Cancer stem cells in solid tumors: elusive or illusive?

Background

The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.

Results

We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44^MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44^MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44^MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.

Conclusions

These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.     

The Hyperthermophilic Euryarchaeon Archaeoglobus fulgidus Repairs Uracil by Single-Nucleotide Replacement

Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transition mutations if uracil is not repaired by the DNA base excision repair (BER) pathway. Since deamination increases rapidly with temperature, hyperthermophiles, in particular, are expected to succumb to such damage. There has been only one report of crenarchaeotic BER showing strong similarities to that in most eukaryotes and bacteria for hyperthermophilic Archaea. Here we report a different type of BER performed by extract prepared from cells of the euryarchaeon Archaeoglobus fulgidus. Although immunodepletion showed that the monofunctional family 4 type of uracil-DNA glycosylase (UDG) is the principal and probably only UDG in this organism, a β-elimination mechanism rather than a hydrolytic mechanism is employed for incision of the abasic site following uracil removal. The resulting 3′ remnant is removed by efficient 3′-phosphodiesterase activity followed by single-nucleotide insertion and ligation. The finding that repair product formation is stimulated similarly by ATP and ADP in vitro raises the question of whether ADP is more important in vivo because of its higher heat stability.After depurination, hydrolytic deamination of cytosine to uracil is the most frequent event that damages DNA (36), and it results in G·C-to-A·T transition mutations if the damage is not repaired. In addition, some dUTP molecules escape hydrolysis by dUTPase, which results in a certain amount of dUMP introduced into DNA opposite adenine during replication (32). Irrespective of the mode of appearance, all cells contain uracil-DNA glycosylase (UDG) (EC 3.2.2.3) enzymes to remove uracil from DNA (17). The resulting abasic or apurinic/apyrimidinic (AP) site can subsequently be removed, and the integrity of the DNA can be restored by the so-called base excision repair (BER) pathway, which consists in its simplest form of the sequential actions of 5′-acting AP endonuclease, 5′-deoxyribose phosphate (dRP) lyase, DNA polymerase, and DNA ligase. The BER pathway can be initiated by one of several DNA glycosylases with different substrate specificities (17, 36, 57), and quantitatively it is the most important repair mechanism for the removal of spontaneously generated base modifications. Genes encoding bacterial and eukaryotic UDGs exhibiting significant selectivity for uracil have been cloned and sequenced in the last 2 decades, and the results have demonstrated that there is a high degree of conservation between distantly related species. Family 1 UDGs (for a review of UDG families 1 to 3, see reference 44), typified by the Escherichia coli Ung enzyme (37), recognize uracil in an extrahelical or flipped-out conformation in double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Several family 1 enzymes have been extensively characterized, both structurally and at the cell and organism levels. Family 2 UDGs, which includes E. coli Mug and mammalian thymine-DNA glycosylase, are mismatch specific and recognize guanine on the complementary strand rather than the lesion itself and thus are inactive with ssDNA. Family 3 UDGs, typified by the SMUG1 enzyme of human cells, have similar substrate requirements but exhibit a stronger preference for uracil in ssDNA than family 1 enzymes (17, 27, 57, 67).UDG activity in hyperthermophilic microorganisms was first reported in 1996 (33). Three years later, Sandigursky and Franklin (47) cloned and overexpressed an open reading frame (ORF) of the hyperthermophilic bacterium Thermotoga maritima that typifies the family 4 UDGs that are able to remove uracil from U·G and U·A base pairs, as well as from ssDNA. By means of homology searches, these workers found ORFs homologous to the T. maritima UDG gene in several prokaryotic genomes, including that of the hyperthermophilic archaeon Archaeoglobus fulgidus, a strict anaerobe that grows optimally at 83°C (60; for a review of DNA repair in hyperthermophilic archaea, see reference 20). Subsequently, they cloned and overexpressed the A. fulgidus ORF in E. coli by producing a His-tagged fusion protein. As expected, the purified A. fulgidus recombinant Afung (rAfung) protein exhibited UDG activity (48). However, whether Afung is the major UDG of A. fulgidus or is just a minor glycosylase with uracil-releasing ability remained to be determined.As a continuation of previous biochemical and physicochemical studies (31) of non-His-tagged rAfung protein, here we characterized a family 4 UDG in archaeon cell extract. The abundance of Afung in vivo was determined, and evidence indicates that this enzyme is the principal UDG of A. fulgidus. Here we also describe the mechanism of dUMP repair employed by this euryarchaeon, which differs in important ways from the mechanism reported for the crenarchaeon Pyrobaculum aerophilum (50).     

