p21WAF1/CIP1 response to genotoxic agents in wild-type TP53 ...

Oncogene (1997) 14, 45 ± 52  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

p21WAF1/CIP1 response to genotoxic agents in wild-type TP53 expressing breast primary tumours CeÂline Guillot1, Nicole Falette1,2, Marie-Pierre Paperin2, SteÂphanie Courtois2, Anne Gentil-Perret1, Isabelle Treilleux3, Mehmet Ozturk1 and Alain Puisieux1,2 1 INSERM U453, 2UniteÂ d'Oncologie MoleÂculaire and 3DeÂpartment d'Anatomie et de Cytologie Pathologiques, Centre LeÂon BeÂrard, 28 rue LaeÈnnec, 69008 Lyon, France

Functional inactivation of the wild-type p53 protein has been described in dierent human cancers. Since a signi®cant proportion of breast tumours express wildtype TP53, the p53 antiproliferative activity could be inactivated in transformed mammary epithelial cells by a mechanism independent on structural alteration of the gene. To test this hypothesis, we analysed the p53 activity in primary breast tumour cells. As a preliminary study, we demonstrated in breast adenocarcinoma cell lines that the nuclear accumulation of the inhibitor of cyclin dependent kinase p21WAF1/CIP1, in response to adriamycin treatment, speci®cally re¯ected the activity of a functional wild-type p53 protein. Then, we used this strategy to study the p53 activity in 23 primary breast tumours. p21WAF1/CIP1 accumulation was detected in all tumours expressing wild-type TP53. In contrast, no p21WAF1/CIP1 response was detected in cells harboring a mutant TP53 gene. This report is the ®rst functional study of p53 in primary breast tumours. The results demonstrate that TP53 mutation represents the only common mechanism leading to an irreversible inactivation of p53 functions in this cancer type. Keywords: breast cancer; p53; functional inactivation; p21WAF1/CIP1

Introduction Wild-type p53 exhibits both growth and transformation suppression activities, giving rise to a G1 block in cell cycle progression (Diller et al., 1990; Lin et al., 1992), and in some cell types leading to apoptosis (Yonish-Rouach et al., 1991). Deletion of the TP53 gene in transgenic mice results in a massive increase tumour incidence (Donehower et al., 1992), supporting a role for p53 as a tumour suppressor. The p53 functions appear to be inactivated in a very large range of human cancers (Hollstein et al., 1991; Soussi et al., 1994). The most frequent form of this inactivation is genetic alteration which results in altered conformation of the protein (Levine et al., 1991). An alternative mechanism is the functional inactivation of wild-type p53 protein by complexation with cellular oncoproteins (MDM-2 gene in soft-tissue sarcomas) (Leach et al., 1993) or by interaction with viral proteins (E6 protein of human papillomavirus in cervical cancer) (Crook et al., 1992). Correspondence: A Puisieux Received 2 April 1996; revised 28 August 1996; accepted 3 September 1996

TP53 gene mutations have been reported in about 25% of breast cancers (Soussi et al., 1994, for review). Recent evidence suggests that sequencing of the TP53 gene is a useful prognostic determinant for this cancer type (Thorlacius et al., 1993; Bergh et al., 1995; Kovach et al., 1996). Indeed, TP53 gene mutation appears to be an independent prognostic marker of early relapse and death. However, sequencing does not detect p53 inactivation independent on structural alteration of the gene. To estimate the magnitude of wild-type p53 protein inactivation in breast cancers, we searched to develop a simple methodology to test the p53 activity in primary tumours. The current and most powerful model of wild-type p53 function describes p53 as a guardian of the genome (Lane, 1992). In response to DNA damage, p53 can function as a sequence-speci®c DNA-binding protein that positively regulates gene expression. Dierent p53target genes have been identi®ed, including the inhibitor of cyclin dependent kinases p21WAF1/CIP1 (Xiong et al., 1993). The expression of P21WAF1/CIP1 is induced by activated wild-type p53 in cell lines and appears to be an essential downstream eector of p53 antiproliferative eect (El-Deiry et al., 1993, 1994; Waldman et al., 1995). Unlike wild-type p53, mutant p53 is unable to induce P21WAF1/CIP1 expression (ElDeiry et al., 1993). In concordance with these results, it was recently reported that breast cancer cells harboring wild-type p53 express higher basal P21WAF1/CIP1 mRNA levels than cells harboring mutant p53 (Sheikh et al., 1994; OÈzcËelik et al., 1995). However, in addition to direct transcriptional induction by p53, P21WAF1/CIP1 expression can be induced by signals involving p53independent mechanisms (Michieli et al., 1994; Steinman et al., 1994). Some of these signals include serum stimulation, treatment with growth factors, cytokines and dierentiation-inducing agents. Furthermore, although the expression of P21WAF1/CIP1 in response to genotoxic treatment is predominantly under p53 control, a p53-independent induction of P21WAF1/CIP1 expression was also reported following treatment with dierent genotoxic drugs (Johnson et al., 1994; Michieli et al., 1994). The speci®city of the P21WAF1/CIP1 response appears to be dependent on the type and the dose of the drug. As an example, the chemotherapeutic drug adriamycin, has been shown to be one of the strongest and most reproducible inducers of p53 protein (El Deiry et al., 1994). It causes P21WAF1/CIP1 induction in a p53 dependent manner at 0.2 mg/ml, inducing both p53 protein accumulation and P21WAF1/CIP1 gene expression only in the cell lines which contained wild-type p53 protein. However, an induction of P21WAF1/CIP1 in p53 null cells was reported at higher doses (Michieli et al.,

