Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model

Abstract

Using an improved system for the functional identification of active antisense fragments, we have isolated antisense fragments which inactivate the p53 tumour suppressor gene. These antisense fragments map in two small regions between nt 350 and 700 and nt 800 and 950 of the coding sequence. These antisense fragments appear to act by inhibition of p53 mRNA translation both in vivo and in vitro. Expression of these antisense fragments overcame the p53-induced growth arrest in a cell line which expresses a thermolabile mutant of p53 and extended the in vitro lifespan of primary mouse embryonic fibroblasts. Continued expression of the p53 antisense fragment contributed to immortalisation of primary mouse fibroblasts. Subsequent elimination of the antisense fragment in these immortalised cells led to restoration of p53 expression and growth arrest, indicating that immortal cells continuously require inactivation of p53. Expression of MDM2 or SV40 large T antigen, but not E7 nor oncogenic ras, overcomes the arrest induced by restoration of p53 expression. Functional inactivation of both p21 and bax (by overexpression of Bcl2), but not either alone, allowed some bypass of p53-induced growth arrest, indicating that multiple transcriptional targets of p53 may mediate its antiproliferative action. The ability to conditionally inactivate and subsequently restore normal gene function may be extremely valuable for genetic analysis of genes for which loss-of-function is involved in specific phenotypes.

Figure 1

Expression of p53 antisense fragments overcomes p53-induced growth arrest. p53ts p53–/– MEF cells were infected with viruses that expressed p53 antisense fragments (numbers 15, 55, 57, 61, 71, 17 and 2) or empty vector (v). After drug selection for virally transduced cells, 5 × 10⁴ cells were plated in 10 cm dishes and grown at restrictive temperature (32°C) for 15 days. Cells were then fixed and stained with crystal violet.

Figure 2

Localisation and activity of p53 antisense fragments. (A) p53 antisense fragments isolated following phenotypic selection mapped to two regions, RI and RII, of the p53 coding sequence. Expression of these antisense fragments reduces p53 protein levels in vivo. p53ts p53–/– MEF cells were infected with viruses expressing different antisense fragments (numbers 15, 55, 57, 61, 71, 17 and 2) or control vector (v). Infectants were selected, cells were lysed and p53 protein levels were assayed by western blot. (B) Expression of p53 antisense fragments reduces the expression of p21, a transcriptional target of p53. Two clones (A and B) of antisense-expressing p53ts p53–/– MEF cells (numbers 15, 55, 71 and 2) or vector alone (c) were selected and shifted to 32°C for induction of p53 activity. After 24 h, cells were lysed and levels of p53 or p21 protein were analysed by western blot. (C) Expression of p53 antisense fragments does not affect levels of p53 mRNA in vivo. Northern blot of p53 mRNA in colonies expressing p53 antisense fragments. Total RNA was prepared from antisense-expressing clones as above, separated by gel electrophoresis, transferred to Hybond membranes and probed with a radiolabelled p53-specific probe (Upper). (Lower) Ethidium bromide stained 28S band of the total RNA. The results of densitometric analysis of p53 versus 28S mRNA bands showed no significant variation with respect to control cells (not shown).

Figure 2

Localisation and activity of p53 antisense fragments. (A) p53 antisense fragments isolated following phenotypic selection mapped to two regions, RI and RII, of the p53 coding sequence. Expression of these antisense fragments reduces p53 protein levels in vivo. p53ts p53–/– MEF cells were infected with viruses expressing different antisense fragments (numbers 15, 55, 57, 61, 71, 17 and 2) or control vector (v). Infectants were selected, cells were lysed and p53 protein levels were assayed by western blot. (B) Expression of p53 antisense fragments reduces the expression of p21, a transcriptional target of p53. Two clones (A and B) of antisense-expressing p53ts p53–/– MEF cells (numbers 15, 55, 71 and 2) or vector alone (c) were selected and shifted to 32°C for induction of p53 activity. After 24 h, cells were lysed and levels of p53 or p21 protein were analysed by western blot. (C) Expression of p53 antisense fragments does not affect levels of p53 mRNA in vivo. Northern blot of p53 mRNA in colonies expressing p53 antisense fragments. Total RNA was prepared from antisense-expressing clones as above, separated by gel electrophoresis, transferred to Hybond membranes and probed with a radiolabelled p53-specific probe (Upper). (Lower) Ethidium bromide stained 28S band of the total RNA. The results of densitometric analysis of p53 versus 28S mRNA bands showed no significant variation with respect to control cells (not shown).

Figure 3

Inhibition of p53 translation by expression of p53 antisense fragments in vitro. (A) p53 protein was translated in vitro in the presence of p53 antisense fragment transcripts (numbers 15, 55, 71 and 2), control vector (v) or no additional vector (c). (B) In vitro translation of p53 protein in the presence of different molar ratios of p53 antisense transcripts (numbers 15 and 2) or control vector. (C) Effect of preincubation of antisense transcripts on translation of p53 protein.

Figure 4

Expression of p53 antisense fragments induces an extended lifespan of primary MEFs. Primary MEFs were infected with antisense-expressing viruses (numbers 15, 55, 57, 61, 71, 17 and 2) or control vector (v) in PD 2. After selection, cells were passaged until PD 12, at which time 10⁵ cells were plated in 10 cm dishes and grown for 15 days. Cells were then fixed and stained with crystal violet. Cells which exhibit an extended lifespan are capable of colony formation under these conditions.

Figure 5

Recovery of p53 expression leads to growth arrest in immortalised cells. Presenescent MEFs were infected with viruses directing the expression of CRE-excisable pMarxIVp53αs71 or pMarxIVp53αs2 and immortalised using a 3T3 immortalisation protocol. Immortalised cells (p53αs^CR) were infected with a control vector or pWZL-Hygro-CRE to excise the integrated p53 antisense construct leading to restoration of p53 expression. p53αs^CR2 cells were infected with a virus expressing dominant negative p53, p53(175H), prior to CRE infection. (A) Levels of expression of wild-type p53 (wt p53) or mutant p53 (mutp53) in the p53αs^CR cells after infection with CRE or vector alone. (B) Colony formation of p53αs^CR cells infected with viruses carrying CRE or vector alone. Cells were plated at equal densities, cultured in the presence of hygromycin for 10–15 days, fixed and stained with crystal violet.

Figure 6

Bypass of growth arrest induced by restoration of p53 function. p53αs^CR cells were infected with viruses directing the expression of various genes or a p53αs^CR cell line was constructed in a p21–/– genetic background. Cells were then infected with a control or CRE-expressing virus, plated and grown for 10–15 days under selective conditions, fixed and stained with crystal violet.