Jacques Lapointe

Genomic Profiling Reveals Alternative Genetic Pathways of Prostate Tumorigenesis

Prostate cancer is clinically heterogeneous, ranging from indolent to lethal disease. Expression profiling previously defined three subtypes of prostate cancer, one (subtype-1) linked to clinically favorable behavior, and the others (subtypes-2 and -3) linked with a more aggressive form of the disease. To explore disease heterogeneity at the genomic level, we carried out array-based comparative genomic hybridization (array CGH) on 64 prostate tumor specimens, including 55 primary tumors and 9 pelvic lymph node metastases. Unsupervised cluster analysis of DNA copy number alterations (CNA) identified recurrent aberrations, including a 6q15-deletion group associated with subtype-1 gene expression patterns and decreased tumor recurrence. Supervised analysis further disclosed distinct patterns of CNA among gene-expression subtypes, where subtype-1 tumors exhibited characteristic deletions at 5q21 and 6q15, and subtype-2 cases harbored deletions at 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion). Lymph node metastases, predominantly subtype-3, displayed overall higher frequencies of CNA, and in particular gains at 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23. Our findings reveal that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating prostate cancer biology and explain in part the clinical heterogeneity of the disease.

Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons.

The integron is a new type of mobile element containing one or more antibiotic-resistance-encoding genes site-specifically integrated as cassettes. The integrated genes are expressed from a common promoter region located in an adjacent conserved segment. Sequence analysis has revealed the existence of four versions of the integron promoters. In this study, we have determined the relative strength of the different integron promoters and compared their activity with that of the tac promoter. Each version of the promoter was cloned upstream from a promoter-less chloramphenicol acetyltransferase-encoding gene (cat) in plasmid pKK232-8. CAT activity was used to measure transcriptional expression from the promoters of the antibiotic-resistance operon. The strongest promoter is the version (TTGACAN17TAAACT) found in plasmid R388 and in transposon Tn1696. This promoter is six times more efficient than the derepressed tac promoter.

A variant TMPRSS2 isoform and ERG fusion product in prostate cancer with implications for molecular diagnosis.

Prostate cancer is the most commonly diagnosed cancer among men in the United States. Recently, fusion of TMPRSS2 with ETS family oncogenic transcription factors has been identified as a common molecular alteration in prostate cancer, where most often the rearrangement places ERG under the androgen-regulated transcriptional control of TMPRSS2. Here, we carried out rapid amplification of cDNA ends (RACE) on a prostate cancer specimen carrying an atypical aberration discovered by array-based comparative genomic hybridization (array CGH), suggesting an alternative fusion partner of ERG. We identified novel transcribed sequences fused to ERG, mapping 4 kb upstream of the TMPRSS2 start site. The sequences derive from an apparent second TMPRSS2 isoform, which we found also expressed in some prostate tumors, suggesting similar androgen-regulated control. In a reverse transcription-polymerase chain reaction (RT-PCR)-based survey of 63 prostate tumor specimens (54 primary and nine lymph node metastases), 44 (70%) cases expressed either the known or novel variant TMPRSS2-ERG fusion, 28 (44%) expressed both, 10 (16%) expressed only the known, and notably six (10%) expressed only the variant isoform fusion. In this specimen set, the presence of a TMPRSS2-ERG fusion showed no statistical association with tumor stage, Gleason grade or recurrence-free survival. Nonetheless, the discovery of a novel variant TMPRSS2 isoform-ERG fusion adds to the characterization of ETS-family rearrangements in prostate cancer, and has important implications for the accurate molecular diagnosis of TMPRSS2-ETS fusions.

ATP binding by glutamyl‐tRNA synthetase is switched to the productive mode by tRNA binding

Aminoacyl‐tRNA synthetases catalyze the formation of an aminoacyl‐AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl‐tRNA synthetases, including glutamyl‐tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl‐AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the ‘non‐productive’ complex of Thermus thermophilus GluRS, ATP and L‐glutamate, together with those of the GluRS·ATP, GluRS·tRNA·ATP and GluRS·tRNA·GoA (a glutamyl‐AMP analog) complexes. In the absence of tRNAGlu, ATP is accommodated in a ‘non‐productive’ subsite within the ATP‐binding site, so that the ATP α‐phosphate and the glutamate α‐carboxyl groups in GluRS· ATP·Glu are too far from each other (6.2 Å) to react. In contrast, the ATP‐binding mode in GluRS·tRNA· ATP is dramatically different from those in GluRS·ATP·Glu and GluRS·ATP, but corresponds to the AMP moiety binding mode in GluRS·tRNA·GoA (the ‘productive’ subsite). Therefore, tRNA binding to GluRS switches the ATP‐binding mode. The interactions of the three tRNAGlu regions with GluRS cause conformational changes around the ATP‐binding site, and allow ATP to bind to the ‘productive’ subsite.

