Structural basis for the regulation of UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells.

The UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 (Gal NAc-T3) gene, a member of the Gal NAc transferase gene family, is expressed in a tissue-specific manner. To elucidate the function of this gene, we have focused on the molecular mechanism underlying regulation of gene expression. We have cloned Gal NAc-T3 cDNA and used it to show that Gal NAc-T3 mRNA is expressed in tumor cell lines derived from secretory epithelial tissue adenocarcinomas but not in cell lines derived from bladder and epidermoid carcinomas. Using a polyclonal antibody to Gal NAc-T3, we observed protein expression in adenocarcinoma but not non-adenocarcinoma cell lines, and in breast carcinoma cells but not in normal breast tissue. We used Gal NAc-T3 cDNA to isolate three overlapping genomic clones containing the 5'-portion of the human Gal NAc-T3 gene, and we sequenced 1.6 kb around the first exon. A transient expression assay using the luciferase gene showed that promoter activity was much higher in MCF-7 cells than in KB cells. In vivo footprint experiments showed significant protection of a distal GC box, an NRF-1 site, and an AP-2 site in MCF-7 cells. A novel stem and loop structure extending from nucleotide -103 to nucleotide -165 and contiguous to these transcription factor binding sites seemed to be functional in regulating Gal NAc-T3 gene transcription, and a KMnO4 footprint experiment showed that this stem and loop structure could be formed in vivo. We also observed dimethyl sulfate hypersensitive sites in the untranslated region around nucleotide +50 in MCF-7 but not in KB cells. These findings indicate that Gal NAc-T3 gene expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure, and that this regulation is restricted to cell lines derived from epithelial gland adenocarcinomas but not cells derived from nonsecretory epithelial tissue carcinomas. In addition, our immunohistochemical results suggest that our anti-Gal NAc-T3 antibody may be useful for diagnostic purposes in the early stages of breast cancer.

Alterative expression of the collagenase and adhesion molecules in the highly metastatic clones of human colonic cancer cell lines.

Human colonic carcinoma cell lines, KM12C, KM12SM and KM12L4, were previously established and their in vivo metastatic potentials have been well evaluated. The highly metastatic cell lines KM12SM and KM12L4 were derived from the parental low metastatic cell line KM12C in vivo. To evaluate the metastatic behavior of these cell lines in vitro, we examined colony formation on monolayers of the pulmonary arterial endothelial (CPAE) cells. On day 4, the highly metastatic cell lines showed an approximately 2-fold increase in number of colonies on CPAE cell monolayers relative to the parental KM12C cell line. To investigate what evidence is correlated with their metastatic and invasive abilities, Northern blot analysis and flow cytometry were performed in all cell lines. According to the results of Northern blot analysis, the levels of matrix metalloproteinase (MMP)-2 and c-met mRNA expression were increased in highly metastatic cell lines as compared with the parental cell line. We also examined the cell-surface expression of several adhesion molecules by flow cytometry. The levels of expression of sialyl Lewisa antigen (sLe(a)) in KM12SM and KM12L4 were twice higher than that in KM12C. However, the levels of expression of E-cadherin in KM12SM and KM12L4 were decreased to half that in KM12C. The alterative expression of the collagenase and adhesion molecules might contribute to their metastatic/invasive abilities of these cell lines both in vivo and in vitro.

Establishment of a human colon cancer cell line (PMF-ko14) displaying highly metastatic activity.

A new human colon cancer cell line (PMF-ko14) derived from a peritoneal disseminated tumor has been established and maintained for over 25 months. In tissue culture, the cells grew in a mainly monolayered sheet with a population doubling time of about 27 h. Chromosome counts at the 60th passage ranged from 79 to 84 with a modal number of 83. Flow cytometry of the cell surface antigen expression indicated that CD49b, CD29, carcinoembryonic antigen (CEA), sialyl Lewis a (sLea), and CD49c were positive in more than 70% of the cells. The nude mouse xenograft models indicated are: subcutaneous or intraperitoneal injection model, spleen injection-liver metastasis model, and orthotopic implantation-spontaneous metastasis model. As PMF-ko14 has highly metastatic activity it should prove to be a useful tool for research in biological behavior of metastatic colon cancer.

Expression of survivin, YB-1, and KI-67 in sporadic adenomas and dysplasia-associated lesions or masses in ulcerative colitis.

