Jan 4, 1995 - This was manifested by the expression of the K light .... 70Z/3 pre-B cells which harbor a wild-type p53 protein ..... the cell type and stage.
The EMBO Journal vol.14 no.7 pp. 1392-1401, 1995
Accumulation of wild-type p53 protein upon y-irradiation induces a G2 arrest-dependent immunoglobulin K light chain gene expression Ronit Aloni-Grinstein, Dov Schwartz and Varda Rotterl Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel 'Corresponding author R.Aloni-Grinstein and D.Schwartz contributed equally to this study Communicated by M.Oren
The exposure of cells to DNA-damaging agents leads to the accumulation of wild-type p53 protein. Furthermore, overexpression of the wild-type p53, mediated by transfection of p53-coding cDNA, induced cells to undergo apoptosis or cell differentiation. In this study we found that the y-irradiation that caused the accumulation of wild-type p53 in 70Z/3 pre-B cells induced, in addition to apoptosis, cell differentiation. This was manifested by the expression of the K light chain immunoglobulin gene that coincided with the accumulation of cells at the G2 phase. Overexpression of mutant p53 in 70Z/3 cells interferes with both differentiation and accumulation of cells at the G2 phase, as well as with apoptosis, which were induced by y-irradiation. Furthermore, the increment in the wild-type p53 protein level following y-irradiation was disrupted in the mutant p53 overproducer-derived cell lines. This suggests that mutant p53 may exert a dominant negative effect in all of these activities. Data presented here show that while p53-induced apoptosis is associated with the G1 checkpoint, p53-mediated differentiation, which may be an additional pathway to escape the fixation of genetic errors, may be associated with the G2 growth arrest phase. Key words: differentiation/y-irradiation/G2/i/pS3
Introduction The p53 tumor suppressor gene plays an important role in the regulation of the normal cell cycle. Taken together, the observations that (i) p53 transactivates the waf-1/p21 (El-Deiry et al., 1993; Hunter, 1993; Wade-Harper et al., 1993), (ii) it is spatially regulated during the cell cycle and (iii) it functions at the boundary of the GO/GI and S phases, confirm the idea that the p53 protein is indeed a cell cycle control factor (Deppert et al., 1990; Diller et al., 1990; Ginsberg et al., 1991; Martinez et al., 1991; Shaulsky et al., 1991c). However, overexpression of p53 may force the cell to exit the cell cycle through either programmed cell death (apoptosis; Yonish-Rouach et al., 1991; Shaw et al., 1992) or cell differentiation (Feinstein et al., 1991; Shaulsky et al., 1991a,b; Aloni-Grinstein et al., 1993; Johnson et al., 1993; Rotter et al., 1993, 1994), depending on the cell type lineage.
Data accumulated from several laboratories have suggested that p53 plays a role in mediating the response to DNA damage by orchestrating both a GI growth arrest and the decision to undergo apoptosis. Levels of p53 rapidly increase upon DNA damage, mainly through the stabilization of p53 protein (Kastan et al., 1991; Kuerbitz et al., 1992; Lane, 1992; Livingstone et al., 1992; Lu and Lane, 1993). The notion that p53 is an important component of the apoptotic pathway was suggested following the observation that the expression of wild-type p53 in the murine Ml myeloid cell line induced cell apoptosis, manifested by specific cell morphology and typical DNA fragmentation (Yonish-Rouach et al., 1991). This conclusion was substantiated further by experiments showing that the induction of wild-type p53 in a human colon tumor-derived cell line (EB) led to apoptosis of in vitro proliferating cells and the development of regressor apoptotic tumors in vivo (Shaw et al., 1992). Results from our laboratory indicated that p53 may be involved in cell differentiation of the B cell lineage. Reconstitution of wild-type p53 expression in an early pre-B non-producer cell line advanced the cells to a more differentiated phenotype (Shaulsky et al., 1991a,b). Furthermore, the overexpression of wild-type p53 protein in Friend virus-transformed erythroleukemia cells, mediated by transfection of the temperature-sensitive p53 construct, caused an arrest in the GO/GI phase of the cell cycle and a rapid loss in viability, as well as the expression of the differentiation marker hemoglobin (Johnson et al., 1993). Similarly, the expression of wild-type p53 in K562, a chronic myelogenous leukemia (CML) blastic crisis cell line, caused the cells to express up to 50-fold more hemoglobin than the controls (Feinstein et al., 1991). Spermatogenesis (Almon et al., 1993; Schwartz et al., 1993) and B cell differentiation (Shaulsky et al., 1991a,b; Aloni-Grinstein et al., 1993) are two physiological pathways in which p53 was proved to be involved (Rotter et al., 1994); both pathways were shown to include DNA reshuffling and rearrangement. The fact that in spermatogenesis the expression of p53 was confined to the pachytene stage, which involves recombination and repair of DNA, suggests a direct role for p53 in these activities (Schwartz et al., 1993). In the B cell differentiation pathway, upregulation of p53 was found in the transition of early pre-B cells to pre-B cells, in the transition of pre-B cells to B cells and during processes that trigger non-secretor B cells to secrete immunoglobulins
(Aloni-Grinstein et al., 1993). The fact that upregulation of p53 was found to be associated with B cell differentiation on the one hand, and the observation that DNA damaging agents induce upregulation of p53 on the other, prompted us to examine whether damaging DNA induced a p53-mediated cell differentiation. To test this notion, we exposed cells to an
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Fig. 1. Effect of y-irradiation on the levels of p53 protein. (A) 70Z/3 cells expressing wild-type p53 were exposed to 400 or 800 rads. At various time points cells were labeled with [35S]methionine and cell extracts were immunoprecipitated with the PAb-421 anti-p53 antibodies. (B) An additional quantification of the newly synthesized p53 protein was performed by increasing the irradiation dose. 6 h later cells underwent a specific immunoprecipitation with PAb-421. (C) Steady-state levels of p53 protein, 6 h following y-irradiation, were estimated by Westem blot analysis using the PAb-421 antibody.
