Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients.

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Co-expression of urokinase with haptoglobin in human carcinomas.

BACKGROUND: We have shown that colon and breast cancer contains large amounts of urokinase (uPA), and that these cells are the actual sites of its synthesis. We isolated a large complex molecule consisting of the beta-chain of uPA, both chains of haptoglobin (Hp), and part or all of an NCAM-like molecule. The question arose whether it would be possible to show the presence of Hp in the same cells where uPA was found. MATERIALS AND METHODS: Human colon and breast adenocarcinomas were investigated for expression of Hp and uPA by immunohistochemistry. Fluorescence in situ hybridization was used to identify the cells of origin of these antigens. RESULTS: Hp was expressed in 8 of 11 colon adenocarcinomas, and in 10 of 12 breast tumors. uPA was demonstrable on the cell membrane and in the cytoplasm of all 11 colon adenocarcinomas studied, and cytoplasmic uPA-mRNA was found in all cases. While uPA was also detected in some stromal and inflammatory cells, Hp was present abundantly in such cells, as well as in capillary endothelial cells. Hp-mRNA was also found in both colon and breast tumors wherever the antigens were expressed. CONCLUSIONS: uPA and Hp are produced by the cancer cells and are not taken up by stromal elements. While the role of uPA in the malignant process is well documented, that of Hp is largely unexplored. Its ubiquity, shown here, suggests that it is also involved in some aspects of that process.

Longitudinal variation in O(6)-alkylguanine DNA-alkyltransferase activity in the human colon and rectum.

In a systematic study of O(6)-alkylguanine DNA-alkyltransferase activity in the human colon and rectum, tumours were found to occur in regions of low activity. These results are consistent with the hypothesis that O(6)-alkylguanine DNA-alkyltransferase levels and alkylating agent exposure may be important determinants of large bowel tumorigenesis.

Enhanced expression of cyclooxygenase-2 and nuclear beta-catenin are related to mutations in the APC gene in human colorectal cancer.

Mutational inactivation of the human tumour suppressor gene adenomatous polyposis coli (APC) results in constitutive activation of beta-catenin/T cell factor-4 (Tcf-4) mediated transcription of target genes. Up-regulation of cyclooxygenase-2 (COX-2) protein is frequently found in human colorectal cancer (CRC). We analysed 38 CRC for mutations in APC and beta-catenin and found an association between APC mutations and elevated COX-2 levels. Furthermore, APC mutations were predominantly observed in tumour tissues from the rectum compared to tumours of colonic origin. Western blot analysis revealed that nuclear beta-catenin levels were generally higher in tumours with APC mutations compared to tumours with wild type APC. However, there was also a higher level of nuclear beta-catenin in tumour compared to normal tissue, but nuclear Tcf-4 protein was constitutively expressed in tumour and normal tissue and showed no differences. An identified putative Tcf-4 binding element in the COX-2 promoter may partly explain the enhanced level of COX-2 and support the idea that COX-2 may be a downstream target of the APC/beta-catenin/Tcf-4 pathway.

N-acetyltransferase 2 (NAT2) genotype and colorectal carcinoma: risk variability according to tumour site?

BACKGROUND: Dietary heterocyclic aromatic amines (HAAs) are members of a family of chemicals that comprise highly mutagenic compounds related to colon cancer. The polymorphic N-acetyltransferase 2 enzyme (NAT2, E.C. 2.3.1.5) plays a key role in the transformation of HAAs to ultimate carcinogens. NAT2 enzyme activity is expressed in a genotype-dependent manner in colon epithelium. Therefore local activation of HAAs in colon, and hence increased risk to develop colon cancer, is likely to be related to high NAT2 enzyme activity. This study is aimed at analysing the association between genotypes leading to high NAT2 activity and colorectal cancer risk. METHODS: Genomic DNA from 120 colorectal cancer patients and 258 healthy individuals were analysed for enzyme-inactivating mutations at the coding region of the NAT2 gene by means of a mutation-specific polymerase chain reaction. RESULTS: Among patients with sigmoid colon cancer, a significant excess of individuals with genotypes leading to high NAT2 activity was observed as compared both to controls and to the rest of patients with colorectal cancer (P < 0.05). CONCLUSIONS: Our findings, which require independent confirmation, suggest that the NAT2 genotype constitutes a secondary risk factor to develop sigmoid colon cancer.

Determinants of O(6)-alkylguanine-DNA alkyltransferase activity in normal and tumour tissue from human colon and rectum.

