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A novel p53-inducible gene coding for a microtubule-localized protein with G2-phase-specific expression

1998; Springer Nature; Volume: 17; Issue: 17 Linguagem: Inglês

10.1093/emboj/17.17.5015

1460-2075

René Utrera, Licio Collavin, Dejan Lazarević, Domenico Delia, Claudio Schneider,

Epigenetics and DNA Methylation

Article1 September 1998free access A novel p53-inducible gene coding for a microtubule-localized protein with G2-phase-specific expression René Utrera René Utrera Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Present address: Departamento Biologia Celular, Universidad Simon Bolivar, Apartado 89.000, Valle de Sartenejas, Caracas, 1081-A Venezuela Search for more papers by this author Licio Collavin Licio Collavin Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Search for more papers by this author Dejan Lazarević Dejan Lazarević Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Search for more papers by this author Domenico Delia Domenico Delia Istituto Nazionale Tumori, Div. Oncologia Sperimentale A, Via G. Venezian 1, 20133 Milano, Italy Search for more papers by this author Claudio Schneider Corresponding Author Claudio Schneider Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Dipartimento Scienze e Tecnologie Biomediche, Universita' degli Studi di Udine, p.le Kolbe 1, 33100 Udine, Italy Search for more papers by this author René Utrera René Utrera Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Present address: Departamento Biologia Celular, Universidad Simon Bolivar, Apartado 89.000, Valle de Sartenejas, Caracas, 1081-A Venezuela Search for more papers by this author Licio Collavin Licio Collavin Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Search for more papers by this author Dejan Lazarević Dejan Lazarević Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Search for more papers by this author Domenico Delia Domenico Delia Istituto Nazionale Tumori, Div. Oncologia Sperimentale A, Via G. Venezian 1, 20133 Milano, Italy Search for more papers by this author Claudio Schneider Corresponding Author Claudio Schneider Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy Dipartimento Scienze e Tecnologie Biomediche, Universita' degli Studi di Udine, p.le Kolbe 1, 33100 Udine, Italy Search for more papers by this author Author Information René Utrera1,2, Licio Collavin1, Dejan Lazarević1, Domenico Delia3 and Claudio Schneider 1,4 1Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano 99, 34012 Trieste, Italy 2Present address: Departamento Biologia Celular, Universidad Simon Bolivar, Apartado 89.000, Valle de Sartenejas, Caracas, 1081-A Venezuela 3Istituto Nazionale Tumori, Div. Oncologia Sperimentale A, Via G. Venezian 1, 20133 Milano, Italy 4Dipartimento Scienze e Tecnologie Biomediche, Universita' degli Studi di Udine, p.le Kolbe 1, 33100 Udine, Italy ‡R.Utrera, L.Collavin and D.Lazarević contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5015-5025https://doi.org/10.1093/emboj/17.17.5015 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Wild-type (wt) p53 can act as a sequence-specific transcriptional activator and it is believed that p53 elicits at least part of its biological effects by regulating the expression of specific target genes. By using a differential subtractive hybridization approach in a murine cell line stably transfected with a temperature-sensitive p53 mutant (Val135), we isolated a set of genes markedly induced by wt p53. One of them, provisionally named B99, was further characterized; its transcriptional induction was dependent on wt p53 function and the corresponding protein product was shown to accumulate after DNA damage in different cell types. Immunofluorescence analysis located the B99 protein to the microtubule network. Flow cytometry revealed that upon activation of p53 function the endogenous B99 protein was selectively induced in the G2 fraction of the cell population. When B99 was ectopically expressed in p53-null murine fibroblasts, B99-transfected cells displayed an increased fraction with a 4N DNA content, indicative of interference with G2 phase progression. Taken together these data suggest that B99 might play a role in mediating specific biological activities of wt p53 during the G2 phase. Introduction One of the most frequent genetic events associated with the development of human cancer is the inactivation or loss of the normal function of the tumour suppressor gene p53 (Hollstein et al., 1991). A large body of evidence has accumulated suggesting wild-type (wt) p53 (but not its mutant derivatives) as a mediator of multiple antiproliferative activities including cell-cycle arrest, induction of apoptotic cell death and suppression of oncogene-mediated transformation (for review see Bates and Vousden, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). The