G. Eric Blair

Prognostic value of p53 overexpression and c-Ki-ras gene mutations in colorectal cancer

BACKGROUND Mutations in Ki-ras codon 12 and the p53 gene are common abnormalities in colorectal cancer. The occurrence of p53 overexpression and/or Ki-ras codon 12 mutations were analyzed in 100 colorectal adenomas to determine if they were related to patient survival. METHODS p53 overexpression was identified by immunohistochemistry, and Ki-ras codon 12 mutations were detected using the polymerase chain reaction and a restriction enzyme digestion method. RESULTS p53 overexpression was identified in 45% of tumors, with a higher frequency identified in DNA aneuploid and left-sided tumors than in DNA diploid and right-sided tumors. Mutations in Ki-ras codon 12 were identified in 24% of carcinomas. Individually, mutations in Ki-ras codon 12 or p53 overexpression were not prognostic indicators of survival. However, a statistically significant difference in survival was identified when these two oncogenic abnormalities were analyzed together. The median survival of patients whose tumors contained both oncogenic abnormalities was less than half of that of patients with either alteration alone or without either abnormality. CONCLUSIONS Screening for multiple genetic abnormalities in colorectal cancers excised at surgery may prove to be a useful tool in determining prognosis.

Periaxin, a novel protein of myelinating schwann cells with a possible role in axonal ensheathment

We report the cloning and subcellular localization of a novel Schwann cell-specific protein of 147 kd that we have named periaxin. Periaxin has a remarkable domain of repetitive pentameric units in the primary sequence. It is expressed in the first uncompacted whorls of membrane that ensheathe the axon, and further synthesis of the protein in the rat sciatic nerve parallels the deposition of myelin. In mature myelin, periaxin colocalizes with the myelin-associated glycoprotein in the cytoplasm-filled periaxonal regions of the sheath but is excluded from compact myelin. We propose that periaxin has a role in axon-glial interactions, possibly by interacting with the cytoplasmic domains of integral membrane proteins such as myelin-associated glycoprotein in the periaxonal regions of the Schwann cell plasma membrane.

Dendritic cells: Immunological sentinels with a central role in health and disease

Immunological effector cells must be sensitive to the antigens or environmental signals that indicate that a pathogen is present. To this end, a group of cells known as the professional antigen‐presenting cells have the ability to educate T, B and NK cells as to the fingerprints of specific infections. The most adept of these cells are a closely related family termed dendritic cells (DC). A subset of these act as peripheral sentinels, specializing in the uptake, processing and presentation of antigenic material combined with an ability to detect a wide variety of ‘danger’ signals. These ‘danger’ or activation signals induce profound changes in dendritic cell physiology, facilitating the efficient stimulation of both adaptive and innate immunity. In the present review, a number of recent advances in the understanding of DC biology are discussed. These advances offer insights into the pathogenesis of a wide variety of diseases and point towards future strategies for immunotherapy.

Transcriptional regulation of the major histocompatibility complex (MHC) class I heavy chain, TAP1 and LMP2 genes by the human papillomavirus (HPV) type 6b, 16 and 18 E7 oncoproteins.

We have examined the possibility that the E7 proteins of the high-risk human papillomavirus (HPV) type 16 and 18 and the oncogenic adenovirus (Ad) type 12 E1A protein share the ability to down-regulate the expression of components of the antigen processing and presentation pathway, as a common strategy in the evasion of immune surveillance during the induction of cell transformation. Expression of the HPV 18 E7 oncoprotein, like Ad 12 E1A, resulted in repression of the major histocompatibility complex (MHC) class I heavy chain promoter, as well as repression of a bidirectional promoter that regulates expression of the genes encoding the transporter associated with antigen processing subunit 1 (TAP1) and a proteasome subunit, low molecular weight protein 2 (LMP2). HPV 16 E7 also caused a reduction in class I heavy chain promoter activity, however it did not have any significant effect on the activity of the bidirectional promoter. Interestingly, expression of the low-risk HPV 6b E7 protein resulted in an increase in MHC class I heavy chain promoter activity, while repressing the TAP1/LMP2 promoter. Interference with the class I pathway could also explain the ability of low-risk HPVs in inducing benign lesions.

A review on viral biosensors to detect human pathogens

Rapid identification of viruses has important implications for medical healthcare. Current methods for identification and quantification of particular virus are time consuming and often expensive. Therefore, demand for sensitive and accurate viral biosensors with rapid detection systems is increasing. A hand held biosensing device would give fast, reliable results for identifying and quantitating the number of virus particles in a sample. Techniques currently being applied to achieve this aim include electrochemical biosensors, based on amperometric, potentiometric and impedance measurement, optical biosensors using surface plasmon resonance (SPR), optical fibers and piezoelectric biosensors based on microcantilevers. Future research also looks to the use of nanoparticles and novel nanomaterials as alternate recognition surfaces for use in a variety of sensor formats.

DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food

The potential for transfer of antibiotic resistance genes from genetically modified (GM) plant material to microbes through genetic recombination in the human or animal gut is a consideration that has engendered caution in the use of GM foods. This study was aimed at defining the optimal physical and chemical conditions necessary to ensure sufficient fragmentation of DNA in plant tissues to a size where it would be unlikely to be stably transferred to bacterial gut microflora. The ribulose 1,5‐bisphosphate carboxylase/oxygenase small subunit (Rubisco SS) genes are of similar size (approximately 1.4 kb) to transgenes present in GM plants. DNA analysis and PCR amplification of Rubisco SS genes showed that fresh maize and maize silage contained high molecular weight DNA and intact Rubisco SS genes. Relatively high temperatures and pressurised steam were necessary to degrade fully genomic DNA and Rubisco SS genes in maize and wheat grains, the source of most animal feedstuffs. Furthermore, chemical expulsion and extrusion of oilseeds resulted in residues with completely degraded genomic DNA. These results imply that stringent conditions are needed in the processing of GM plant tissues for feedstuffs to eliminate the possibility of transmission of transgenes.

Novel molecular approaches to cystic fibrosis gene therapy.

Gene therapy holds promise for the treatment of a range of inherited diseases, such as cystic fibrosis. However, efficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of cystic fibrosis pulmonary disease has proved elusive. There are many reasons for this lack of progress, both macroscopically in terms of airway defence mechanisms and at the molecular level with regard to effective cDNA delivery. This review of approaches to cystic fibrosis gene therapy covers these areas in detail and highlights recent progress in the field. For gene therapy to be effective in patients with cystic fibrosis, the cDNA encoding the cystic fibrosis transmembrane conductance regulator protein must be delivered effectively to the nucleus of the epithelial cells lining the bronchial tree within the lungs. Expression of the transgene must be maintained at adequate levels for the lifetime of the patient, either by repeat dosage of the vector or by targeting airway stem cells. Clinical trials of gene therapy for cystic fibrosis have demonstrated proof of principle, but gene expression has been limited to 30 days at best. Results suggest that viral vectors such as adenovirus and adeno-associated virus are unsuited to repeat dosing, as the immune response reduces the effectiveness of each subsequent dose. Nonviral approaches, such as cationic liposomes, appear more suited to repeat dosing, but have been less effective. Current work regarding non-viral gene delivery is now focused on understanding the mechanisms involved in cell entry, endosomal escape and nuclear import of the transgene. There is now increasing evidence to suggest that additional ligands that facilitate endosomal escape or contain a nuclear localization signal may enhance liposome-mediated gene delivery. Much progress in this area has been informed by advances in our understanding of the mechanisms by which viruses deliver their genomes to the nuclei of host cells.

Restricted replication of human adenovirus type 5 in mouse cell lines.

Infection of mouse BALB/c 3T3 cells by adenovirus 5 resulted in at least 1000-fold lowered yields of virus compared to human cells. The molecular basis of this restriction was analysed at the level of viral gene expression. Steady-state levels of viral DNA and RNA were greatly reduced in infected mouse, compared to human cells. Both early region 1A (E1A) and E1B mRNAs were decreased in mouse cells and their protein products were barely detectable by metabolic labelling of infected cells. The E2A-72 kDa protein and the hexon protein were detected by metabolic labelling, and immunocytochemical analysis showed that they were correctly located in nuclei of infected mouse cells. Only a minor proportion of infected mouse 3T3 cells expressed the E2A-72 kDa or hexon proteins. Low yields of virus were obtained by infection of SV40 transformed BALB/c 3T3 cells showing that SV40 does not provide a helper function for adenovirus 5 growth in this cell system.

A Human NK Cell Activation/Inhibition Threshold Allows Small Changes in the Target Cell Surface Phenotype To Dramatically Alter Susceptibility to NK Cells

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing’s sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing’s sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.

Expression of the CUB domain containing protein 1 (CDCP1) gene in colorectal tumour cells

Expression of CUB domain containing protein 1 (CDCP1) is upregulated in carcinoma cells. We quantitated CDCP1 gene expression in matched normal colon and tumour tissue and compared the level of expression to other genes upregulated in colorectal tumourigenesis. Furthermore, we show that the CDCP1 gene generates two transcripts which are co‐expressed in normal and matched tumour tissue as well as in the majority of cell lines analysed. However, intracellular localisation studies revealed that only one of these transcripts encodes a protein that is localised to the cell surface.