Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining two-dimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis.

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.

Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer.

PURPOSE: The goal of this study was to identify and validate novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm. EXPERIMENTAL DESIGN: Employing two-dimensional gel electrophoresis and mass spectrometry, we analyzed 16 matched colorectal cancer and adjacent normal tissue samples. Proteins found to be elevated in cancer tissue were further validated by generating antibodies which were used for immunoblotting of tissue samples and for the development of highly sensitive immunoassays for assessment of serum samples. RESULTS: In total, 735 different proteins were identified in colon tissue. Strong elevation in colorectal cancer for five proteins was confirmed by immunoblot analysis: transforming growth factor-beta induced protein ig-h3 (betaIG-H3), nicotinamide N-methyltransferase (NNMT), nucleoside diphosphate kinase A (nm23-H1), purine nucleoside phosphorylase (PNPH), and mannose-6-phosphate receptor binding protein 1 (M6P1). Elevated levels of NNMT, which is not predicted to be secreted but is known as a cytoplasmic protein, were found in serum from patients with colorectal cancer. Employing a receiver-operating characteristic curve based on the measurement of 109 patients with colorectal cancer and 317 healthy controls, we obtained an area under the curve of 0.84 for NNMT, which was superior to the established tumor marker carcinoembryogenic antigen with an area under the curve of 0.78. CONCLUSIONS: It is proposed that NNMT serum levels may have significance in the early detection and in the management of patients with colorectal cancer.

Expression of TGF-alpha in neuroendocrine tumours of the distal colon and rectum.

AIM: Transforming growth factor alpha (TGF-alpha) has been localized in neuroendocrine L-cells of the colon and rectum in previous studies. We examined whether neuroendocrine tumours with L-cell differentiation express TGF-alpha. EXPERIMENTAL DESIGN: Immunohistochemistry was performed for proglucagon- and pro-pancreatic polypeptide derivatives, as well as for TGF-alpha, and epidermal growth factor receptor (EGFR) using paraffin sections from 16 neuroendocrine tumours of the colon and rectum. Also, in situ hybridization for TGF-alpha and proglucagon was carried out. MAIN RESULTS: A strong expression of TGF-alpha at the protein level can be shown for neuroendocrine tumours of the hindgut. In one third of our cases we found a strong hybridization signal and in two thirds a moderate signal for TGF-alpha. The immunohistological phenotype concerning gut hormones is highly heterogeneous. Glucagon-like peptide 2 (GLP2) in our series was the most sensitive immunohistological hormone marker. MAJOR CONCLUSIONS: The immunophenotype of colorectal neuroendocrine tumours regarding hormone markers is heterogeneous. The expression of TGF-alpha corresponds to the immunohistological profile of normal L-cells. TGF-alpha, especially in the neuroendocrine L-cells, most probably acts as a multifunctional trophic factor responsible for cellular integrity and survival, and not as an oncogenic growth factor.

High resolution proton magnetic resonance spectroscopy of human cervical mucus.

High resolution 1H NMR spectroscopy is a powerful method for qualitative and quantitative analysis of the highly viscous human cervical mucus (CM). Up to 23 compounds could be identified in this study and can be observed in the 1H NMR spectra of native mucus without the need of any complicated preparative chemistry. Storage conditions could be excluded as a possible reason for variations observed between different samples. pH values decreased after freezing and storing at 253 K. For NMR studies, lyophilization proved to be most useful, allowing the determination of the water content, replacement of H2O by D2O, and most importantly, absolute quantification of low molecular mass compounds. In a small collective of women the concentrations of some of the small constituents of the CM are strongly correlated; an example is the mutual positive correlation of taurine, citrate and creatinine. In conclusion, high resolution 1H NMR spectroscopy is a valid method to investigate mucus composition and to determine absolute concentrations of low molecular mass compounds in CM.

Seminal antibodies to human 60kd heat shock protein (HSP 60) in male partners of subfertile couples.

BACKGROUND: Heat shock proteins (HSP) are essential mammalian and bacterial stress proteins. At the cellular level, they act as chaperones, have important regulatory functions, and are considered to be an essential factor for reproduction. Scarce information exists on the role of sensitization to HSP and the potential role in the aetiology of male infertility. METHODS: The potential association of immunoglobulin (Ig)A antibodies (Ab) to the human 60 kDa heat shock protein (HSP 60) with several parameters of subclinical male genital tract infection/inflammation and with semen quality and sperm fertilizing capacity was analysed in a prospective study. IgA Ab to human HSP 60 were determined in seminal plasma of 202 randomly chosen male partners of subfertile couples with a median duration of infertility of 4 years (range 1-15 years), who were asymptomatic for genital tract infection. After medical history and clinical examination, a comprehensive evaluation of semen quality, in aliquots of the same ejaculates used for HSP Ab determination, included: sperm analysis; local antisperm antibody (ASA) screening; standardized sperm-cervical mucus (CM) penetration testing; immunocytochemical round cell differentiation to determine seminal leukocyte counts; evaluation of complement fraction C(3) and of some pro-inflammatory cytokines; and microbial screening. Subsequent fertility was recorded after 6 months. RESULTS: The presence of HSP 60 IgA Ab in seminal fluid (total positive 6.9%) was significantly associated with leukocytospermia, the presence of C(3), and also with high interleukin (IL) levels in seminal plasma. HSP 60 Ab were not related to the bacterial colonization of ejaculates. There was no association of seminal IgA Ab to human HSP 60 with semen quality, determined with microscopical semen analysis, nor with local IgG- or IgA-class ASA. There was no relationship with sperm intrinsic motility and duration of motility in the sperm CM-penetration test, nor with sperm fertilizing capacity. CONCLUSIONS: The combined presence of IgA Ab to human 60 kDa HSP, leukocytes and other established infection/inflammation markers in semen might suggest a potential role of the immune response to heat shock proteins (HSP) in cases of silent male genital tract infection, but the results do not indicate a marked relationship of HSP 60 Ab in seminal fluid with standard parameters of semen quality.

Are zinc levels in seminal plasma associated with seminal leukocytes and other determinants of semen quality?

OBJECTIVE: To evaluate a potential association of zinc levels with seminal leukocytes, the outcome of semen cultures; and semen quality and sperm fertilizing capacity. DESIGN: Prospective study. SETTING: Outpatient infertility clinic of a university hospital. PATIENT(S): Two hundred fifty-six randomly chosen asymptomatic males from subfertile couples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Determination of zinc in seminal plasma by flame atomic absorption spectroscopy. In aliquots of the same ejaculates the following tests were performed: immunocytochemical round cell differentiation to determine leukocyte counts and ratios, microbial screening, and comprehensive evaluation of semen quality (sperm analysis, biochemical parameters, antisperm antibody testing, and in vitro examination of sperm ability to penetrate cervical mucus). The patients underwent medical history, clinical examination, and postcoital testing. Subsequent fertility was determined (controlled for female infertility factors). RESULT(S): The concentration of zinc in seminal plasma did not correlate in a statistically significant way with leukocytes in semen, nor was it associated with bacterial colonization. There was no statistically significant relationship of zinc in seminal plasma or serum with semen quality parameters nor with local antisperm antibody testing of the IgG or IgA class. Zinc levels did not influence sperm capacity to penetrate cervical mucus in vitro or in vivo, and did not affect subsequent fertility. CONCLUSION(S): The zinc level in seminal fluid and serum is not associated with silent male genital tract infection (indicated by seminal leukocytes); nor is it related to semen cultures in asymptomatic individuals. The lack of association with other semen quality parameters indicates that the routine determination of zinc levels during infertility investigation is not recommended.