Article Figures & Data
Figures
- Figure 1.
A, morphology of parental and cisplatin-resistant UM-SCC cell lines. Parental cell lines UM-SCC-5 and UM-SCC-10B (top) and corresponding cisplatin-resistant cell lines UM-SCC-5PT and UM-SCC-10BPT (bottom) are shown at ×20 magnification by phase-contrast microscopy. B, cell proliferation in parental and cisplatin-resistant cell lines. Cell counts using trypan blue exclusion to assess viable cells were used to determine the doubling time (proliferation rate) for each cell line. Doubling times were calculated as described in Materials and Methods. Data are from one representative experiment of three that showed the same results.
- Figure 2.
A, Western blot analysis of p53 expression in parental and cisplatin-resistant cell lines. High p53 expression in the parental lines corresponds to mutant p53 and low expression in the resistant lines corresponds to wild-type p53. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) shows equal loading. B, DNA sequencing confirms that parental cell line UM-SCC-5 contains a transition mutation at Val157 of exon 5 and UM-SCC-10B contains a transversion mutation at Gly245 of exon 7. Cisplatin-selected cell lines UM-SCC-5PT and UM-SCC-10BPT contain only the wild-type p53 allele in each case. C, immunohistochemical staining for p53 (top) demonstrating a nuclear staining pattern in a heterogeneous population of cells in the parental lines and low staining in cisplatin-resistant lines. IgG2a isotype control (bottom) staining indicates no nonspecific antibody staining. Magnification, ×40.
- Figure 3.
Western blot analysis of Bcl-2 and Bcl-xL in parental and cisplatin-resistant cell lines. Bcl-xL is expressed at higher levels in the cisplatin-resistant cell lines than in the parental lines. Bcl-2 expression is inversely correlated with cisplatin resistance and is higher in parental lines than the cisplatin-resistant cell lines. Glyceraldehyde-3-phosphate dehydrogenase shows equal loading. Western blots were repeated a minimum of thrice. Bands were quantified for fold change using ImageJ software (n = 3).
- Figure 4.
MTT cell survival assays of parental and cisplatin-resistant cell lines. A, response to cisplatin. IC50 values were obtained for each cell line. Fold change in sensitivity was determined by comparing IC50 values (P = 0.0091, 5 versus 5PT; P < 0.0001, 10 versus 10BPT). B, response to (−)-gossypol (estimated IC50 values of 1.5–2 μmol/L versus 3.7–5.5 μmol/L, P = 0.0091, 5 versus 5PT; P < 0.0001, 10 versus 10BPT). Data represent five replicate wells. Experiments were repeated thrice.
- Figure 5.
TUNEL assay of parental and cisplatin-resistant cell lines following 48-h treatment with vehicle, cisplatin, or (−)-gossypol. The level of TUNEL-positive cells is determined as a percent of total cells above the gated untreated control cells. Data represents three independent experiments. Significant difference between cisplatin-resistant and parental cell lines, (*, **P < 0.0001).
Volume 4, Issue 7