O(6)-Alkylguanine-DNA-alkyltransferase (ATase) is an important modulator of alkylating agent-induced toxicity and carcinogenicity, but those factors which influence the expression of this repair protein in human tissues are poorly characterised. In this study, we have determined ATase levels in macroscopically normal and tumour tissues from 76 individuals with benign or malignant colorectal disease. All tissue samples had detectable ATase activity, with values ranging from 35 to 451 fmol/mg protein. ATase activity in normal rectal tissue was significantly higher than that in normal tissue from the sigmoid colon (148 +/- 76 vs. 100 +/- 40 fmol/mg protein, p = 0.01), whereas ATase levels within different regions of the colon (proximal vs. sigmoid colon) were similar. In normal tissue, inter-individual variation in ATase activity was 4-fold in the colon and 6-fold in the rectum, whereas in tumour tissue the corresponding figures were approx. 13.0- and 7-fold, respectively. There was no detectable difference in normal tissue ATase activity between individuals with benign or malignant disease of the colon. Normal and tumour tissue ATase activities were strongly correlated in the sigmoid colon (r = 0.80) and rectum (r = 0.59) but not the caecum (r = -0.03). In a multivariate analysis, ATase activity in normal colon tissue increased with age (p = 0.01) and current smoking (p = 0.06), whereas tumour ATase activity increased only with use of anti-histamines (p = 0.05). In rectal tumour tissue, activity decreased with age (p = 0.05) and use of anti-muscarinic medications (p = 0.01): in normal rectal tissue, no modulating factors were identified.

Evaluation of cathepsin D immunostaining in colorectal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Cathepsin D (CD), an estrogen-regulated lysosomal protease, has been detected in a variety of tissues. CD expression has been correlated with the invasive potential of breast cancer, acting as an autocrine mitogen or as a protease that degrades the extracellular matrix. The role of CD expression in predicting prognosis or invasive potential in colorectal carcinomas is mostly unknown. METHODS: CD immunohistochemical expression was studied in 60 surgical specimens of colon adenocarcinomas. A three-step avidin biotinylated, horseradish immuno-peroxidase (ABC-HRP) staining technique was performed on 4 microm paraffin-embedded tissue sections with a polyclonal antibody to CD. RESULTS: Carcinoma cells showed positive CD immunostaining in 41.6% of adenocarcinomas (50%, 43.7%, 37.5%, and 25% of Dukes' Stage A, B, C, and D, respectively). Nonneoplastic stromal cells demonstrated positive staining in 68.3% of the adenocarcinoma specimens (37.5%, 62.5%, 91.6%, and 75% of Stage A, B, C, and D, respectively). Patients with colorectal carcinomas exhibiting simultaneously negative and positive CD expression in malignant and stromal cells, respectively, had a worse 5-year overall survival (P < 0.05). The mean 5-year survival of the 16 patients overexpressing CD in nonneoplastic stromal cells (>15% of stromal cells positive for CD) was significantly worse in comparison with the rest of the adenocarcinomas (n = 44) (27.6 +/- 4.6 vs. 46 +/- 2.7 months, respectively, P < 0.01). CONCLUSIONS: Expression of CD immunoreactivity by the stromal cells may be associated with a more invasive phenotype. Therefore, CD expression in tumor and stromal cells may serve as an important indicator of progression and guide postoperative treatment.

Increased mRNA expression of the receptor-like protein tyrosine phosphatase alpha in late stage colon carcinomas.

The protein tyrosine phosphatase alpha (PTP alpha) mRNA level in paired samples of late stage (Dukes' D) colorectal tumors and adjacent normal colon mucosa was quantified by RNase protection assays. After normalization against 18S RNA or beta-actin mRNA level, a 2-10-fold increase in PTP alpha mRNA was detected in 10 of 14 tumors (approximately 70%) compared to mucosa. In situ hybridization of digoxigenin-labelled antisense PTP alpha RNA to tumor and mucosa sections produced a signal only in neoplastic cells of the tumor sample, consistent with the high increase in PTP alpha mRNA detected by RNase protection assays of some of the tumors. This is the first report suggesting an association of a protein tyrosine phosphatase with colorectal carcinoma. PTP alpha is a receptor-like PTP thought to be involved in regulating cell proliferation. Its oncogenic properties when overexpressed in cultured fibroblasts suggest that PTP alpha overexpression could contribute to the tumorigenic process in colon carcinoma.

Identification and characterization of alkaline phosphatase isozymes in human colorectal adenocarcinomas.

Using alkaline phosphatase isozyme-specific immunocatalytical assays, the content of isozymes was determined in normal mucosas and adenocarcinomas from human colon or rectum. Tumor levels of both the tissue (liver)-unspecific and the placental-like alkaline phosphatase (PLAP-like) were elevated compared to normal mucosas of the same patients. Such elevations have been reported previously, particularly in seminomas and ovarian tumors. In several tumors, moreover, the intestinal isozyme was expressed in lesser amounts than in the adjacent mucosa. The present results indicate that the activation of two of the phosphatase isozymes, including expression of the typical germ cell line phosphatase (the PLAP-like isozyme), may occur even in nongonadal tumors. This may reflect an induction pattern of phosphatase isozymes, with implications for malignant transformation also in other tumors.

Epidemiology of polyps in the rectum and sigmoid colon. Size, enzyme levels, DNA distributions, and nuclear diameter in polyps of the large intestine.