biochemical mechanisms by which wt p53 can elicit all of these activities have not been completely defined. However, an essential aspect of p53 function depends on it being a transcription factor. In fact, p53 contains a strong transcriptional activation domain within its N-terminus, it binds DNA with a well-defined cognate binding sequence and activates transcription of genes carrying such consensus sites in their promoter sequences (Lin et al., 1994; Levine, 1997). Apart from its function as a transcriptional activator, p53 can also repress the transcription of several genes (Sabbatini et al., 1995; Murphy et al., 1996 and references therein) and important aspects of its biological functions might be mediated through direct interaction with specific protein partners (Ko and Prives, 1996; Levine, 1997 and references therein). Much evidence indicates that p53 acts primarily by arresting cells at the G1/S restriction point and this effect has been clearly correlated to induction of Waf-1, a potent inhibitor of G1-specific cyclin-dependent kinases (El-Deiry et al., 1993; Xiong et al., 1993; Waldman et al., 1996). Nevertheless, studies conducted on cells derived from p21Waf1−/− mice showed that loss of Waf-1 only partially abolishes the G1 arrest function associated with wt p53 (Brugarolas et al., 1995; Deng et al., 1995). So this is probably mediated by multiple pathways in which other p53-regulated genes might play a role (Levine, 1997). It has also been suggested that wt p53 plays a role in control of the G2 phase. p53 has been shown to interact with the centrosomes (Brown et al., 1994) and p53-null cells frequently develop an aberrant number of centrosomes (Fukasawa et al., 1996). Fibroblasts from p53−/− mice are deficient in the checkpoint that blocks cell-cycle and prevents S phase entry when cells are treated with spindle-inhibitory drugs such as nocodazole (Cross et al., 1995). Furthermore, in several cell lines carrying an inducible p53 allele, it has been shown that p53 activation can arrest at both G1/S and G2/M phases of the cell cycle (Agarwal et al., 1995; Stewart et al., 1995). While the importance of p21Waf1 in G1 arrest is well established, the role of p21Waf1 in such G2-specific functions is still unclear (Deng et al., 1995; Lanni and Jacks, 1998) and it is reasonable to hypothesize that other p53-target genes could be involved. A recent report showed that wt p53 specifically upregulates the σ member of the 14-3-3 protein family, and that 14-3-3σ is capable of inducing G2/M cell-cycle arrest, possibly by targeting cdc25c phosphatase (Furnari et al., 1997; Hermeking et al., 1997). It is also worth noting that p53 negatively regulates the expression of MAP4, a microtubule stabilizing protein whose intracellular relocalization has been correlated to p53-dependent apoptosis (Olmsted, 1991; Murphy et al., 1996). This links p53 to microtubule dynamics and cytoskeletal functions, but also suggests a possible correlation to G2-specific cell-cycle events; in fact, MAP4 is phosphorylated by cdc2/B kinase at the G2/M transition (Ookata et al., 1995), and progression of the cell cycle through G2/M is linked to the status of microtubules (Andreassen and Margolis, 1994). To further characterize p53 functions with respect to cell-cycle control, we screened for p53 target genes in a cellular system in which regulated induction of wt p53 causes an efficient and reversible growth arrest with no significant evidence of apoptosis. In this study we report the cloning of six cDNAs to be considered as potential p53-responsive genes. We focus on one of these, provisionally named B99, and provide evidence that it is a direct target for transcriptional activation by p53. Clone B99 encodes a novel protein that is localized to microtubules. When we characterized the p53-dependent regulation of B99, we found that induction of B99 protein was restricted to the G2 population of cells, providing a notable example of a p53 target gene with cell-cycle-dependent expression. Ectopic expression of clone B99 reduced cell growth and caused a delay at the G2/M phase of the cell cycle, as determined by flow cytometry. This evidence suggests that B99 could be involved in mediating the G2-specific biological activities of wt p53. Results Isolation of novel p53-regulated genes by subtractive hybridization In order to identify novel transcripts regulated upon induction of biochemically active p53, we used a subtractive hybridization approach in Val5 cells (Wu and Levine, 1994). The Val5 cell line is derived from p53-deficient Balb/c (10)1 mouse fibroblasts (Harvey and Levine, 1991) by stable transfection of the temperature-sensitive Val135 allele of murine p53 (Michalovitz et al., 1990; Martinez et al., 1991); at the permissive temperature of 32°C these cells upregulate p53 transcriptional targets like waf-1 or mdm-2 (Figure 1) and undergo an efficient and reversible G1 arrest (Del Sal et al., 1996). Figure 1.Northern blot analysis of cDNA clones isolated during the screening. Total RNA was prepared from Val5 cells cultured at either 32 or 37°C. Lanes labelled 32°C contain a 1:1 mixture of RNA extracted 12 and 24 h after temperature down shift. 