Functional wild-type p53 in breast cancer C Guillot et al

46

1994; Gartenhaus et al., 1996). As a preliminary study, we investigated the P21WAF1/CIP1 response in a series of adenocarcinoma cell lines following adriamycin treatment (0.2 mg/ml) and ionizing radiation. Con®rming previous results, following these genotoxic treatments induction of P21WAF1/CIP1 occurred only in cell lines with a functional wild-type p53. Based on this observation, we studied p53 and p21WAF1/CIP1 response in primary breast tumours.

a

wtp53/ sv40Ag wtp53 MCF–10A MCF–7 ZR75–1 HBL–100 MB231 c Ad c Ad c Ad c Ad c Ad

— p53

— p21

b

Results p53 response to adriamycin treatment in breast cell lines In wild-type TP53 expressing cell lines, genotoxic treatment leads to an increase of p53 protein levels and a subsequent transcription of the P21WAF1/CIP1 gene (El-Deiry et al., 1994). To better de®ne the speci®city of p21WAF1/CIP1 response, we tested the eects of ionizing radiation and adriamycin treatment on p53 and p21WAF1/CIP1 protein levels in a panel of established human breast cell lines (Table 1). The MCF-10A cell line was established from mammary tissue of a patient with ®brocystic breast disease (Soule et al., 1990). These cells have a normal or near normal karyotype and are therefore considered as a good culture model of non-transformed mammary epithelial cells. MCF10A cells express wild-type TP53 (Diella et al., 1993). As expected, basal levels of p53 and p21WAF1/CIP1 protein levels were low. Accumulation of both proteins was easily detectable by Western blot and immunocytochemistry after genotoxic treatment. This cellular response was particularly high following adriamycin treatment (Figure 1a.). Similar results were obtained in ZR75-1 cells (Figures 1a and 2). Exponentially growing breast adenocarcinoma MCF-7 cells were recently described to express a wild-type p53 protein detectable in the cytoplasm (Takahashi et al., 1993). However, this particular feature was not reported by others (Bartek et al., 1990a,b). In our experiments, immunocytochemical studies revealed a diuse nuclear and cytoplasmic pattern of p53 staining. In these cells, the induction of genetic alterations by genotoxic agents generated an appropriate p53-mediated response: nuclear accumulation, induction of P21WAF1/CIP1 expression (Figures 1 and 2) and cell cycle arrest (data not shown). The presence of a functional wild-type p53 in MCF-7 and ZR-75-1 cells was also reported by others (Skeikh et al., 1994).

