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Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis
Nata
a Levi
ar, Ricardo A Dewey, Emma Daley, Timothy E Bates, Derek Davies, Janko Kos, Geoffrey J Pilkington and Tamara T Lah
Figure 1.
A: Flow cytometric analysis of intracellular content of cathepsin L in human glioblastoma IPTP cells and IPTP-transfected clones. Cells were stably transfected with PreproCatL cDNA in antisense and sense orientation. Cathepsin L content was determined using monoclonal antibodies and flow cytometric analysis. The relative amounts of cathepsin L in the transfected IPTP cells are presented as the overlaid histogram and compared with the control parental IPTP cells (thick black line). The IPTP4asn clone (dark grey line) showed the lowest content of cathepsin L (shift to the left), whereas the IPTP24sn (light grey line) cells showed the highest cathepsin L protein levels (shift to the right) compared to the parental IPTP cells. IPTP cells where primary antibodies were omitted represented negative control (thin black line). B: Cathepsin L RNA quantification in IPTP parental and IPTP-transfected clones. Cathepsin L RNA content was measured by real-time PCR in the parental IPTP, and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin L RNA levels were expressed relative to GADPH levels. Significantly (29%) lower cathepsin L RNA levels in IPTP4asn cells and significantly (66%) higher cathepsin L RNA levels in IPTP24sn cells were found compared to untransfecetd IPTP cells. Bars represent mean value of two independent experiments and standard error of the mean (mean
SEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line. C: Cathepsin L protein concentration in cell lysates of selected clones of IPTP cells. Cathepsin L protein was measured by ELISA in the parental IPTP, and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin L protein concentrations were expressed relative to total protein concentration in the cell lysates. Significantly (41%) lower cathepsin L concentrations in IPTP4asn cells and significantly (150% higher) cathepsin L concentrations in IPTP24sn cells were found compared to untransfecetd IPTP cells. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.
Figure 2.
Cathepsin L-type activity in cell lysates of selected clones of IPTP cells. Cathepsin L-type activity was measured using the fluorogenic substrate Z-Phe-Arg-AMC. The mean values of specific enzymatic activities in IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were compared. Cathepsin L-type activity was reduced by 50% in IPTP4asn and significantly elevated by 143% in IPTP24sn cells compared with the parental cells. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). ***Indicates a statistically significant (P<.05) difference compared with the parental IPTP cell line.
Figure 3.
Cathepsin B protein concentration in cell lysates of selected clones of IPTP cells. Cathepsin B protein was measured by ELISA in the parental IPTP and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin B protein concentrations were expressed relative to total protein concentration in the cell lysates. No significant changes in cathepsin B concentrations were observed between cell lines analyzed. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM).
Figure 4.
Panel A: Adhesion of parental and transfected glioblastoma cell lines in vitro. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were tested for adhesion on the Matrigel substratum. No significant differences in adhesion were observed between all four types of cell lines. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). Panel B: Motility of parental and transfected glioblastoma cell lines in vitro. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were tested for motility in transwell chambers, using 8-
m polycarbonate filters. No significant differences in motility were observed among all four types of cell lines. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). Panel C: Invasion of parental and transfected glioblastoma cell lines in vitro. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were tested for penetration into Matrigel in transwell chambers with 8-m polycarbonate filters. The relative invasive potential of IPTP4asn was significantly reduced by 70%, compared with the parental IPTP cells. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.
Figure 5.
Apoptosis of human glioblastoma cell lines induced by STS. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were treated with 1 M STS and the relative rate of apoptosis was determined after 24 hours, using acridine orange/ethidium bromide staining. IPTP4asn cells had significantly higher rate of apoptosis (54% of cells), whereas IPTP24sn cells were more resistant to apoptosis (13% of cells). Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.
Figure 6.
Analysis of cellular DNA content in human glioblastoma cell lines. Cell cycle analysis in IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells was performed during 24-hour exposure to 1 M STS and the amount of the apoptotic cells in sub-G1 region was determined by flow cytometry, ranging between 1% and 25%, 1% and 23%, 2% and 45%, and 1% and 14%, respectively. Relative percentages of apoptotic cells in sub-G1 peak are presented after 24 hours of exposure to STS. Histograms are from a representative experiment.
Figure 7.
Time course of caspase-3 activation after STS exposure of human glioblastoma clones. Caspase-3 activity was measured in the lysates of IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells during exposure to 1 M STS at the indicated time intervals. Data were normalized for easier comparison. For each cell line, mean value of peak activity was taken as 1. For each time point, the mean value was than calculated as a percentage of peak activity. IPTP, IPTPvgfp, and IPTP4asn cells showed a peak of caspase-3 activity at 8 hours, whereas IPTP24sn showed delayed peak at 16 hours.
Figure 8.
Flow cytometric analyses of intracellular amount of Bcl-2 in human glioblastoma cell lines. Relative amounts of Bcl-2 protein were assessed by flow cytometry using specific Bcl-2 antibodies in IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells. The relative amounts of Bcl-2 are presented in two overlaid histograms (A,B) and compared with the control parental IPTP cells (thick black line, 63% of positive cells). A: Bcl-2 was significantly less expressed in IPTP4asn cells (grey line, 44% of positive cells). B: IPTP24sn cells expressed significantly more Bcl-2 protein (grey line, 81% of positive cells) compared to parental IPTP cells.
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