Genetic polymorphism in UDP-glucuronosyltransferase 2B7 and colorectal cancer risk.

Colorectal cancer (CRC) is one of the most common malignancies in the Western world. CRC is strongly associated with lifestyle factors. Susceptibility to CRC may be partly due to deficient detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes result in variations in detoxification activities, which might influence the levels of carcinogens in the gastrointestinal tract, influencing the risk for CRC. To determine whether a genetic polymorphism in the detoxification enzyme UDP-glucuronosyltransferase 2B7 (UGT2B7) predisposes to CRC, 411 Caucasian patients with sporadic CRC and 600 Caucasian controls recruited from the same geographic area were genotyped for the functional UGT2B7 H268Y polymorphism. DNA was isolated and tested by a dual-color real-time polymerase chain reaction assay. Overall, no differences in genotype distributions between patients with CRC and controls were observed. When analyzed with respect to tumor location, a shift from the UGT2B7*I *2 into the UGT2B7*2*2 genotype was seen in patients with proximal CRC (OR 1.80, 95% CI 1.11-2.89). In the male patient subpopulation an even stronger association was observed (*1*1 + *1*2 vs. *2*2: OR 2.17, 95% CI 1.11-4.04; *1*2 vs. *2*2: OR 2.19, 95% CI 1.10-4.37). No associations with respect to tumor stage were seen. In conclusion, the frequency of the UGT2B7*2*2 genotype is higher in CRC patients with proximal location of the tumor, especially in males, which suggests that this genotype is associated with an increased risk for proximal CRC.

Hypercholesterolaemia and hepatosplenomegaly: two manifestations of cholesteryl ester storage disease.

Cholesteryl ester storage disease (CESD) is a rare autosomal recessive disease caused by mutations in LIPA. Here we describe two different clinical presentations of this disease: one case with a clear phenotype of familial hypercholesterolaemia and one case with hepatosplenomegaly from childhood onwards. These two cases exemplify the diversity of clinical phenotypes of patients with CESD. Knowledge on the phenotypic variability of the disease is of clinical relevance in light of enzyme replacement therapy (sebelipase alpha) for patients with mutations in LIPA, which is currently under development.

Altered insulin-like growth factor-II (IGF-II) level and IGF-binding protein-3 (IGFBP-3) protease activity in interstitial fluid taken from the skin lesion of psoriasis.

In the present study, we have investigated insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in serum and artificially raised blister fluid from uninvolved and involved areas of nine patients with psoriasis. Both levels of IGFs and IGFBP-3, and profiles of IGFBP in serum and fluid from the uninvolved areas of these patients were comparable to those seen in normal subjects. In fluid from the involved areas, the IGF-II but not IGF-I level was significantly elevated. Among five molecular forms of IGFBP, the density of 41.5- and 38.5-kDa forms of IGFBP-3 were apparently increased in fluid from the involved areas, shown by Western ligand blotting. Radioimmunoassay further showed that the IGFBP-3 concentration in the involved areas was significantly raised. Immunoblotting revealed that the predominant form of IGFBP-3 in fluid from the uninvolved areas was a 29-kDa proteolytically modified product. In contrast, intact doublet IGFBP-3 was the main form of IGFBP-3 in fluid from the involved areas. Fluid from the involved areas but not the matched serum concentration-dependently inhibited the degradation of 125I-labeled nonglycosylated IGFBP-3 (ngIGFBP-3) caused by fluid from the uninvolved areas, suggesting the presence of an IGFBP-3 protease inhibitor(s) in psoriatic skin lesion. These findings suggest that the alterations in IGF/IGFBP system may contribute to the pathogenesis of psoriasis.

Effects of treatment with megestrol acetate, aminoglutethimide, or formestane on insulin-like growth factor (IGF) I and II, IGF-binding proteins (IGFBPs), and IGFBP-3 protease status in patients with advanced breast cancer.

