Probasin promoter-driven expression of ID1 is not sufficient for carcinogenesis in rodent prostate

Abstract

Inhibitor of DNA-binding-1 (ID1) negatively regulates cell differentiation and senescence, and enhances cellular proliferation and angiogenesis. Elevated levels of ID1 have been found in a variety of cancers, including prostate cancer, but whether ID1 has a tumourigenic role remains to be established. We established heterozygous and homozygous ID1-transgenic mouse lines driven by the prostate-specific probasin promoter (-426 to +28 bp). Although elevated levels of ID1 were confirmed by RT-PCR, immunohistochemistry, and Western blot analysis, there were no morphological changes identified in the prostate of transgenic mice at 26 and 52 weeks. Thus, overexpression of ID1 alone is not sufficient to drive neoplastic change in mouse prostate.

Figure 1

Id1 transgene construction. (A) The Id1 coding region was isolated from mouse skin, sequenced, and used in the construction of the Pb-ID1 vector. (B) This vector was then digested with the restriction enzymes SalI and MluI to produce the transgene.

Figure 2

A breeding diagram and confirmation of heterozygous and homozygous status with PCR analysis using transgene-specific primers. (A) When heterozygous animals were crossed with wild-type animals, only half of the offspring incorporated the transgene. (B) When homozygous animals were crossed with wild-type animals, all offspring incorporated the transgene.

Figure 3

Identification of Id1 mRNA expression in transgenic animals. (A) Heterozygous animals exhibited higher levels of total Id1 mRNA than did their wild-type counterparts, and the homozygous animals exhibited a further increase. National Institutes of Health image analysis was used to quantitate the intensity of the Id1 and housekeeping bands. (B) The ratio of Id1 band intensity to the intensity of the housekeeping band is depicted in the bar graph.

Figure 4

Detection of ID1 protein in transgenic animals. Top: Immunohistochemistry was performed on homozygous transgenic animals (A,B) and wild-type animals (C). An isotype control shows no nonspecific binding to the tissue (D). The majority of stain is restricted to epithelial cells of the prostate. Bottom: Western blot for ID1 was performed with proteins taken from the ventral lobes of two wild-type (Wt1 and Wt2) and three homozygous transgenic animals (Tr1, Tr2, and Tr3). HeLa cell lysates were used as a positive control and GAPDH as a loading control.

Figure 5

Histological assessment of transgenic animals. Hematoxylin and eosin–stained sections from transgenic and wild-type animals at 52 weeks. A and B represent a wild-type animal, whereas C–F represent two lines of ID1-homozygous transgenic animals. There are no observable morphological differences in transgenic animals when compared with age-matched wild-type animals.