Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer

Abstract

PKCε is a transforming oncogene and a predictive biomarker of various human cancers. However, a precise in vivo link of PKCε to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice (PKCε-CKO) using cre-lox technology. Homozygous PKCε(LoxP/LoxP) mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostate, the PKCε(LoxP/LoxP) mice were bred to probasin cre (PB-Cre4+) mice which express cre specifically in the prostate epithelium of postnatal mice. Western blot and immunohistochemical analyses showed reduced levels of PKCε specifically in the prostate of PKCε-CKO mice. Histopathological analyses of prostate from both PKCε(LoxP/LoxP) and prostate PKCε-CKO mice showed normal pathology. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMPC1, 2×106) were implanted in the prostate of PKCε-CKO mice. Mice were sacrificed at 6th week post-implantation. Results demonstrated a significant (P<0.05) decrease in the growth of TRAMPC1 cells-derived xenograft tumors in PKCε-CKO mice compared to wild type. To determine a link of PKCε to ultraviolet radiation (UVR) exposure-induced epidermal Stat3 phosphorylation, PKCε(LoxP/LoxP) mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of UVR-induced Stat3 phosphorylation. In summary, our novel PKCε(LoxP/LoxP) mice will be useful for defining the link of PKCε to various cancers in specific organ, tissue, or cells.

Keywords: PKCεLoxP/LoxP mice; transgenic mice.

Figure 1. Generation of floxed PKCε (PKCε^LoxP/LoxP) mice

A. Schematic diagram showing generation and characterization of tissue specific PKCε knockout mice. B. Targeting vector diagram showing LoxP sites flanking Exon 4 of PKCε gene that are the targets of Cre recombinase enzyme and neo flanking with FRT. Exon 4 was deleted leaving a mutant form of PKCε allele and neo cassette was removed by FLP recombinase. C. Southern blot analysis to confirm incorporation of targeting vector containing LoxP-PKCε Exon 4-LoxP into the endogenous PKCε locus in embryonic stem (ES) cells. Briefly, DNA was taken from 6 representative wells (numbered 1-6 in the gel) of ES cells containing LoxP-PKCε Exon 4- LoxP targeting vector. On the left side of the blot, DNA was digested with Nde1 and probed with the 5′ flanking probe. The blot shows the wild type target allele at 15 kb and the targeted allele at 8.9 kb. On the right side of the blot, DNA was digested with BamH1 and probed with the 3′ flanking probe. The blot shows the wild type allele at 11 kb and the targeted allele at 7.6 kb. Abbreviations: M=molecular weight marker.

Figure 2. Characterization of floxed PKCε (PKCε^LoxP/LoxP) mice using prostate specific Cre (PB^Cre+)

A. Breeding strategy to generate prostate specific conditional PKCε knockout mice. Female founders (F0) PKCε^LoxP/LoxP were crossbred with male prostate specific Cre transgenic (PB^Cre4/+) mice. B. PCR gel picture showing genomic DNA tail genotyping for PKCε^LoxP/LoxP, PKCε^LoxP/+/PB^Cre4/+ (Pr-PKCε-CHet), and PKCε^LoxP/LoxP/PB^Cre4/+ (Pr-PKCε-CKO) mice. C. Representative picture of nine week old control floxed PKCε and Pr-PKCε-CKO mice. Bar graph representing the body weight D. and prostate weight E. of control and Pr-PKCε-CKO mice. Value in bar graphs has shown the mean±SE of five mice in each group. No statistical significant difference was observed. Abbreviations: M=molecular weight marker; NC = negative control without DNA.

Figure 3. Histopathological analysis of PKCε^LoxP/LoxP/PB^Cre4/− (Control) and PKCε^LoxP/LoxP/PB^Cre4/+ (Pr-PKCε-CKO) mice

A. H&E staining of control mice anterior prostate (i), dorsal prostate (ii), lateral prostate (iii), and ventral prostate (iv). B. H&E staining of Pr-PKCε-CKO mice anterior prostate (i), dorsal prostate (ii), lateral prostate (iii), and ventral prostate (iv). The prostate of Pr-PKCε-CKO mice was indistinguishable from that of the control.

