Detection of circulating crosslinked fibrin derivatives by a heat extraction-SDS gradient gel electrophoretic technique

A technique has been developed to identify and quantitate unique plasmic degradation products of crosslinked fibrin in plasma. In this method, fibrin derivatives are extracted by heat precipitation and dissolved with disulfide bond reduction, after which the crosslinked gamma-gamma chain remnants are identified by SDS-polyacrylamide gradient gel electrophoresis and quantitated by densitometric analysis. A heterogenous group of gamma-gamma chains with molecular weights between 100,000 and 76,000 daltons was identified in lysates of crosslinked fibrin during plasmic degradation in vitro. Three stages of crosslinked fibrin degradation have been arbitrarily defined based primarily on the extent of degradation of these gamma-gamma polypeptide chains. As little as 20 microgram of crosslinked fibrin digests added to 1 ml of normal plasma could be detected by the heat-extraction--gel- electrophoresis technique, identifying the gamma-gamma derivatives with molecular weights of 96,000, 86,000, 82,000, and 76,000 daltons. Plasmic derivatives of gamma-gamma chains were not found in normal plasma, but they were identified in the plasma of patients with disseminated intravascular coagulation and deep-vein thrombosis, both before and in increased quantity during successful thrombolytic therapy.     

Acute lymphoblastic leukemia of childhood: identification of two distinct regions of deletion on the short arm of chromosome 12 in the region of TEL and KIP1

Cytogenetic analysis of acute lymphoblastic leukemia (ALL) of childhood identified nonrandom chromosomal abnormalities of the short arm of chromosome 12. The alterations include deletions that are thought to be indicative of the presence of a tumor suppressor gene that is mutated on the remaining allele. To refine further the chromosomal localization of this gene, we analyzed the loss of heterozygosity (LOH) of chromosome 12 in 100 primary ALL samples using 22 polymorphic markers and identified two distinct smallest common deleted regions on chromosome 12p13. One region is flanked by D12S77 and D12S98 and has a size of 4 cM. Twenty-six percent of informative patients showed LOH in this region. This region may contain the TEL gene. The other region is flanked by D12S269 and D12S308 including the KIP1 gene. Forty-four percent of informative patients showed LOH in this second region. Mutational analysis of KIP1 using polymerase chain reaction-single- strand conformation polymorphism analysis and Southern blot analysis showed no homozygous deletions and point mutations suggesting that the altered gene in this second region is not the KIP1. Clinical data showed that LOH of 12p was demonstrated more frequently in precursor-B ALLs (32 of 80; 40%) than in T-ALLs (1 of 20; 5%) (P = .0027). Furthermore, patients with 12p LOH were younger (P = .013), with a lower DNA index (P = .046), but they had the same survival rates at 3 years. In summary, these data suggest that two different tumor suppressor genes are on chromosome arm 12p, which act separately in the development of childhood precursor-B ALLs. One of the tumor suppressor genes is in the region the KIP1 gene, but our data suggest this gene is not abnormal. The other target is in the region of the TEL gene; and this candidate deserves further study.     

Aven‐mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma

Conventional high‐grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis‐free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti‐apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G₂ cell‐cycle arrest; Chk1 protein levels were attenuated and ATR–Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven‐depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven‐controlled ATR–Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix‐embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR–Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Radiation-guided gene therapy of cancer

Gene therapy has been proposed as a means to combat cancer. However, systemic toxicity observed in preclinical trials suggested the importance of selectively targeted delivery and inducible gene expression in tumor tissues. Discovery of radiation-inducible promoter sequences provides one way to minimize inadvertent toxicity from gene therapy in normal tissues. Radiation is administered to selectively induce cytotoxic gene expression in the targeted tumor tissues. With promising results from phase II clinical trials using TNF-expressing adenovirus, it is possible to have radiation-guided gene therapy regimes once the tumor-targeted delivery has been achieved. Tumor endothelium is an attractive biological target for gene therapy, because it has the advantage of stability, accessibility, and bioavailability for therapeutic agents. Technological development of DNA microarray, proteomic profiling, and phage-displayed libraries accelerates the identification of tumor-specific endothelial biomarkers and discovery of its relevant affinity reagents for targeted delivery. The application of radiation-guided gene delivery, its amplification, as well as expression of gene therapy presents great opportunities to be employed as an alternative cancer treatment.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

Molecular strategies targeting the host component of cancer to enhance tumor response to radiation therapy

The tumor microenvironment, in particular, the tumor vasculature, as an important target for the cytotoxic effects of radiation therapy is an established paradigm for cancer therapy. We review the evidence that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated in endothelial cells exposed to ionizing radiation (IR) and is a molecular target for the development of novel radiation sensitizing agents. On the basis of this premise, several promising preclinical studies that targeted the inhibition of the PI3K/Akt activation as a potential method of sensitizing the tumor vasculature to the cytotoxic effects of IR have been conducted. An innovative strategy to guide cytotoxic therapy in tumors treated with radiation and PI3K/Akt inhibitors is presented. The evidence supports a need for further investigation of combined-modality therapy that involves radiation therapy and inhibitors of PI3K/Akt pathway as a promising strategy for improving the treatment of patients with cancer.     

银杏叶提取物改善反复脑缺血再灌注小鼠血液流变学的作用

目的：研究银杏叶提取物（ＥＧＢ）改善反复脑缺血再灌注小鼠血液流这的作用。方法：采用反复脑缺血再注模型鼠,应用毛细管微量热沉法和毛细管微量法分别检测血纤维蛋白原含量和红细胞压积数值,并将结果输入全自动血液流变仪得出血浆粘度、血液粘度、血细胞聚集系数,血栓形成系数及微循环滞留时间（ＭＳＴ）。结果：ＥＢＧ２５￣１００ｍｇ／ｋｇ均可不同程度地降低Ｆｉｂ、ＨＣＴ、降低ηｂ、ηｐ、ηｈ,缩小ＶＡＬ及ＴＷＥＬ,     

Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models

PURPOSE: Integrins alpha v beta 3 and alpha v beta 5 are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation. METHODS AND MATERIALS: Levels of alpha v beta 3 were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non-small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment. RESULTS: Human umbilical vein endothelial cells had the highest alpha v beta 3 level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of alpha v beta 3 in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment. CONCLUSIONS: We conclude that radiation induces expression of alpha v beta 3 integrin in endothelial and non-small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.