Lab Anim Res.  2018 Sep;34(3):118-125. 10.5625/lar.2018.34.3.118.

Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA

• Choi JY
• Yun WB
• Kim JE
• Lee MR
• Park JJ
• Song BR
• Kim HR
• Park JW
• Kang MJ
• Kang BC
• Lee HW
• Hwang DY

• KMID: 2420824
• DOI: http://doi.org/10.5625/lar.2018.34.3.118

Abstract

To evaluate the carcinogenicity of p27 knockout (KO) mice with RNA-guided endonuclease (RGENs)-mediated p27 mutant exon I gene (IΔ), alterations in the carcinogenic phenotypes including tumor spectrum, tumor suppressor proteins, apoptotic proteins and cell cycle regulators were observed in p27 (IΔ) KO mice after treatment with 7,12-Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)(DT) for 5 months. The target region (544~571 nt) in exon I of the p27 gene was successfully disrupted in p27 (IΔ) KO mice using the RGEN-induced non-homologous end joining (NHEJ) technique. After DT exposure for 5 months, a few solid tumors (identified as squamous cell carcinoma) developed on the surface of back skin of DT-treated p27 (IΔ) KO mice. Also, squamous cell hyperplasia with chronic inflammation was detected in the skin dermis of DT-treated p27 (IΔ) KO mice, while the Vehicle+p27 (IΔ) KO mice and WT mice maintained their normal histological skin structure. A significant increase was observed in the expression levels of tumor suppressor protein (p53), apoptotic proteins (Bax, Bcl-2 and Caspase-3) and cell-cycle regulator proteins (Cyclin D1, CDK2 and CDK4) in the skin of DT-treated p27 (IΔ) KO mice, although their enhancement ratio was varied. Taken together, the results of the present study suggest that squamous cell carcinoma and hyperplasia of skin tissue can be successfully developed in new p27 (IΔ) KO mice produced by RGEN-induced NHEJ technique following DT exposure for 5 months.

Keyword

p27; tumorigenesis; DMBA; TPA; carcinoma; hyperplasia

MeSH Terms

9,10-Dimethyl-1,2-benzanthracene*
Animals
Carcinogenesis
Carcinoma, Squamous Cell*
Cell Cycle
Dermis
Epithelial Cells*
Exons
Hyperplasia*
Inflammation
Mice*
Phenotype
Skin
Tumor Suppressor Proteins
9,10-Dimethyl-1,2-benzanthracene
Tumor Suppressor Proteins

Figure

• 

  Figure 1 Scheme for deletion of the p27 gene, strategy for DT treatment, and evaluation of organ weights of DT-treated p27 (IΔ) KO mice. (A) Specific region (28 bp) of exon I in p27 (IΔ) KO mice were deleted with RGENs specific targeting. (B) After DMBA (25 µg) application on day 1, TP (10−4 M) was treated on the back skin of p27 (IΔ) KO mice for 5 months. (C) The weight of 9 organs collected from DT-treated p27 (IΔ) KO mice were measured using an electrical balance. The data shown represents the means±SD of three replicates. BR, Brain; LV, Liver; KD, Kidney; HT, Heart; LU, Lung; SP, Spleen; AD, Adrenal gland; TH, Thymus; GO, Gonad.

• 

  Figure 2 Tumor formation on the skin surface of DT-treated p27 (IΔ) KO mice. (A) Tumor morphology. Solid tumors were observed on the surface of the back skin. (B) Histopathology of solid tumors. H&E stained sections of skin and solid tumor from the DT-treated p27 (IΔ) KO mice were observed at 100× (left column) and 400× (right column) using a light microscope. Ep, epidermis; De, dermis; Hd, hypodermis; Ms, muscle layer.

• 

  Figure 3 Expression analysis of tumor suppressor protein. An alteration in the expressions of p53 and p27 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P<0.05 compared to the WT mice. # indicates P<0.05 compared to the Vehicle-treated p27 (IΔ) KO mice.

• 

  Figure 4 Expression analysis of apoptotic proteins. Alterations in the expressions of Bax, Bcl-2 and Caspase-3 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P<0.05 compared to the WT mice. # indicates P<0.05 compared to the Vehicle-treated p27 (IΔ) KO mice.

• 

  Figure 5 Expression analysis of cell-cycle regulator proteins. An alteration in the expressions of Cyclin D1, CDK2 and CDK4 proteins were determined in DT-treated p27 (IΔ) KO mice by Western blot assays using HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and the expression level of 6 proteins were evaluated relative to the intensity of actin bands. The data represents the means±SD (n=8). *, indicates P<0.05 compared to the WT mice. # indicates P<0.05 compared to the Vehicle-treated p27 (IΔ) KO mice.

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