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Results: 3

1.
Figure 2

Figure 2. Invasion is inhibited by Met-RANTES and by a monoclonal antibody against CCR5. From: Lung Adenocarcinoma Invasion in TGFβRII Deficient Cells is Mediated by CCL5/RANTES.

We tested the requirement for RANTES in increasing invasion of TGFβRII deficient cells by using a competitive inhibitor (Met-RANTES) and a monoclonal antibody against CCR5 (α-CCR5). H23, SKLU, and H522 lung adenocarcinoma cells transfected with siRNATGFβRII-A, siRNATGFβRII-B, or control siRNA were incubated with drug or control IgG for 24 hours prior to seeding on the upper well of a matrigel coated transwell migration chamber. Results for assays performed in triplicate using siRNATGFβRII-A and representative photomicrographs of invasive H23 cells (magnification x40) are shown. * indicates P<.05 vs. siRNATGFβRII-A. All results are presented in Table 1. In each instance, invasion in TGFβRII knockdown cells was significantly reduced to levels near control after treatment with Met-RANTES and α-CCR5.

Alain C. Borczuk, et al. Oncogene. ;27(4):557-564.
2.
Figure 3

Figure 3. RANTES and CCR5 expression in lung adenocarcinoma tumors is associated with Invasion and with clinical outcome. From: Lung Adenocarcinoma Invasion in TGFβRII Deficient Cells is Mediated by CCL5/RANTES.

A. Immunohistochemistry for CCR5 and RANTES in representative lung adenocarcinoma specimens. Panels A,C,E: CCR5 immunohistochemistry is strongly positive (3+) in invasive adenocarcinoma (Panel A) and weakly positive (1+) in BAC (Panel C). Fibroblasts (indicated by arrows in Panel E) are positive (2+), with the inset showing isotype and concentration matched negative control (lung). Panels B,D,F: RANTES is positive (2+) in invasive adenocarcinoma (Panel B) and weakly positive (1+) in BAC (Panel D). Fibroblasts (indicated by arrow in Panel F) are positive (2+), with the inset showing isotype matched negative control (lung).
B. The percentage of BAC and invasive tumors staining negatively or weakly (scores 0 and 1) and moderately or strongly (scores 2 and 3) for RANTES (left panel) and CCR5 (right panel) is indicated. A majority of invasive tumors stain moderately or strongly for both molecules, whereas a minority of BAC tumors stains moderately or strongly. The probabilities of these percentages occurring by chance by the test for difference between the two proportions is .03 for RANTES and 1.60×10−8 for CCR5, indicating that the results are statistically significant.
C. Kaplan-Meier survival plots of RANTES and CCR5 expression in tumor cells from 162 patients with resected lung adenocarcinoma. For log rank analysis of survival, specimens were classified as low expression (scores 0 and 1) or high expression (scores 2 and 3).

Alain C. Borczuk, et al. Oncogene. ;27(4):557-564.
3.
Figure 1

Figure 1. RANTES mRNA and protein secretion in invasive lung cancer cells. From: Lung Adenocarcinoma Invasion in TGFβRII Deficient Cells is Mediated by CCL5/RANTES.

A. Relative change in CCL5 mRNA expression in cells after transfection with siRNA TGFβRII - A and siRNA TGFβRII -B vs. with control siRNA was determined by qRT-PCR, normalized to copies of Actin. Relative quantification was used to determine fold change in mRNA copy number (log base 2). RANTES mRNA expression was significantly higher in TGFβRII knock-down cells compared with control (P <.05 in each instance), thus confirming the microarray results.
B. RANTES secretion in cells transfected with siRNATGFβRII-A was measured in cell supernatants by ELISA. Only small amounts of RANTES were detected in control cells, but RANTES secretion was strongly induced by repression of TGFβRII in each cell line. The P value of the ratio of RANTES expression in siRNATGFβRII-A cells vs. control was 1×10−2 for H23, 1×10−5 for SKLU, and 1×10−3 for H522 cells. Results for RANTES secretion in cells transfected with siRNATGFβRII-B vs. control were similar in regards to ratio and significance (data not shown).
C. RANTES increases invasiveness of lung adenocarcinoma cells with wildtype expression of TGFβRII. Serum starved H23, SKLU, and H522 cells were pretreated with recombinant human (rh) RANTES at 100 pg/ml for 60 minutes placed in the upper well of a matrigel coated transwell migration chamber. Values are representative of three matrigel experiments.

Alain C. Borczuk, et al. Oncogene. ;27(4):557-564.

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