Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping.

Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P<0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR (P<0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

EGFR-TKI and lung adenocarcinoma with CNS relapse: interest of molecular follow-up.

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib improves survival of lung cancer as second- or third-line therapy. However, after an initial response, most patients will recur, particularly within the central nervous system. The present study reports the case of a 27-yr-old nonsmoking male presenting with a metastatic lung adenocarcinoma with EGFR exon 19 deletion, associated with sensitivity to EGFR-TKI. Gefitinib, followed by chemotherapy and finally erlotinib resulted in prolonged disease control, until multiple liver metastases were detected. After stopping EGFR-TKI, brain metastases with carcinomatous meningitis were diagnosed. A secondary T790M mutation, associated with resistance to EGFR-TKI, was found on the liver biopsy but not in the cerebrospinal fluid. Erlotinib was reintroduced and allowed a quick neurological improvement, even though the extra-cranial disease remained resistant to erlotinib. The present report underscores the interest of molecular monitoring in lung cancer. Persistent cerebral tyrosine kinase inhibitor sensitivity should be considered in patients presenting with an early central nervous system relapse after stopping epidermal growth factor receptor tyrosine kinase inhibitor, even with a T790M-resistant mutation in noncerebral metastases. Questions remain concerning the selection of sub-clones during epidermal growth factor receptor tyrosine kinase inhibitor therapy, which could differ according to metastatic sites, especially in the central nervous system.

ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase IIalpha expression.

The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase IIalpha gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase IIalpha and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase IIalpha. Immunohistochemistry experiments showed that topoisomerase IIalpha and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase IIalpha expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.

Evaluation of microsatellite analysis in urine sediment for diagnosis of bladder cancer.

Alterations at microsatellite DNA markers in cells exfoliated in urine have been correlated to the presence of bladder cancer. To check the feasibility of such noninvasive analysis to routinely diagnose bladder cancers, we have developed a highly sensitive method using fluorescent PCR to search for DNA microsatellite alterations in urine sediment compared with a blood paired sample. One hundred eighty-three patients were included in our study. This population comprised 103 bladder cancers (64 pTa stages), the complement representing controls and other benign or malignant diseases. Results of the analysis at 17 loci in a blinded study were compared with cystoscopy and/or pathology. The high reproducibility of this technique and the analysis of 26 control patients allowed us to determine for each microsatellite a cutoff characterizing a significant allelic imbalance. For bladder cancer detection, the overall sensitivity of the test was 84%. Using this procedure, we identified alterations in 81%, 84%, 91%, and 100% of pTa, pT1, pT2, and >pT2 stages, respectively. This corresponds to 79%, 82%, and 96% sensitivity for grades I, II, and III, respectively. Interestingly, for routine purposes, we observed an overall sensitivity of 80% (76% for pTa stages) when only the eight most rearranged microsatellites were considered. In conclusion, the noninvasive feature combined with the rapidity of this fluorescent and highly sensitive technique for the detection of early stages provides us with a useful help for the diagnosis of bladder cancer.

Systematic study of phospholipids linked to a steroid derivative, spread into a monolayer at the air/water interface.

Isothermal pressure-area curves of different phospholipids linked to a cortisol derivative, spread into monolayers at the air/water interface are studied. It is shown that derivatives containing saturated lipid chains and those with unsaturated chains present quite different behaviours. With saturated derivatives, the main phase transition plateau and the stability of the fluid phase are very sensitive to the length of the lipid chains, the presence of a spacer between the lipid and the steroid moieties, the temperature and the presence of di- and trivalent cations in the aqueous subphase; the calcium ion shows an especially high effect, compared to the other ions studied. The presence of the steroid on the lipid modifies the specific area of the molecules of unsaturated lipids, which is not the case with saturated lipids, probably due to differences in the lipophilic cohesion.

Linker histone-dependent DNA structure in linear mononucleosomes.