Sporadic adenomas are said to exhibit an orderly growth pattern with a reversal of proliferative and apoptotic cell distribution as compared with normal colonic crypts. Dysplastic polyps of patients with ulcerative colitis (UC) may represent dysplasia-associated lesions or masses (DALM) with a high associated cancer risk, or, alternatively, may represent sporadic adenomas. Histologic criteria to differentiate between sporadic adenomas and DALM have not focused on the balance between cell renewal and cell loss. The expression of the novel anti-apoptosis gene product, survivin, and the proliferation markers, Ki-67 and Y-box binding protein (YB-1), were investigated by immunohistochemical localization in sporadic adenomas and DALM lesions of patients with UC. In adenomas, KI-67 was expressed preponderantly at the luminal aspect of the polyp, whereas its expression was diffuse in DALM. Survivin was detected diffusely in both adenomas and DALM. YB-1 showed positive staining in the deep aspect of adenomatous glands but only to a minor degree at the surface, whereas both deep and diffuse expression patterns of YB-1 were seen in DALM. The authors conclude that DALM and sporadic adenomas exhibit different patterns of cellular proliferation and that molecular markers of cell proliferation, Ki-67 and YB-1, may be useful to distinguish sporadic adenomas from DALM. However, the similar expression of survivin suggests that the underlying mechanisms that regulate apoptotic cell death are uniform in these lesions.

Serum levels of interleukin-12 in patients with gastrointestinal cancer.

Interleukin 12(IL-12) is a heterodimeric cytokine that has potent anti-tumor and anti-metastatic activities. Although clinical trials of recombinant human IL-12 have begun in patients with several advanced malignancies, very few studies have investigated the preoperative serum levels of IL-12 in patients with gastric or colorectal cancer. The purpose of the present study was to investigate the relationships between the preoperative serum levels of IL-12 and clinicopathological factors in patients with gastric or colorectal cancer. Blood was obtained before surgery from 14 patients with gastric cancer and 15 patients with colorectal cancer. Serum levels of IL-12 was assessed using the quantitative sandwich enzyme immunoassay technique. Although not statistically significantly, low serum levels of IL-12 tended to be associated with gastric cancer patients who were node-positive, CEA positive, had tumors that penetrated the serosa, had tumors greater than 5 cm in diameter, were more than 60 years-old, or were more advanced than stage IIIA(TNM) or stage IIIa(Japanese Research Society for Gastric Cancer). Patients with colorectal cancer who were node-positive, had tumors that penetrated the serosa, were more than 60 years-old, or were more advanced than stage III(TNM), stage IIIa(Japanese Society for Cancer of the Colon and Rectum) and Dukes' C also tended to have low serum IL-12 levels. These results suggest that low serum levels of IL-12 may be observed in more advanced gastric and colorectal cancer patients. Thus, patients with low serum levels of IL-12 in gastric or colorectal cancer may require additional immunochemotherapy after surgery.

Relationship between plasma levels of vascular endothelial growth factor and serum levels of interleukin-12 in patients with colorectal cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) is thought to be the most potent angiogenic factor in the numerous malignant tumors and a prognostic indicator for cancer patients. Interleukin 12 (IL-12) is a heterodimeric cytokine that has potent anti-tumor and anti-metastatic activities. Recently, it has been suggested that IL-12 regulates VEGF in a murine breast cancer model. MATERIALS AND METHODS: Blood from 26 patients with colorectal cancer was obtained prior to surgery. Plasma levels of VEGF and serum levels of IL-12 were assessed using the quantitative sandwich enzyme immunoassay technique. We investigated the preoperative relationships between plasma levels of VEGF, serum levels of IL-12 and clinicopathological factors in patients with colorectal cancer. RESULTS: Although not statistically significantly, high plasma levels of VEGF and low serum levels of IL-12 tended to occur with more advanced colorectal cancer. Plasma levels of VEGF in patients who had circumferential involvement of the tumor greater than 1/2 were only significantly increased. The preoperative relationship between plasma levels of VEGF and serum levels of IL-12 tended to be negatively correlated. CONCLUSION: These results suggest that high plasma levels of IL-12 or low serum levels of IL-12 may be observed in more advanced colorectal cancer patients. Thus, these patients may require additional immunochemotherapy after surgery. IL-12 may regulate VEGF in the patients diagnosed with colorectal cancer.

[Evaluation of the quality of mortality information by unspecified accidents and events of undetermined intent]. / Avaliação da qualidade das informações de mortalidade por acidentes não especificados e eventos com intenção indeterminada.

