Gastrointestinal tract wall visualization and distention during abdominal and pelvic multidetector CT with a neutral barium sulphate suspension: comparison with positive barium sulphate suspension and with water.

OBJECTIVE: When examining patients with contrast-enhanced multidetector-row CT, we determined if the stomach and small bowel were visualized and distended better with a neutral barium sulphate suspension than with positive barium sulphate suspension or water. MATERIALS AND METHODS: After obtaining approval from our institutional review board, 156 patients (women: 84; mean age: 54 yrs) with no history of gastrointestinal tract disease were randomized prospectively to receive orally either 900 ml of neutral (0.1% w/v) barium sulphate suspension (n = 53), 900 ml of positive (2.1% w/v) barium sulphate suspension (n = 53), or 900 ml of water (n = 50), prior to undergoing contrast-enhanced abdominal and pelvic multidetector-row CT. Two independent radiologists evaluated the stomach, and small bowel, for luminal distension and wall visualization, using a five point scale. Results were compared using Kruskal-Wallis and Mann-Whitney U tests. RESULTS: The walls of the stomach, and small bowel were visualized better in patients who were administered neutral barium sulphate suspension than those who were administered either positive barium sulphate suspension (p < 0.01) or water (p < 0.01). In patients who received neutral barium sulphate suspension, the stomach and small bowel were distended better compared to patients administered water (p < 0.01); the stomach, duodenum, and ileum were distended better compared to patients administered positive barium sulphate suspension (p < 0.05). CONCLUSIONS: When examining patients with intravenous contrast-enhanced abdominal and pelvic multidetector-row CT, orally administered neutral barium sulphate suspension allows the gastrointestinal tract to be visualized and distended better than either positive barium sulphate suspension, or water.

Glomerulocystic kidney disease: MRI findings.

Glomerulocystic kidney disease (GCKD) is a rare form of renal cystic disease characterized by cystic dilation of Bowman's capsule. The imaging findings of small renal cysts with a predominant cortical and subcapsular distribution allows for distinction from other, more common, polycystic kidney diseases. The appearance and distribution of the renal cysts by magnetic resonance imaging allow for a definitive diagnosis of GCKD.

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells.

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125 mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2Kd, H-2Dd and H-2Ld molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2Ld molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NF kappa B. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism. Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2'-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.

Differences between cysteine and homocysteine in the induction of deoxyribose degradation and DNA damage.

The effect of two naturally occurring thiols, such as cysteine and homocysteine, has been examined for their ability to induce deoxyribose degradation and DNA damage. Copper(II) ions have been added to incubation mixtures and oxygen consumption measurements have been performed in order to correlate the observed damaging effects with the rate of metal catalyzed thiol oxidation. Ascorbic acid plus copper has been used as a positive control of deoxyribose and DNA oxidation due to reactive oxygen species. Cysteine or homocysteine in the presence of copper ions induce the degradation of deoxyribose and the yield of 8-hydroxy-2'-deoxyguanosine (8-OHdG), although important differences are observed between the two thiols tested, homocysteine being less reactive than cysteine. DNA cleavage is induced by cysteine in the presence of copper(II) ions but not by homocysteine. Catalase and thiourea, but not superoxide dismutase (SOD), were shown to inhibit the damaging effects of cysteine on deoxyribose or DNA suggesting that H(2)O(2) and *OH radicals are responsible for the observed induced damage. The results indicate that there are differences between the damaging effects of the two thiols tested towards deoxyribose and DNA damage. The pathophysiological importance will be discussed.

Age-related changes of liver antioxidant enzymes and 8-hydroxy-2'-deoxyguanosine during fetal-neonate transition and early rat development.

