Integrin αvß6 sets the stage for colorectal cancer metastasis.

The ß6 subunit of the αvß6 integrin heterodimer has long been an enigma in cancer biology though recent research has provided many new insights into its biology. Collectively, these findings include discovery of the transcriptional, translational and cell biological mechanisms by which ß6 acts, the identification of the cellular influences ß6 exerts upon the cell proteome, the characterisation of multiple ß6-centric pro-metastatic signalling systems and the search for pharmacological therapies (industry and academia) targeted against ß6. Once expressional restriction is overcome in early colorectal cancer (CRC), epithelial cell surface restricted αvß6 can physically interact with, and activate, known oncoproteins, and has the potential to enable the cross-talk through non-canonical signal transduction pathways, resulting in the adoption of an invasive/metastatic phenotype. This recent research has identified numerous interconnections and potential feedback loops, highlighting the fact that the expression of the ß6 subunit may initiate a cascade of downstream effects on the CRC cell rather than acting through a single mechanism. We here review these recent studies and postulate that the existence of a cell surface uPAR/αvß6/TGFß "metastasome" interactome in/on a proportion of colorectal cancer cells, where ß6 expression sequesters and activates multiple systems at the invasive front of tumour lesions, promoting cancer metastasis and hence explaining why ß6 has been correlated with reduced patient survival in CRC.

Fetal growth restriction promotes physical inactivity and obesity in female mice.

BACKGROUND: Environmental exposures during critical periods of prenatal and early postnatal life affect the development of mammalian body weight regulatory mechanisms, influencing lifelong risk of obesity. The specific biological processes that mediate the persistence of such effects, however, remain poorly understood. OBJECTIVE: The objectives of this study were to determine the developmental timing and physiological basis of the obesity-promoting effect previously reported in offspring of obese agouti viable yellow (A(vy)/a) mothers. DESIGN: Newborn offspring of obese A(vy)/a and lean (a/a) mothers were cross-fostered shortly after birth to study separately the effects of in utero or suckling period exposure to A(vy)/a dams. Body composition, food intake, physical activity and energy expenditure were measured in offspring shortly after weaning and in adulthood. RESULTS: Offspring of obese A(vy)/a dams paradoxically experienced fetal growth restriction, which was followed by adult-onset obesity specifically in females. Our main analyses focused on wild-type (a/a) offspring, because a subset of adult A(vy)/a offspring contracted a kidney disease resembling diabetic nephropathy. Detailed physiological characterization demonstrated that, both shortly after weaning and in adulthood, female wild-type mice born to A(vy)/a mothers are not hyperphagic but have reduced physical activity and energy expenditure. No such coordinated changes were detected in male offspring. Mediational regression analysis of our longitudinal data supported a causal pathway in which fetal growth restriction persistently reduces physical activity, leading to adult obesity. CONCLUSIONS: Our data are consistent with several recent human epidemiological studies showing female-specific effects of perinatal nutritional restriction on later obesity, and provide the novel mechanistic insight that this may occur via permanent and sex-specific changes in one's inherent propensity for physical activity.

RNA metabolism in tracheal epithelium: alteration in hamsters deficient in vitamin A.

The electrophoretic pattern of RNA molecules that are synthesized in vitro in tracheal epithelium from hamsters deficient in vitamin A differs from that of RNA synthesized in normal, pair-fed control hamsters. There is less RNA of low electrophoretic mobility in the epithelial cells deficient in vitamin A. This alteration is reversed after the deficient animals have been treated with vitamin A.

Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease.