INTRODUCTION: Mortality data are important for monitoring violence, making it possible to assess the trends and the impact of interventions towards its reduction. The objective of the study is to assess the quality of the filling out and codification of the death certificates for unspecified accidents and events of undetermined intent in the city of S. Paulo in 1996. METHODS: Death certificates on which the underlying cause of death (UCD) given was an unspecified accident (ICD-10 X59) or an event of undetermined intent (ICD-10 Y10-Y34) were selected for investigation at the Legal Medicine Institute (IML). After consulting the police reports which accompany the corpses to the IML, the autopsy reports and other legal forms, these were analysed and the UCD was recoded. RESULTS: For unspecified accidents, 53.2% were changed to a specified cause: 15.1% due to pedestrians injured in traffic accidents, 17.5% due to other traffic accidents and 14.5% due to falls. Homicides and suicides constituted 9.8%. In 20.9% no additional information was found. For events of undetermined intent, 2/3 had no clarification; in 1/3 of the cases, the underlying cause changed to falls (10.6%), homicides (7.5%) and pedestrians injured in transport accidents (6. 7%). CONCLUSIONS: The quality of mortality information by external causes in the City of S. Paulo is not satisfactory. The IML has not used all the available information to fill out the death certificates. The findings reveal that the instruction of the World Health Organization and the Brazilian Center for the Classification of Diseases to codify as accidents those events for which there is no information on the death certificate about the external cause, does not seem to be appropriate. In that category 66.0% of the deaths were found to have been inferred incorrectly as accidental. The improvement of the quality of mortality data due to external causes may contribute to the monitoring of violence and may give support to decisions leading to its reduction whatever the form that violence may take.

[The clinicopathological features and operative results in a cancer of the remnant stomach--risk and prevention of cancer after partial gastrectomy].

The clinicopathological features and operative results were analyzed in 21 patients undergoing an operation for cancer of the remnant stomach between 1979 and 1997. The twenty-one patients were divided into two groups: Group A; n = 9: who had undergone a gastrectomy for benign gastric disease, Group B; n = 12: who had undergone the same operation for gastric cancer. In Group A, the interval between the first gastrectomy and the second was longer than in Group B. In both groups, a large number of advanced cancers (n = 16, 76.2%) and undifferentiated carcinomas (n = 15, 71.4%) were seen. Five-year-survival rates in Group A and Group B were 23.7%, 19.0%, respectively. The incidence of gastric stump cancer following partial gastrectomy was 80% in the patients on whom Billroth II reconstruction had been performed after gastrectomy. It is suggested that the residual stomach in the Billroth II reconstruction patients is susceptible to cancer development. Consideration of the reconstruction method and a systemic follow-up is needed to improve a prognosis.

Non-icteric fulminant hepatitis and Reye's syndrome: comparison of laboratory data.

Twenty-eight patients with suspected Reye's syndrome (RS) were seen in our Department from 1974 through 1987. Liver biopsy confirmed the diagnoses of RS and non-icteric fulminant hepatitis (NIFH) in 7 and 5 cases, respectively. NIFH was the most common RS mimicker. Total bilirubin, LDH and serum ammonia levels showed no significant differences between RS and NIFH. However, the levels of serum GOT and GPT were significantly higher in the NIFH group. Serum and urinary carnitine levels were measured in both groups, but the results were inconclusive. Amino acid analysis in one RS and two NIFH patients showed no significant differences in the ratio of branched chain to aromatic amino acids. However, one RS patient showed a high level of lysine. Histological findings in the liver of two NIFH patients showed minor mitochondrial swelling and microvesicular fat, but the major finding was hepatic necrosis. Our experience indicates that NIFH and RS cannot be differentiated by routine laboratory tests. Liver biopsy is essential for the accurate diagnosis of RS.

Impaired myocardial perfusion in patients with hypertrophic cardiomyopathy: assessment with digital subtraction coronary arteriography.