We have studied the pro-antioxidant status of the rat liver on the last day of gestation and at 1, 15, and 30 days of extrauterine life. Representative variables, such as activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase and concentrations of reduced glutathione and 8-hydroxy-2'-deoxyguanosine, were determined in liver to assess the degree of birth-associated oxidative stress during the fetal-neonatal transition and early development of the rat. Percentages by which liver Cu/ZnSOD activity increased over the basal value of the fetal liver were 54%, 95%, and 127% at neonatal days 1, 15, and 30, respectively. There was a lack of induction in the development profile of MnSOD. Catalase activity was clearly and progressively induced with time from the fetal state up to the neonatal age of 1 month. Glutathione peroxidase activity and glutathione content showed a tendency to decline during the first day after birth, though they increased to significantly higher values on days 15 and 30. However, the amount of rat liver 8-hydroxy-2'-deoxyguanosine did not increase. These results suggest that the induced antioxidant activities may be responsible for maintaining DNA stability during the perinatal development of the rat liver.

Beta2-microglobulin gene mutation is not a common mechanism of HLA class I total loss in human tumors.

One hundred and sixty-two tumor samples were analyzed for HLA class I expression using immunohistological techniques. HLA class I total loss (phenotype no. I) was detected in 31 cases (19%), comprising 20 colorectal, 3 laryngeal, and 2 bladder carcinomas and 6 melanomas. Twenty-one cases were selected for molecular analysis due to a higher proportion of tumor cells versus stroma cells (75%). We investigated whether beta2-microglobulin mutation was responsible for HLA downregulation. Single-strand conformation polymorphism and sequencing analysis of DNA samples was performed. Alterations were detected only in melanomas M78 (a point mutation in the initiation ATG sequence), M79 (a mutation in codon 31 producing a stop codon), and M34 (a TTCT deletion introducing a termination codon signal). We found no beta2-microglobulin gene mutation in the other 18 samples. Loss of heterozygosity in 15q close to the beta2-microglobulin gene was found in 5 cases. We conclude that HLA class I total loss can frequently occur without beta2-microglobulin gene mutations.

A new beta 2 microglobulin mutation found in a melanoma tumor cell line.

Beta2 microglobulin mutations are an important mechanism for HLA class I total loss, (phenotype No. I) and have been described in colon carcinomas, melanomas and lymphomas. We describe a new beta2 microglobulin mutation detected in the melanoma cell line GR-34. The new mutation reported here was identified as a deletion of 4 bases (TTCT) in the highly repetitive sequence CTCTCTCTTTCT located in the leader sequence of the beta2 microglobulin gene at codon 15-16 of exon 1. The mutation produces a frameshift in the open reading frame sequence with the appearance of a stop codon at position 42. We also demonstrate that the second beta2 microglobulin gene is deleted. Comparisons with beta2 microglobulin mutations in other tumor cell lines suggest a mutation hot spot in exon 1.

Mutations of the beta2-microglobulin gene result in a lack of HLA class I molecules on melanoma cells of two patients immunized with MAGE peptides.

Mutations have been identified in the beta2-microglobulin gene of tumor cells of two metastatic melanoma patients who received immunizations with MAGE peptides. One mutation abolishes the start codon whereas the other introduces a premature stop codon. The second beta2-microglobulin allele of both tumors appears to be lost on the basis of sequence data and loss of microsatellite heterozygosity. The lack of beta2-microglobulin gene product results in the absence of HLA class I antigens on the surface of the tumor cells. This may explain why the tumors of both patients progressed despite the immunization treatment and shows the necessity of analyzing in depth the antigen presentation capability of the tumor cells for the interpretation of clinical trials involving anti-tumor vaccination.

Genetic alterations and oxidative metabolism in sporadic colorectal tumors from a Spanish community.

