Two micromethods for measuring proteolytic activity were developed. A semiquantitative assay on microtitre plates with granular Azocoll as substrate is based on the determination of the time of first appearance of pink colour, t, in the reaction mixture and proteolytic activity is expressed as 1/t. This simple, sensitive and economical method is convenient for preliminary testing of a large number of small samples with unknown proteolytic activity, such as fractions after chromatography.
It speeds up considerably proteinase purification. The other test, a quantitative microassay, is a low-cost and time-saving version of the classical method which reduces material consumption and includes automated measurement of absorbance in microtitre plates by a Multiscan reader. Application of these methods are demonstrated.
Diagnostic and prognostic impact of Cytokeratin 19 expression analysis in human tumors: A tissue microarray study of 13,172 tumors
To evaluate cytokeratin 19 expression in normal and cancerous tissues, 15,977 samples from 122 tumor types and 608 samples of 76 normal tissue types were analyzed by immunohistochemistry. In normal tissues, CK19 expression occurred in epithelial cells of most glandular organs but was strictly limited to the basal cell layer of non-keratinizing squamous epithelium and absent in the skin. CK19 expression in ≥90% of cases was seen in 34% of the tumor entities including the adenocarcinomas of the pancreas (99.4%), colorectum (99.8%), esophagus (98.7%), and stomach (97.7%) as well as breast cancer (90.0%-100%), high grade serous (99.1%) or endometroid (97.8%) ovarian cancer, and urothelial carcinoma (92.6%-100%). A low CK19 positivity rate (0.1-10%) was seen in 5 of 122 tumor entities including hepatocellular carcinoma and seminoma.
A comparison of tumor versus normal tissue findings demonstrated that up-regulation and down-regulation of CK19 can occur in cancer and that both alterations can be linked to unfavorable phenotype. CK19 down regulation was linked to high grade (p=0.0017) and loss of ER- and PR-expression (p<0.0001 each) in invasive breast carcinoma of no special type. CK19 up regulation was linked to nodal metastases in neuroendocrine tumors and papillary thyroid carcinomas (p<0.05 each) and to poor grade in clear cell renal cell carcinoma (p<0.05). CK19 up regulation was particularly common in squamous cell carcinomas. We concluded that CK19 immunohistochemistry might separate primary liver cell carcinoma from liver metastases, seminoma from other testicular tumors, and helps in the detection of early neoplastic transformation in squamous epithelium.
Tissue microarray profiling and integrative proteomics indicate the modulatory potential of Maytenus royleanus in inhibition of overexpressed TPD52 in prostate cancers
Maytenus roylanus (MEM) is a plant with anti-proliferative effects against prostate cancer. We aimed to explore the mechanism of action of MEM in prostate cancer (PCa) by employing an in vitro global proteome approach to get useful information of various signaling pathways and effected genes to define the mechanism of MEM action in prostate cancer.
We conducted a global proteome analysis of CWR22Rv1after treatment with methanolic extract of MEM. The result of the proteomic profiling of in vitro PCa cells demonstrated the reduction in tumor protein D52 (TPD52) expression after treatment with methanolic extract of MEM.
Down-regulation of TPD52 expression at mRNA level was observed by MEM treatment in CWR22Rν1 and C4-2cells in a dose-dependent fashion probably by cleavage of Caspase 3 and PARP, or by modulation of cyclin-dependent kinases in CWR22Rν1 and C4-2
The progressive character of the TRAMP model demonstrates a chance to evaluate the potential of chemo-preventive agents for both initial and late stages of prostate cancer development, and induction in TPD52 protein expression with development as well as the progression of prostate cancer was observed in the TRAMP model.
Analyses of the tissue microarray collection of 25 specimens confirmed the clinical significance of our findings identifying TPD52 as a potential marker for PCa progression.
We determined that knockdown of TPD52 (CWR22Rν1 cells), a considerable downregulation was seen at the protein level.
Downregulation of TPD52 inhibited the migration and invasive behavior of prostate cancer cells as observed. Moreover, we observed that the siRNA-TPD52 transfection of CWR22Rν1 cells resulted in tumor growth inhibition with a marked reduction in the secretion of prostate–specific antigen (PSA) in the serum.