10 μg of RNA were loaded on each lane and hybridizations were performed under high stringency conditions. Exposure times varied between overnight and 7 days at −80°C with intensifying screens. Blots were also probed for GAPDH as a control. Download figure Download PowerPoint Two subtracted cDNA probes, one representing mRNAs expressed at the permissive temperature (target probe) and the other representing mRNAs expressed at the restrictive temperature (driver probe), were used in a differential-subtractive screening of a cDNA library constructed from mRNA of Val5 cells grown at 32°C. Enrichment and specificity of target and driver probes were evaluated by slot-blot analysis on a panel of sample genes, before and after subtractive hybridization. Transcripts abundantly expressed, such as GAPDH and 28S RNA, were significantly subtracted; in contrast, known p53-induced genes such as waf-1 and mdm-2, were significantly enriched in the subtracted target probe (data not shown). More than 500 plaques corresponding to differential signals were picked from the primary screening and analysed in a secondary screening by Southern blots. Polyclonal plaques corresponding to primary signals were excised in vivo, digested at the cloning sites, run in duplicate on the same agarose gels and blotted. Each of the duplicate blots was hybridized either with the target or the driver probes used for the primary screening. DNA fragments corresponding to differential signals were excised from agarose gels and used as probes to evaluate the expression of the corresponding transcripts in Val5 cells at 37 and 32°C. During the screening several clones corresponding to waf-1 and mdm-2 were identified, thus confirming the reliability of the technique. Figure 1 displays a panel of Northern blots corresponding to six regulated cDNA clones detected in Val5 cells, together with waf-1 and mdm-2. The clones appear heterogeneous in the level of expression, corresponding to medium to low abundance mRNAs. Inserts corresponding to purified plaques were sequenced at both ends and compared with nucleic acid and protein databases using the NCBI blast server. Three clones showed significant homology to known genes (not shown). Three failed to match any sequence in the databases and to date are to be considered potential new genes. We hereby report the characterization of the insert from clone B99, while the other isolated clones will be described elsewhere. p53-dependent regulation of B99 mRNA in murine fibroblasts We analysed the mRNA expression of clone B99 by Northern blot, comparing its regulation in Val5 cells with its expression in the recipient Balb/c (10)1 and in Balb/c Val135(25–26) cells. The latter is a cell line derived from p53-deficient Balb/c (10)1 fibroblasts, stably expressing the temperature sensitive (ts) p53 mutant (Val135) additionally carrying a double point mutation in the transactivation domain that renders it transcriptionally inactive (Lin et al., 1994). As reported in Figure 2A, column 2, B99 mRNA was strongly induced in Val5 cells after 12 h at the permissive temperature, with a decrease after 24 h. No significant variations in the mRNA levels of B99 were observed in the recipient p53-null cells at 32 or 37°C (Figure 2A, column 1), thus indicating that upregulation is not a consequence of temperature shift. Importantly, no induction of B99 mRNA was observed in Val135 (25–26) cells shifted to 32°C (Figure 2A, column 3), where p53 is in wt conformation, binds to target sites on DNA, but is incapable of transcriptional activity (Lin et al., 1994), thus suggesting that transactivation by wt p53 is essential for enhanced expression of this clone. As a control, the same Northern blots were hybridized with a waf-1 probe; induction of waf-1 in Val5 was observed both after 12 and 24 h at 32°C, while no waf-1 mRNA could be detected at either 37 or 32°C in both the Balb/c (10)1 or the Val135(25–26) cells. Figure 2.Northern blot analysis of B99 mRNA expression in murine cell lines. 