Deletions of loci on chromosomes 5q, 17p, 18q, and 22q, together with the incidence of p53 mutations and amplification of the double minute-2 gene were investigated in the sporadic colorectal tumors of 44 patients from a Spanish community. Chromosome deletions were analyzed by means of loss of heterozygosity analysis using a restriction fragment length polymorphism assay. Allelic losses were also detected by polymerase chain reaction (PCR)-single-stranded conformation polymorphism (SSCP) analysis of a polymorphic site in intron 2 of the p53 gene. The percentages of genetic deletions on the screened chromosomes were 39.3% (5q), 58.3% (17p), 40.9% (18q), and 40% (22q). Mutations in p53 exons 2-9 were examined by PCR-SSCP analysis and direct sequencing of the mutated region. Twenty of 44 tumor samples (45.45%) showed mutations at various exons except for exons 2, 3, and 9, the most frequent changes being G-->T transversion and C-->T transition. Because oxygen-free radicals play a role in the carcinogenesis process, we evaluated the oxidative status of the colorectal tumors. Antioxidant activities, lipid peroxidation, and DNA-damaged product concentrations in colon tumors and normal mucosa were compared. In tumor tissues, superoxide dismutase and catalase decreased fourfold and twofold, respectively, whereas glutathione peroxidase and reduced glutathione increased threefold. Malondialdehyde and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were twofold higher in colorectal tumors than in normal mucosa. Seven of 10 DNA tumor samples (70%) showing higher values of 8-OHdG also had genetic alterations at different chromosomal loci. In these samples, the p53 gene was deleted or mutated in 71.4% of cases. We concluded that the observed changes in the oxidative metabolism of the tumor cells and the consecutive increase in DNA damage may potentiate the genomic instability of different chromosomal regions, leading to further cell malignancy and tumor expansion.

Differential expression of the H-ras mutated and normal alleles in rabbit DMBA-induced keratoacanthomas.

Keratoacanthomas (KAs) are benign and self-regressing tumors in which a high incidence of the mutated H-ras oncogene has been observed both in humans and in experimental models. To determine the level of expression of the mutated H-ras allele with respect to its normal counterpart in 7,12-dimethylbenz(a)anthracene (DMBA)-induced KAs in rabbit skin, we have utilized a quantitative technique based on reverse transcription polymerase chain reaction (RT-PCR) and selective cleavage of the mutated molecules of the H-ras gene. Analysis of 16 KAs showed that the mutated H-ras transcripts were up to 3-fold more abundant than the non-mutated H-ras transcript in the different tumors. This higher expression of the mutated allele appears to correlate with increased differentiation in the KAs and in turn may contribute to tumor regression.

The role of 8-hydroxy-2'-deoxyguanosine in rifamycin-induced DNA damage.

The effect of rifamycin SV on the formation of 8-hydroxy-2'-deoxyguanosine (8-0HdG) has been investigated in vitro and in vivo. Oxidative modification of 2'-deoxyguanosine has been measured as an indication of DNA damage using high-performance liquid chromatography with electrochemical detection. Rifamycin SV in the presence of copper(II) ions induces the formation of 8-0HdG in calf thymus DNA. The effect is enhanced by increasing the antibiotic concentration and inhibited by catalase and hydroxyl radical (.0H) scavengers, such as thiourea and ethanol, in a rifamycin SV concentration-dependent manner. The reduced glutathione (GSH) inhibits DNA damage, and this effect is proportional to the final concentration of the tripeptide in the incubation medium. A significant increase in the formation of 8-0HdG and of malondialdehyde (MDA) in rat liver DNA was observed only in GSH-depleted animals after 5 days of rifamycin SV treatment. These results support the involvement of hydrogen peroxide (H2(0)2) and .0H in the mechanism of the oxidative modification of DNA achieved by rifamycin SV. The role of other reactive species and the antioxidant properties of GSH against oxidative damage is also discussed.

A new polymorphic site in intron 2 of TP53 characterizes LOH in human tumors by PCR-SSCP.

