Table 1.
Sequences of oligonucleotides used as primers and probes in various PCR assays.
Fig 1.
Association between P16 expression and methylation and palbociclib sensitivity in cancer cell lines.
(a and b) Correlations between the palbociclib IC50 levels and CDKN2A/P16 mRNA levels and copy number in cancer cell lines (n = 522). The p-values were measured by a nonparametric Spearman correlation test and linear regression model. (c) The methylation status of the P16 gene in different cell lines as assessed by MSP. (d) The palbociclib IC50 values in 22 cancer cell lines with different P16 alterations, including P16 copy number deletion (P16-D), P16 methylation (P16-M), and P16 unmethylated and undeleted (16-U). Student’s t-test, **p<0.01.
Fig 2.
Effect of P16-specific methylation on the sensitivity of cancer cells to palbociclib.
(a) Methylation of the CpG P16 island in three cancer cell lines stably transfected with an engineered P16-specific DNA methyltransferase (P16-Dnmt) by MSP. Genomic DNA samples from RKO and MGC803 cells were used as the positive and negative controls for P16 methylation, respectively. (b) Effect of P16-specific methylation on the levels of P16 expression in cell lines stably transfected with P16-Dnmt, as assessed by qRT-PCR. (c) Effect of P16-specific methylation on the viability (survival rate) of cells treated with palbociclib for 48 hrs, as assessed with the IncuCyte ZOOM system. Each point represents the mean ± SD of 4 wells. Student’s t-test, *p<0.05, **p<0.01.
Fig 3.
Selective inhibition of the proliferation of P16-methylated cancer cells by palbociclib in vitro.
After palbociclib treatment (H661 and BGC823 cells: 10 μM; HCC827 cells: 7 μM) for 48 hrs, cells were harvested at 70–80% confluence. (a and b) Effect of palbociclib treatment on the levels of P16-methylated alleles and P16 mRNA in cell lines stably transfected with P16-Dnmt, as assessed by MSP and qRT-PCR assays, respectively. The data are presented as the means ± SDs. (c) The results of immunofluorescence and confocal microscopy analyses to directly detect changes in the subpopulation of cells staining positive for P16 protein within the H661 cell line stably transfected with P16-Dnmt. (d) Illustration of the proliferation of P16 methylated cells selectively inhibited by palbociclib treatment. Scale bar, 400 μm. Student’s t-test, *p<0.05, **p<0.01.
Fig 4.
Effect of palbociclib treatment on the growth of tumors derived from HCC827 cancer cells with and without stable transfection of P16-Dnmt in nude mice.
(a) Images of HCC827 xenograft tumors in different groups of mice (6 mice/group). These mice were treated with palbociclib (100 mg/kg b.w., i.g.) or control buffer for 3 weeks. A tumor weight chart is inserted on the right side. The data are expressed as the medians ± interquartile ranges; Mann–Whitney test, **p<0.01. (b) Immunohistochemical staining for Ki-67 in HCC827 xenograft tumors in the different groups (scale bar, 100 μm). The percentage of Ki-67+ cells is presented as the mean ± SD of three sections. Ki-67+ cells were counted in 5 random fields per section. (c) The levels of methylated P16 alleles (left chart) and P16 expression (middle chart) in three representative tumors from each group, as assessed by MethyLight and qRT-PCR assays. (d) The levels of RB, phosphorylated RB (p-RB), and P16 proteins in three representative tumors from each group, as assessed by Western blot. Student’s t-test, *p<0.05, **p<0.01.