10 μg of total RNA were loaded on each lane and hybridization was performed under high-stringency conditions. Blots were hybridized with a GAPDH probe as a loading control. (A) Regulation in Val5 and control cell lines. RNA was prepared from the indicated cell lines cultured at 37°C or maintained at 32°C for the indicated time. Balb/c (10)1 are murine fibroblasts with both p53 alleles deleted. Val5 are Balb/c (10)1 cells stably expressing the ts Val135 mutant of murine p53. Val135(25–26) are Balb/c (10)1 cells stably transfected with a derivative of the ts p53 Val135 mutant carrying two additional point mutations (residues 25 and 26) that abrogate its transcriptional activation function. (B) Regulation in the absence of protein neosynthesis. Total RNA was prepared from Val5 cells grown at 37°C or kept for 6 h at 32°C in the absence or in the presence of 5 μg/ml of the protein synthesis inhibitor cycloheximide. (C) Regulation of B99 mRNA in UV-treated mouse fibroblasts. Total RNA was prepared from NIH 3T3 cells or p53−/− mouse embryo fibroblasts at the indicated time points after UV irradiation (10 J/m2). waf-1 mRNA was analysed in the same experiment as a control of p53 activation. Levels of B99 and waf-1 are not comparable since exposure times were different. Download figure Download PowerPoint To test whether induction of B99 might be a secondary consequence of p53 activation, expression of B99 mRNA was analysed in Val5 cells in the absence of de novo protein synthesis. No significant B99 mRNA stabilization could be detected upon cycloheximide treatment of Val5 cells maintained at 37°C (not shown). As can be observed in Figure 2B, B99 mRNA was clearly induced after 6 h at 32°C both in the absence or in the presence of 5 μg/ml cycloheximide, as was waf-1 mRNA, analysed as a control. Under these conditions protein synthesis was efficiently inhibited, as determined by the lack of p21Waf1 protein induction observed by immunofluorescence (not shown). We conclude that de novo protein synthesis is not required for induction of B99 transcription thus providing further evidence that B99 might be a transcriptional target of p53 in these cells. To test whether B99 might be regulated by p53 under more physiological conditions, B99 mRNA expression was analysed in mouse fibroblasts subjected to stimuli known to activate endogenous p53 function. For this purpose NIH 3T3 cells, which are wild-type for p53 (Hermeking and Eick, 1994; Del Sal et al., 1995), and fibroblasts from p53-nullizygous (p53−/−) mice were treated with UV light (10 J/m2) and B99 mRNA levels were analysed by Northern blot at 12 and 18 h post-irradiation. As shown in Figure 2C, significant induction of B99 mRNA expression was detected in UV-irradiated NIH 3T3 cells as compared with the basal level in untreated cells. On the contrary, B99 mRNA was not significantly induced by UV treatment in p53−/− MEFs. As a control, waf-1 mRNA was analysed in the same experiments as a marker of p53-dependent transcriptional activity. Thus, UV-induced DNA damage causes upregulation of B99 mRNA in NIH 3T3, but not in p53-null fibroblasts. Identification of a p53-responsive element within the B99 gene To better characterize the p53 dependency of B99 transcriptional regulation a screening was performed on a mouse genomic library. A clone was selected containing the 5′ end of the cDNA. Restriction digests of this clone were Southern blotted and hybridized at low stringency with a labelled degenerated oligonucleotide probe corresponding to the consensus p53 binding sequence described by El-Deiry (1992). A 2.8 kb BglII fragment resulted positive to hybridization; this fragment was isolated and fully sequenced. This genomic segment contained the most 5′ sequence of B99 cDNA and its structure is schematically summarized in Figure 3A. An intron was found starting at nucleotide 26 after the first ATG and extending to the 3′ end of the analysed fragment. A tripartite sequence conforming to the consensus p53 binding site was identified at position −127 from the ATG. Interestingly this sequence contains three half-site decamers, separated respectively by 0 and 1 nucleotides (Figure 3A). Figure 3.Identification of a p53-responsive element within the B99 gene. (A) Schematic representation of the genomic clone analysed and structure of the CAT reporter constructs. The position of the p53-responsive site identified is indicated, together with its alignment to the consensus p53-binding sequence (El-Deiry et al., 1992). (B) p53 responsiveness of B99 promoter fragments. The constructs indicated were cotransfected in p53-null Balb/c(10)1 cells with either wild-type p53, mutant p53 or empty expression plasmids. CAT activity was assayed 24 h after transfection. An MDM-2 promoter construct (pBP100CAT) was used as a positive control. Download figure Download PowerPoint The entire BglII fragment and corresponding 5′ deletions were cloned in front of a CAT reporter. The constructs were transfected in p53-null Balb/c (10)1 cells alone or together with an expression vector containing either wt p53 or the KH215 mutant version (Finlay et al., 1988). As shown in Figure 3B, the genomic segment (33.2) contains a powerful promoter, capable of driving abundant expression of the reporter gene in (10)1 cells. As expected, cotransfection of wt p53 enhanced such expression. Further