David H.-F. Teng

Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers

Deletions involving regions of chromosome 10 occur in the vast majority (>90%) of human glioblastoma multiformes. A region at chromosome 10q23–24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.

A candidate prostate cancer susceptibility gene at chromosome 17p

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3′ end cleavage and polyadenylation specificity factor (CPSF73).

FilmArray, an Automated Nested Multiplex PCR System for Multi-Pathogen Detection: Development and Application to Respiratory Tract Infection

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

Characterization of a carboxy-terminal BRCA1 interacting protein.

There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain. Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602–1863. A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP. The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription. A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants. In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele. Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1.

Phenotypic analysis of human glioma cells expressing the MMAC1 tumor suppressor phosphatase.

MMAC1, also known as PTEN or TEP-1, was recently identified as a gene commonly mutated in a variety of human neoplasias. Sequence analysis revealed that MMAC1 harbored sequences similar to those found in several protein phosphatases. Subsequent studies demonstrated that MMAC1 possessed in vitro enzymatic activity similar to that exhibited by dual specificity phosphatases. To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression. While expression of wild-type MMAC1 in these cells significantly reduced their growth rate and saturation density, expression of enzymatically inactive MMAC1 significantly enhanced growth in soft agar. Our observations indicate that while wild-type MMAC1 exhibits activities compatible with its proposed role as a tumor suppressor, cellular expression of MMAC1 containing mutations in the catalytic domain may yield protein products that enhance transformation characteristics.

Enrichment during transdominant genetic experiments using a flow sorter

BACKGROUND Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. METHODS Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. RESULTS MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. CONCLUSIONS Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS.

Creation of a Stable Human Reporter Cell Line Suitable for FACS-Based, Transdominant Genetic Selection

Quality bioassays are central to all approaches directed at understanding or perturbing the function of proteins. One type of cell-based bioassay involves an engineered reporter whose transcriptional activity serves as a readout for upstream signals of a biochemical pathway(s) that feeds into the reporter. We describe a general strategy for creating a mammalian reporter line with attributes suitable for a high complexity, en masse transdominant genetic screen. The basic criteria required of the mammalian cells engineered with the reporter include ease of maintenance, ease of sorting by FACS, ability to be transduced by retroviruses, and high expression of transduced peptides or cDNAs. For maximal enrichment during selection, the reporter line should have a relatively homogeneous response and a high signal-to-background ratio. We use a melanoma cell line transduced with a retinoic-acid-responsive promoter coupled to a GFP reporter as a case study to demonstrate the strategy. We characterize an optimized retinoic-acid-responsive reporter clone to determine the kinetics of reporter induction and decay in the presence and absence of retinoids. Dose-response studies reveal that the reporter responds to all-trans retinoic acid with an EC50 of approximately 1 nM. The strategy described is general and may be applied to create other reporter lines that respond to a specific stimulus.

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.