Evidence of in vivo oxidant-induced injury in inflammatory bowel disease (IBD) is largely indirect. Colon epithelial crypt cells (CEC) from paired specimens of histologically normal and inflamed bowel from IBD patients with active disease were examined for altered protein thiol redox status as an indicator of oxidative damage. When CEC preparations from 22 IBD patients were labeled with the reduced-thiol-specific probe [14C]-iodoacetamide (IAM), there was decreased labeling of a number of proteins indicating oxidation of thiol groups in CEC from inflamed mucosa compared to paired normal mucosa, especially the loss of thiol labeling of a 37-kD protein which was almost completely lost. The loss of reduced protein thiol status for the 37-kD band was paralleled by loss of epithelial cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) enzyme activity, an enzyme known to contain an essential reduced cysteine (Cys149) at the active site. The identity of the 37-kD protein as GADPH monomer was confirmed by NH2-terminal amino acid sequence analysis. To examine whether this type of in vivo injury could be attributed to biologically relevant oxidants produced by inflammatory cells, CEC prepared from normal mucosa were exposed to H2O2, OCl-, nitric oxide (NO), and a model chloramine molecule chloramine T (ChT) in vitro. Dose-dependent loss of IAM labeling and GAPDH enzyme activity was observed. The efficacy (IC50) against IAM labeling was OCl- >> ChT > H2O2 > NO (52 +/- 3, 250 +/- 17, 420 +/- 12, 779 +/- 120 microM oxidant) and OCl- >> ChT > NO > H2O2 (89 +/- 17, 256 +/- 11, 407 +/- 105, 457 +/- 75 microM oxidant), respectively, for GAPDH enzyme activity. This study provides direct evidence of in vivo oxidant injury in CEC from inflamed mucosa of IBD patients. Oxidation and inhibition of essential protein function by inflammatory cells is a potential mechanism of tissue injury that may contribute to the pathogenesis of the disease and supports the exploration of compounds with antioxidant activity as new therapies for IBD.

Mass Spectrometry-Based Analysis for the Discovery and Validation of Potential Colorectal Cancer Stool Biomarkers.

Colorectal cancer (CRC) is the third leading cause of cancer mortality for both men and women, and the second leading cause of cancer death for men and women combined. If detected early, before metastasis has occurred, survival following surgical resection of the tumor is >90%. Early detection is therefore critical for effective disease surveillance. Unfortunately, current biomarker assays lack the necessary sensitivity and specificity for reliable early disease detection. Development of new robust, non- or minimally invasive specific and sensitive biomarkers or panels with improved compliance and performance is therefore urgently required. The use of fecal samples offers several advantages over other clinical biospecimens (e.g., plasma or serum) as a source of CRC biomarkers, including: collection is noninvasive, the test can be performed at home, one is not sample limited, and the stool effectively samples the entire length of the inner bowel wall contents (including tumor) as it passes down the gastrointestinal tract. Recent advances in mass spectrometry now facilitate both the targeted discovery and validation of potential CRC biomarkers. We describe, herein, detailed protocols that can be used to mine deeply into the fecal proteome to reveal candidate proteins, identify proteotypic/unitypic peptides (i.e., peptides found in only a single known human protein that serve to identify that protein) suitable for sensitive and specific quantitative multiplexed analysis, and undertake high-throughput analysis of clinical samples. Finally, we discuss future directions that may further position this technology to support the current switch in translation research toward personalized medicine.

The effect of 1-beta-D-arabinofuranosylcytosine on cell viability, DNA synthesis, and chromatid breakage in synchronized hamster fibrosarcoma cells.

Hamster fibrosarcoma cells were synchronized by mitotic selection and exposed to varying concentrations of 1-beta-D-arabinofuranosylcytosine (ara-C) for 2 hr in mid-S phase. There was a direct relationship between DNA synthesis inhibition and cytotoxicity produced by ara-C once DNA synthesis was decreased by over 85%. The noncytotoxic concentration of 10(-5) M ara-C produced little chromatid breakage; but extensive chromatid breakage and chromosomal rearrangement were seen in cells treated with the cytotoxic concentration of 10(-3) M ara-C, thus supporting earlier observations that chromatid breakage is highly correlated with cytotoxicity. Predominantly small DNA was synthesized when cells were treated with both 10(-5) and 10(-3) M ara-C, and this DNA could be completely chased into high-molecular-weight DNA after addition of deoxycytidine. Both concentrations of ara-C also inhibited, to different degrees, the joining of intermediate-size DNA fragments into larger DNA; thus neither parameter appeared directly related to the ara-C-produced cytotoxicity.

Inhibition of cancer cell urokinase plasminogen activator by its specific inhibitor PAI-2 and subsequent effects on extracellular matrix degradation.

