Assessment of genomic damage in colorectal cancer by DNA fingerprinting: prognostic applications

PURPOSE: Here we evaluate the prognostic significance of the relative value of genomic damage assessed by DNA fingerprinting in colorectal cancer. MATERIALS AND METHODS: Sixty-three tumor and paired normal mucosa samples were included in the study. Genomic damage was assessed by comparative analysis of paired normal and tumor tissue DNA fingerprints by the arbitrarily primed polymerase chain reaction (AP-PCR). Decreases and increases of intensity in bands were computed and referred to the total number of visualized bands per case. An index reflecting the genomic damage fraction (GDF), with separated values for losses and gains, was obtained for each tumor. This index was used to determine molecular and clinicopathologic correlates after exclusion of eight cases displaying microsatellite instability. RESULTS: Fifty-five cases were considered for the statistical analysis. The average fraction of altered bands per tumor was 0.287+/-0.121. When losses and gains were computed separately, the average fraction of changes was 0.126+/-0.113 and 0.161+/-0.120, respectively. Tumors lacking a ras mutation showed an increased GDF, primarily because of a higher fraction of gains. Tumors that were at advanced Dukes' stages and that were poorly differentiated also displayed a higher GDF. Finally, disease-free survival was significantly diminished in tumors with a GDF greater than 0.314 (P < .001). The prognostic significance of the GDF was independent of Dukes' stage (Cox multivariate analysis, P = .005). CONCLUSION: The degree of genomic damage assessed by unbiased DNA fingerprinting correlates with genotypic, phenotypic, and clinical variables in colorectal carcinoma and may be useful in assessing prognosis in colorectal cancer.     

Management considerations for treating vesicoureteral reflux in children with solitary kidneys

OBJECTIVES: To evaluate the management approach for vesicoureteral reflux (reflux) into a solitary kidney. METHODS: Outcomes of all children with solitary kidneys and reflux managed between 1981 and 1996 were reviewed. Solitary kidneys were documented by nuclear renography and ultrasonography; reflux was graded after cystography. Management consisted of observation and antimicrobial prophylaxis or surgery by ureteroneocystostomy or subureteric injection of polytetrafluoroethylene (STING). Follow-up ranged from 3 months to 14 years and included serial cystography, sonography, and serum creatinine measurement. RESULTS: Twenty-one patients with a median follow-up of 26 months were identified. Etiologies included contralateral renal agenesis (14 children), multicystic dysplastic kidney (5 children), or nonfunctioning ureteropelvic junction obstruction (2 children). Low-grade (I to II) reflux was identified in 6 children, and high grade (III to V) was identified in 15. Reflux resolved in 20 patients. Five children with low-grade reflux were managed without surgery and demonstrated reflux resolution after a mean of 20.5 months. Renal function deteriorated in only 1 child. Ureteroneocystostomy was performed in 13 children with grades III to V reflux, and STING was performed in 1 child with grade II reflux. Every surgical patient maintained stable renal function and was infection-free during a mean follow-up of 56 months. Management by observation in 2 children with grades IV to V reflux resulted in spontaneous resolution in one and stable grade IV in the other. CONCLUSIONS: Reflux into the solitary functioning kidney may be managed by the same strategies used to manage unilateral reflux in children with two normally functioning kidneys: low-grade reflux by observation/ chemoprophylaxis until spontaneous resolution occurs, and higher grades by surgery to protect renal function; however, chemoprophylaxis and serial imaging may be used until well-defined indications for surgery are satisfied. Renal function should be monitored diligently.     

A potential role for interleukin-15 in the regulation of human natural killer cell survival

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

Characterization of the major bovine brain Go alpha isoforms. Mapping the structural differences between the alpha subunit isoforms identifies a variable region of the protein involved in receptor interactions

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.     

Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.     

In vitro genotoxicity studies of chrysotile asbestos fibers dispersed in simulated pulmonary surfactant

Micronucleus (MN) formation and sister-chromatid exchange (SCE) assays were performed for asbestos in cultured Chinese hamster lung (V79) cells to determine the effect of surfactant treatment on the genotoxicity of two chrysotile asbestos samples of different fiber lengths. The cells were challenged in vitro with NIEHS intermediate- and short-length chrysotile fibers in both their native state and with surfactant pretreatment. For the surfactant pretreatment, the fibers were incubated in a simulated pulmonary surfactant which was prepared by ultrasonically dispersing dipalmitoyl lecithin (DPL), a primary component of pulmonary surfactant, in minimal essential medium (MEM). Chrysotile asbestos was ultrasonically mixed into the prepared surfactant dispersion or into MEM. V79 cells were exposed to DPL-treated intermediate-length chrysotile (TICA), intermediate-length chrysotile (ICA), DPL-treated short-length chrysotile (TSCA) or short-length chrysotile (SCA) fibers for 48 h. For each treatment, 2000 mononucleated cells were scored for MN formation, and 30 M2 metaphase cells were scored for SCE induction. The results showed that all samples, TICA, ICA, TSCA and SCA, caused significant elevation in the frequency of cells with micronuclei and of cells with two or more nuclei. The increase in micronucleus frequency was greatest in cells challenged with untreated intermediate-length fibers, and was greater for untreated than for DPL-treated short-length fibers. For the short-length fiber samples, DPL surfactant treatment decreased activity for multiple nucleus formation, while DPL treatment did not result in consistent changes in that activity for intermediate-length fibers. Results of SCE assays were either negative or inconclusive. Cells were more viable following TICA and TSCA than following ICA and SCA challenge as measured by cell counts after 48 h of incubation.