To study the clinical significance of abnormal myocardial perfusion in patients with hypertrophic cardiomyopathy (HCM), we performed a computerized washout analysis of digital subtraction coronary arteriograms in 28 patients with HCM and 16 control subjects. The contrast disappearance half-life (T1/2) was calculated from a time-density curve generated in the four sectors of the myocardium perfused by the left anterior descending coronary artery and the mean T1/2 was calculated by averaging T1/2 values for these four sectors. Patients with HCM demonstrated longer T1/2 in the ventricular septal region than control subjects. Thirteen (46%) of the patients with HCM presented abnormally longer mean T1/2 values, suggesting impaired myocardial perfusion. Family histories of HCM were more frequent in patients with abnormal mean T1/2 values (92% vs 47%; p less than 0.05). On the exercise stress test, patients with abnormal T1/2 values presented significantly lower exercise tolerance with more frequent exercise-induced ST segment depression (62% vs 13%; p less than 0.05). However, there were no significant differences between the two groups with regard to ventricular wall thickness, left ventricular end-diastolic pressure, or the severity of systolic narrowing of the coronary arteries. These findings suggest that 13 (46%) of the patients with HCM have impaired myocardial perfusion, which may be a manifestation of intramural coronary artery disease in addition to left ventricular hypertrophy, elevated left ventricular end-diastolic pressure, or systolic narrowing of the coronary arteries. Additionally, significant association of the prolonged T1/2 with a familial occurrence of HCM and depressed exercise tolerance with ST segment depression imply that impaired myocardial perfusion could be an important inherent pathophysiological state leading to myocardial ischemia during exercise.

Solitary "amyloid ulcer" localized in the sigmoid colon without evidence of systemic amyloidosis.

In a 51-yr-old man, colonoscopy for heme-positive stools revealed a solitary "amyloid ulcer" localized in the sigmoid colon. There were no clinical symptoms suggesting amyloidosis, and additional examination revealed no findings characteristic of amyloidosis or any chronic inflammatory disease. Ischemic change as a result of amyloid infiltration into the vessel wall may lead to formation of an ulcer in the affected bowel. Although local resection can be considered for a symptomatic and well defined lesion, it should be kept in mind that amyloid can present in various forms and follow various courses, such as the spontaneous healing that occurred in our patient.

Enhancement of metastatic activity of colon cancer as influenced by expression of cell surface antigens.

BACKGROUND: Cell surface antigens are contributory factors toward metastatic activity. There have been no detailed studies on changes in cell surface antigens of colon cancer cell lines. To control life-threatening metastasis, it is necessary to evaluate what types of changes in cell surface antigens exert an influence on metastatic activity. MATERIALS AND METHODS: In vivo selection was performed using the human colon cancer-derived cell line KM12SM to obtain variants of metastatic activity. A murine spleen injection-liver metastasis procedure reflecting the latter half of the metastatic process was adopted and repeated four times. Flow cytometric analyses were carried out to detect expression of antigens: Lewis a (Lea), Lewis x (Lex), sialyl Lewis a (sLea), sialyl Lewis x (sLex), E-cadherin, CD44v6, integrin alpha2 (CD49b), integrin alpha3 (CD49c), integrin alpha4 (CD49d), integrin alpha5 (CD49e), and integrin beta1 (CD29). RESULTS: In vivo selection produced variants with higher metastatic activity. In the original line KM12SM, sLea, E-cadherin, CD49b, CD49c, or CD29 were positive in more than 40% of the cells. After selection, the percentage of cells positive for Lea, sLea, and all examined integrins significantly increased. Lex, sLex, and CD44v6 increased slightly, while E-cadherin decreased slightly. CONCLUSIONS: In vivo selection and flow cytometric analysis revealed that Lea, sLea, CD49b, CD49c, and CD29 appear to be involved in the increase of metastatic activity. The changes of integrin expression in this study suggest that integrins collaborate in the promotion of adhesion to an extracellular matrix.

[Quantitative evaluation of regional myocardial blood flow by digital subtraction angiography: correlations with exercise electrocardiography and Tl-201 myocardial scintigraphy].

We previously reported that the contrast disappearance half-life (T1/2) derived by the computerized washout analysis of digital subtraction coronary arteriograms provides a useful index for quantitatively evaluating regional myocardial blood flow. In the present study, we further evaluated the clinical usefulness of T1/2, comparing it with exercise electrocardiography and exercise thallium-201 myocardial scintigraphy. The study subjects consisted of 25 patients with angina pectoris and 14 patients with normal coronary arteries. Following the manual injection of contrast media into the left anterior descending coronary artery (LAD), a time-density curve was generated in the sectors of the myocardium which were perfused by the LAD and the T1/2 was calculated. T1/2 values correlated closely with double product (r = -0.73). They were significantly greater in patients with exercise-induced ST depression (8.3 +/- 1.0 vs 5.8 +/- 0.7, p less than 0.005). In addition, there was a good correlation between T1/2 values and washout ratio as determined by exercise thallium-201 myocardial scintigraphy, with r = -0.83. Although T1/2 values were within the normal range (mean +/- 2SD of control subjects) in all patients with LAD stenosis of 50 percent or less, these values were abnormally increased, exceeding the normal range, in 11 of the 12 patients with stenosis of 90 percent or more. Compared with exercise electrocardiography, T1/2 values were abnormally prolonged in 11 of the 13 patients with exercise-induced ST depression. Compared with exercise thallium-201 myocardial scintigraphy, T1/2 values were abnormally prolonged in seven of the nine patients with transient perfusion defects.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal Dubin-Johnson syndrome with severe cholestasis: effective phenobarbital therapy.