Many human cancers present deletions of the short arm of chromosome 17, which includes the TP53 locus. We detected a new polymorphism in intron 2 of the TP53 gene using PCR-SSCP and used this polymorphic site as a marker to detect loss of heterozygosity in 135 human tumors (73 soft tissue sarcomas, and 48 colorectal and 14 bladder carcinomas). Heterozygosity for this site was 41.5% in this study group and tumor-specific loss of alleles occurred in 43% of informative cases. Allelic losses were more frequently detected at this site than at that in which restriction fragment length polymorphism (RFLP) is located, as detected by the pHp53B probe. It is concluded that this novel approach has several advantages, including detection of a high incidence of informative cases and minimal tissue requirements.

Altered patterns of MDM2 and TP53 expression in human bladder cancer.

BACKGROUND: The TP53 gene maps to the short arm of chromosome 17 (17p13.1) and encodes for a nuclear phosphoprotein of 53 kd (p53) involved in cell cycle control. The MDM2 gene is located on the long arm of chromosome 12 (12q13-14), and it encodes for a nuclear protein (Mdm2) of 90 kd of molecular mass. Genetic alterations in the TP53 gene have been reported as frequent events in bladder cancer and are associated with disease progression. The MDM2 gene has been shown to be amplified and overexpressed in sarcomas; however, these changes have not yet been analyzed in neoplastic lesions of the urinary bladder. PURPOSE: We undertook the present study in order to determine the frequency of MDM2 and TP53 abnormalities in bladder tumors, as well as to examine the clinical relevance of identifying their altered patterns of expression in patients affected with bladder cancer. METHODS: We analyzed a cohort of 87 patients affected by bladder tumors. Altered patterns of expression of Mdm2 proteins were determined using an immunohistochemical assay with monoclonal antibody 2A10, and MDM2 gene amplifications were studied by Southern blotting. Mutant p53 proteins were identified using monoclonal antibody PAb1801. The presence of intragenic mutations in the TP53 gene were assessed utilizing single-strand conformation polymorphism and further characterized by sequencing. Associations were assessed statistically by the two-tailed Fisher's exact test. RESULTS: Twenty-six of 87 cases had abnormally high levels of Mdm2 proteins; however, only one case showed an MDM2 amplification. Thirty-six of 87 cases displayed p53 nuclear overexpression. Sixteen cases had abnormally high levels of both Mdm2 and p53 proteins. There was a strong statistical association between Mdm2 and p53 overexpression (Fisher's exact test: P = .018). Moreover, there was a striking association between Mdm2 overexpression and low-stage, low-grade bladder tumors (Fisher's exact test: P = .0005). CONCLUSIONS: The results suggest that aberrant Mdm2 and p53 phenotypes are frequent events in bladder cancer and may be involved in tumorigenesis or tumor progression in urothelial neoplasias. IMPLICATIONS: This study is the first to report altered patterns of MDM2 expression in human bladder tumors and demonstrates that aberrant Mdm2 and p53 phenotypes may be important diagnostic and prognostic markers in patients affected by bladder cancer.

Chromosome 17 abnormalities and TP53 mutations in adult soft tissue sarcomas.

This study was designed to determine the frequency of structural genetic abnormalities of chromosome 17 and the incidence of TP53 mutations as they relate to the biological behavior of adult soft tissue sarcomas. We analyzed a group of 73 soft tissue sarcomas of adults that were clinically and pathologically well characterized using molecular genetic techniques and expression studies. We then correlated genotype and phenotype with pathological parameters. Overall, allelic loss of 17p and 17q was identified in 53 and 29% of informative cases, respectively. p53 nuclear overexpression was detected in 34% of the tumors analyzed. We observed an association between 17p deletions and tumor presentation being more frequent in recurrent and metastatic tumors than primary lesion. p53 nuclear overexpression was associated with tumor grade, size, and more frequently detected in metastatic than primary sarcomas. The 11 intragenic mutations characterized included 10 cases of single base substitution and one single base deletion; 8 were of the missense type and 3 were nonsense. It is concluded that 17p deletions and TP53 mutations are common events in adult soft tissue sarcomas and that due to the trends observed with the cohort of patients analyzed they may become prognostic markers for patients affected with these tumors.

p53 mutations in human bladder cancer: genotypic versus phenotypic patterns.

