Flexible sigmoidoscopy training and its impact on colorectal cancer screening by primary care physicians.

BACKGROUND: Colorectal cancer is an ideal disease for prevention with screening programs. Efforts to increase compliance with screening recommendations have included training primary care physicians to perform flexible sigmoidoscopy. OBJECTIVE: To assess the impact of flexible sigmoidoscopy training on compliance with current screening recommendations. METHODS: We performed a cross-sectional study of 232 patients cared for by physicians in a primary care network. MAIN OUTCOME MEASURES: Rates of screening for colorectal cancer and rates of undergoing flexible sigmoidoscopy were compared across patient groups according to the physician's training and whether the physician performs flexible sigmoidoscopy in his or her practice. RESULTS: Among 217 patients included in the analysis, 122 (56%) were cared for by physicians who were trained in flexible sigmoidoscopy, of whom 79 (36%) were cared for by physicians who perform flexible sigmoidoscopy in their practice. Patients cared for by physicians trained in flexible sigmoidoscopy were not significantly more likely to receive any colorectal cancer screening than were patients cared for by physicians not trained in flexible sigmoidoscopy (odds ratio, 1.16; 95% confidence interval, 0.67-2.01). However, patients cared for by physicians who perform flexible sigmoidoscopy in their practice were more likely to have undergone any colorectal cancer screening (odds ratio, 1.73; 95% confidence interval, 1.02-2.95) and flexible sigmoidoscopy (odds ratio, 2.69; 95% confidence interval, 1.14-6.36). CONCLUSION: Performance of flexible sigmoidoscopy by primary care physicians has the potential to increase the rate of colorectal cancer screening with flexible sigmoidoscopy.

Primary care physicians' decisions to perform flexible sigmoidoscopy.

OBJECTIVE: This study was designed to identify factors that influence primary care physicians' willingness to perform flexible sigmoidoscopy. MEASUREMENTS: Using a mailed questionnaire, we surveyed all 161 primary care physicians participating in a large health care system. We obtained information on training, current practice patterns, beliefs about screening for colorectal cancer, and the influence of various factors on their decision whether or not to perform flexible sigmoidoscopy in practice. MAIN RESULTS: Of the 131 physicians included in the analysis, 68 (52%) reported training in flexible sigmoidoscopy, of whom 36 (53%) were currently performing flexible sigmoidoscopy in practice. Time required to perform flexible sigmoidoscopy, availability of adequately trained staff, and availability of flexible sigmoidoscopy services provided by other clinicians were identified most often as reasons not to perform the procedure in practice. Male physicians were more likely than female physicians to report either performing flexible sigmoidoscopy or desiring to train to perform flexible sigmoidoscopy (odds ratio 2.61; 95% confidence interval 1.10, 6.23). This observed difference appears to be mediated through different weighting of decision criteria by male and female physicians. CONCLUSIONS: Approximately half of these primary care physicians trained in flexible sigmoidoscopy chose not to perform this procedure in practice. Self-perceived inefficiency in performing office-based flexible sigmoidoscopy deterred many of these physicians from providing this service for their patients.

Molecular pathogenesis of colorectal cancer.

The enormous progress made in the identification of genes that are involved in colon carcinogenesis has provided the foundation for further understanding the biology of both normal and cancer cells and for targeted therapeutic strategies. In one sense, the genes described in this review are only the building blocks of a larger puzzle that constitutes the integrated metabolic function of a cell. The current challenge is to understand the functional role of these genes in normal cellular physiology and make the connections between pathways that knit together integrated cellular homeostasis. A complete understanding of the regulatory pathways, and the synthesis and modifications of the proteins involved, will provide novel targets for therapeutic agents.

Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of the pancreatic membrane protein GP-2 into secretory granules in exocrine but not endocrine cells.