We have examined the binding of the linker histone H5 (LH) to mononucleosomes. Mononucleosomes reconstituted on short DNA fragments display a series of discrete bands on a gel corresponding to various nucleosome positions along the DNA. When a series of engineered H5s with differing extents of the C-terminal tail are bound to these mononucleosomes, the electrophoretic mobilities of the resulting complexes are altered. Not only is there a general increase in mobility upon complex formation, but there is a reduction in the differences in mobility of the most distal nucleosomes. The complexes were also visualized by electronmicroscopy. From these two complementary studies, we conclude the following. (1) Entering and exiting DNAs are uncrossed in the LH-free particles, despite a DNA wrapping of 1.65 to 1.7 turns around the histone core. This results from a bending of the entering and exiting DNA away from each other and the histone surface, presumably as a consequence of electrostatic repulsion. This confirms and extends conclusions derived from our recent examination of the same particles in 3D through cryo-electron microscopy. (2) Binding of the globular domain of H5 increases DNA wrapping to 1.8 to 1.9 turns, but fails to induce a crossing due to an accentuation of the bends. (3) The C-terminal tail of H5 bridges entering and exiting DNAs together into a four-stranded stem over a distance of about 30 bp. The occurrence of such a stem may introduce constraints on models of the 30 nm chromatin fiber.

Chromatin reconstitution on small DNA rings. III. Histone H5 dependence of DNA supercoiling in the nucleosome.

Mononucleosomes were reconstituted on small DNA rings in the presence of histone H5 and relaxed to an equilibrium using calf thymus topoisomerase I. DNA products, when compared to the equilibria observed with the same minicircles in the absence of histones, showed that a linking number reduction of 1.6 to 1.7 was associated with this reconstitution, in contrast with the 1.1 to 1.2 figure reported in our recent study of the H5-free nucleosome. Gel electrophoretic properties and electron microscopic visualization of the nucleosomes suggest a correlation between this increase and a further wrapping of the DNA around the histone core from less than 1.5 turns of the superhelix in the absence of H5, to close to two turns in its presence. Implications for DNA topology in chromatin are discussed.

Basal promoter and enhancer element of yeast U6 snRNA gene.

The yeast U6 snRNA gene, SNR6, transcribed by RNA polymerase III or C, is shown to have a mixed promoter with upstream, intragenic and downstream elements. The distant downstream B block behaves as a typical enhancer element. Required in vivo, and for transcription of chromatin templates in vitro, it was also active in reversed orientation. As shown by footprinting and electron microscopy, the factor TFIIIC, or tau, bound the B block in an oriented manner and was able to induce DNA looping. The factor TFIIIC appeared to act via a weak A block located at position +21. This A block-related motif was essential in vivo and with chromatin templates. When changed into a consensus A block it favored DNA looping by TFIIIC firmly anchored on the B block, and activated a B block lacking gene in vivo and in vitro. The role of the TATA box at -30 was most apparent using a purified transcription system. With the A block, it appeared to contribute to start site selection. The results suggest a model where three weak promoter elements collaborate to assemble the transcription complex by DNA looping and synergistic protein-DNA interactions.

Three-dimensional model of Escherichia coli gyrase B subunit crystallized in two-dimensions on novobiocin-linked phospholipid films.

Two-dimensional crystals of the Escherichia coli DNA gyrase B subunit were obtained upon specific interactions with novobiocin linked phospholipid films. A three-dimensional surface model of the protein was generated by analysing images of tilted negatively stained crystals. The structure showed, at 2.5 to 3.0 nm resolution, two elongated arms organised as a V-shaped protein: the bottom of the V contains the novobiocin binding site, and the extremities of the arms mediate protein-protein interactions between the two monomers in the unit cell. Image analysis of frozen hydrated two-dimensional crystals resulted in a 1.0 nm resolution projection map that shows structural elements not revealed with negative staining. Electron microscopic structural data were compared with the crystallographic structure of the 43 kDa N-terminal fragment of the B subunit complexed with a non hydrolysable ATP analogue.

Structural study of the yeast RNA polymerase A. Electron microscopy of lipid-bound molecules and two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.

Modification of topoisomerase genes copy number in newly diagnosed childhood acute lymphoblastic leukemia.

Topoisomerase genes were analyzed at both DNA and RNA levels in 25 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL). The results of molecular analysis were compared to risk group classification of children in order to identify molecular characteristics associated with response to therapy. At diagnosis, allelic imbalance at topo-isomerase IIalpha (TOP2A) gene locus was found in 75% of informative cases whereas topoisomerase I and IIbeta gene loci are altered in none or only one case, respectively. By semi-quantitative Polymerase chain reaction, we found a 2.5 to 8-fold TOP2A gene amplification in 72% of the children, which was correlated to gene overexpression in every case. These results show that TOP2A gene amplification is a frequent event in ALL at diagnosis. Interestingly, we also identified a small population of children that do not present TOP2A gene amplification or gene overexpression and who are significantly associated with very high risk classified patients showing glucocorticoid resistance. In conclusion, characterization of TOP2A gene status in childhood ALL at diagnosis provides useful complementary information for risk assessment.