Isotopically labeled [( 3H]serine, [3H]proline, and [35S]sulfate) subendothelial cell basement membranes were used to determine the role of urokinase plasminogen activator (uPA) and its specific inhibitor plasminogen activator inhibitor 2 (PAI-2) in colon cancer cell extracellular matrix degradation. Recombinant PAI-2 irreversibly inhibited low and high molecular weight purified human uPA in addition to both colon cancer cell-associated and secreted uPA, particularly if pro-uPA had been preactivated. Two selected lines (COLO394 and LIM1215) preferentially degraded differently labeled matrices in a time- and plasminogen-dependent manner. This process was inhibitable by PAI-2 in the medium at levels which suggested that some degree of "shielding" of cell surface uPA from inhibitor occurred. The ability of PAI-2 to regulate the invasive phenotype of cells which express cell surface or receptor-bound uPA is discussed.

Cell cycle-specific oncogenic transformation of C3H/10T1/2 clone 8 mouse embryo cells by 1-beta-D-arabinofuranosylcytosine.

1-beta-Darabinofuranosylcytosine at concentrations ranging from 10(-3) to 10(-6) M induces oncogenic transformation in the C3H/10T1/2 clone 8 mouse embryo cell line. Cell lines derived from type III transformed foci grew in soft agarose and produced tumors in immunosuppressed syngeneic mice. With cells synchronized by postconfluent inhibition of growth or isoleucine deprivation, transformation was cell cycle dependent. Maximal transformation was seen in cells treated when in S phase, although some transformation was seen in cells treated in G1 phase of the cell cycle.

Oncogenic transformation of C3H/10T1/2 clone 8 mouse embryo cells by halogenated pyrimidine nucleosides.

Oncogenic transformation has been induced in vitro in the C3H/10T1/2 clone 8 line of mouse cells by exposure to 5-fluoro-2'-deoxyuridine (FUdR) or 5-fluorouracil. This transformation is both dose and time dependent and can be markedly decreased by simultaneous exposure of the cells to thymidine. The transformation induced by 5-fluorouracil is probably due to its intracellular conversion to FUdR or its monophosphate. Transformation by FUdR was found to be cell cycle dependent with maximum sensitivity to transformation occurring in early S phase. Cell lines that produced sarcomas in antithymocyte-treated syngeneic mice were isolated from FUdR-transformed cultures. Trifluorothymidine, 5-bromo-2'-deoxyuridine, and 5-iodo-2'-deoxyuridine induced no transformed foci in the C3H/10T1/2 clone 8 cell line. Thus, not all mutagens produce oncogenic transformation nor does the lack of mutagenicity, as classically measured, completely exclude the possibility that a given agent is oncogenic. Also, there was no evidence of the "switch on" of oncornaviral information in the FUdR-transformed cell lines.

Mutagenicity of cancer chemotherapeutic agents in the Salmonella/microsome test.

Seventeen cancer chemotherapeutic agents were tested for their ability to mutate Salmonella typhimurium tester strains in the Salmonella/microsome mutagenicity test. There was a high correlation between the mutagenicity and carcinogenicity of a given agent. Carcinogens positive in the test were Adriamycin, daunomycin, 1-propanol-3,3'-iminodimethanesulfonate, cyclophosphamide, isophosphamide, hycanthone, chlornaphazin, nitrogen mustard, uracil mustard, melphalan, and thio-tepa. Two carcinogesn, actinomycin D and bleomycin, were not detected as mutagens. The presumptive noncarcinogen, methotrexate, was negative in the test. Tilorone and 6-mercaptopurine, tentatively classified as noncarcinogens, were mutagenic. The carcinogenicity of cis-dichlorodiammineplatinum(II), which was positive in the test, has not been determined.

Occupancy of the cancer cell urokinase receptor (uPAR): effects of acid elution and exogenous uPA on cell surface urokinase (uPA).

The development of a simple, sensitive fluorimetric assay for the measurement of cell surface-associated urokinase plasminogen activator (uPA) on viable, adherent HCT116 cells in microtitre plates, after a preincubation with purified human plasminogen is described. The assay determines plasmin activity by the cleavage of H-D-Val-Leu-Lys 4-aminomethyl coumarin under near physiological pH and ionic conditions with a sensitivity in the range of 5-100 mIU uPA/well at excitation 355 nm and emission 460 nm. Plasmin generated during the assay converted all cell-surface sc-uPA to tc-uPA, allowing the determination of total uPA activity. Inhibitor studies (PAI-2, amiloride or Glu-Gly-Arg chloromethylketone) confirmed the specificity of the uPA assay. Removal of these agents prior to assay allowed determination of the cell surface sc-uPA:tc-uPA ratio. Cell surface activity was only partially removed by acid elution. This corresponded with the loss of a number of proteins and uPA-containing species as detected by SDS-PAGE, gelatin enzymography and Western blotting. Although the major protein species eluted had a M(r) of 55 kDa, reacted with a commercial anti-human uPA mAb and correlated with the main lytic zone, other higher M(r) species were also eluted from HCT116 cells. Exogenous uPA increased cell-surface activity markedly on cells previously treated with acid. Following acid elution, cell surface uPA activity was restored after 30h in culture suggesting either de novo synthesis or release of pre-formed uPA with subsequent secretion and binding to uPAR. The assay has enabled studies on adherent cells to address questions about the regulation and expression of cell-surface uPA.

