16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

AIMS: Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. METHODS AND RESULTS: Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. CONCLUSIONS: Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. SIGNIFICANCE AND IMPACT OF THE STUDY: We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

Fn14 receptor promotes invasive potential and metastatic capacity of non-small lung adenocarcinoma cells through the up-regulation of integrin α6.

UNLABELLED: Lung cancer is one of the leading cause of cancer-related death around the world with the majority of diagnoses being non-small cell lung cancer (NSCLC). Given the poor survival rate and efficacy of current therapy for NSCLC, there is a need to identify and develop new therapeutic targets for treatment. We have observed significantly up-regulated levels of Fn14 in clinical samples of lung cancer relative to normal adjacent tissue. However, the functional role of Fn14 in these tumors is not understood yet. We used RT-PCR to establish the Fn14 expression profile in various NSCLC cell lines. Using isogenic variants of H460 NSCLC cell line with low, intermediate and high Fn14 expression as a cellular model, we determined that increased levels of integrin α6 in cells over-expressing Fn14 is suggestive of an important role of α6ß1-fn14 interactions in motility of lung carcinoma and formation of metastases. Enhanced levels of Fn14 correlated with higher tumor cell migration and invasion in an MMP-1 dependent manner. Cells over-expressing Fn14 showed increased in vivo tumor formation with metastatic capacity to lymph nodes, lungs and liver. Thus, this research may be a step toward developing improved treatment strategies for NSCLC by improved detection and inhibition of metastases. KEYWORDS: Fn14, TNFRSF12A, non-small cell lung cancer, H460 cells, motility, tumor formation and metastasis, integrin α6.

AP-2alpha and AP-2gamma are transcriptional targets of p53 in human breast carcinoma cells.

Activating enhancer-binding protein 2alpha (AP-2alpha) and activating enhancer-binding protein 2gamma (AP-2gamma) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2alpha and AP-2gamma interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2gamma as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2gamma but also AP-2alpha mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2alpha and AP-2gamma promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2alpha or gamma inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation.

cDNA microarray analysis of multidrug resistance: doxorubicin selection produces multiple defects in apoptosis signaling pathways.

Doxorubicin plays an important role in the treatment of leukemias, lymphomas, and a variety of carcinomas. Tumor cell resistance to doxorubicin is often associated with expression of the multidrug resistance gene MDR1, which codes for the drug efflux pump P-glycoprotein, and a multidrug-resistant phenotype. Evidence from multiple sources suggests, however, that additional genes besides MDR1 are involved in development of multidrug resistance. To identify genes involved in the multidrug resistance phenotype, we created a 5760-gene cDNA microarray to search for differentially expressed genes between the human multiple myeloma cell line RPMI 8226 and its doxorubicin-selected sublines 8226/Dox6 and 8226/Dox40, both of which express MDR1 and are multidrug-resistant. The cDNA microarray results identified a set of differentially expressed genes, which included MDR1 as expected. Thirty Northern analyses were used to confirm the results of the cDNA microarrays; comparison with the microarray results showed a 90% agreement between the two techniques. Within the set of differentially expressed genes identified by the cDNA microarrays, 29 were of particular interest as they can participate in apoptotic signaling, particularly as mediated by ceramide and the mitochondrial permeability transition. The functional importance of these changes in gene expression is supported by their explanation of the 8226/Dox cell lines' cross-resistance to substances that are not P-glycoprotein substrates, such as Fas/CD95 ligand and staurosporine. We conclude that doxorubicin selection led to changes in gene expression that reduce the apoptotic response to death-inducing stimuli and thus contribute to the multidrug resistance phenotype.

Laminin-5-mediated gene expression in human prostate carcinoma cells.

Interactions between extracellular matrix (ECM) proteins and prostate carcinoma cells provide a dynamic model of prostate tumor progression. Previous work in our laboratory showed that laminin-5, an important member of a family of ECM glycoproteins expressed in the basal lamina, is lost in prostate carcinoma. Moreover, we showed that the receptor for laminin-5, the alpha6beta4 integrin, is altered in prostate tumors. However, the genes that laminin-5 potentially regulates and the significance of its loss of expression in prostate cancer are not known. We selected cDNA microarray as a comprehensive and systematic method for surveying and examining gene expression induced by laminin-5. To establish a definitive role for laminin-5 in prostate tumor progression and understand the significance of its loss of expression, we used a cDNA microarray containing 5289 human genes to detect perturbations of gene expression when DU145 prostate carcinoma cells interacted with purified laminin-5 after 0.5, 6, and 24 h. Triplicate experiments showed modulations of four, 61, and 14 genes at 0.5, 6, and 24 h, respectively. Genes associated with signal transduction, cell adhesion, the cell cycle, and cell structure were identified and validated by northern blot analysis. Protein expression was further assessed by immunohistochemistry. Mol. Carcinog. 30:119-129, 2001.

Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.