DNA-damaging stress, such as y-irradiation, and examined whether this modified their p53-dependent cell differentiation. To that end, we took advantage of the 70Z/3 pre-B cell line which expresses wild-type p53 and can be induced to undergo cell differentiation, manifested by the expression of the K light chain gene (AloniGrinstein et al., 1993). We found that y-irradiation of 70Z/ 3 pre-B cells induced an increase in the p53 protein levels and a change in the cell cycle pattern that were followed by cell differentiation. These wild-type p53-mediated activities were abrogated by the overexpression of mutant p53. exogenous
Results Effect of y-irradiation on p53 protein expression in the 70Z/3 pre-B cell line 70Z/3 pre-B cells which harbor a wild-type p53 protein were exposed to 400 and 800 rads. At various time points following irradiation, cells were metabolically labeled and examined for p53 protein expression. Immunoprecipitation of cell extracts with PAb-421 anti-p53 monoclonal antibodies showed a marked increase in the synthesis of the p53 protein as early as 3 h post-irradiation which decreased with time (Figure LA). Because equal amounts of radioactively labeled protein were examined in each of the experiments (400 and 800 rads), it appears that the effect of y-irradiation was more pronounced at higher levels of
y-irradiation. To quantify this correlation further between the increased levels of p53 and irradiation doses, 70Z/3 cells were exposed to gradually increasing doses of irradiation (200-800 rads), and p53 expression was evaluated 6 h post-irradiation by either immunoprecipitation (Figure iB) or Western blot analysis (Figure IC). As can be seen, an increase in the irradiation dose correlated with increased synthesis of the p53 protein (Figure 1B), as well as with higher levels of steady state p53 protein (Figure IC).
Induction of differentiation in 7OZ/3 cells following y-irradiation In the following experiment we examined whether the increased p53 protein expression mediated by y-irradiation in the 70Z/3 pre-B cells induced their differentiation. Cells were y-irradiated at 400 rads and were drawn 3 and 6 h post-irradiation. Differentiation of the 70Z/3 pre-B cells is expected to induce the expression of the rearranged Kc light chain immunoglobulin gene that can be measured by PCR (LeBoeuf et al., 1989; Aloni-Grinstein et al., 1993). PCR analysis was performed using a specific pair of primers mapping to the sites bordering the rearrangement junctions (see Materials and methods). In the event of a productive K light chain gene rearrangement, an amplified fragment of 400 bp is expected (LeBoeuf et al., 1989; Aloni-Grinstein et al., 1993). Using this approach we detected the expected PCR fragment as early as 3 h post1393
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Fig. 2. Induction of K light chain expression following y-irradiation. (A) 70Z/3 cells were exposed to 400 rads. At various time points following irradiation, cells were examined by PCR analysis for the specific expression of K light chain immunoglobulin. Control cells were treated with LPS; M, molecular size marker. (B) An evaluation of mRNA content was carried out by PCR analysis of the same cDNA with HPRT-specific primers. (C) Confirmation of the identity of the PCR-derived K light chain product was carried out by hybridization with a radioactively labeled 'K light chain-specific DNA probe.
y-irradiation, which was further augmented as time elapsed. cDNA produced from cells which were treated for 24 h with lipopolysaccharide (LPS) served as a positive control (Figure 2A). Non-irradiated 70Z/3 cells exhibited no K light chain-specific PCR product under the same conditions. When the same cDNA samples were primed with hypoxanthine phosphoribosyl transferase (HPRT)-specific primers, an expected band with the same intensity was found in all samples (Figure 2B). Blotting the PCR product and hybridizing the filter with a K light chain-specific radiolabeled probe confirmed that the PCR-amplified band represents the K light chain gene product (Figure 2C). Induction of apoptosis in 70Z/3 cells following y-irradiation In the following experiment we examined y-irradiated 70Z/3 cells for their content of fragmented DNA which is indicative for apoptosis. To that end, 70Z/3 cells were exposed to increasing doses of y-irradiation and 6 h later DNA was extracted and analyzed for the appearance of fragmented DNA. Figure 3 shows that a ladder of fragmented DNA starts appearing in the DNA of cells that were exposed to ;700 rads. When the same cells were treated with LPS, a differentiation inducer, no ladder of fragmented DNA was detected as late as 23 h following treatment. As a response to DNA damage, pre-B cells such as the 70Z/3 were induced to undergo an apoptotic process. LPS treatment does not seem to involve apoptosis under the present experimental conditions.
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