The human ENO1 gene product (recombinant human alpha-enolase) displays characteristics required for a plasminogen binding protein.

Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an alpha-enolase-like molecule. The aims of this study were to determine whether recombinant alpha-enolase (r-alpha-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-alpha-enolase with a Kd 1.9 microM and approached saturation at 10 microM. Lysine-dependent plasminogen binding to r-alpha-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues epsilon-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-alpha-enolase with carboxy-peptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-alpha-enolase. Binding to r-alpha-enolase enhanced the activation rate of plasminogen by urokinase but prevented alpha 2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.

Granulocyte activation induced by intense interval running.

Activation of granulocytes has been associated with normal immune function, inflammation, and exercise-induced muscle damage. The effect of intense interval running on granulocyte activation was examined by use of flow cytometry, monoclonal antibodies, and spectrophotometric techniques. Eight trained males [maximal oxygen uptake VO2max, mean (SD) = 64.4 (3.6) ml/kg/min; age 30.1 (4.8) years] undertook an intense interval exercise (treadmill running) protocol to exhaustion. Subjects completed an average of 16.5 one-minute runs. Granulocyte expression of CR3 (CD11b), receptor for complement component C3bi (6 and 24 h post-test), and Fc gamma RIII (CD16) (24 h post-test) and the plasma concentration of elastase-inhibitor complex (1 h post-test) increased significantly (all P < .05). Subjects (8 of 8) exhibited a post-test decrease at either 1 or 6 h (P < .01) and a 24-h post-test significant increase (7 of 8; P < .05) in granulocyte 90 degrees light scattering (LS). Plasma lactoferrin (Lf) concentration, although increased by 17% at 6 h post-test, was not significantly different from resting values at any sampling point. Changes in plasma Lf and median channel 90 degrees LS were significantly correlated (r = -.43, P = .04), raising the possibility of monitoring exercise-induced granulocyte activation (degranulation) by flow cytometry. Intense interval exercise appears to induce granulocyte activation, as manifested by release of granule proteins and changes in 90 degrees LS and expression of both Fc and complement receptors.

Is isolation of comprehensive human plasma peptidomes an achievable quest?

The low molecular weight (LMW; <10kDa)* plasma peptidome has been considered a source of useful diagnostic biomarkers and potentially therapeutic molecules, as it contains many cytokines, peptide hormones, endogenous peptide products and potentially bioactive fragments derived from the parent proteome. The small size of the peptides allows them almost unrestricted vascular and interstitial access, and hence distribution across blood-brain barriers, tumour and other vascular permeability barriers. Therefore, the peptidome may carry specific signatures or fingerprints of an individual's health, wellbeing or disease status. This occurs primarily because of the advantage the peptidome has in being readily accessible in human blood and/or other biofluids. However, the co-expression of highly abundant proteins (>10kDa) and other factors present inherently in human plasma make direct analysis of the blood peptidome one of the most challenging tasks faced in contemporary analytical biochemistry. A comprehensive compendium of extraction and fractionation tools has been collected concerning the isolation and micromanipulation of peptides. However, the search for a reliable, accurate and reproducible single or combinatorial separation process for capturing and analysing the plasma peptidome remains a challenge. This review outlines current techniques used for the separation and detection of plasma peptides and suggests potential avenues for future investigation. This article is part of a Special Issue entitled: HUPO 2014.

Serpins in the human hair follicle.