deletions abolished constitutive transcription from this promoter, unveiling a strong regulation by p53. As shown in Figure 3B, a shorter segment (PvuII) starting at nucleotide −312 with respect to the first ATG was sufficient to confer p53-dependent expression to the CAT reporter. This transactivation was strictly dependent on wt p53 function and was not observed with a mutant p53 protein. When the p53 binding sequence was removed by restriction (ΔPstI) the observed regulation was lost (Figure 3B). We can conclude that a functional p53-responsive element is located in the close vicinity of the B99 promoter, providing the molecular basis for the observed regulation by p53. B99 encodes a novel protein whose expression is regulated by wt p53 Several signals corresponding to clone B99 were isolated during the screening. Of 30 plaques analysed, two appeared to contain a near full length cDNA, predicted to be ∼2.8 kb by Northern blot analysis. DNA sequencing revealed an open reading frame encoding a polypeptide of 741 amino acids, with an ATG codon at position 96 and an in-frame termination codon at nucleotide 2319 (Figure 4A). The protein is rich in positively charged residues and has a predicted isoelectric point of 10.17. Homology search of protein databases revealed no strong similarities to any known gene product. A low homology was found with the proline-rich domain of microtubule-associated protein MAP4 (Figure 4B), a region which has been reported to mediate MAP4 interaction with cyclin B (Ookata et al., 1995). Figure 4.(A) cDNA sequence of clone B99. The amino acid sequence was obtained by conceptual translation of the cDNA, starting at nucleotide 96 and terminating at nucleotide 2319. (B) Schematic structure of MAP4 and similarity with B99. MAP4 is divided into an N-terminal projection domain and a C-terminal region involved in interaction with microtubules, further subdivided in a conserved proline-rich domain, and an Assembly Promoting domain (AP) containing the sequences required for MAP4-microtubule interaction (Ookata et al., 1995 and references therein). The alignment between B99 and human MAP4 in the region of similarity is also reported. Download figure Download PowerPoint An affinity-purified rabbit polyclonal antibody was obtained against the central part of B99 (amino acids 255–474) produced as a 6×His-tagged bacterially-expressed recombinant protein. In order to characterize biochemically the expression of B99 protein in Val5 cells, a kinetic analysis was performed by Western blot on extracts prepared 12 and 24 h after temperature shift at 32°C. Extracts were also prepared 6, 12 and 24 h after shifting the temperature back to 37°C (i.e. p53 to a mutant conformation), a condition in which the cells exit p53-induced arrest and promptly re-enter the cell cycle. As reported in Figure 5A, the antibody detected a specific protein with an apparent molecular weight of 110 kDa. B99 protein was strongly induced after 12 h at 32°C, while at the 24 h time point a noticeable decrease could be observed, in line with the regulation of B99 mRNA (Figure 2). A polyclonal antibody to p21Waf1 was used in the same analysis as a control of p53 activation. When Val5 cells were shifted back to 37°C, B99 protein expression was efficiently downregulated within 6 h, slightly accumulating again at 12 h and returning to basal levels after 24 h. For a preliminary understanding of the transient re-appearance of B99 after release from p53-mediated cell-cycle arrest, cyclin B was analysed in the same blot as a marker of G2 phase. As shown in Figure 5A, cyclin B was efficiently downregulated at 32°C, as expected in arrested cells. When Val5 cells were returned to 37°C, cyclin B re-appeared at 12 h, as observed for B99. This observation suggests that a transient increase in B99 might be associated with passage through the G2 phase during cell-cycle re-entry. Figure 5.Western blot analysis of B99 protein expression in murine cells. (A) B99 protein regulation in Val5. Cells grown at 37°C for 18 h after plating (time 0) were shifted to 32°C for 12 and 24 h. Cells arrested by 24 h culture at 32°C were then shifted back to 37°C for the indicated times. Cyclin B was analysed as a marker of cell-cycle. The asterisk indicates a cross-reacting protein that serves as internal loading control. (B) Accumulation of B99 protein upon DNA damage. NIH 3T3 fibroblasts were treated with 400 rad ionizing radiation (IR), MMS (100 μg/ml for 4 h), or UV-light (10 J/m2). Primary fibroblasts (MEF) from p53 and p21 knockout mice were exposed to UV-light or MMS. Cells were collected 18 h (UV and MMS) or 24 h (IR) after treatment, and total lysates were analysed by Western blotting with the indicated antibodies. Download figure Download PowerPoint B99 protein expression was also analysed upon DNA damage in mouse fibroblasts with or without