Table 1

Characteristics of breast cancer cell lines Origin

TP53 status

MCF-10A

fibrocystic disease

Wild-type

MCF-7

breast carcinoma

Wild-type

ZR75-1

breast carcinoma

Wild-type

Cell line

HBL-100

milk

MDA-MB231

breast carcinoma

BT-20 T47-D MDA-MB157

? ? ? 77

Wild-type m280 Arg

Lys

breast carcinoma

m132 Lys

Glu

breast carcinoma

m194 Leu

Phe

breast carcinoma

p53

Comments

SV-40 large T

/

The status of endogenous p53 in each cell line is from: Diella et al., 1993; Takahashi et al.,1993; Thor et al., 1992; Bartek et al., 1990a; Nigro et al., 1989

mp53 p53–/– BT–20 T47–D MB157 c Ad c Ad c Ad

wtp53 MCF–7 c Ad RX

wtp53/ sv40Ag p53–/– mp53 HBL–100 MB157 BT–20 c Ad RX c Ad RX c Ad RX

— p53

— p21 Figure 1 Western blot analysis of p53 and p21WAF1/CIP1 response to a genotoxic agent in human breast cell lines. Cells were seeded 24 h before drug addition or X-ray irradiation in order to obtain 50 to 70% con¯uence. Cells extracts were prepared 18 h after continuous treatment by adriamycin (0.2 mg/ ml) or 6 h after X-ray irradiation. Each lane contains 100 mg of protein. (a) p53 and p21WAF1/CIP1 response to adriamycin treatment. (b) p53 and p21WAF1/CIP1 response to adriamycin treatment and X-ray irradiation. c: untreated cells; Ad: adriamycin treated cells; RX: X-ray irradiated cells. wtp53: wild-type TP53; mp53: mutant TP53; p537/7: null TP53. MB231: MDA-MB231 cell line. MB157: MDA-MB157 cell line

The HBL-100 cell line was derived from milk of an apparently healthy woman. The SV40 genome is stably integrated and cells produce the SV40 large T-antigen that is co-immunoprecipitated with wildtype p53 (Caron de Fromentel et al., 1985). This cell line is therefore an interesting model of wild-type p53 functional inactivation by interaction with a viral protein. As described previously, HBL-100 cells displayed a high p53 protein level due to its stabilization by SV40 large T-antigen. In response to adriamycin treatment, accumulation of p21 WAF1/CIP1 occurred only in 5% of HBL-100 cells as compared to more than 70% in MCF-10A, MCF7 or ZR75-1 cells (Figure 2). Supporting this observation, the increase of p53 levels, as assessed by Western blot, was weaker as compared to other wild-type TP53 expressing cell lines (Figure 1a). As expected, basal levels of p53 were high in the three studied breast adenocarcinoma cell lines expressing mutant p53 (MDA-MB231, BT-20, T47-D). Neither p53 nor p21WAF1/CIP1 accumulation was detectable after adriamycin treatment (Figures 1 and 2). Similarly, no p21WAF1/CIP1 response was detected in the p53 null MDA-MB157 cells (Figure 1a). The P21 WAF1/CIP1 overexpression following ionizing radiation was reported to be speci®cally p53-dependent (Johnson et al., 1994). We compared, p53 and p21WAF1/CIP1 response to ionizing radiation and adriamycin treatment in breast adenocarcinoma cell lines. As expected, both treatments led to an accumulation of p53 and p21WAF1/CIP1 only in cells with a functional wild-type p53. However, induction was consistently stronger following adriamycin treatment (Figure 1b). To con®rm our observations, seven dierent hepato-


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Figure 2 Immunocytochemical detection of p21WAF1/CIP1 in breast cancer cell lines in response to adriamycin treatment. (a ± b): MCF-7 cells (wtp53); (c ± d): ZR75-1 cells (wtp53); (e ± f): HBL-100 cells (wtp53 - SV40TAg); (g ± h): BT-20 cells (mp53). (a, c, e, g) untreated cells; (b, d, f, h) adriamycin (0.2 mg/ml) treated cells. Cultures were counterstained with hematoxylin

carcinoma cell lines were studied in the same series of experiments (data not shown). Similar results were obtained: p21WAF1/CIP1 induction was detected only in cell lines expressing wild-type TP53 (HepG2 and HepG2/2215). No induction was observed in cell lines harboring either a mutant TP53 (Mahlavu, PLC/PRF/5, HuH7, FOCUS) or a homozygous deletion of the TP53 gene (Hep3B). Taken together these results con®rm that nuclear p21WAF1/CIP1 accumulation in response to adriamycin treatment (0.2 mg/ml) or ionizing radiation is highly dependent on wild-type p53 activity. As previously described, the chemotherapeutic drug adriamycin appears to be a strong and reproducible inducer of p53 (El-Deiry et al., 1994). Based on these preliminary observations, we carried out p53 and p21WAF1/CIP1 immunostaining following