Enzyme activity and cell cycle variables were measured in 38 adenomas and 9 hyperplastic large-intestinal polyps equal to or larger than 5 mm in diameter. The polyps were resected endoscopically from patients 50-59 years old. A significantly higher activity of lactate dehydrogenase (LD) was found in polyps from women than in those from men. A higher LD and activity was also observed in adenomas with moderate to severe dysplasia than in those with mild dysplasia. A significantly higher activity was found for LD and glucose-6-phosphate dehydrogenase (G6PD) in adenomas greater than or equal to 10 mm than in adenomas less than 10 mm in diameter. DNA flow cytometry showed that all hyperplastic polyps were diploid and that two of the adenomas had an aneuploid DNA stemline in addition to the diploid one. The S-phase fraction varied from 3.5% to 26.5% and the G2 fraction from 0.4% to 6.7%. Two overlapping populations were found, based on nuclear size measurements. Hyperplastic polyps had almost only small nuclei, whereas adenomas had both small and large nuclei in various ratios. No statistical correlations were found between the S-phase or G2-phase fractions and polyp size or the presence of dysplasia. The number of adenomas with aneuploidy was too small to disclose a relationship to polyp size or enzyme activity. The increased enzyme activity in larger polyps and in polyps from women may point to certain risk factors in these special groups. The results indicate a further need for studies of combination of markers for prognostic evaluation of large-intestinal adenomas.

The role of peptidases in cancer of the rectum and sigmoid colon.

Increased levels of peptidases are found in some human carcinomas and may be related to invasive potential. We therefore measured the activity of four peptidases in 50 specimens of tumour and normal colonic wall from patients with a rectal or sigmoid carcinoma, and correlated this with the stage, differentiation, fixity of the tumour and presence of venous invasion, determined histologically. Since acute phase reactant proteins (APRP) may inhibit these proteolytic enzymes we have also measured serum levels of two relevant APRPs, alpha 1 acid glycoprotein (AGP) and C-reactive protein (CRP) pre-operatively. Activity of cathepsin B, cathepsin H and collagenase-like peptidase (CLP) was determined fluorimetrically and collagenase photometrically. Significantly elevated activity of cathepsin B, CLP and collagenase was found in tumour compared with normal colonic wall (median values: (nmol (mg protein)-1 min-1) Cat B 0.71 and 0.42 (P less than 0.001), CLP 25.24 and 12.25 (P less than 0.0001) and collagenase 0.49 and 0.31 (P less than 0.001). There was no correlation between the activity of these enzymes expressed as a ratio of tumour/colonic wall, and differentiation or Dukes' stage of the tumour. However, there was significant elevation of activity of cathepsin B in tumours with local spread (n = 13) compared with those with no spread (n = 37) (median values 2.76 and 1.36 respectively (P less than 0.001] and also in tumour with venous invasion (n = 24) compared with tumours without (n = 26) (median values 1.82 and 1.18 respectively (P less than 0.01]. Pre-operative serum levels of CRP were inversely correlated with the activity of CLP and cathepsin H and collagenase in the tumours (rs = 0.332, 0.359 (P less than 0.05) and 0.302 (P = 0.05) respectively). Thus certain peptidases are raised in rectal and sigmoid tumours. Activity of cathepsin B appears related to local tumour invasion. APRP may have a role in inhibiting the activity of these enzymes. These findings may have therapeutic implications.

[Early judgement on the results in radiation therapy of colorectal cancer: determination of carcinoembryonic antigen (CEA) in blood serum at daily intervals from the beginning of irradiation (author's transl)]. / Frühzeitige Erfolgsbeurteilung der Strahlentherapie von rektosigmoidalen Karzinomen. Die Bestimmung des Karzinoembryonalen Antigens (CEA) im Serum in Tagesabständen zu Beginn der Bestrahlung.

Carcinoembryonic antigen (CEA) and lactatedehydrogenase (LDH) as tumor cell markers have been determined in the blood serum of 10 patients with colorectal cancer, who underwent preoperative irradiation with 200 to 250 rad daily (total focal dose 2000 to 2500 rad). Daily analysis was made during the irradiation, and, moreover, a determination 2 weeks after the resection of the tumor. Simultaneously CEA and LDH were determined in 10 controls. Serum CEA and LDH levels were found significantly increased in tumor patients as compared to controls. The course was characterized by a peak of LDH on days 4 and 7 from the onset of radiation treatment, while serum CEA did not show a peak in the total of the tumor patients; but in 3 individual patients a peak was detected. In 5 patients, with low CEA values from the beginning, no significant modifications of CEA did appear. Thus, some of the patients undergoing irradiation revealed CEA kinetics conform to theoretical considerations, suggesting a response to irradiation of the colorectal carcinoma. The prognostic value of the early CEA kinetics during radiation treatment should be examined in patients being irradiated for inoperable local recurrence.

Histochemical demonstration of alkaline phosphatase in human large intestine, normal and diseased.

Alkaline phosphatase activity has been investigated by histochemical methods in normal and diseased human large intestine. The tissues were constantly maintained at 4 degrees C or below. Specimens were either frozen in liquid nitrogen, freeze-dried and embedded in glycol methacrylate for sectioning at 2 mu, or, fixed in ice-cold formol-calcium for frozen sectioning at 10 mu. The simultaneous coupling azo dye method using the substrates sodium alpha-naphthyl phosphate and Naphthol AS-BI phosphate, resulted in the demonstration of alkaline phosphatase activity in the surface epithelial cells, and the middle and upper crypts, of normal and transitional mucosa.