Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15–80 of cases and red CK18 staining in almost 100 of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (106 bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1–280 in 106; double staining 808, range 0–253) demonstrated relative reproducibility of APAAP results by double staining of 97. Correlation of results between both methods was significant (p<0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5 uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients

Prognostic relevance of MMP-2 (72-kD collagenase IV) in gastric cancer

The association of MMP-2 (matrix metalloproteinase 2, 72-kD collagenase IV) with invasive and metastatic capacity of tumor cells has implicated a potential role in the prognosis for cancer patients. However, no larger study has been done to prove this hypothesis. The present study was therefore designed to investigate the prognostic impact of MMP-2 in a prospective series of 203 gastric cancer patients. MMP-2 expression was measured immunohistochemically and scored semiquantitatively (score 0-3) in carcinoma cells, and results were correlated with clinicopathological tumor parameters and parameters of the urokinase-type plasminogen activator (uPA) system. Survival analyses were done using the Kaplan-Meier method (log-rank statistics) and multivariate Cox analysis. Significant correlations were found for MMP-2 and Lauren's classification, M stage and proteases/inhibitors of the uPA system in the primary tumor. Kaplan-Meier analysis revealed an association of increasing MMP-2 expression with worse prognosis. This was especially seen in patients with a parallel high expression of uPA receptor. However, differences in survival probabilities between low and high MMP-2 levels were not significant. In a separate analysis of diffuse-type cancers, MMP-2 was significantly associated with disease-free (p = 0.0056) and overall survival (p = 0.0426). Multivariately, MMP-2 was not an independent parameter. Our results demonstrate that there is an association of immunohistochemical detection of MMP-2 with prognosis of cancer patients. For diffuse gastric cancers, it is a significant prognostic parameter, however, not of independent impact. The study further suggests that consideration of interrelated tumor-associated proteases like uPA receptor in combination with MMP-2 may improve its prognostic power

Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

BACKGROUND: Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. METHODS: Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. RESULTS: Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. CONCLUSIONS: Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors

Tackling tumour cell heterogeneity at the super-resolution level in human colorectal cancer tissue

Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT

<p>Abstract</p> <p>Background</p> <p>T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-γ-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level.</p> <p>Results</p> <p>The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-γ producing T-cells were also producing MIP-1β whereas T-cells characterized by the sole production of IFN-γ were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-γ+ MIP-1β+ T-cells was equivalent to the measurement of the total IFN-γ+ T-cells, we adopted the IFN-γ+ MIP-1β+ data analysis system to evaluate IFN-γ-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-γ+ MIP-1β+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay.</p> <p>Conclusion</p> <p>The IFN-γ+ MIP-1β+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.</p

Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells

Abstract Although methylglyoxal (MGO) has emerged as key mediator of diabetic microvascular complications, the influence of MGO on the vascular transcriptome has not thoroughly been assessed. Since diabetes is associated with low grade inflammation causing sustained nuclear factor-kappa B (NF-κB) activation, the current study addressed 1) to what extent MGO changes the transcriptome of human umbilical vein endothelial cells (HUVECs) exposed to an inflammatory milieu, 2) what are the dominant pathways by which these changes occur and 3) to what extent is this affected by carnosine, a putative scavenger of MGO. Microarray analysis revealed that exposure of HUVECs to high MGO concentrations significantly changes gene expression, characterized by prominent down-regulation of cell cycle associated genes and up-regulation of heme oxygenase-1 (HO-1). KEGG-based pathway analysis identified six significantly enriched pathways of which the p53 pathway was the most affected. No significant enrichment of inflammatory pathways was found, yet, MGO did inhibit VCAM-1 expression in Western blot analysis. Carnosine significantly counteracted MGO-mediated changes in a subset of differentially expressed genes. Collectively, our results suggest that MGO initiates distinct transcriptional changes in cell cycle/apoptosis genes, which may explain MGO toxicity at high concentrations. MGO did not augment TNF-α induced inflammation

CD24 Is Not Required for Tumor Initiation and Growth in Murine Breast and Prostate Cancer Models