adriamycin treatment to test directly the functional activity of p53 in primary breast tumours. Functional wild-type p53 in primary breast tumours Primary cultures were prepared from twenty-four breast tumour samples on the day of surgery. Forty-eight hours later, islands of epithelial cells were observed for 23 tumours. In all cases, the entire cDNA (residues 1 ± 393) was sequenced from adjacent sections. As shown in Table 2, 19 tumours expressed a wild-type TP53 gene, three expressed a mutant TP53 gene with a ratio between mutant and normal allele from 5 : 1 to 9 : 1 and one tumour (AG362) expressed both mutant and wild-type TP53 with a ratio of 1 : 1.


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p21WAF1/CIP1 response to adriamycin treatment as related to histological type and TP53 status p53 Primocultures immunohistochemistry p21 immunostaining Tumour Histology % of positive cells TP53 status c Ad AF099 invasive ductal carcinoma 0 Wt ± 3 AF116 invasive ductal carcinoma 0 Wt ± 3 AF225 invasive ductal carcinoma 0 Wt ± 3 AF250 invasive ductal carcinoma 5% Wt 1 4 AF361 invasive ductal carcinoma 80% m248 Arg?Gln ± ± AF372 pseudosarcomatous carcinoma 90% Wt ± 3 AF373 invasive ductal carcinoma 90% m273 Arg?His ± ± AF410 invasive ductal carcinoma 0 Wt ± 3 AF420 invasive ductal carcinoma 50% Wt ± 3 AF521 invasive lobular carcinoma 0 Wt ± 3 AG070 invasive ductal carcinoma 0 Wt ± 3 AG075 invasive ductal carcinoma 0 Wt ± 3 AG096 invasive ductal carcinoma 0 Wt ± 3 AG105 invasive ductal carcinoma 5% Wt ± 3 AG127 invasive ductal carcinoma 50% Wt ± 3 AG167 invasive ductal carcinoma 70% m239 Asn?Ser ± ± AG200 mucinous carcinoma 0 Wt ± 3 AG229 invasive ductal carcinoma 0 Wt ± 3 AG309 invasive ductal carcinoma 0 Wt ± 3 AG310 invasive ductal carcinoma 5% Wt ± 3 AG334 invasive ductal carcinoma 0 Wt ± 3 AG356 invasive ductal carcinoma 0 Wt ± 3 AG362 invasive ductal carcinoma 90% Wt/m237 Met?Ile ± 3 c: untreated primary cultures. Ad: adriamycin (0.2 mg/ml) treated primary cultures. Wt: wild-type tp53. m: mutant TP53. ±: undetectable. 1: weak or moderate staining in less than 50% of epithelial cells. 2: intense staining in less than 50% of epithelial cells. 3: moderate staining in more than 50% of epithelial cells. 4: intense staining in more than 50% of epithelial cells Table 2

p53 immunohistochemical staining was performed in standard conditions on paran sections from each tumour specimen. Increased stability leading to higher steady-state levels of p53 protein has been observed to result from mutations that change the coding sequence of the conserved region of the gene (Hinds et al., 1989). In concordance with this previous observation, all tumours harboring a mutant p53 exhibited intense nuclear staining. Among wild-type TP53 expressing tumours, three patterns of nuclear staining were observed. (i) No p53 protein was detected in 13 cancer specimens. (ii) Three cancer specimens had detectable p53 protein but only in a very small percentage of the malignant epithelial cells. (iii) Three of the tumours exhibited strong nuclear staining in up to 90% of malignant cells. Tumour AG362 which expressed both wild-type and mutant p53 expressed elevated levels of p53 protein in 90% of epithelial cells. To investigate the endogenous activity of p53 in mammary adenocarcinoma cells, primary cultures were analysed by immunocytochemistry using four dierent p53-speci®c antibodies (PAb1801, PAb122, HR231 and PAb240) and one p21WAF1/CIP1-speci®c monoclonal antibody, before and after adriamycin treatment. Staining patterns were examined independently by two observers. Samples were scored with respect to the intensity and the percentage of stained cells. In the absence of genotoxic treatment, both p53 and p21 WAF1/CIP1 immunostainings were weak or undetectable in wild-type TP53 expressing tumours. Tumour AG200 was notable in that it showed an exclusive cytoplasmic staining with Pab122 antibody and a nuclear and cytoplasmic staining with the three other anti-p53 antibodies. Such a discrepancy has also been reported by other investigators after immunohis-