The effects of treatment with the aromatase inhibitors aminoglutethimide (AG) and formestane or the synthetic progestin megestrol acetate (MA) on plasma levels of insulin-like growth factor I (IGF-1), IGF-II, IGF-binding proteins (IGFBPs), and IGFBP-3 protease status were investigated in 39 patients suffering from advanced breast cancer. Treatment with AG and MA elevated plasma levels of IGF-I by mean values of 27% (n = 15; P < 0.025) and 81% (n = 7; P < 0.025), respectively, whereas treatment with formestane had no effect (n = 13). Treatment with AG increased plasma levels of IGFBP-2, as evaluated by Western blotting (P < 0.01). MA caused a significant reduction in IGFBP-3 protease activity (mean reduction, 69%; P < 0.05). These alterations in plasma IGF-I and IGFBP-3 protease activity were reversed 4 weeks after terminating MA therapy (n = 8; P < 0.025). Taken together, 13 of 15 patients had reduced IGFBP-3 protease activity during treatment with MA compared to the control situation (P < 0.0025). Total levels of IGFBP-3 as measured by RIA were moderately elevated by treatment with MA (mean increase, 19%; P < 0.05), and Western immunoblotting revealed an increase in the amount of intact IGFBP-3 and reduced amounts of IGFBP-3 in the modified form. None of the treatment modalities had any influence on plasma levels of IGF-II. The increase in the plasma IGF-I concentration seen during treatment with MA may be secondary to an increased level of intact IGFBP-3. This could reflect an alteration in IGF availability that contributes to the antitumor effect of MA.

Insulin-like growth factors (IGFs) and IGF-binding proteins in human skin interstitial fluid.

Despite extensive investigation of the insulin-like growth factor (IGF)/IGF-binding protein (IGFBP) system in the circulation and body fluids, there is no information on this in interstitial fluid. We have compared the IGF/IGFBP system in the circulation with that in fluid obtained from blisters artificially raised by negative pressure in 10 healthy volunteers. IGFBP-1, -2, -3, and -4 were all found in blister fluid, but in concentrations much lower than those in matched serum. The IGF-I, IGF-II, and IGFBP-3 levels measured by RIA were 18%, 14%, and 16% of those in serum, respectively. Fast protein liquid chromoatography showed that both IGF-I and IGFBP-3 in 150- and 50-kilodalton complexes were approximately 13% and 37%, respectively, of the corresponding peaks found in matched serum. Compared to that in serum, the IGFBP-3 in the blister fluid was predominantly in a modified 29-kilodalton form, and there was increased activity of an IGFBP-3 protease. Therefore, although IGF concentrations are much lower in interstitial fluid than in the circulation, a greater proportion of this IGF is in forms more readily available for interaction with tissue receptors. The blister fluid appears to represent physiological interstitial fluid and may provide a model for studying the physiology and pathophysiology of growth factors in the interstitial environment.

A study on post-synthetic modifications in alfa-alfa enolase (EC 4.2.1.11) brought about by a human serum protein.

Human alfa-alfa enolase is modified in the blood circulation by a serum protein for which the name 'modifying protein' is proposed. The protein occurs in every human serum tested and appears to be the same protein that is responsible for the post-synthetic modification in the M subunit of creatine kinase. Three alfa-alfa enolase forms, the original one plus two modified forms can be found in serum both in vivo and after in vitro incubation. The original alfa-alfa enolase is modified completely within a few hours in vitro in a pH-controlled human serum matrix at 37 degrees C. As the modification also takes place in vivo it is theoretically possible to acquire information about the activity of a disease process by doing one single determination of the amount of circulating alfa-alfa 3 enolase. A mechanism is proposed for the modification, where at first one of the two alfa chains is modified resulting in the alfa-alfa 2 form. Ultimately the second alfa chain is also modified. The alfa-alfa 1 form seems to be the completely modified alfa-alfa form. The three enolase forms differ in their isoelectric points but have similar Michaelis-Menten constants.

Correlation between collagenolytic activity and grade of histological differentiation in colorectal tumors.

Collagenolytic activity, extracted from 55 tumor and healthy corresponding intestinal control samples, was determined by 3 different assays using soluble type I and fibrillar type I and III collagen, respectively, as substrate. The enzyme extracted from tumor-digested collagen type I reconstituted fibrils and yielded the three-quarter segments characteristic for the action of one of the matrix metalloproteinases: MMP-I or mammalian collagenase. Metal-chelating agents such as EDTA and O-phenanthrolin indeed inhibited this activity. Collagenolytic activities were calculated on the basis of wet weight, total DNA and total extracted protein. Correlations were sought between levels of activity and both clinicopathological stage (Dukes' staging) and grade of histological differentiation. In all the assays applied, significant correlations were found between grade of histological differentiation and collagenolytic activity expressed as the tumor/control ratios: poorly differentiated tumors exhibited a higher tumor/control ratio than well-differentiated tumors. Also, tumors penetrating into the serosa showed a higher tumor/control ratio than tumors invading the muscularis propria only. A relation between collagenolytic activity and clinico-pathological stage was observed only if activities were calculated on a DNA basis. These results confirm a relationship between the histological appearance of a tumor and its enzymatic potential to degrade interstitial collagens.

