Paul T. Morrison

Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition

Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.

Genetic recombination of bacterial plasmid DNA: Analysis of the effect of recombination-deficient mutations on plasmid recombination☆

Abstract The recombination of two plasmids, pRDK101 and pACYC184, was examined in wild-type Escherichia coli and in various E. coli strains containing different recombination-deficient mutations. Recombination-induced oligomer formation from monomeric plasmids was found to be dependent on the functions of the recA, recB, recC and recF genes. Intramolecular recombination of tetrameric plasmids to form lower-order oligomers and monomers also required the functions of the recA and recF genes but did not require the function of the recB and recC genes. In all cases where recA, recB, recC and recF mutations appeared to block the formation or reduction of plasmid circular oligomers, these effects could be reversed by the presence of an sbcA mutation. Further studies on recombination in vivo were carried out utilizing two tetracycline-sensitive derivatives of the compatible plasmids pACYC184 and pBR322. Recombination events between these two plasmids could be quantitated by measuring the production of tetracycline-resistant cells during the growth of transformants. Plasmid recombination was found to be reduced by recA and recF mutations, and the effect of these two mutations was reversed by the presence of an sbcA mutation. Plasmid recombination measured by this genetic assay was stimulated by recB recC mutations, and electrophoretic analysis of the recombination products demonstrated that they were primarily circular monomers. A role for the recB recC gene product, exonuclease V, in the resolution of recombination intermediates is discussed.

Genetic recombination of bacterial plasmid DNA: Physical and genetic analysis of the products of plasmid recombination in Escherichia coli

Derivatives of plasmid pBR322 DNA containing tet mutations were constructed by inserting XhoI linkers at various sites in the tetracycline resistance gene. Monomer plasmids containing either the tet-10 allele located at nucleotide position 23 or the tet-14 allele located at nucleotide position 1267 were used to construct a circular dimer containing one copy of each allele and a circular trimer containing one copy of the tet-10 allele and two copies of the tet-14 allele. Genetic recombination of these plasmid DNAs to produce a functional tetracycline resistance gene could be detected as the production of tetracycline-resistant progeny during the growth of transformants or using a restriction mapping assay which detected the rearrangement of the mutant alleles. The structure of individual tetracycline-resistant recombination products was determined by restriction mapping. This analysis suggested that as many as 70% of the plasmid recombination events in Escherichia coli AB1157 could have involved gene conversion events. The formation of these recombination products was most easily predicted by a model involving figure 8 recombination intermediates and the formation of symmetric regions of heteroduplex. Recombination in JC10287 delta(srlR-recA)304 occurred at 5% of the wild-type frequency and appeared to occur by a similar mechanism. Recombination in JC9604 recA56 recB21 recC22 sbcA23 occurred at 20 times the wild-type frequency and appeared to involve multiple independent recombination events.

Intramolecular recombination of linear DNA catalyzed by the Escherichia coli RecE recombination system

Transformation of different Escherichia coli strains by linear dimers of pBR322 containing different tet alleles was investigated. Linear dimers transformed wild-type strains 0.1 to 1% as efficiently as circular dimers. In contrast, linear dimers transformed recBrecCsbcA strains, where the RecE recombination system is functional, as efficiently as circular dimers. The transformants contained plasmids that had a single recombinant monomer genotype, indicating that transformation was mediated by a recombination-dependent cyclization reaction. Altering the position of the double-strand break changed the frequency of recovering different recombination products, but had no effect on the frequency of transformation. Both the frequency of transformation and the production of Tcr recombinants were decreased by recE mutations, while recA and recF mutations were slightly stimulatory (twofold). Several recombination models consistent with these results are presented.

Multimodality Non-rigid Image Registration for Planning, Targeting and Monitoring During CT-Guided Percutaneous Liver Tumor Cryoablation

RATIONALE AND OBJECTIVES The aim of this study was to develop non-rigid image registration between preprocedure contrast-enhanced magnetic resonance (MR) images and intraprocedure unenhanced computed tomographic (CT) images, to enhance tumor visualization and localization during CT imaging-guided liver tumor cryoablation procedures. MATERIALS AND METHODS A non-rigid registration technique was evaluated with different preprocessing steps and algorithm parameters and compared to a standard rigid registration approach. The Dice similarity coefficient, target registration error, 95th-percentile Hausdorff distance, and total registration time (minutes) were compared using a two-sided Students t test. The entire registration method was then applied during five CT imaging-guided liver cryoablation cases with the intraprocedural CT data transmitted directly from the CT scanner, with both accuracy and registration time evaluated. RESULTS Selected optimal parameters for registration were a section thickness of 5 mm, cropping the field of view to 66% of its original size, manual segmentation of the liver, B-spline control grid of 5 × 5 × 5, and spatial sampling of 50,000 pixels. A mean 95th-percentile Hausdorff distance of 3.3 mm (a 2.5 times improvement compared to rigid registration, P < .05), a mean Dice similarity coefficient of 0.97 (a 13% increase), and a mean target registration error of 4.1 mm (a 2.7 times reduction) were measured. During the cryoablation procedure, registration between the preprocedure MR and the planning intraprocedure CT imaging took a mean time of 10.6 minutes, MR to targeting CT image took 4 minutes, and MR to monitoring CT imaging took 4.3 minutes. Mean registration accuracy was <3.4 mm. CONCLUSIONS Non-rigid registration allowed improved visualization of the tumor during interventional planning, targeting, and evaluation of tumor coverage by the ice ball. Future work is focused on reducing segmentation time to make the method more clinically acceptable.