Genomic structure and chromosomal mapping of the gene coding for ICBP90, a protein involved in the regulation of the topoisomerase IIalpha gene expression.

We have recently identified a novel CCAAT box binding protein (ICBP90) involved in the regulation of topoisomerase IIalpha gene expression. We have observed that it is expressed in non-tumoral proliferating human lung fibroblast cells whereas in HeLa cells, a tumoral cell line, ICBP90 was still present even when cells were at confluence. In the present study, we have determined the ICBP90 gene structure by screening of a human placenta genomic library and PCR analysis. We report that the ICBP90 gene spans about 35.8 kb and contains six coding exons named A to F. In the 5' upstream sequence of the region containing the coding exons, two additional exons (I and II) were found. Additionally, an internal splicing site was found in exon A. A promoter region, including three putative Sp1 binding sites between exons I and A, was identified by transient transfection. Northern blot analysis of several cancer cell lines revealed the existence of two ICBP90 mRNA species of 5.1 and 4.3 kb that are transcribed from the gene. The relative amounts of these mRNAs depended on the cell type. In MOLT-4 cells and Burkitt's lymphoma Raji cells, the 4.3 kb or the 5.1 kb transcripts were mainly observed, respectively. In other cell lines, such as HL-60 cells, chronic myelogenous leukaemia K-562, lung carcinoma A549, HeLa or colorectal SW480, both 4.3 and 5.1 kb forms of ICBP90 mRNA could be detected. Interestingly, western blot analysis showed several ICBP90 protein bands in HeLa but only a single band in MOLT-4 cell extracts. Taken together our results are consistent with the ICBP90 gene exhibiting alternative splicing and promoter usage in a cell-specific manner.

Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes.

Plasmid DNA was in vitro assembled into chromatin using an S-150 extract of Xenopus laevis oocytes. By varying the assembly temperature and DNA concentration it is possible to generate fully or partially assembled molecules. The fate of the in vitro preassembled molecules injected into X. laevis oocyte nuclei and their transcriptional activity were studied. Completely reconstituted molecules underwent a rearrangement of their chromatin structure after injection and showed reduced transcriptional activity compared to protein-free DNA or partially reconstituted chromatin.

Two-dimensional crystallization of DNA gyrase B subunit on specifically designed lipid monolayers.

The B subunit of DNA gyrase formed two-dimensional crystals when bound to a specifically recognized phospholipid spread into a monolayer at the air/water interface. The especially designed lipids consisted of novobiocin coupled through the 3' or 2" hydroxyl group and a hydrophilous linker of a given length to dioleoylphosphatidic acid. Two-dimensional crystals of the gyrase B subunit are formed under physiological conditions of pH and ionic strength, with no precipitant added to the solution. Crystal diffraction extended to a 2.7 nm resolution in negative stain, with unit cell parameters a = 6.1 nm, b = 7.6 nm and gamma = 64 degrees.

Allelic imbalance at loci containing FGFR, FGF, c-Met and HGF candidate genes in non-small cell lung cancer sub-types, implication for progression.

Fibroblast growth factors (FGF), hepatocyte growth factor (HGF) and their receptors, FGFR and c-Met, are essential components of the regulatory networks between the epithelium and mesenchyme in embryonic lung, but their respective roles in tumour growth are not clear. We performed allelotyping at loci containing the candidate genes FGFR-1-2-3-4, FGF-1-2-7-10, c-Met and HGF in 36 non-small cell lung cancer (NSCLC) (20 squamous-cell carcinomas (SQC) and 16 adenocarcinomas (ADC)), by surrounding each locus with two microsatellites (MS), as close as possible to the genes of interest. Unexpectedly, SQC and ADC were frequently altered at all of these loci, and SQC showed more simultaneously altered loci. In ADC, alterations at the 15q13-22 locus (FGF7 candidate gene) were significantly more frequent. Thus, these loci showed different patterns of molecular alterations between SQC and ADC. Finally, alterations at loci containing FGFR and HGF candidate genes were inversely correlated to the lymph node status in SQC and ADC, respectively.

Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli.