Intraperitoneal injection of MEM considerably slowed tumor growth in athymic mice, inhibited TPD52 expression, and caused a marked reduction in PSA levels of serum as demonstrated by immunoblot screening and immune-histochemical staining. This report illustrates a molecular overview of pathological processes in PCa, indicating possible new disease biomarkers and therapeutic targets.
Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry
Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells.
The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.
Value of chromosomal microarray analysis for the prenatal diagnosis of pregnancy with high risk signaled by non-invasive prenatal testing
Objective: To explore the value of chromosomal microarray analysis (CMA) for the diagnosis of fetuses with high risk signaled by non-invasive prenatal testing (NIPT).
Methods: From June 2017 to August 2019, 628 pregnant women with high risk signaled by NIPT underwent invasive prenatal diagnosis. Amniotic fluid or cord blood samples were subjected to chromosomal karyotyping analysis or CMA. Pregnancy outcome and postnatal conditions of the fetuses were followed up.
Results: The positive predictive value for trisomy 21, trisomy 18, trisomy 13, sex chromosome aneuploidy, other rare trisomies and copy number variants (CNVs) among the 628 women were 86.4% (127/147), 41.7% (30/72), 12.9% (4/31), 43.7% (101/231), 16.5% (14/85) and 52.2% (35/67), respectively. In 218 samples with normal karyotype, 5.5% (12/218) of additional pathogenic CNVs and 2.3% (5/218) of loss of heterozygosity were detected by CMA.
expressionpathology
E-Cadherin expression in human tumors: a tissue microarray study on 10,851 tumors
Background: The E-Cadherin gene (CDH1, Cadherin 1), located at 16q22.1 encodes for a calcium-dependent membranous glycoprotein with an important role in cellular adhesion and polarity maintenance.
Methods: To systematically determine E-Cadherin protein expression in normal and cancerous tissues, 14,637 tumor samples from 112 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format.
Results: E-Cadherin was strongly expressed in normal epithelial cells of most organs. From 77 tumor entities derived from cell types normally positive for E-Cadherin, 35 (45.5%) retained at least a weak E-Cadherin immunostaining in ≥99% of cases and 61 (79.2%) in ≥90% of cases. Tumors with the highest rates of E-Cadherin loss included Merkel cell carcinoma, anaplastic thyroid carcinoma, lobular carcinoma of the breast, and sarcomatoid and small cell neuroendocrine carcinomas of the urinary bladder.
Reduced E-Cadherin expression was linked to higher grade (p = 0.0009), triple negative receptor status (p = 0.0336), and poor prognosis (p = 0.0466) in invasive breast carcinoma of no special type, triple negative receptor status in lobular carcinoma of the breast (p = 0.0454), advanced pT stage (p = 0.0047) and lymph node metastasis in colorectal cancer (p < 0.0001), and was more common in recurrent than in primary prostate cancer (p < 0.0001). Of 29 tumor entities derived from E-Cadherin negative normal tissues, a weak to strong E-Cadherin staining could be detected in at least 10% of cases in 15 different tumor entities (51.7%).
Tumors with the highest frequency of E-Cadherin upregulation included various subtypes of testicular germ cell tumors and renal cell carcinomas (RCC). E-Cadherin upregulation was more commonly seen in malignant than in benign soft tissue tumors (p = 0.0104) and was associated with advanced tumor stage (p = 0.0276) and higher grade (p = 0.0035) in clear cell RCC, and linked to advanced tumor stage (p = 0.0424) and poor prognosis in papillary RCC (p ≤ 0.05).
Conclusion: E-Cadherin is consistently expressed in various epithelial cancers. Down-regulation or loss of E-Cadherin expression in cancers arising from E-Cadherin positive tissues as well as E-Cadherin neo-expression in cancers arising from E-Cadherin negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.
Description: Stomach carcinoma with matched stomach tissue microarray, containing 94 cases of adenocarcinoma, 1 each of signet ring cell carcinoma and squamous cell carcinoma, duplicated cores per case
Normal human tissue microarray from 12 organs from autopsy
Description: Brain primary tumor high density (69 cases/208 cores) tissue microarray of astrocytoma, glioblastoma, glioblastoma multiforme (GBM) and normal tissue
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