tochemistry on paran sections (Fisher et al., 1994; Jacquemier et al., 1994). Following adriamycin treatment, a striking nuclear accumulation of p21WAF1/ CIP1 was observed in up to 80% of epithelial cells (Figure 3a ± d). The same observation was made on tumour AG362 that expresses an equal ratio of wildtype and mutant TP53. Although at a lesser degree, a similar nuclear accumulation of p53 was detected in these tumours (Figure 4). Conversely, no increase of p21WAF1/CIP1 or p53 levels was observed in epithelial cells of the three tumours expressing a prominent mutant TP53 gene (Figures 3e ± f). Discussion We have examined the relationship of TP53 gene mutation, p53 protein levels and p53 response to DNA damage in 23 primary human breast tumours. To our knowledge, this study is the ®rst report addressing directly the issue of p53 activity in primary breast cancer. An interesting characteristic of p53 mutations lies in the increased stability of the mutated protein. This leads to the accumulation of mutant p53 in the cell nucleus, which makes it detectable by standard immunostaining procedure. Using this methodology, all mutant TP53-expressing tumours that we studied exhibited elevated levels of p53 protein. However, three tumours negative for mutation by sequencing of the entire TP53 cDNA were also positive by immunohistochemical staining. Such an apparent discrepancy was reported by other investigators (SjoÈrgen et al., 1996; Kovach et al., 1996). In recent studies, the observation of an abnormal pattern of expression of wild-type p53 protein (increased nuclear and/or cytoplasmic levels) led to the suggestion of a regulatory defect of p53. As


49

Figure 3 Immunocytochemical detection of p21WAF1/CIP1 in response to adriamycin treatment in three dierent breast tumour primary cultures. (a ± b): tumour AG334 (wtp53); (c ± d): tumour AG356 (wt53); (e ± f): tumour AG167 (mp53). (a; c; e): untreated primary cultures; (b; d; f) primary cultures treated with adriamycin (0.2 mg/ml). Epithelial cells were identi®ed by immunostaining with a cytokeratin antibody and alkaline phosphatase/fast red system. Note the accumulation of p21WAF1/CIP1 in response to genotoxic treatment in epithelial cells of tumours AG334 and AG356 (b; d) and in ®broblasts of the three tumours (b; d; f)

an example, based on immunohistochemistry studies, Moll et al. proposed a mechanism of functional inactivation by cytoplasmic sequestration of wild-type p53 in in¯ammatory breast cancers (Moll et al., 1992). However, due to the lack of functional studies on these tumours, the biological signi®cance of such observations remains unclear. The incidence of TP53 alteration has been reported to be dependent on the histological type (Domagala et al., 1993). In¯ammatory breast carcinomas represent 1 ± 2% of primary breast cancers (Jaiyesimi et al., 1992). Due to the low frequency of these cancers, none were studied in our work. Most of the tumours (20/23) were invasive ductal carcinomas. This tumour type represents about 70% of breast cancers. Therefore, we searched to evaluate, in this representative population of breast cancers, the magnitude of wild-type p53 protein inactivation. By using, eight breast adenocarcinoma cell lines and seven hepatocarcinoma cell lines, we ®rst con®rmed that p21WAF1/CIP1 accumulation following adriamycin treatment (0.2 mg/ml) occurs only in epithelial cells expressing a functional wild-type p53. Of particular interest, very weak p21WAF1/CIP1 accumulation was observed in the HBL-100 cell line that expresses the SV40 large T-antigen. SV40 large T-antigen is known to complex to wild-type p53 protein and to inactivate its transcriptional activity (Farmer et al., 1992). This demonstrates that a test based on the p21WAF1/CIP1

