant cells and TP53(-/-) cells was not increased by the radiation treatment, suggesting that NAG1 was required for apoptosis. The expression of NAG1 increased apoptosis in HCT116 cells, but radiation treatment did not further increase apoptosis. The transfection of a NAG1 siRNA into HCT116 cells suppressed radiation-induced apoptosis and inhibited the induction of NAG1 protein without altering the expression of TP53. a NAG1 luciferase promoter construct that included both of the TP53 binding sites, was activated by radiation in dose-dependent manner, while the promoters lacking one or both of the TP53 binding sites in the NAG1 promoter activity either was less responsive or did not respond. The findings reported here indicate that gamma radiation activates the TP53 tumor suppressor, which then increases the expression of NAG1. NAG1 mediates the induction of apoptosis in human colorectal cells.     

Restoration of mutant TP53 to normal TP53 function by glycerol as a chemical chaperone

We have previously reported that heat stress induces expression of wild-type TP53 (formerly known as p53) activated factor 1 (CDKN1A, formerly known as WAF1) only when TP53 protein is wild-type using cells of a human glioblastoma cell line (A-172) and cells of its transformant (A-172/mp53/ 143) with a mutant TP53 (point mutation at codon 143 from Val to Ala) vector. Transfection of A-172 cells with the mutant TP53 vector abolished the heat-induced expression of CDKN1A, demonstrating the dominant negative nature of this TP53 mutant over the endogenous wild-type TP53. This kind of dominant negative TP53 mutant occurs frequently in various types of cancer. Overcoming this dominance or restoring the normal functions to these TP53 mutants is a new strategy for TP53-targeted cancer therapies. We examined whether glycerol can act as a chemical chaperone to correct the mutant TP53 conformation. No CDKN1A expression was induced after heating or treatment with glycerol at concentrations of 0.6 and 1.2 M in these transformants. In contrast, A-172/mp53/ 143 cells showed CDKN1A expression when they were heated in the presence of glycerol at 0.6 or 1.2 M, which was similar to the response of the parental and neo vector-transfected control cells. To test the generality of the effects of glycerol on mutant TP53, we used human osteosarcoma Saos-2 cells (lacking TP53) transfected with mutant TP53 and cells of two other human glioblastoma cell lines carrying mutant TP53. These cells showed similar CDKN1A expression when heated in the presence of glycerol at 0.6 or 1.2 M. These results suggest that glycerol is effective in restoring several TP53 mutants to normal TP53 function, leading to normal CDKN1A expression after heat stress. This observation provides a novel tool for correction of mutant TP53 conformation and may be applicable for TP53-targeted cancer therapy.