Studies of the response of p53-lacZ transgenic mice have uncovered an unexpected induction of endogenous acid-β-galactosidase activity following whole body irradiation. Strong induction of endogenous enzyme activity is seen in a variety of mouse strains commonly used in the production of transgenes. The induction of endogenous enzyme activity therefore complicates the analysis of p53-lacZ transgenes and may also influence the analysis of radiation responses in other lacZ-reporter mice.
As an approach to study the transcriptional activation of the p53 tumour suppressor protein in vivo we produced transgenic mice containing a lacZ reporter gene under the control of a p53-response element (MacCallum et al., 1996). Transgenic animals were characterized by PCR and Southern blot analysis of the bacterial lacZ gene, which was shown to be present at multiple copies in the genome. Expression of the lacZ reporter was studied by the standard histochemical method for bacterial β-galactosidase using X-gal as substrate at pH 7.3 and reactions were performed overnight at 37°C. We reported that in adult p53-lacZ mice, β-galactosidase is essentially undetectable under normal conditions, which correlated with a lack of detectable p53 protein by immunohistochemistry. After irradiation, β-galactosidase was detected in specific areas of certain tissues (e.g. red pulp of the spleen and ductal epithelium of salivary gland), indicating that radiation-induced activation of p53 had caused transcriptional activation of the lacZ reporter gene. Differences were noted in the patterns of p53 protein and lacZ activation, suggesting that not all p53 protein is transcriptionally active following radiation exposure. For instance, p53 protein was induced in both the red- and white-pulp areas of the spleen following ionizing radiation, whereas β-galactosidase activity was confined to the red pulp. β-galactosidase activity was also seen in the developing brains of transgenic embryos, in keeping with the immunohistochemical identification of p53 protein in this embryonic tissue (MacCallum et al., 1996). These data suggested that the p53-lacZ transgenic mice represented a useful tool for determining transcriptionally active p53 in vivo. Two other groups also reported similar findings using independently generated p53-lacZ transgenic mice (Gottlieb et al., 1997; Komarova et al., 1997), supporting both the validity of our data and the value of this approach to study p53 transcriptional activity in vivo.
Results and Discussion
We are interested in genetic influences on the response to ionizing radiation and therefore began to cross the p53-lacZ transgenic mice onto different genetic backgrounds. Mice were analysed by PCR for the presence of the transgene and by enzyme histochemistry for β-galactosidase activity at pH 7.3. During the analysis of these mice, we were surprised to find that non-transgenic mice of the C57BL/6 strain showed β-galactosidase activity in the spleen and salivary gland following irradiation, in a remarkably similar pattern to that previously seen for the transgenic animals. Since these mice do not contain a bacterial lacZ gene, we reasoned that the histochemical staining must have been due to the induction of an endogenous enzyme activity following radiation exposure. The presence of endogenous β-galactosidase is well recognized in assays of transgenic mice (Hendrikx et al., 1994; Weiss et al., 1999) and is caused by the presence of the mammalian lysosomal enzyme in tissues including spleen, salivary gland and brain (Hatton and Lin, 1992; Nowroozi et al., 1998; Weiss et al., 1999). However, the detection of mammalian β-galactosidase does not usually cause problems in lacZ transgenes because of the relatively high level expression of the transgene and the different assay conditions used; mammalian lysosomal β-galactosidase has an acid pH optimum and exhibits greatly reduced activity under the standard lacZ conditions of neutral pH. To investigate whether the staining we had seen in non-transgenic mice was due to mammalian β-galactosidase, we irradiated groups of the original p53-lacZ strain and non-transgenic C57BL/6 animals and removed the spleens and salivary glands 24 h later from these and unirradiated control animals. Tissues were stained for β-galactosidase activity at 37°C using either acidic (pH 4.0) or neutral (pH 7.3) buffers. We found that non-irradiated spleens from transgenic or non-transgenic mice showed little enzyme staining. In contrast, intense β-galactosidase reactions were seen in the red pulp of irradiated transgenic and non-transgenic animals within 1 h of incubation at pH 4.0. Reactions performed at pH 7.3 showed the same distribution of staining but were much