The objective of this study was to characterize the pattern of p53 mutations in bladder cancer. The sensitivity and specificity to detect these mutations using clinical material was assessed for the following assays: immunohistochemistry, restriction-fragment-length polymorphism, single-strand-conformation polymorphism, and sequencing. Discrepancies of reported results aimed at the identification of genetic alterations in the p53 gene may be due to differences in methodology, as well as to deficient morphological evaluation of the source of tissue utilized. In order to address these critical issues, we have implemented a novel experimental design that permits analysis by molecular genetics and immunopathology techniques in any given tissue specimen, allowing morphological correlation with genotypic and phenotypic characteristics of the tissue analyzed. Forty-two patients affected with bladder tumors in whom paired normal and tumor tissues were available were used for the present study. Nuclear immunoreactivities were observed in 26 out of 42 bladder tumors analyzed. Abnormal shifts in mobility were noted in 14 of the 42 cases in distinct exons, with one tumor revealing 3 mutations. There was a strong association between p53 nuclear over-expression and 17p LOH, as well as p53 nuclear over-expression and detection of mutations by SSCP and sequencing. According to receiver-operating-curve statistical analysis, the accuracy of detecting p53 mutations by IHC was estimated to be 90.3%. It is our conclusion that, when properly used, this is a highly sensitive and specific method with simple application using clinical material.

Molecular abnormalities of mdm2 and p53 genes in adult soft tissue sarcomas.

Genetic alterations in the p53 and mdm2 genes have been reported to occur in soft tissue sarcomas. This study was designed to determine the prevalence and potential clinical value of detected molecular abnormalities and altered patterns of expression of mdm2 and p53 genes in adult soft tissue sarcomas. A cohort of 211 soft tissue sarcomas from adults that were both clinically and pathologically well characterized was analyzed. Monoclonal antibodies directed against mdm2 and p53 proteins were used to measure overexpression of these proteins in the nuclei of cells from sections of these tumors. Seventy-six of 207 tumors had abnormally high levels of mdm2 proteins and 56 of 211 tumors overexpressed p53 protein. Twenty-two cases had abnormally high levels of both mdm2 and p53 proteins based upon immunoreactivity with these antibodies. There was a striking statistically significant correlation between the overexpression of p53 and mdm2 proteins in the same tumor and poor survival (P < 0.05) of the patients. A group of 73 soft tissue sarcomas was chosen for analysis using Southern blots, single strand conformation polymorphisms, and direct DNA sequencing to confirm mdm2 gene amplifications and p53 mutations and correlate these with the results of the immunoreactivities. The overexpression of p53 and mdm2 proteins in the nuclei of tumor cells did not always correlate well with gene amplification at the mdm2 locus or mutation at the p53 gene. The possible reasons for these discrepancies are discussed.

Absence of MDM-2 gene amplification in experimentally induced tumors regardless of p53 status.

To assess the generality of the hypothesis that murine double-minute-2 (MDM-2) gene amplification complements the absence of p53 mutation during tumor development, we analyzed 143 murine tumors induced by a variety of carcinogenic agents in two different mouse strains. Only three of 143 tumors showed p53 genetic alterations and none showed MDM-2 amplification, indicating the existence of alternative pathways that permit tumor cells to bypass p53-MDM-2 control.

HLA class I expression and HPV-16 sequences in premalignant and malignant lesions of the cervix.