CD24 is a small, heavily glycosylated, GPI-linked membrane protein, whose expression has been associated with the tumorigenesis and progression of several types of cancer. Here, we studied the expression of CD24 in tumors of MMTV-PyMT, Apc1572/T+ and TRAMP genetic mouse models that spontaneously develop mammary or prostate carcinoma, respectively. We found that CD24 is expressed during tumor development in all three models. In MMTV-PyMT and Apc1572T/+ breast tumors, CD24 was strongly but heterogeneously expressed during early tumorigenesis, but decreased in more advanced stages, and accordingly was increased in poorly differentiated lesions compared with well differentiated lesions. In prostate tumors developing in TRAMP mice, CD24 expression was strong within hyperplastic lesions in comparison with non-hyperplastic regions, and heterogeneous CD24 expression was maintained in advanced prostate carcinomas. To investigate whether CD24 plays a functional role in tumorigenesis in these models, we crossed CD24 deficient mice with MMTV-PyMT, Apc1572T/+ and TRAMP mice, and assessed the influence of CD24 deficiency on tumor onset and tumor burden. We found that mice negative or positive for CD24 did not significantly differ in terms of tumor initiation and burden in the genetic tumor models tested, with the exception of Apc1572T/+ mice, in which lack of CD24 reduced the mammary tumor burden slightly but significantly. Together, our data suggest that while CD24 is distinctively expressed during the early development of murine mammary and prostate tumors, it is not essential for the formation of tumors developing in MMTV-PyMT, Apc1572T/+ and TRAMP mice

Molecular targeted therapies for breast cancer treatment

Targeting the oestrogen receptor, HER2 (human epidermal growth factor receptor 2) and vascular endothelial growth factor has markedly improved breast cancer therapy. New targeted therapeutic approaches to induction of apoptosis or inhibition of anti-apoptosis, cell cycle progression, signal transduction and angiogenesis are described. The molecular pathways and their inhibitory or repair mechanisms are discussed in the preclinical and clinical settings

A urokinase receptor promoter element (-152/-135) bound with an AP-2-alpha-related protein and SP1 is required for the induction of gene expression by PMA and SRC

The urokinase-type plasminogen activator receptor (u-PAR) promotes extracellular matrix degradation, invasion and metastasis. A first objective of this dissertation was to identify cis-elements and trans-acting factors activating u-PAR gene expression through a previously footprinted (–148/–124) promoter region. Mobility shifting experiments on nuclear extracts of a high u-PAR-expressing colon cancer cell line (RKO) indicated Sp1, Sp3 and a factor similar to, but distinct from, AP-2α bound to an oligonucleotide spanning –152/–135. Mutations preventing the binding of the AP-2α-related factor reduced u-PAR promoter activity. In RKO, the expression of a dominant negative AP-2 (AP-2αB) diminished u-PAR promoter activity, protein and u-PAR mediated laminin degradation. Conversely, u-PAR promoter activity in low u-PAR-expressing GEO cells was increased by AP-2αA expression. PMA treatment, which induces u-PAR expression, caused an increased amount of the AP-2α-related factor-containing complex in GEO, and mutations preventing AP-2α-like and Sp1/Sp3 binding reduced the u-PAR promoter stimulation by PMA. In resected colon cancers, u-PAR protein amounts were related to the amount of the AP-2α-related factor-containing complex. In conclusion, constitutive and PMA- inducible u-PAR gene expression and -proteolysis are mediated partly through transactivation via a promoter sequence (–152/435) bound with an AP-2α-related factor and Sp1/Sp3. A second interest of this dissertation was to determine if a constitutively active Src regulates the transcription of the u-PAR gene, since c-src expression increases invasion in colon cancer. Increased u-PAR protein and laminin degradation paralleling elevated Src activity was evident in SW480 colon cancer cells stably expressing a constitutively active Src (Y- c-src527F). Nuclear run-on experiments indicated that this was due largely to transcriptional activation. While transient transfection of SW480 cells with Y-c-src527F induced a u-PAR-CAT-reporter, mutations preventing Sp1-binding to promoter region –152/435 abolished this induction. Mobility shift assays revealed increased Sp1 binding to region –152/135 with nuclear extracts of Src-transfected SW480 cells. Finally, the amounts of endogenous u-PAR in resected colon cancers significantly correlated with Src-activity. These data suggest that u-PAR gene expression and proteolysis are regulated by Src, this requiring the promoter region (–152/–135) bound with Sp1, thus, demonstrating for the first time that transcription factor Sp1 is a downstream effector of Src

An Editorial View on the Special Issue &ldquo;Colorectal Cancers: From Present Problems to Future Solutions&rdquo;

Colorectal cancer (CRC) represents one of the most frequent human cancer entities and is still amongst the &ldquo;top killers&rdquo; in human cancer, although fundamental progress has been made in recent years in CRC prevention, early diagnosis, basic and translational research, and (targeted) therapy [...