Proteinases and their inhibitors are very likely to function as mediators or regulators of the hair growth cycle. Very little information is currently available, however, regarding the specific inhibitors present in human hair follicles at defined stages of their growth cycle. In this study we have analyzed two proteinase inhibitors, plasminogen activator inhibitor type 2 and protease nexin 1, in human hair follicles using in situ hybridization and/or immunohistochemistry. Protease nexin 1 mRNA was found only in the mesenchymal population of the hair follicle, i.e., the follicular papilla cells, during the anagen but not the catagen phase. In contrast, plasminogen activator inhibitor type 2 was localized to several epithelial populations in the follicle: the more differentiated cells of the infundibulum; the companion layer in anagen follicles; and the single layer of outer root sheath cells directly abutting the club hair in telogen follicles. At least some of the plasminogen activator inhibitor type 2 in human follicles appears to be in the relaxed form, as evidenced by strong staining with an antibody that is specific for this form of the inhibitor. This suggests that plasminogen activator inhibitor type 2 interacts with and is cleaved by an endogenous follicular proteinase and supports a constitutive role for this inhibitor in human follicular epithelia.

Localization of plasminogen activator inhibitor type 2 (PAI-2) in hair and nail: implications for terminal differentiation.

Plasminogen activator inhibitor type 2 (PAI-2) is an unusual serine proteinase inhibitor in that it is largely retained within the cell and is found in high concentrations in the upper viable layers of human epidermis. Studies using transfected cell lines that express high levels of PAI-2 have suggested that this inhibitor may confer protection against programmed cell death. To test the hypothesis that PAI-2 may protect epithelial cells in vivo from premature programmed cell death, we determined expression patterns of PAI-2 in murine hair and nail. These epidermal derivatives are comprised of numerous epithelial cell types with distinct differentiation pathways. Furthermore, the cyclic nature of the follicular epithelium makes it ideal for studying sequential stages of cell differentiation and death. PAI-2 mRNA and protein were detected in the differentiating cells of the outer root sheath and medulla of the follicle during the anagen phase of the hair growth cycle. PAI-2 was also detected in the permanent portion of the catagen follicle. In the telogen phase of the hair growth cycle, PAI-2 was limited to the postmitotic cells of the outer root sheath directly abutting the club hair. In the nail, PAI-2 was detected in the differentiating cells of the matrix and nail bed. This consistent, selective distribution of PAI-2 in the postmitotic, maturing cells prior to terminal keratinization and death suggests that (i) PAI-2 may be considered as a differentiation marker for many epithelial cell types, and (ii) PAI-2 is appropriately positioned to protect epithelial cells from premature demise.

The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate.

The myeloperoxidase-derived oxidant, hypochlorite (OCl-) was shown to be able to degrade proteoglycan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl- concentrations less than 10(-4) M resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [35S]-labeled core proteins by SDS/polyacrylamide electrophoresis and fluorography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl- below 10(-3) M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (greater than 10(-3)) of OCl- caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl- cleaves glycosaminoglycan (GAG) chains.

Evaluation of a test for abnormal rectal mucus for early detection of colon cancer.

This report describes the evaluation of a chemical test for T-antigen in rectal mucus as a screening test for colon cancer. The test, called the Mucus Strip Test, detects the disaccharide residue sialic acid-free beta-D-Gal(1-->3)-D-GalNAc or T-antigen, which accumulates in mucus from malignant cells and colonic mucosa adjacent to cancer but not in normal mucosa. Participants were an unselected case series of 660 persons undergoing colonoscopy, excluding those with ulcerative colitis, polyposis, Crohn's disease, or nonspecific inflammatory bowel disease. In the first study (n = 608) rectal mucus was collected after preparation of the bowel for colonoscopy; in the second study (n = 52) a modified protocol was used to collect mucus approximately 2 weeks before colonoscopy and again following preparation for the procedure. Mucus Strip Test results were compared to the diagnosis received after colonoscopy, which was classified as cancer, adenomatous polyp(s), and others (normal). Analyses were also stratified by previous history of large intestinal disease, classified as previous cancer; previous diagnosis of adenomatous polyp(s); or others. In the first study, T-antigen was detected in approximately 30% of mucus samples, and test results were independent of both diagnosis at colonoscopy and previous medical history. In the second study, T-antigen was detected in 85% of samples collected before and 96% of samples collected after preparation for colonoscopy, but test results were again independent of diagnosis and medical history.(ABSTRACT TRUNCATED AT 250 WORDS)