Human Mismatch Repair Genes and Their Association with Hereditary Non-Polyposis Colon Cancer

Hereditary non-polyposis colon cancer (HNPCC) may affect up to 1 in 200 people in industrialized nations (Bishop and Thomas 1990; Lynch et al. 1991, 1993; Peltomaki et al. 1993b). Four genes have been identified in which inherited mutations appear to cause HNPCC. hMSH2 on chromosome 2p21-22 appears to account for up to 60% of HNPCC (Fishel et al. 1993; Leach et al. 1993; Sandkuijl and Bishop 1993; Nystrom-Lahti et al. 1994), hMLH1 on chromosome 3p21 appears to account for up to 30% of HNPCC (Bronner et al. 1994; Nystrom-Lahti et al. 1994; Papadopoulos et al. 1994), and hPMS1 on chromosome 2q31-33 and hPMS2 on chromosome 7p21 may account for 5% of HNPCC (Nicolaides et al. 1994).

Estimated effective dose of CT-guided percutaneous cryoablation of liver tumors

PURPOSE To estimate effective dose during CT-guided cryoablation of liver tumors, and to assess which procedural factors contribute most to dose. MATERIALS AND METHODS Our institutional review board approved this retrospective, HIPAA-compliant study. A total of 20 CT-guided percutaneous liver tumor cryoablation procedures were performed in 18 patients. Effective dose was determined by multiplying the dose length product for each CT scan obtained during the procedure by a conversion factor (0.015mSv/mGy-cm), and calculating the sum for each phase of the procedure: planning, targeting, monitoring, and post-ablation survey. Effective dose of each phase was compared using a repeated measures analysis. Using Spearman correlation coefficients, effective doses were correlated with procedural factors including number of scans, ratio of targeting distance to tumor size, anesthesia type, number of applicators, performance of ancillary procedures (hydrodissection and biopsy), and use of CT fluoroscopy. RESULTS Effective dose per procedure was 72±18mSv. The effective dose of targeting (37.5±12.5mSv) was the largest component compared to the effective dose of the planning phase (4.8±2.2mSv), the monitoring phase (25.5±6.8mSv), and the post-ablation survey (4.1±1.9mSv) phase (p<0.05). Effective dose correlated positively only with the number of scans (p<0.01). CONCLUSIONS The effective dose of CT-guided percutaneous cryoablation of liver tumors can be substantial. Reducing the number of scans during the procedure is likely to have the greatest effect on lowering dose.

Model-guided therapy for hepatocellular carcinoma: a role for information technology in predictive, preventive and personalized medicine

Predictive, preventive and personalized medicine (PPPM) may have the potential to eventually improve the nature of health care delivery. However, the tools required for a practical and comprehensive form of PPPM that is capable of handling the vast amounts of medical information that is currently available are currently lacking. This article reviews a rationale and method for combining and integrating diagnostic and therapeutic management with information technology (IT), in a manner that supports patients through their continuum of care. It is imperative that any program devised to explore and develop personalized health care delivery must be firmly rooted in clinically confirmed and accepted principles and technologies. Therefore, a use case, relating to hepatocellular carcinoma (HCC), was developed. The approach to the management of medical information we have taken is based on model theory and seeks to implement a form of model-guided therapy (MGT) that can be used as a decision support system in the treatment of patients with HCC. The IT structures to be utilized in MGT include a therapy imaging and model management system (TIMMS) and a digital patient model (DPM). The system that we propose will utilize patient modeling techniques to generate valid DPMs (which factor in age, physiologic condition, disease and co-morbidities, genetics, biomarkers and responses to previous treatments). We may, then, be able to develop a statistically valid methodology, on an individual basis, to predict certain diseases or conditions, to predict certain treatment outcomes, to prevent certain diseases or complications and to develop treatment regimens that are personalized for that particular patient. An IT system for predictive, preventive and personalized medicine (ITS-PM) for HCC is presented to provide a comprehensive system to provide unified access to general medical and patient-specific information for medical researchers and health care providers from different disciplines including hepatologists, gastroenterologists, medical and surgical oncologists, liver transplant teams, interventional radiologists and radiation oncologists. The article concludes with a review providing an outlook and recommendations for the application of MGT to enhance the medical management of HCC through PPPM.

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

Prolonged Dominance of Clonally Restricted CD4+ T Cells in Macaques Infected with Simian Immunodeficiency Viruses

ABSTRACT The repertoire of functional CD4+ T lymphocytes in human immunodeficiency virus type 1-infected individuals remains poorly understood. To explore this issue, we have examined the clonality of CD4+ T cells in simian immunodeficiency virus (SIV)-infected macaques by assessing T-cell receptor complementarity-determining region 3 (CDR3) profiles and sequences. A dominance of CD4+ T cells expressing particular CDR3 sequences was identified within certain Vβ-expressing peripheral blood lymphocyte subpopulations in the infected monkeys. Studies were then done to explore whether these dominant CD4+ T cells represented expanded antigen-specific cell subpopulations or residual cells remaining in the course of virus-induced CD4+ T-cell depletion. Sequence analysis revealed that these selected CDR3-bearing CD4+ T-cell clones emerged soon after infection and dominated the CD4+ T-cell repertoire for up to 14 months. Moreover, inoculation of chronically infected macaques with autologous SIV-infected cell lines to transiently increase plasma viral loads in the monkeys resulted in the dominance of these selected CDR3-bearing CD4+ T cells. Both the temporal association of the detection of these clonal cell populations with infection and the dominance of these cell populations following superinfection with SIV suggest that these cells may be SIV specific. Finally, the inoculation of staphylococcal enterotoxin B superantigen into SIV-infected macaques uncovered a polyclonal background underlying the few dominant CDR3-bearing CD4+ T cells, demonstrating that expandable polyclonal CD4+ T-cell subpopulations persist in these animals. These results support the notions that a chronic AIDS virus infection can induce clonal expansion, in addition to depletion of CD4+ T cells, and that some of these clones may be SIV specific.