A series of 10 normal cervix epithelia, 38 condylomas, 17 CIN (cervical intraepithelial neoplasm) I/II (low-grade CIN), 10 CIN III (high-grade CIN), 27 squamous cell carcinomas and 7 adenocarcinomas of the cervix were studied in paraffin-embedded sections for the expression of MHC class I antigens, using antibodies against HLA antigens and the immunoperoxidase technique. A PCR technique was also used to evaluate the presence of HPV-16 DNA. All samples from normal tissue, benign, premalignant and CIN III lesions expressed HLA class I antigens. However, 15% of the invasive carcinomas completely lacked HLA-B and HLA-C antigen expression, 20% presented a heterogeneous pattern and 2 cases lacked HLA-B and HLA-C heavy chain but retained beta 2-microglobulin. MHC class I antigen expression on tumors was compared with clinical-pathological parameters. The absence of expression of HLA class I molecules was significantly associated with the Glanz histoprognostic index of malignancy. HPV-16 sequences were detected in 60% of the condylomas, 88% of the CIN I/II, 80% of the CIN III and 82% of the cervical carcinomas. Eight-six per cent of the tumors expressing HLA class I antigen presented HPV-16, whereas only 40% of the nonexpressing tumors did. Our results lead us to the following conclusions: a) HLA class I losses occurred when the tumor became invasive, and in tumors of a more aggressive histological type; b) The presence of HPV-16 was associated with tumors expressing HLA class I antigens.

The role of glutathione in protection against DNA damage induced by rifamycin SV and copper(II) ions.

Incubation of calf thymus DNA in the presence of rifamycin SV induces a decrease in the absorbance of DNA at 260 nm. The effect, was found to be proportional to the antibiotic concentration and enhanced by copper(II) ions. In the presence of rifamycin SV and copper(II), a significant increase in thiobarbituric acid-reactive (TBA-reactive) material is also observed. This effect is inhibited to different degrees by the following antioxidants: catalase 77%; thiourea 72%; glutathione (GSH) 62%; ethanol 52%; and DMSO 34%, suggesting that both hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) are involved in DNA damage. Rifamycin SV-copper(II) mixtures were also found to induce the production of peroxidation material from deoxyribose and, in this case, glutathione and ethanol were the most effective antioxidant substrates with inhibition rates of 91% and 88% respectively. Electrophoretic studies show that calf thymus DNA becomes damaged after 20 min. incubation in the presence of both agents together and that the damaged fragments run with migration rates similar to those obtained by the metal chelating agent 1,10-phenanthroline. Normal DNA electrophoretic pattern was found to be preserved by catalase, and GSH at physiological concentrations and by thiourea. No protection is observed in the presence of ethanol or DMSO. The results obtained indicate the involvement of different reactive species in the degradation process of DNA due to rifamycin SV-copper(II) complex and emphasize the role of reduced glutathione as an oxygen free radical scavenger.

HLA molecules in basal cell carcinoma of the skin.

Fifty basal cell carcinomas (BCC) and 8 samples of healthy skin were studied for HLA class I and class II antigen expression and for the presence of mutations in codon 12 of the K-ras and H-ras genes. All samples of healthy skin and of epithelium near the tumor showed high levels of class I molecules, whereas 38% of the tumors showed complete absence. Sixty-two percent of the tumors presented positive class I expression with heterogeneous staining. These losses were due to the simultaneous lack of heavy chain and beta 2-microglobulin. Selective losses of HLA-A or HLA-B antigens were not detected. Class II antigens were absent in most of the tumors, only two tumors showing a few weakly positive cells with anti-HLA-DR mAb. The loss of class I expression correlated significantly with the degree of histological differentiation and aggressiveness. We were unable to correlate class I expression with clinical size, depth of invasion or the extent of leukocytic infiltrate surrounding the tumor. Analysis by PCR amplification of codon 12 of the K-ras and H-ras oncogenes detected H-ras mutations in 1 out of 50 cases, and no K-ras mutations in any of the tumors studied. Thus, a positive relationship between K-ras and H-ras mutations and BCC tumorigenesis or MHC alterations seems unlikely in this neoplasia.