Cellular Localization of Enzymatically Active Thrombin in Intact Human Tissues by Hirudin Binding

1995 ◽  
Vol 73 (05) ◽  
pp. 793-797 ◽  
Author(s):  
Leo R Zacharski ◽  
Vincent A Memoli ◽  
William D Morain ◽  
Jean-Marc Schlaeppi ◽  
Sandra M Rousseau
Keyword(s):  
Tumor Cell ◽  
Cell Carcinoma ◽  
Cellular Localization ◽  
Thrombin Generation ◽  
Normal Lung ◽  
Cancer Tissue ◽  
Immunohistochemical Techniques ◽  
Tumor Types ◽  
Negative Controls

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

scholarly journals NDUFA4L2 promotes glioblastoma progression, is associated with poor survival, and can be effectively targeted by apatinib

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Zheng Chen ◽  
Xiangyu Wei ◽  
Xueyi Wang ◽  
Xuan Zheng ◽  
Bowen Chang ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Hypoxia Inducible Factor ◽  
Mitochondrial Respiratory Chain ◽  
Metabolic Reprogramming ◽  
Tumor Cell Apoptosis ◽  
Survival Gene ◽  
A Subunit ◽  
Tumor Types

AbstractNADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) is a subunit of Complex I of the mitochondrial respiratory chain, which is important in metabolic reprogramming and oxidative stress in multiple cancers. However, the biological role and molecular regulation of NDUFA4L2 in glioblastoma (GBM) are poorly understood. Here, we found that NDUFA4L2 was significantly upregulated in GBM; the elevated levels were correlated with reduced patient survival. Gene knockdown of NDUFA4L2 inhibited tumor cell proliferation and enhanced apoptosis, while tumor cells initiated protective mitophagy in vitro and in vivo. We used lentivirus to reduce expression levels of NDUFA4L2 protein in GBM cells exposed to mitophagy blockers, which led to a significant enhancement of tumor cell apoptosis in vitro and inhibited the development of xenografted tumors in vivo. In contrast to other tumor types, NDUFA4L2 expression in GBM may not be directly regulated by hypoxia-inducible factor (HIF)-1α, because HIF-1α inhibitors failed to inhibit NDUFA4L2 in GBM. Apatinib was able to effectively target NDUFA4L2 in GBM, presenting an alternative to the use of lentiviruses, which currently cannot be used in humans. Taken together, our data suggest the use of NDUFA4L2 as a potential therapeutic target in GBM and demonstrate a practical treatment approach.

scholarly journals Extra-gastric expression of the proton pump H+/K+-ATPase in the gills and kidney of the teleost Oreochromis niloticus

2020 ◽  
Vol 223 (16) ◽  
pp. jeb214890
Author(s):  
Ebtesam Ali Barnawi ◽  
Justine E. Doherty ◽  
Patrícia Gomes Ferreira ◽  
Jonathan M. Wilson
Keyword(s):  
Oreochromis Niloticus ◽  
Cellular Localization ◽  
Ex Vivo ◽  
Uptake Inhibition ◽  
Custom Made ◽  
Potassium Regulation ◽  
Immunohistochemical Techniques ◽  
Collecting Tubules ◽  
First Time

ABSTRACTPotassium regulation is essential for the proper functioning of excitable tissues in vertebrates. The H+/K+-ATPase (HKA), which is composed of the HKα1 (gene: atp4a) and HKβ (gene: atp4b) subunits, has an established role in potassium and acid–base regulation in mammals and is well known for its role in gastric acidification. However, the role of HKA in extra-gastric organs such as the gill and kidney is less clear, especially in fishes. In the present study in Nile tilapia, Oreochromis niloticus, uptake of the K+ surrogate flux marker rubidium (Rb+) was demonstrated in vivo; however, this uptake was not inhibited with omeprazole, a potent inhibitor of the gastric HKA. This contrasts with gill and kidney ex vivo preparations, where tissue Rb+ uptake was significantly inhibited by omeprazole and SCH28080, another gastric HKA inhibitor. The cellular localization of this pump in both the gill and kidney was demonstrated using immunohistochemical techniques with custom-made antibodies specific for Atp4a and Atp4b. Antibodies against the two subunits showed the same apical ionocyte distribution pattern in the gill and collecting tubules/ducts in the kidney. Atp4a antibody specificity was confirmed by western blotting. RT-PCT was used to confirm the expression of both subunits in the gill and kidney. Taken together, these results indicate for the first time K+ (Rb+) uptake in O. niloticus and that HKA is implicated, as shown through the ex vivo uptake inhibition by omeprazole and SCH28080, verifying a role for HKA in K+ absorption in the gill's ionocytes and collecting tubule/duct segments of the kidney.

scholarly journals Dominant-negative ATF5 rapidly depletes survivin in tumor cells

