Method for testing a subject thought to be predisposed to having metastatic cancer using delta133p53beta
A technology for metastatic cancer and subjects, applied in the direction of material inspection products, biochemical equipment and methods, and microbial determination/inspection, etc.
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Embodiment 1
[0147] Example 1: Materials and methods
[0148] DNA constructs, reagents and antibodies:
[0149] The human p53 isotype construct was a gift from J.C. Bourdon. They were subcloned into the EcoRI and BamHI sites of pEGFPCl (Clonetech) to give GFP-tagged proteins or into the BamHI and EcoRI sites of pLPCmyc to give myc-tagged proteins. Constructs were amplified using the Nucleobond PC 500 kit (Macherey-Nagel) according to the manufacturer's instructions.
[0150] Y27632 was purchased from calbiochem and used at 10 μM in all experiments.
[0151] Mouse anti-E-cadherin (clone 36), mouse anti-β1-integrin, mouse anti-ROCK I and mouse anti-ROCK II antibodies were purchased from BD-Transduction Laboratories and were divided into 1 / 400 °, 1 / 2500°, 1 / 250° and 1 / 250° for dilution. Mouse anti-RhoA and rabbit anti-ECT2 antibodies were purchased from Santa Cruz (26C4 and C-20, respectively) and diluted 1 / 500° and 1 / 200°, respectively. Mouse anti-GEF-H1 antibody was a gift from K.Matte...
Embodiment 2
[0179] Example 2: Analysis of Δ133p53β expression in breast cancer patients
[0180] Analysis of primary, previously untreated operable from 171 Caucasian women (age range 24-89 years, median age 64 years) with sufficient tumor tissue remnant for diagnosis and complete clinical and pathologic data of breast tumors.
[0181] result
[0182] In 171 breast cancers, Δ133p53 expression was identified in 50 / 171 (29%), Δ133p53β expression in 20 / 171 (11%) and Δ133p53γ expression in 27 / 171 (16%), while it was not detected in normal breast tissue to these three isotypes.
[0183] Δ133p53β expression correlated with axillary lymph node metastasis (Mann-Whitney analysis, exact one-tailed, p<0.036), and consistent with this, patients with tumors expressing Δ133p53β had significantly worse disease free survival (log -rank, Cox-Mantel, p<0.025) (Figure 1a) and overall survival (log-rank, Cox-Mantel, p<0.025) (Figure 1b).
[0184] Δ133p53β was significantly associated with low ER expressi...
Embodiment 3
[0187] Example 3: Major role of the Δ133p53β isoform during invasion
[0188] First, expression of GFP-tagged Δ133p53, Δ133p53γ, and Δ133p53β isoforms was examined using an invasion assay (see Methods) for colon and breast cancer cells that retained epithelial features and expressed wild-type p53 (hct116 for colon cancer and MCF7 for breast cancer). cancer) invasion.
[0189] Cells transfected with GFP alone were used as a negative control. Cells were plated on top of a thick layer of matrigel reproducing the physiological basement membrane. This in vitro assay aims to mimic the progression of tumor cells across the basement membrane.
[0190] Figure 3a showed that expression of the Δ133p53β isoform significantly increased invasion. Similar results were obtained with MCF7 (data not shown). This implies that overexpression of ectopic Δ133p53β isoforms can overcome the anti-migratory activity of endogenous p53.
[0191]To determine which mode of migration cells expressing...
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