The pancreatic zymogen granule membrane protein GP-2 was introduced into cells of exocrine or endocrine origin by transfection of its cDNA in order to investigate the mechanisms by which proteins are specifically incorporated into the membranes of secretory granules. Permanent transformants expressing GP-2 were isolated from exocrine pancreatic-derived AR42J cells as well as AtT20 cells of anterior pituitary origin and insulinoma-derived Rin5F cells. In AR42J cells, GP-2 was localized by immunofluorescence and immunoelectron microscopy to the endogenous zymogen-like granules as well as to the plasma membrane. In experiments supporting the localization data, incubation of the AR42J transformants with the secretagogue cholecystokinin (CCK8) resulted in enhanced release of a shed form of GP-2 into the medium in parallel with amylase, suggesting that the two proteins were secreted from the same compartment. By contrast, when expressed in AtT20 cells, the protein was found by immunofluorescence microscopy on the plasma membrane as well as in intracellular vesicles that differed in size and location from the endogenous secretory vesicles. By electron microscopy, large (approximately 0.5 micron) multivesicular structures were observed. Single- and double-label immunoelectron microscopy demonstrated that these large organelles labeled with anti-GP-2 antibodies, whereas the smaller adrenocorticotropic hormone (ACTH)-containing secretory vesicles did not. In permanent transformants of Rin5F cells, GP-2 was also excluded from the insulin-containing granules and found in multivesicular bodies similar to those in the AtT20 cells and containing the endosomal/lysosomal marker endolyn-78. Despite the apparent accumulation of GP-2 in lysosome-like structures, it turned over slowly and did not undergo rapid endocytosis from the cell surface. We conclude that GP-2 is targeted to secretory granule membranes by cell type-specific mechanisms that likely involve its interaction with other membrane or content proteins expressed only in the exocrine cells.

Biosynthesis and oligosaccharide processing of human Tamm-Horsfall glycoprotein permanently expressed in HeLa cells.

Human Tamm-Horsfall glycoprotein (T-H) is produced by renal cells of ascending limb of loop of Henle and is largely excreted in urine. N-linked glycans account for close to 30% of the weight of T-H. We studied the biosynthesis of recombinant T-H permanently expressed in HeLa cells. The conversion from the precursor (84 kDa) to the mature form (97 kDa) mainly depends on the processing of glycans from the high-mannose to polyantennary type. The conversion from precursor to mature form is very slow and the glycan structure of precursor appears to be that of a glycoprotein not yet processed by Golgi alpha 1,2 mannosidase. Since T-H has a very high number of disulfide bridges (more than 50 cysteine residues/mol) one may infer that the rate limiting step for the precursor export out of ER is the formation of a correct set of disulfide bonds. Mature T-H isolated from HeLa cells retained one N-linked chain with the high-mannose structure similarly to urinary T-H. This result indicates that the occurrence of one unprocessed high-mannose chain in mature T-H is host-cell independent and very likely related to the T-H primary structure.

Isolation of the cDNA encoding glycoprotein-2 (GP-2), the major zymogen granule membrane protein. Homology to uromodulin/Tamm-Horsfall protein.

GP-2, a 78-kDa glycoprotein, is the major component of zymogen granule membranes of the exocrine pancreas. We report the isolation of the cDNA encoding for GP-2 from a rat pancreatic lambda gt-11 cDNA library. The cDNA is 1921 base pairs in length with an open reading frame encoding for a protein of 530 amino acids containing eight potential N-glycosylation sites. It encompasses the amino terminus of the protein including the signal sequence as evidenced by in vitro transcription/translation experiments conducted in the presence of dog pancreas rough microsomes in which the protein underwent apparent core glycosylation. A hydrophobic stretch of amino acids is present at the carboxyl terminus which is likely to serve as a signal for attachment of a glycosyl phosphatidylinositol (GPI) membrane anchor as has been described for GP-2. When the cDNA was introduced into HeLa cells by transfection, the expressed protein was located on the cell surface and could be released by incubation of the cells with phosphatidylinositol-specific phospholipase C, confirming that it is GPI-linked. Upon searching through the GenBank database, the GP-2 amino acid sequence was found to have a 53% identity and 85% similarity to human uromodulin/Tamm-Horsfall protein (THP) over a 450-amino acid stretch that encompassed all 28 cysteines after the signal sequence of GP-2. Uromodulin/Tamm-Horsfall protein, an 85-kDa glycoprotein synthesized by the kidney, shares several characteristics with GP-2 in addition to its sequence similarity. Both are attached to the membrane by a GPI anchor, but are also released from the apical surface of their respective cells and subsequently form large aggregates. Together they may define a new gene family.

Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein.

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.

The pancreatic membrane protein GP-2 localizes specifically to secretory granules and is shed into the pancreatic juice as a protein aggregate.

GP-2 is the major secretory granule membrane glycoprotein of the exocrine pancreas and appears in the pancreatic juice in a modified sedimentable form. We have localized GP-2 in the rat pancreas at the electron microscopic level using affinity-purified antibodies and found it to be concentrated in the zymogen granules and in the acinar lumen. Label was also present on the apical and basolateral plasma membranes but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties reduced substantially the staining of the basolateral surface. Approximately 45% of the GP-2 in the granules was not membrane-associated but appeared instead in the granule lumen. Parallel biochemical characterization of GP-2 in isolated secretory granules demonstrated that 60% fractionated with the membranes after granule lysis while 40% remained in the content fraction. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl-phosphatidylinositol (GPI), GP-2 in the content did not enter the detergent phase upon Triton X-114 extraction; nor was it sedimentable at 200,000g, as is characteristic of the form collected in the pancreatic juice. In addition, GP-2 in the pancreatic juice was recovered in the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. These results are consistent with a life cycle for the protein that begins with synthesis of a membrane-associated precursor that can be converted by lipolytic or proteolytic cleavage to a soluble form within the zymogen granule. Further modification to a sedimentable form may then occur in the pancreatic juice.

Uncoupling of the secretory pathways for IgA and secretory component by cholestasis.

Circulating polymeric immunoglobulin A (IgA) binds to secretory component (SC) on the surface of rat hepatocytes and is internalized and transported by vesicles to the canalicular membrane where the IgA-SC complex is secreted into bile. To further characterize this transport pathway, we examined the effects of bile flow reduction or transient bile duct obstruction on the secretion of IgA and SC into bile. In response to gradually increasing resistance to bile flow, the biliary concentration of IgA decreased as bile flow decreased, whereas total biliary protein concentration was little changed. After 2 h of bile duct clamping, the amount of IgA secreted into bile during the postclamp period was decreased to one-tenth of control values. Similarly, transport of biosynthetically labeled monoclonal IgA ([3H]MoIgA) during the postclamp period was reduced three-fold. In contrast to the impairment in IgA secretion, secretion of SC continued at nearly normal levels after resumption of bile flow. The reduced transport of IgA was not due to a failure of IgA to reach the hepatocyte, a functional alteration of the IgA, or a decrease in the number of hepatic IgA receptors. Our studies indicate that secretion of IgA is sensitive to bile flow and that the biliary secretory pathways for IgA and SC are dissociated after brief periods of cholestasis.

Posttranslational processing of secretory component in the rat jejunum by a brush border metalloprotease.

Secretory component (SC) is a glycoprotein that mediates the transcellular transport of polymeric immunoglobulins into external secretions. SC is synthesized and inserted into the plasma membrane of epithelial cells and hepatocytes as a transmembrane protein, where it serves as a receptor for polymeric immunoglobulins. SC is posttranslationally cleaved to a soluble protein before secretion into external fluids. In the rat jejunum, we observed that the molecular weights of both the major membrane and soluble forms of SC were 10,000-20,000 smaller than the comparable hepatic forms of the glycoprotein. We therefore set out to determine the reason for the differences in size of SC between these two tissues. The smaller size of jejunal SC was not due to the action of pancreatic proteases or differential glycosylation but was due to proteolysis by a jejunal brush border protease. The protease was characterized as a metalloprotease, with a pH optimum of approximately 5. It is present in jejunal, ileal, and renal tubular brush borders as an integral membrane constituent. When the protease was inhibited in vivo, conversion of jejunal secretory component to the smaller size was partially prevented. Thus, in the rat jejunum, SC undergoes two posttranslational proteolytic events: conversion of membrane secretory component to the soluble form and conversion of soluble SC to a smaller size by a previously undescribed brush border protease.