Recurrent deletion of CHD1 in prostate cancer with relevance to cell invasiveness

Though prostate cancer is often indolent, it is nonetheless a leading cause of cancer death. Defining the underlying molecular genetic alterations may lead to new strategies for prevention or treatment. Towards this goal, we performed array-based comparative genomic hybridization (CGH) on 86 primary prostate tumors. Among the most frequent alterations not associated with a known cancer gene, we identified focal deletions within 5q21 in 15 out of 86 (17%) cases. By high-resolution tiling array CGH, the smallest common deletion targeted just one gene, the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1). Expression of CHD1 was significantly reduced in tumors with deletion (P=0.03), and compared with normal prostate (P=0.04). Exon sequencing analysis also uncovered nonsynonymous mutations in 1 out of 7 (14%) cell lines (LAPC4) and in 1 out of 24 (4%) prostate tumors surveyed. RNA interference-mediated knockdown of CHD1 in two nontumorigenic prostate epithelial cell lines, OPCN2 and RWPE-1, did not alter cell growth, but promoted cell invasiveness, and in OPCN2-enhanced cell clonogenicity. Taken together, our findings suggest that CHD1 deletion may underlie cell invasiveness in a subset of prostate cancers, and indicate a possible novel role of altered chromatin remodeling in prostate tumorigenesis.

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 μg ml-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.

Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine

Abstract: Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro‐2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10‐fold increase in glutamine synthetase specific activity (15‐fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 mM glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 mM). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C‐6 glioma cells and glial cells in primary cultures.

Direct Glutaminyl-tRNA Biosynthesis and Indirect Asparaginyl-tRNA Biosynthesis in Pseudomonas aeruginosa PAO1

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA(Gln), that AspRS is nondiscriminating, and that its Asp-tRNA(Asn) product is transamidated by AdT. On the other hand, tRNA(Gln) is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.

Role of the Cyclin-Dependent Kinase Inhibitor p27Kip1 in Androgen-Induced Inhibition of CAMA-1 Breast Cancer Cell Proliferation

Androgens are known to inhibit the growth of breast cancer cells, but the molecular mechanism of androgen-induced growth inhibition remains unknown. To address this question, we examined functional and quantitative alterations in cell cycle regulators in the E-responsive CAMA-1 breast cancer cell line. We report here that the androgen 5α-dihydrotestosterone inhibits the proliferation of CAMA-1 breast cancer cells. This inhibition of cell proliferation was dose dependent, and maximal inhibition of E2-stimulated proliferation was observed at the concentration of 1 nm 5α-dihydrotestosterone. 5α-Dihydrotestosterone-induced growth arrest was accompanied by an increase in the proportion of cells in the G1 phase of the cell cycle. Compared with control cells, 5α-dihydrotestosterone-treated cells showed an increase in the relative proportion of hypophosphorylated retinoblastoma protein consistent with G1 arrest. In CAMA-1 cells, 5α-dihydrotestosterone caused an accumulation of the cyclin-dependent kinase inhibito...

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. I: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques.

The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E·Ile-AMP; Scheme FS1). Isoleucine (Ile) binds to the E·ATP complex (K 4 = 1.7 ± 0.9 μm) ∼35-fold more tightly than to E(K 1 = 50–100 μm), primarily due to a reduction in the Ile dissociation rate constant (k -1 ≈ 100–150 s−1,cf. k -4 = 3 ± 1.5 s−1). Similarly, ATP binds more tightly toE·Ile (K 3 = ∼70 μm) than to E (K 2 = ∼2.5 mm). The formation of the E·isoleucyl adenylate intermediate, E·Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k +5 = ∼60 s−1) and the reverse rate constant (k -5 = ∼150–200 s−1) to be determined from pyrophosphorolysis of a pre-formed E·Ile-AMP complex (K 6= ∼0.25 mm). Scheme FS1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme FS1could also provide an adequate description of the kinetics of tRNA aminoacylation (k cat,tr = ∼0.35 s−1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.