Transcriptomic response to differentiation induction

Background  

Microarrays used for gene expression studies yield large amounts of data. The processing of such data typically leads to lists of differentially-regulated genes. A common terminal data analysis step is to map pathways of potentially interrelated genes.     

Synthesis and Characterization of Novel Quinazoline Type Inhibitors for Mutant and Wild-Type EGFR and RICK Kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.     

A simple and sensitive enzyme-mediated assay of biotin.

Free biotin was quantitated by a competition by coating biotin-bovine serum albumin conjugate on a polystyrene microplate for binding to avidin-beta-galactosidase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by reaction with one of the following fluorogenic substrates, resorufin beta-D-galactoside and fluorescein di-beta-D-galactoside, in a fluorescence plate reader. Free biotin as little as 0.1 nmol can be routinely detected.     

The GINS complex from the thermophilic archaeon, <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis> may function as a homotetramer in DNA replication

The eukaryotic GINS heterotetramer, consisting of Sld5, Psf1, Psf2, and Psf3, participates in “CMG complex” formation with mini-chromosome maintenance (MCM) and Cdc45 as a key component of a replicative helicase. There are only two homologs of the GINS proteins in Archaea, and these proteins, Gins51 and Gins23, form a heterotetrameric GINS with a 2:2 molar ratio. The Pyrococcus furiosus GINS stimulates the ATPase and helicase activities of its cognate MCM, whereas the Sulfolobus solfataricus GINS does not affect those activities of its cognate MCM, although the proteins bind each other. Intriguingly, Thermoplasma acidophilum, as well as many euryarchaea, have only one gene encoding the sequence homologous to that of archaeal Gins protein (Gins51) on the genome. In this study, we investigated the biochemical properties of the gene product (TaGins51). A gel filtration and electron microscopy revealed that TaGins51 forms a homotetramer. A physical interaction between TaGins51 and TaMcm was detected by a surface plasmon resonance analysis. Unexpectedly, TaGins51 inhibited the ATPase activity, but did not affect the helicase activity of its cognate MCM. These results suggest that another factor is required to form a stable helicase complex with MCM and GINS at the replication fork in T. acidophilum cells.     

A basal sphenodontian (Lepidosauria) from the Jurassic of Patagonia: new insights on the phylogeny and biogeography of Gondwanan rhynchocephalians

Herein we describe a new rhynchocephalian taxon from the Middle Jurassic of Patagonia, Argentina, representing the first Jurassic record of the group in South America. The new taxon, consisting of a complete dentary, is ascribed to Sphenodontia based on the presence of a deep and wide Meckelian groove, long posterior process, well‐developed coronoid process, and acrodont teeth showing dental regionalization including successional, alternate hatchling, and additional series. This allocation is reinforced by a phylogenetic analysis that places the new taxon in a basal position within a clade of sphenodontians that excludes Diphydontosaurus and Planocephalosaurus. Additionally, the new taxon clusters within a Gondwanan clade with the Indian Godavarisaurus from the Jurassic Kota Formation, sharing the presence of recurved and relatively large posterior successional teeth that are ribbed and bear a peculiar anterolingual groove. This sister‐group relationship is intriguing from a palaeobiogeographical viewpoint, as it suggests some degree of endemism during the initial stages of the breakup of Pangaea. We also discuss the ontogenetic stage of the new taxon and provide insights on the evolution of successional dentition in rhynchocephalians. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 342–360.