DNA gyrase is an essential enzyme that regulates the DNA topology in bacteria. It belongs to the type II DNA topoisomerase family and is responsible for the introduction of negative supercoils into DNA at the expense of hydrolysis of ATP molecules. The aim of the present work was to study the contribution of I10, one of the most important residues responsible for the stabilization of GyrB dimer and involved in the ATP-binding step, in the ATP-hydrolysis reaction and in the DNA supercoiling mechanism. We constructed MBP-tagged GyrB mutants I10G and Delta4-14. Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA supercoiling. Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activity and DNA supercoiling. Interestingly, the DNA-relaxation activity of MBP-GyrB mutants and wild type was completely inhibited by ATP. Binding of ADPNP to MBP-tagged mutants was significantly decreased. ADPNP had no effect on DNA-relaxation activity of MBP-tagged mutants but was able to inhibit MBP-tagged wild type enzyme. Our results demonstrate that GyrB N-terminal arm, and specially I10 residue is essential for ATP binding/hydrolysis efficiency and DNA transfer through DNA gyrase.

Rational design and synthesis of phospholipids for the two-dimensional crystallization of DNA gyrase, a key element in chromosome organization.

Properties required of lipids for two-dimensional crystallization of proteins on lipid layers at the air/water interface are discussed in terms of molecular structure. These properties are related to essential features of the overall system such as (i) the fluidity and stability of the lipid film, (ii) the affinity of the protein to be crystallized for the lipids and (iii) the accessibility of the protein to the ligand in the lipid layer as well as (iv) technical constraints of the crystallization technique. The resulting ideas were tested through the rational design and synthesis of original phospholipid structures linked to novobiocin subsequently used in the production of two-dimensional crystals of DNA gyrase (B subunit), a prokaryotic type II DNA topoisomerase.

Self-assembly of soluble proteins on functionalized lipid layers: a tentative correlation between the fluidity properties of the lipid film and protein ordering.

New series of amphiphilic structures are designed to exhibit various fluidity properties when spread at the air-water interface. The influence of the molecular structure of these lipids on the process of two-dimensional (2D) crystallization of the B subunit of DNA gyrase, a soluble protein, is investigated in terms of size of the crystals produced, protein ordering, and crystallization kinetics. Whereas no difference is observed concerning the mean size of the protein 2D crystals obtained on the different lipid supports, the ultimate protein ordering observable by electron microscopy using the negative-staining technique is more regularly attained with some of these new lipids. The most interesting point results from large discrepancies in crystallization kinetics as highly-ordered protein 2D crystals form within 6-24 h depending on the lipid layer structure. Thus, these new lipids reveal of special interest when studying proteins that suffer from extended incubation time at 4 degrees C or higher temperature and lose their functionality.

The SRA protein UHRF1 promotes epigenetic crosstalks and is involved in prostate cancer progression.

Epigenetic silencing of tumour suppressor genes is an important mechanism involved in cell transformation and tumour progression. The Set and RING-finger-associated domain-containing protein UHRF1 might be an important link between different epigenetic pathways. Here, we report that UHRF1 is frequently overexpressed in human prostate tumours and has an important role in prostate cancer pathogenesis and progression. Analysis of human prostate cancer samples by microarrays and immunohistochemistry showed increased expression of UHRF1 in about half of the cases. Moreover, UHRF1 expression was associated with reduced overall survival after prostatectomy in patients with organ-confined prostate tumours (P < 0.0001). UHRF1 expression was negatively correlated with several tumour suppressor genes and positively with the histone methyltransferase (HMT) EZH2 both in prostate tumours and cell lines. UHRF1 knockdown reduced proliferation, clonogenic capability and anchorage-independent growth of prostate cancer cells. Depletion of UHRF1 resulted in reactivation of several tumour suppressor genes. Gene reactivation upon UHRF1 depletion was associated with changes in histone H3K9 methylation, acetylation and DNA methylation, and impaired binding of the H3K9 HMT Suv39H1 to the promoter of silenced genes. Co-immunoprecipitation experiments showed direct interaction between UHRF1 and Suv39H1. Our data support the notion that UHRF1, along with Suv39H1 and DNA methyltransferases, contributes to epigenetic gene silencing in prostate tumours. This could represent a parallel and convergent pathway to the H3K27 methylation catalyzed by EZH2 to synergistically promote inactivation of tumour suppressor genes. Deregulated expression of UHRF1 is involved in the prostate cancer pathogenesis and might represent a useful marker to distinguish indolent cancer from those at high risk of lethal progression.