Collagenolytic activity extracted from intestinal anastomoses of the rat.

The post-operative degradation of collagen has been postulated to play an important role in the development of anastomotic leakage in the intestine. However, collagenolytic activity in intestinal anastomoses has hardly been studied so far. We have measured collagenolytic activity, after extraction in an urea-containing medium, in both ileal and colonic anastomoses in the rat, from 12 hours to 31 days after operation. In ileum collagenolytic activity increased significantly, from 2 to 4 (average 2.7) times the control value, at 12 hours post-operatively followed by a steady decline to original levels. Four weeks after surgery the activity was still slightly, but significantly, enhanced. In colon collagenolytic activity also increased up to 4 times the pre-operative level (average 3.0) 12 hours after operation. Return to original levels was delayed in colon compared to ileum but here activities were similar to control values after one month. In both parts of the intestine there was only a small increase in activity at a segment proximal to the anastomosis during the first 24 hours after operation. The amount of protein extracted did not vary significantly between control and anastomotic samples. These data are the first to show a transiently increased extractable collagenolytic activity in intestinal anastomoses.

Activation of cathepsin B, secreted by a colorectal cancer cell line requires low pH and is mediated by cathepsin D.

The aim of our study was to identify changes in secreted procathepsin B levels in a model of the human colorectal adenoma to carcinoma sequence and to determine the factors required for its extracellular activation. Conversion of the non-tumorigenic adenoma-derived cell line PC/AA to a highly tumorigenic phenotype (designated AA/CI/SB10/M) was associated with an 8-fold increase in the presence of the proform of cathepsin B in 24 hr conditioned serum-free medium (SFM). In addition, mature enzyme was only detected in the cell lines of this model with increased malignant potential. This is in agreement with the findings of a previous study, in which mature cathepsin B was only present in the 24 hr conditioned SFM of cancer-derived cell lines and not in SFM from adenoma-derived cell lines. Having demonstrated a reduction in the pH of conditioned medium from cell lines with increased malignant potential, we used a range of specific proteinase inhibitors to show that an aspartyl proteinase was involved in the initial activation of procathepsin B. Consistent with this finding, we subsequently demonstrated an increased secretion of the aspartyl proteinase cathepsin D in the medium of the AA/CI/SB10/M adenocarcinoma cells compared with the non-tumorigenic AA/Cl cell line. Therefore, the presence of mature cathpsin B in the conditioned medium of the more malignant cell lines coincided with a reduction in pH and an increase in the amount of cathepsin D secreted. Data from the human colorectal derived adenoma to carcinoma sequence indicate that an in vivo mechanism may exist that, dependent on the simultaneous presence of both a tumour-generated acidic extracellular environment and an elevated secretion of procathepsin D, could result in the activation of latent procathepsin outside the cell.

Escape from negative regulation of growth by transforming growth factor beta and from the induction of apoptosis by the dietary agent sodium butyrate may be important in colorectal carcinogenesis.

There are a number of lines of evidence suggesting that transforming growth factor beta (TGF beta) has an important role in the control of intestinal growth and differentiation. In vivo localization studies show that TGF beta expression occurs predominantly in the differentiated non proliferating cells of the intestinal epithelium. The use of an antisense expression vector for TGF beta resulted in an increased tumorigenicity in an antisense-transfected cancer cell line. In vitro proliferation studies showed colorectal premalignant adenoma cells to be more sensitive to the growth inhibitory effects of TGF beta than colorectal cancer cells. Furthermore the conversion of an adenoma to a carcinoma was accompanied by a reduced response to the inhibitory effects of TGF beta. The acquisition of partial or complete resistance to the inhibitory effects of TGF beta may be an important late event in colorectal carcinogenesis. Of further interest is the possibility that clonal selection could occur even more rapidly in colorectal tumour cells which not only had lost response to TGF beta inhibition but produced TGF beta and were growth stimulated by it. This could have the advantage of not only inhibiting the growth of surrounding less malignantly advanced cells but of also escaping from their potential growth suppressive influence. Carcinogenesis is not, however, simply losing response to negative regulators of growth; the fully malignant cell has to acquire new characteristics of invasiveness and metastatic potential. Growth factors including TGF beta may have a role in the complex cascade of events leading to the activation of proteolytic enzymes which are involved in progression to an invasive phenotype. Cell proliferation in the large bowel, as well as being under the control of endogenous growth factors, is also under the influence of dietary components in the lumen such as the naturally occurring fatty acid sodium butyrate. Sodium butyrate at physiological concentrations induces apoptosis (programmed cell death) in colonic tumour cell lines. Since sodium butyrate occurs naturally in the colorectum, being produced by bacterial fermentation of dietary fibre, it may be involved in the control of cell death in human colorectal epithelium. This could, in part, explain the apparent protective effects of dietary fibre. Clonal evolution and tumour progression in colorectal carcinogenesis could therefore involve loss of response to endogenous growth factors such as TGF beta and an escape from the induction of programmed cell death by dietary factors.