functional p53. Cells were exposed to UV light, ionizing radiation or the alkylating agent methyl methane sulfonate (MMS). Total lysates were prepared 18 h after treatment and B99 protein levels were analysed by immunoblotting. As reported in Figure 5B, B99 clearly accumulated upon DNA damage in NIH 3T3 and p21−/− MEF, which are wt for p53. Interestingly, B99 protein also accumulated in MEF from p53−/− mice, suggesting that B99 might be subject to multiple regulations. Since B99 mRNA appears not to be increased by UV treatment in the same p53−/− fibroblasts (Figure 2C), it is likely that the observed B99 accumulation is due to translational or post-translational regulation. Significantly, as shown in Figure 5, the same behaviour was also observed for p21Waf1. These results indicate that similarly to Waf-1, B99 can be specifically induced by wt p53 but can also respond to other signalling pathways. B99 protein is localized to the microtubule network The affinity purified anti-B99 polyclonal antibody was employed to determine the intracellular localization of B99 protein by indirect immunofluorescence. In Val5 cells cultured at the permissive temperature for 12 h, the antibody to B99 revealed a distribution coincident with the microtubule network as defined by anti-tubulin staining (Figure 6a and b), suggesting that B99 protein may be associated with the microtubules. This localization was reproducibly observed both when the cells were fixed with cold methanol or 3% paraformaldehyde. Ectopic expression of B99 in Balb/c (10)1 mouse fibroblasts confirmed the specificity of the signal; as shown in Figure 6c and d, the transfected exogenous B99 protein colocalized with tubulin, displaying a distribution similar to the endogenous protein observed in Val5 cells at 32°C. Figure 6.Immunological definition of B99 endocellular localization. B99 protein was detected using a rabbit anti-B99 affinity-purified polyclonal antibody followed by a FITC-conjugated anti-rabbit antibody. Tubulin was stained using a monoclonal antibody (Sigma) followed by RITC-conjugated anti-mouse antibody. Images were taken with a laser scan confocal microscope. (a) Detection of endogenous B99 protein in Val5 cells cultured at 32°C for 12 h. (b) The same microscopic field as in (a), stained for tubulin. (c) Detection of ectopically expressed B99 protein in p53-null Balb/c (10)1 cells 48 h after transfection. (d) The same microscopic field as in (c), stained for tubulin. Download figure Download PowerPoint Endogenous B99 is expressed in the G2/M fraction of the cell population When Val5 cells were shifted to the permissive temperature of 32°C and B99 induction was observed by immunofluorescence, expression appeared heterogeneous in the cell population analysed. Figure 7A shows two fields of Val5 cells cultured at 37°C or kept for 12 h at 32°C and stained with the affinity-purified anti B99 antibody; as can be seen, B99 protein was clearly induced, but not in all the cells. Double immunofluorescence staining revealed that there was no correlation between B99 expression and fluctuations in the levels of p53 within individual cells (not shown). A flow-cytometric analysis was therefore performed to measure the DNA content of the B99-expressing sub-population of Val5 cells as shown in Figure 7B. Fixed cells were stained with the anti-B99 antibody followed by a FITC-conjugated anti-rabbit antibody, and DNA was stained with propidium iodide (PI). An appropriate gating was applied to the specific protein fluorescence, based on the background signal detected in the 37°C sample. In the 32°C sample, the gated B99 positive cells displayed a 4N DNA content, indicating that B99 expression was restricted to the G2/M subpopulation. The same correlation could also be observed at longer periods after temperature shift (i.e. 24 and 48 h), when Val5 still display a significant, albeit lower, fraction of cells with 4N DNA content (not shown). It should be stressed here that the outgated B99-negative cells showed a markedly lower 4N fraction with respect to the total population (see Figure 7B), indicating that the majority of G2 cells expressed high levels of B99 protein. Figure 7.B99 is selectively expressed in the G2/M fraction of arrested Val5 cells. (A) Expression of endogenous B99 protein in Val5 fibroblasts. Val5 cells growing at 37°C or kept for 12 h at 32°C were stained with the affinity-purified anti B99 antibody followed by a FITC-conjugated anti-rabbit secondary antibody. (B) Flow-cytometric analysis of Val5 cells corresponding to the same growth conditions as in (A). A gating on the specific protein fluorescence was applied to sort the cells expressing high levels of B99 (B99+) from the total population. The profile of the outgated, B99 negative cells is also reported (B99−). Download figure Download PowerPoint To understand whether t