Summary
Transgenic mice overexpressing a rWAP-p53 minigene containing an Arg-Leu mutation at amino acid 172 exhibit decreased lobuloalveolar development during late pregnancy and a failure of lactation. This is due to extensive apoptosis induced by this mutant p53 with pseudo-wildtype properties. Mice bearing pituitary isografts treated with the chemical carcinogen, DMBA, display a significant reduction in mammary tumor development compared to non-transgenic controls.
p53 knockout mice while of enormous utility present some limitations for the study of the role of p53 in breast cancer. Specifically when homozygous p53 knockout mice were administered the chemical carcinogen DMBA, the mice died from lymphomas and sarcomas before they developed mammary tumors. In order to study the role of wildtype p53 as well as specific p53 mutations in the initiation and progression of breast cancer, the rat whey acidic regulatory sequences were used to target a p53 minigene containing specific point mutations at amino acid 172(equivalent to human p53 aa175) to the mammary gland of transgenic mice. Lines of mice bearing two different mutations with different phenotypic and genotypic properties have been generated. The 172Arg-Leu mice express p53 with pseudo-wildtype properties capable of inducing p21 and mdm-2 and inhibiting PCNA expression. These mice displayed altered lobuloalveolar development and an increase in gamma-irradiation induced apoptosis. Females from the highest expressing line were unable to lactate. Mice expressing a dominant negative 172Arg-His mutation have also been generated. These mice do not exhibit any detectable alterations in mammary gland development and have a very low incidence of spontaneous mammary tumors. Mice bearing the 172Arg-Leu transgene and a pituitary isograft displayed a marked increase in apoptosis and a significant delay in DMBA-induced tumorigenesis, while those bearing the 172Arg-His dominant negative p53 transgene were more susceptible to DMBA-induced tumorigenesis.
Baolin Li, Norman Greenberg, L. Clifton Stephens, Raymond Meyn, Daniel Medina, and Jeffrey M. Rosen. (1994) Preferential overexpression of a 172Arg-Leu mutant p53 in the mammary gland of transgenic mice results in altered lobuloalveolar development. Cell Growth & Diff. 5:711-721.
Baolin Li, Frances S. Kittrell, Daniel Medina and Jeffrey M. Rosen (1995). Delay of dimethylbenz(a)anthracene-induced mammary tumorigenesis in transgenic mice by apoptosis induced by an unusual mutant p53 protein. Mol. Carcinogenesis 14:75-83.
Altered lobuloalveolar development and increased apoptosis during late pregancy. Failure of lactation. No apparent alteration in ductal development. Heterogeneous, interspersed expression of the transgene observed at day 15 of pregnancy in lobuloalveolar cells by IHC. The few normal lobules observed during early lactation did not express the p53 transgene suggesting that they arose by clonal expansion of cells not expressing the transgene during mid-pregnancy.
Histology
from 17 day pregnant mouse mammary glands of the p53Arg-Leu transgenic (left) and a
control (left) mouse stained using the TUNEL assay indicate apoptotic cells. Only one
apoptotic cell is present in the upper right hand corner of the control, but approximately
20% of the cells stained positively in the line expressing the p53Arg-Leu transgene.
Lines of mice bearing the Arg-Leu and Arg-His mutations are currently being crossed with mice bearing WAP-bcl-2 to determine if the effects of p53 on apoptosis are sufficient to explain its effect on DMBA-induced mammary carcinogenesis or if additional effects of p53 on DNA repair and genomic instability play an important role as well. The p53 transgenic are also being crossed with mice bearing WAP-desIGF-I and WAP-TGFalpha transgenes both of which develop multiple mammary tumors after several pregnancies and lactations. Overexpression of both of these growth factors has been shown to inhibit normal mammary gland involution.
The two different 172 p53 mutants exhibit markedly different genotypes and phenotypes. Induction of p21/ mdm-2 and inhibition of PCNA is observed for the Arg-Leu mutant characteristic of wildtype p53 transactivation properties. Decreased expression of milk protein mRNAs for mWAP and mfl-casein is also observed during late pregnancy and early lactation. The 172Arg-His dominant negative mutant did not significantly effect milk protein mRNA levels, no significant changes in p21 and PCNA mRNA levels was observed as a function of p53 expression.
Recent X-ray crystallographic analysis of the human p53 DNA binding region has indicated that aa 175(172 in the mouse) is located in the L2 loop in the vicinity of a Zn++ binding site that exerts major effects on the conformation of p53. Amino acid 175 is one of the hotspots in p53 accounting for almost 8% of the known mutations. While the Arg-His mutation is the prevalent mutation, the Arg-Leu mutation has also been observed in several breast cancers. These studies in transgenic mice have established that the functions of all mutant p53 are not equivalent, and that they may exert markedly different effects on mammary carcinogenesis. These mouse models should be invaluable in helping to define the role of p53 in the initiation and progression of breast cancer, specifically to determine the role that p53-induced apoptosis as contrasted to its effects on genomic instability and DNA repair in mammary carcinogenesis.
0.95 kb of the 5' flanking sequence of the rat WAP (-949/+33) gene fused to a murine p53 minigene as described in Lozano, G. and Levine, A.J. Mol. Carcinog.4:3-9, 1991. Removal of the ts mutation in the original p53 minigene at codon 135(Ala-Val) and introduction of either an Arg-Leu or an Arg-His mutation at aa 172 was accomplished using recombinant PCR mutagenesis.. Preferential expression in the mammary gland with some expression in the lung, intestine, spleen and ovary. Transgene expression is similar to that of most rWAP-based transgenes, i.e. expression increases at approximately day 10 of pregnancy through late pregnancy.
FVB
key words transgenic, p53, DMBA, lobuloalveolar development.
Jeffrey Rosen
Department of Cell Biology
Baylor College of Medicine
DeBakey M638
1 Baylor Plaza
Houston TX 77030-3498
Phone: 713-798-6210
Fax: 713-798-8012
e-mail: jrosen@mbcr.bcm.tmc.edu
More info about the Rosen lab.
last update: September 1998 |