Figure 4. PKCε expression in the prostate of PKCε^LoxP/LoxP/PB^Cre4/− (Control), and PKCε^LoxP/LoxP/PB^Cre4/+ (Pr-PKCε-CKO) mice

A. Protein levels PKCε, PKCα, PKCβII and PKCζ in the prostate lysates of control PKCε-Het (PKCε^LoxP/+/PB^Cre4/+) and PKCε-KO PKCε^LoxP/LoxP/PB^Cre4/+ mice as analyzed by Western blot analysis. B. Protein level of PKCε in the spleen, liver and lungs (PKCε^LoxP/LoxP), PKCε-Het (PKCε^LoxP/+/PB^Cre4/+) and Pr-PKCε-CKO PKCε^LoxP/LoxP/PB^Cre4/+ mice as analyzed by Western blot analysis. Fifty μg protein of each sample was loaded on the gel. Each lane of the blots in Figure 4A and Figure 4B represents an individual mouse sample. In Figure 4B, PC denotes the positive control where epidermal lysates (10 μg protein) from PKCε transgenic overexpressing mice (224) was loaded. Equal loading of protein was confirmed by stripping and re-probing the blots with an anti-β-actin antibody. C. Immunohistochemistry analysis of PKCε in the prostate tissues of PKCε^LoxP/LoxP (control), and PKCε-CKO PKCε^LoxP/LoxP/PB^Cre4/+ mice, and PKCε blocking peptide. Abbreviations: BP = blocking peptide.

Figure 5. Inhibition of TRAMPC1 cells orthotopic xenograft tumors in prostate specific PKCε knockout (Pr-PKCε-CKO) mice

A. Representative pictures of TRAMPC1 xenograft tumor bearing PKCε^LoxP/LoxP and Pr-PKCε-CKO mice. Representative pictures of excised urogenital apparatus of PKCε^LoxP/LoxP and Pr-PKCε-CKO mice at 8 weeks. Arrows indicate the development of xenograft tumors in the anterior prostate lobes. B. Bar graph showing prostate tumor weight of PKCε^LoxP/LoxP and Pr-PKCε-CKO mice. Values in bar graph represent mean±SE of 4 mice. P<0.05 was considered as significant value.

Figure 6. Specific deletion of PKCε in mouse epidermis inhibits phosphorylation of Stat3 in response to UVR irradiation

A. Breeding strategy to generate skin specific conditional PKCε knockout mice. PKCε^LoxP/LoxP mice were crossbred with skin specific Cre transgenic (K14^Cre+) mice to produce PKCε^LoxP/LoxP/K14^Cre/+ (Sk-PKCε-CKO) in F2 generation. PKCε^LoxP/LoxP (Control), PKCε^LoxP/+/K14^Cre/+ (Sk-PKCε-CHet), and PKCε^LoxP/LoxP/K14^Cre/+ (Sk-PKCε-CKO) mice were confirmed by genomic DNA tail genotyping as described in material and methods. Eight week old PKCε^LoxP/LoxP (n=4), and Sk-PKCε-CKO (n=4) mice were treated once with tamoxifen (75 mg/kg, i.p.) before UVR exposure (2 kJ/m²). Forty eight hours post-UVR treatment, mice were sacrificed and epidermal lysates prepared. B. PKCε expression in the epidermal lysates of tamoxifen treated, irradiated PKCε^LoxP/LoxP and Sk-PKCε-CKO mice as determined by Western blot analysis. Blots were stripped and reprobed for β-actin as a loading control. C. Expression of pStat3Ser727 and total Stat3 in the epidermal protein lysates of tamoxifen treated, irradiated, PKCε^LoxP/LoxP and Sk-PKCε-CKO mice as determined by immunoprecipitation (IP)/Western blot (WB) analysis. PC indicates the positive control where 10μg of epidermal protein lysates of PKCε transgenic overexpressing mice (224) was used.