weaker and required an overnight incubation, the time of reaction used in our original report (MacCallum et al., 1996). In the salivary gland, staining was seen without irradiation, but was much stronger after radiation exposure. The reactions occurred much faster at pH 4.0 than pH 7.3, and again the distribution of staining appeared identical between transgenic and non-transgenic mice. Taken together, these observations demonstrate that endogenous acid β-galactosidase activity is induced following exposure to ionizing radiation, and this enzyme activity interferes with the identification of p53-driven lacZ activity in our transgenic mice. Indeed, the data suggest that the majority of β-galactosidase activity we found following radiation exposure in p53-lacZ transgenic mice is not due to p53-driven expression of the transgene, but is caused by endogenous acid β-galactosidase activity. Various methods can be used to differentially suppress endogenous β-galactosidase activity to help discriminate between the bacterial and mammalian enzymes when endogenous activity is a problem in the analysis of lacZ transgenes. In our mice, neither further manipulation of pH nor addition of D-galactose to the reaction mixture (Hendrikx et al., 1994; Weiss et al., 1999) provided a differential enhancement of staining in the p53-lacZ animals.
Since we could not observe any difference in the staining patterns between p53-lacZ transgenic mice and non-transgenic C57BL/6 mice, we considered whether the animals actually expressed the bacterially encoded enzyme, even though they contained multiple copies of the transgene. Adult transgenic mice were irradiated and the spleens removed at various times thereafter. As a control, murine T22 cells stably transfected with the identical p53-lacZ construct were also analysed. Northern blots were probed for lacZ or mdm-2 as a control mRNA which is a target for transcriptional up-regulation by p53. We could demonstrate an induction of both mdm-2 and lacZ mRNA in the T22 cell line after irradiation. In contrast, no lacZ mRNA was seen in irradiated transgenic spleens, although radiation increased the levels of mdm-2 mRNA in these samples.
Consequently, we conclude that our p53-lacZ transgenic mice do not express lacZ activity in a p53-dependent manner. The reason for this error lies in the unexpected finding of high level induction of endogenous β-galactosidase activity following irradiation. We have proposed elsewhere that the induction of this enzyme activity after irradiation in vivo reflects increased macrophage lysosomal activity required for efficient clearance of cells dying by radiation-induced apoptosis (Lorimore et al., 2001). An analysis of different strains of mice has shown that the level of acid β-galactosidase and the degree of enzyme induction after irradiation is highly variable. Of particular relevance is that three outbred strains of mice tested, CD1, Swiss and FVB, which are often used for breeding of transgenic mice, all show high levels of induction, similar to C57BL/6. Of other strains examined, both CBA/Ca and 129/Sv show lower levels of endogenous enzyme activity, and 24 h post-irradiation acid β-galactosidase activity is induced to a lesser degree than C57BL/6 and the above outbred strains. Consequently, these observations have importance not only for our mice, but also for any other lacZ-based reporter systems that are used to study radiation effects in vivo, including the other p53-lacZ transgenics reported at the same time as our original publication. We therefore suggest that alternative reporters, such as GFP and related fluorescent proteins, are more suitable for analysing radiation responses in transgenic animals. In addition, when using transgenic mice for these types of studies, the importance of genetic background on the measured response must be taken into account, and this will be particularly important when using crosses of different transgenes prepared with different and uncharacterized mixtures of genotypes. Finally, we would like to emphasize that these findings do not affect the validity of the immunohistochemical and Western blotting data contained in our original study (MacCallum et al., 1996). Indeed, subsequent work by ourselves and others has confirmed the major conclusions of the study regarding the tissue- and cell type-specific complexity of p53 responses to radiation.
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Coates, P., Lorimore, S., Rigat, B. et al. Induction of endogenous β-galactosidase by ionizing radiation complicates the analysis of p53-LacZ transgenic mice. Oncogene 20, 7096–7097 (2001). https://doi.org/10.1038/sj.onc.1204904
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