Collagenases from human and rat skin fibroblasts purified on a zinc chelating column reveal marked differences in latency as a result of serum culture conditions.

1. Fibroblasts from both human and rat skin were grown in the presence or absence of serum and the collagenase activity in the medium was partially purified on zinc-Sepharose. 2. During chromatography, using a discontinuous elution gradient, the rat collagenase elutes at different pH and ionic strength than the human collagenase. Both latent and active collagenases of both species are retarded by the affinity matrix. 3. Latency of collagenase in media obtained from fibroblast cultures appears to be influenced by the presence of a serum component in the culture medium. 4. The results demonstrate that collagenases secreted by fibroblast cultures established from the same tissue but obtained from different species are biochemically diverse and that, within one species, the amount of active enzyme depends on the presence of a serum factor.

In vitro models of human colorectal cancer.

Epithelial cell lines that differentiate in vitro have been isolated from hereditary and sporadic colorectal adenomas representing different stages in tumour progression, from small adenomas with a low malignant potential to large adenomas with a relatively high malignant potential. The majority of cell cultures derived from small adenomas senesced, whereas the larger adenomas were more likely to give rise to an immortal cell line. Karyotypic analysis has shown that specific abnormalities of chromosomes 1, 6, 7, 13, 14, 17, 18 and 22 occur in these adenoma cell lines. Abnormalities of chromosome 1 have been implicated in tumour progression and the in vitro immortalization of colorectal adenomas. Molecular and cellular changes involving abnormalities of chromosomes 1 and 18, TP53 and ras gene mutations and reduced response to the growth inhibitory effects of TGFB and sodium butyrate, which occur during tumour progression, suggest that the in vitro model has relevance to in vivo carcinogenesis.

Biological consequences of the genetic changes which occur during human colorectal carcinogenesis.

Colorectal carcinogenesis is a complex multistage process and occurs through the accumulation of gene mutations in both oncogenes and tumour suppressor genes. Frequent genetic abnormalities include mutation of the familial adenomatous polyposis (APC) and/or the mutated in colorectal cancer (MCC) genes on chromosome 5q21, activation of K-ras and loss of the tumour suppressor genes p53 and DCC (deleted in colorectal cancer). In our laboratory we have developed human in vitro colonic cell culture model systems, to determine the biological consequences of these well characterised genetic changes, and how such changes can uncouple proliferation from differentiation and ultimately lead to the malignant phenotype.

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles.

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles. The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.

Collagenolytic activity in experimental intestinal anastomoses. Differences between small and large bowel and evidence for the presence of collagenase.

Collagen degradation is thought to be an integral part of the healing sequence of intestinal anastomoses, but almost nothing is known about the enzyme activities involved. We have studied collagenolytic activities, extracted from 1 day-old intestinal anastomoses in the rat. Using either soluble type I collagen or fibrillar type I or type III collagen as a substrate, activities measured in extracts from anastomotic segments were compared to those in extracts from uninjured intestine, removed at operation: in all cases, the collagenolytic activity in anastomotic extracts was significantly higher. This increase was significantly more pronounced in large bowel than in small bowel. The activities were strongly inhibited by serum and metallo-chelating compounds. Analysis, by means of SDS-polyacrylamide gel electrophoresis, of the reaction products of the degradation of fibrillar type I collagen by the extracts revealed the presence of a multitude of fragments, amongst them TcA fragments characteristic for the activity of mammalian collagenase. Thus, the degradative capacity towards various collagen substrates is enhanced in the anastomotic area during the first postoperative period and a true mammalian collagenase is one of the enzymes present.