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Xiaotian Sun ◽  
James M. Angelastro ◽  
David Merino ◽  
Qing Zhou ◽  
Markus D. Siegelin ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Cancer Cells ◽  
Dominant Negative ◽  
Tumor Cell Death ◽  
Cell Penetrating ◽  
Selective Death ◽  
Tumor Types

Abstract Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

scholarly journals Inhibition of Lung Cell Carcinoma Malignity in Vivo via Tumor Cell Transduction by Interferon-β Gene

2018 ◽  
Vol 28 (1) ◽  
pp. 074-078
Author(s):  
O. O. Lykhova ◽  
◽  
N. O. Bezdenezhnykh ◽  
K. O. Saulenko ◽  
L. I. Strokovska ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Carcinoma ◽  
Interferon Β

Cellular Localization of Activated Factor X by Xa-Specific Probes

1991 ◽  
Vol 65 (05) ◽  
pp. 545-548 ◽  
Author(s):  
Leo R Zacharski ◽  
Christopher Dunwiddie ◽  
Elka M Nutt ◽  
Jane Hunt ◽  
Vincent A Memoli
Keyword(s):  
Cell Carcinoma ◽  
Tumor Cells ◽  
Cellular Localization ◽  
Factor Xa ◽  
Normal Liver ◽  
Factor X ◽  
Melanoma Tumor ◽  
Cancer Tumor ◽  
Tumor Types ◽  
Coagulation Pathway

SummaryA probe, recombinant antistasin, that reacts specifically with the activated form of factor X (Xa) was used in immunohistochemical procedures to detect cellular sites of Xa generation within intact tissues. Factor Xa was detected on tumor cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma. Tumor-associated macrophages (but not tumor cells) expressed Xa in adenocarcinoma and squamous cell carcinoma of the lung, and Hodgkin’s disease. Factor Xa in these locations corresponded to evidence reported previously for an intact coagulation pathway and thrombin formation associated with these tumor cells and macrophages. By contrast, only rare connective tissue cells stained for Xa in breast and colon cancer, tumor types shown previously to lack an intratumoral coagulation pathway and thrombin generation, and in normal liver, lung, breast, kidney, and placental tissues. Hepatocytes did not stain. These results suggest that such probes may be useful for studying the activation state of cell-associated factor X in situ within intact tissues.

Simultaneous, single cell, flow cytometric quantification of PD-L1/TILs/cell cycle in non-small cell lung cancer tissue biopsies: Concordance of PD-L1 expression detection between flow cytometry and immunohistochemistry.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 159-159
Author(s):  
Stephen Young ◽  
Christen Griego-Fullbright ◽  
Aaron Wagner ◽  
Amanda Chargin ◽  
Bruce Kendrick Patterson
Keyword(s):  
Cell Cycle ◽  
Tumor Cell ◽  
Single Cell ◽  
Lung Tissue ◽  
Dna Content ◽  
Immune Cell ◽  
Normal Lung Tissue ◽  
Normal Lung ◽  
Cell Populations ◽  
Cancer Tissue

159 Background: Tumor cell expression of PD-L1 leads to the inhibition of immune responses specifically inactivation of cytotoxic T-cells. PD-L1 expression on tumor cells by immunohistochemistry does not provide the complete picture of therapeutic (PD-L1) and prognostic (TILs, aneuploidy) markers. Here, we report a clinical assay that quantifies tumor infiltrating lymphocytes (TILs), cell cycle/DNA content as well as PD-L1 expression on tumor and aneuploid tumor cell populations. Methods: Punch biopsies were taken from 19 fresh tissues specimens and were processed into single cell suspensions using the IVD/CE-IVD IncellPREP Single Cell Preparation Kit. Cells were fixed and permeabilized in 1 mL IncellMax. Cells were tested with the OncoTect iO Lung Assay which contains antibodies directed against PD-L1 (clone 28-8), CD45, CD3, and CD8, and a cell cycle dye. Concordance to IHC was tested by the Dako PD-L1 IHC 28-8 PharmDx Kit. Immune cell populations were quantified and aneuploidy was determined using a DNA index > 1.05. Results: The number of CD8+ CTL were significantly increased (P = 0.02) in tumor compared to normal lung tissue (37.4% to 28.2%). The number of CTL in aneuploid tumors was twice the number of CTL in diploid tumors. The percentage of antigen presenting cells (CD45+, high SSC) was decreased in the tumor cell samples relative to the normal lung tissue (P = 0.01). PD-L1 expression varies in cells depending on the cell cycle. Conclusions: In this study, the concordance between Oncotect iO and IHC was 95% (NPA-97%, PPA-89%). PD-L1 expression varies with DNA content, PD-L1 expression decreases with increasing DNA content. Though CTL are increased in aneuploid tumors, PD-L1 expression decreasing with DNA content may contribute to the association of aneuploidy with distant metastases. PD-L1 expression is a powerful determinant of treatment and aneuploidy is a powerful prognostic factor that combined yield important clinical information.

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