We described Dubin-Johnson syndrome (DJS) with severe cholestasis in a 20-day-old Japanese boy. Although neonatal DJS has been sporadically reported. DJS with severe cholestasis has not to our knowledge been described in the English literature. The ratio of urinary coproporphyrin isomer I to urinary total coproporphyrin in our patient was high (93%). Liver histology showed cytoplasmic pigment granules in the liver cells. Administration of phenobarbital (PB) significantly decreased the levels of bilirubin and bile acids in the serum. There was a significant elevation of 1 beta-hydroxylated bile acids in the urine. It is predicted that severe cholestasis in neonatal DJS may cause metabolic abnormalities in both bilirubin and bile acids transport.

Enhanced coexpression of YB-1 and DNA topoisomerase II alpha genes in human colorectal carcinomas.

The transcription factor YB-1 is expressed in a wide range of cell types and has been implicated in the regulation of various genes involved in cell proliferation. Nuclear expression of YB-1 is correlated with MDR-1 gene expression in breast cancer and osteosarcoma. In this study, we asked whether YB-1 expression is enhanced in human colorectral carcinoma and if it is associated with the expression of target genes such as MDR-1, DNA topoisomerase II alpha and PCNA. YB-1, DNA topoisomerase II alpha, PCNA and MDR-1 expression were assessed by Western blotting, Northern blotting and immunohistochemistry in 26 human colorectal carcinomas. The involvement of YB-1 in DNA topoisomerase II alpha gene expression was examined by transient DNA transfection assays. YB-1 was overexpressed in almost all cancerous lesions in comparison with normal mucosa in surgically resected colorectal carcinomas of 26 patients. YB-1 expression correlated well with both DNA topoisomerase II alpha and PCNA expression. In contrast, no correlation was observed between YB-1 and MDR-1 expression. We also found that a transient co-transfection with a DNA topoisomerase II alpha promoter-luciferase plasmid and an antisense YB-1 expression construct resulted in a significant reduction of the promoter activity in KM12C human colon cancer cells. YB-1 may be an excellent proliferation-associated marker and may be a transcription factor regulating DNA topoisomerase II alpha gene expression in human colorectal carcinoma.

[Quantitative assessments of regional myocardial perfusion by digital subtraction angiography].

Regional myocardial perfusion was evaluated by computerized washout analysis of digital subtraction angiography (DSA) images. Diatrizoate meglumine (76% Urografin), 2 to 3 ml, was manually injected into the left main coronary artery. For 26 patients with ischemic heart disease (IHD), 14 patients with cardiomyopathy, and eight patients with normal coronary angiograms, DSA images of myocardial perfusions were obtained in the right anterior oblique projection. These were digitized into an image-processing computer. Time-density curves were constructed in four segments of the left ventricle perfused by the left anterior descending coronary artery (LAD) and the contrast decay half-lives (T1/2) were calculated from the decay phases of the curves, using mono-exponential least square fits. The mean T1/2 was significantly longer in patients with 75% or more LAD narrowing than in those with normal coronary arteries. By contrast, patients with 50% or less LAD narrowing had T1/2 comparable to those with normal coronary arteries. In patients with IHD, there was a significant curvilinear relationship of T1/2 with percent stenosis of the LAD. This indicates that a decrease in regional myocardial flow develops rapidly in coronary stenosis of 70-80% or more. In patients with comparable coronary stenosis, T1/2 was significantly longer in the asynergic regions than in those with normal wall motion, but T1/2 was shorter in regions perfused by collateral vessels. These findings indicate that left ventricular contraction and collateral flow could contribute to regional myocardial perfusion. In addition, patients with hypertrophic and dilated cardiomyopathy had prolonged T1/2 despite normal coronary angiograms, suggesting abnormalities in intramural coronary arteries. Thus, T1/2 derived by computerized washout analysis of DSA myocardial image proved to be a useful index for quantitative evaluation of regional myocardial perfusion.