An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control - PubMed (original) (raw)

An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control

A Gualberto et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Although it is well-established that p53 functions as a tumor suppressor gene, certain mutations exhibit gain-of-function activities that increase oncogenic transformation. We have found a common class of p53 missense mutation that exhibits a dominant, gain-of-function activity that generates genomic instability. Fibroblasts from Li-Fraumeni syndrome heterozygotes with such mutations generate polyploid cells when exposed to spindle depolymerizing agents. Expression of such mutant alleles in normal fibroblasts yields the same phenotype. This class of dominant, gain-of-function p53 mutation (p53(RSC), relaxed spindle checkpoint allele) does not require the transcriptional activation function of p53 for this behavior. Thus p53 mutations can contribute to progression of a cancer cell not only by absence of p53 tumor suppressor activity but also by the presence of an activity that promotes genetic instability.

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Figures

Figure 1

Analysis of the status of the spindle cell cycle checkpoint in human fibroblasts. Cell cycle was analyzed by flow cytometry to determine the distribution of DNA content in NHF3 incubated without (a) or with (b) 500 ng/ml of colcemid for two population doublings. With increasing time in colcemid, an increasing fraction of normal cells accumulate in the G2/M peak. LFS fibroblasts (c_–_h) are described in Table 1 and ref. . LFS cells were processed for cell-cycle distribution of DNA content as for NHF, except for JML cells, which were incubated for 1 population doubling time. The three-dimensional plots represent cell number (y axis) versus BrdUrd (BUDR) incorporation (z) and PI (x). Plots show 104 cells. (i and j) Metaphase spreads from NHF cells expressing a p53RSC mutation (p53–143A) after two population doubling times in colcemid. Metaphase spreads with 92 (i) and 182 (j) chromosomes are presented.

Figure 2

The expression of a p53RSC (p53–143A) in NHFs (NHF29) alters the spindle cell cycle checkpoint. Flow cytometric analysis was performed to obtain the cell cycle distribution of DNA content in colcemid-treated NHFs infected with retroviruses that produce p53–143A mutant protein (b), HPV16 E6 protein (c), both p53–143A and HPV16 E6 proteins (d), or neither (a). Polyclonal populations at passage 1 were incubated and processed for cell cycle distribution of DNA content as in Fig. 1. Population doubling times for NHF29, NHF29-E6, NHF29-p53–143A, and NHF29-p53–143A+E6 were 30, 24, 30, and 36 h, respectively. wt refers to the presence of the wild-type allele, and ∗ indicates the polyploid population of cells. The x axis represents PI intensity and the y axis represents cell number.

Figure 3

Characterization of reporter activity and spindle cell cycle checkpoint induced by a transcription-deficient p53 mutant protein. (A) NHF3 cells were transiently cotransfected with 5 μg of MDR1 CAT reporter plasmid and 10 μg of either p53–281G or p53–281G,22,23 using Lipofectamine. After 48 h, cells were lysed and CAT activity was assayed using 14C-labeled chloramphenicol (31). CAT activity was observed only in cells cotransfected with the reporter alone nor cells cotransfected with p53-281G,22,23, and MDR1 CAT (lanes 1 and 3). (B) Flow cytometric analysis of NHF stably transfected with either p53–281G or p53–281G,22,23 in the presence (b and d) or absence (a and c) of colcemid. Colonies expressing mutant p53 were pooled and processed for cell cycle distribution of DNA content after colcemid treatment for 48 h as described in Fig. 1. Flow cytometry documented a decrease in apoptotic cells but no increase in polyploid cells.

Figure 4

(A) Loss of wild-type p53 activity in human cells does not generate polyploid cells. NHFs expressing HPV16 E6 protein [NHF1-E6 (a), NHF3-E6 (b), and NHF29-E6 (c)] were incubated without (−) or with (+) colcemid and analyzed as in Fig. 2. There is no change in the fraction of polyploid cells. Expression of the p53RSC 143A allelle in colcemid-treated cells produces polyploid populations (denoted by ∗) [NHF3–143A (d), NHF29–143A (e), and FF6–143A (f)]. (B) Western analysis of p53 protein in NHF3 fibroblasts after expression of an empty vector containing the neomycin resistance gene, or the HPV16 oncoproteins E6, E7, or E6/E7 together. (C) Production of E1B 19k affects cell death but not ploidy in human p53 null cells. JML cells were stably transfected by the calcium phosphate method with plasmids containing the neo resistance gene alone (a) or with E1B 19k (b). Drug-resistant colonies were pooled and subject to colcemid treatment for two population doubling times (48 h), and analyzed for DNA content by PI staining.

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An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control - PubMed (original) (raw)