Transgenic CHD1L expression in mouse induces spontaneous tumors

Abstract

Background: Amplification of 1q21 is the most frequent genetic alteration in hepatocellular carcinoma (HCC), which was detected in 58-78% of primary HCC cases by comparative genomic hybridization (CGH). Using chromosome microdissection/hybrid selection approach we recently isolated a candidate oncogene CHD1L from 1q21 region. Our previous study has demonstrated that CHD1L had strong oncogenic ability, which could be effectively suppressed by siRNA against CHD1L. The molecular mechanism of CHD1L in tumorigenesis has been associated with its role in promoting cell proliferation.

Methodology/principal findings: To further investigate the in vivo oncogenic role of CHD1L, CHD1L ubiquitous-expression transgenic mouse model was generated. Spontaneous tumor formations were found in 10/41 (24.4%) transgenic mice, including 4 HCCs, but not in their 39 wild-type littermates. In addition, alcohol intoxication was used to induce hepatocyte pathological lesions and results found that overexpression of CHD1L in hepatocytes could promote tumor susceptibility in CHD1L-transgenic mice. To address the mechanism of CHD1L in promoting cell proliferation, DNA content between CHD1L-transgenic and wildtype mouse embryo fibroblasts (MEFs) was compared by flow cytometry. Flow cytometry results found that CHD1L could facilitate DNA synthesis and G1/S transition through the up-regulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, and CDK4, and down-regulation of Rb, p27(Kip1), and p53.

Conclusion/significance: Taken together, our data strongly support that CHD1L is a novel oncogene and plays an important role in HCC pathogenesis.

Figure 1. Generation of CHD1L-transgenic mouse model.

(A) Construction of human CHD1L gene in pCAGGS for the generation of CHD1L-transgenic mouse. (B) Four CHD1L-transgenic mouse (#3, 21, 26, and 38) founders were identified by PCR screening. Genomic DNA from cloned CHD1L was used as positive controls. (C) CHD1L expression in transgenic mouse (#38) was confirmed by Northern blot analysis. (D) Two founders (lines 21 and 38) were able to transmit the transgene to their offspring (P1). Genomic DNA was digested with BamHI and DNA fragment containing transgene CHD1L was detected by Southern blot analysis. The size of BamHI-DNA fragment was 6 kb in line 21 and 8 kb in line 38, implying their integrated sites in host genomic DNA are different .

Figure 2. Characterization of CHD1L-transgenic mice.

(A) Endogenous mouse CHD1L expression was tested by RT-PCR using mouse-specific primers. Weak expression of CHD1L was detected in liver and spleen. GAPDH was used as internal control. (B) Expression of transgene CHD1L in multiple tissues of transgenic and wildtype mice was studied by RT-PCR using human-specific primers in adult mice. GAPDH was used as internal control. (C) Expression of CHD1L in liver at different ages was tested by RT-PCR. 18S rRNA was used as loading control.

Figure 3. Ethanol intoxication promotes the susceptibility of liver tumor in CHD1L-transgenic mice.

(A) A visible liver tumor (left) and adipoma (right) were found in CHD1L-transgenic mice following the ethanol exposure. (B) Histological study confirms the liver tumor is HCC. (C) Representative example of severe dysplasia lesion observed in a CHD1L- transgenic mouse following the ethanol exposure. (D) Representative example of normal liver tissue observed in a wildtype mouse following the ethanol exposure. (E) PCNA staining results showed that the frequency of cell proliferation (PCNA positive staining cells, indicated by arrows) was significantly higher in CHD1L-transgenic mice than that in wildtype littermates. (F) Representative example of AFP positive staining detected in a CHD1L- transgenic mouse and AFP negative staining in a wildtype mouse, respectively, after the ethanol exposure.

Figure 4. Detection of spontaneous liver tumors in CHD1L-transgenic mice.

(A) Representative example of a visible liver tumor in one CHD1L-transgenic mouse (left), which was diagnosed as HCC by histological study (right). (B) Two liver tumors were found in one CHD1L-transgenic mouse (left, indicated by arrows) and histological study confirmed they are HCCs. (C and D) Representative examples of other two HCCs observed in CHD1L-transgenic mice.

Figure 5. Detection of spontaneous tumors in other organs in CHD1L-transgenic mice.

Besides HCC, several other different tumors were found in CHD1L-transgenic mice including salivary acinic cell adenocarcinoma (A and B), rhabdomyosarcoma (C), and colon adenocarcinoma (D).

Figure 6. Overexpression of CHD1L promotes cell proliferation in CHD1L-transgenic MEF.

(A) Detection of CHD1L expression in MEFs from CHD1L-transgenic (Tg) and wild type (Wt) mice by RT-PCR. GAPDH was used as internal control. (B) Examples of DNA content in CHD1L-transgenic and wild type MEFs detected by flow cytometry. (C) Western blot analyses indicated that Cyclin D1, Cyclin A and CDK2, 4 were up-regulated, whiles p53, Rb and p27^Kip1 were down-regulated in CHD1L-transgenic MEFs compared with their wild type MEFs (pool of two MEFs). β-actin was used as loading control.