CENP-50 is required for papilloma development in the two-stage skin carcinogenesis model

Abstract

CENP-50/U is a component of the CENP-O complex (CENP-O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP-50/U has been reported in many types of cancers. However, as Cenp-50/U-deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp-50/U in skin carcinogenesis, we generated Cenp-50/U conditional knockout (K14Cre^ER -Cenp-50/U^fl/fl ) mice and subjected them to the 7,12-dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early-stage papillomas decreased in Cenp-50/U-deficient mice. In contrast, Cenp-50/U-deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA-induced papillomas and carcinomas revealed that Cenp-50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp-50/U functions mainly in early papilloma development and it has little effect on malignant conversion.

Keywords: CENP; malignant conversion; mouse models; papilloma; two-stage skin carcinogenesis.

FIGURE 1

The CENP‐O family is expressed differently in skin tumors. mRNA expression levels in skin tumors (Papillomas/Skin (red) (n = 10), Carcinomas/Skin (blue) (n = 10)). mRNA expression levels detected by qRT‐PCR. A, Cenp‐50/U (P = .0253). B, Cenp‐r (P = .00140). C, Cenp‐o (P = .255). D, Cenp‐p (P = .771) and E, Cenp‐q (P = .212). These expression levels were normalized by Gapdh in skins, papillomas and carcinomas first and then the ratios between papillomas and skins and between carcinomas and skins are presented. The P‐values were calculated by two‐way ANOVA (**P < .01; *P < .05). n.s., not significant. Error bars represent the standard deviation (SD)

FIGURE 2

Generation of Cenp‐50/U knockout mice. A, Targeting strategy to generate mice lacking Cenp‐50/U. Blue boxes indicate the positions of exons. loxP was inserted into the genomic region in exons 4‐6 of the Cenp‐50/U gene. Red arrows indicate the construction primers for detection of the Cenp‐50/U ^fl/fl‐allele and Cenp‐50/U knockout allele. B, Genotype analysis of genomic DNA from Cenp‐50/U ^fl/fl mice. The floxed allele and wild‐type allele were detected by 3F and 3R primers (floxed allele: approximately 500 bp, wild‐type allele: 350 bp). C, Genotype analysis of Cenp‐50/U knockout (KO) allele. The KO allele was detected by 5F and 3Rb primers (approximately 500 bp) using genomic DNA from mouse skins and tail. D, Cenp‐50/U expression levels detected by RT‐PCR. RT‐PCR analysis using mouse skins from Cenp‐50/U ^fl/fl and K14Cer^ER‐ Cenp‐50/U ^fl/fl mice. Gapdh expression is shown as an internal control. E, Cenp‐50/U protein expression levels measured by western blot analysis using mouse skins from Cenp‐50/U ^fl/fl and K14Cer^ER‐Cenp‐50/U ^fl/fl mice. Actin expression is shown as an internal control

FIGURE 3

Cenp‐50/U‐deficient mice have a decrease in cell proliferation in normal skins. A, B, Immunostaining patterns of Ki67 (green) in skins from (A) Cenp‐50/U ^fl/fl and (B) K14Cer^ER‐Cenp‐50/U ^fl/fl mice. C, D, Representative TUNEL staining pattern of TdT (green) in skins from (C) Cenp‐50/U ^fl/fl and (D) K14Cer^ER‐Cenp‐50/U ^fl/fl mice. Cells were counterstained with Hoechst (red). E, The number of Ki67‐positive cells in skins from Cenp‐50/U ^fl/fl (n = 12) (red bar) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 12) (blue bar) mice (P = 4.93E‐8). F, The number of TUNEL‐positive cells in skins from Cenp‐50/U ^fl/fl (n = 7) and K14Cer^ER‐Cenp‐50/U ^fl/fl mice (n = 7) (blue bar). G, Cell cycle analysis of mouse normal skins from Cenp‐50/U ^fl/fl (n = 6) (red bar) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 5) (blue bar) mice (G₀/G₁ phase: P = .00424, subG₁ phase: P = .000203). DNA content was measured by propidium iodide (PI) staining. H, Trypan blue exclusion test of cell viability. Cells were isolated from Cenp‐50/U ^fl/fl (n = 6) (red bar) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 5) (blue bar) mice (P = .002013). The P‐value was calculated by t test (**P < .01). n.s., not significant. n.d., not detected. Error bars represent the standard deviation (SD)

FIGURE 4

Cenp‐50/U functions in early tumor development. A, Comparison of DMBA/TPA‐induced papilloma numbers per mouse between Cenp‐50/U ^fl/fl (n = 22) (red line) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 17) (blue line) mice. B, Number of papillomas < 2 mm per mouse. C, Number of papillomas 2‐6 mm per mouse. D, Number of papillomas >6 mm per mouse. Red bars represent Cenp‐50/U ^fl/fl mice. Blue bars represent K14Cer^ER‐Cenp‐50/U ^fl/fl mice. E, Representative photographs of Cenp‐50/U ^fl/fl (left) and K14Cer^ER‐Cenp‐50/U ^fl/fl (right) mice at 20 wk after initiation. The P‐values were calculated by t test (**P < .01; *P < .05). n.s., not significant. Error bars represent the standard deviation (SD)

FIGURE 5

Cenp‐50/U deficiency induces cell death in papillomas. A, B, Immunostaining patterns of Ki67 (green) and K14 (red) in papillomas from (A) Cenp‐50/U ^fl/fl and (B) K14Cer^ER‐Cenp‐50/U ^fl/fl mice. C, D, Representative TUNEL staining pattern of TdT (green) in papillomas from (C) Cenp‐50/U ^fl/fl and (D) K14Cer^ER‐Cenp‐50/U ^fl/fl mice (P = .00217). Cells were counterstained with Hoechst (blue). White inset boxes indicate the magnified region. E, The number of Ki67‐positive cells in papillomas from Cenp‐50/U ^fl/fl (n = 12) (red bar) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 12) (blue bar) mice (P = .144). F, The number of TUNEL‐positive cells in papillomas from Cenp‐50/U ^fl/fl (n = 8) (red bar) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 8) (blue bar) mice. The P‐value was calculated by t test (**P < .01). n.s., not significant. Error bars represent the standard deviation (SD)

FIGURE 6

Cenp‐50/U has little effect on malignant conversion. A, Comparison of the incidence of DMBA/TPA‐induced carcinoma between Cenp‐50/U ^fl/fl (n = 22) (red line) and K14Cre^ER‐Cenp‐50 ^fl/fl (n = 17) (blue line) mice. B, Comparison of the incidence of DMBA/TPA‐induced carcinomas per papilloma between Cenp‐50/U ^fl/fl (n = 172) (red line) and K14Cer^ER‐Cenp‐50/U ^fl/fl (n = 99) (blue line) mice

FIGURE 7

Schematic drawing of the functions of Cenp‐50/U in the process of skin carcinogenesis. Cenp‐50/U regulates early papilloma development. Conversely, Cenp‐50/U has little effect on papilloma growth and malignant conversion