immunostaining after genotoxic treatment allows detection of functional inactivation of p53. By using this simple strategy, we were able to evaluate the p53 transcriptional activity in 23 human primary breast tumours. Con®rming the speci®city of the methodology, no p21WAF1/CIP1 accumulation was observed, in response to genotoxic treatment, in malignant epithelial cells of tumours harboring only a mutant p53. This con®rms that mutations altering the coding sequence in a conserved region of the TP53 gene leads frequently to a loss of p53 activity in human tumours. In contrast, regardless of dierences in their protein expression pattern, all primary tumours expressing wild-type TP53 displayed an easily detectable accumulation of p21WAF1/CIP1 in more than 80% of transformed epithelial cells. This striking observation demonstrates that wild-type p53 is able to respond to DNA damage in breast cancer cells. An interesting tumour specimen was the sample AG362. This tumour expressed equal amounts of wildtype and mutant TP53 mRNA. The observation of wild-type and mutant TP53 in the same specimen can result from dierent phenomenons: (i) the tumour samples can be contaminated with non-tumour tissue. However, adjacent tumour sections of all samples were ®rst examined by haematoxylin and eosin staining and it seems very unlikely that this tumour specimen contained an equivalent amount of normal, non-


50

Figure 4 Immunocytochemical detection of p53 in response to adriamycin treatment in tumour AF250 which express wild-type p53. (a) Untreated cells; (b) adriamycin (0.2 mg/ml) treated cells. Fibroblasts were identi®ed by immunostaining with a vimentin antibody and alkaline phosphatase/fast red system

cancerous tissue. (ii) Two dierent tumour cell populations can be equally present in the tumour sample. The homogeneity of p53 immunohistochemical staining, as well as the high percentage of p21WAF1/CIP1 positive cells after adriamycin treatment, do not support this hypothesis. (iii) There is a co-expression in tumour cells of both wild-type and mutant TP53. The retention of the wild-type TP53 allele in primary breast tumours has been previously reported by other investigators (Davido et al., 1991; Mazars et al., 1992; Thompson et al., 1992; Thor et al., 1992). If this were the case in this tumour, it would indicate that the mutant p53 (237 Ile?Met) is recessive over the wildtype. The observation of a transcriptional activity of p53 in response to DNA damage in breast cancer cells,

makes unlikely an irreversible mechanism of functional inactivation by complexation of p53 with either viral proteins or overexpressed cellular proteins, such as MDM2, that have been shown in other cell types to inhibit the eector functions of wild-type p53. Therefore, our results strongly suggest that TP53 genetic alterations are the only common mechanisms leading to a de®nitive loss of p53 transcriptional activity in breast cancer cells. As an important implication, these data may explain the recent clinical observation of a greater prognostic value of the detection of a mutation relative to positive p53 nuclear immunostaining (Kovach et al., 1996; SjoÈgren et al., 1996). Our results raise the question of the biological importance of p53 inactivation during the process of breast carcinogenesis. p53 is thought to be a critical growth limiting control after the initial stages of tumour development have taken place (Lane, 1992). Nevertheless, 75% of breast cancers do not display any TP53 genetic alteration, suggesting that breast cancer cells can acquire the genetic events needed for malignancy with such an invasion, without needing to de®nitely get rid of a potentially tumour suppressing form of p53. Either these cells can exhibit a rate of DNA damage suciently low to not invoke the growth inhibitory response mediated by p53, or p53 abnormally remains in a `dormant' phase during cell transformation. The former hypothesis may involve a mechanism of partial and reversible inactivation of wild-type p53 protein. Recently, Lutzker and Levine presented evidence suggesting the presence of a latent form of murine p53 in teratocarcinoma cell lines (Lutzker and Levine, 1996). These authors characterized two cell lines expressing high levels of nuclear wild-type p53 protein but found no evidence of upregulation of the p53-target genes, suggesting the presence of a transcriptionally inactive form of p53. This apparent inactivation of p53 was reversed by cellular dierentiation or by treatment with DNA damaging agents. These observations are reminiscent of the three human breast tumours (AF372, AF420, AG127) that exhibited high nuclear levels of wild-type p53 protein. In the absence of genotoxic treatment, we did not detect any p21WAF1/CIP1 protein overexpression in these tumour cells. However, adriamycin treatment led to a normal p53 response, compatible with a phenomenon of `p53 rescue'. p53 transcriptional activity appears to be controlled by post-translational mechanisms, such as phosphorylation (Hecker et al., 1996). Genotoxic treatment, by increasing the rate of genetic alterations in cancer cells, could activate a latent form of p53 protein which was unable to control tumour development. At last, an alternative hypothesis of a reversible p53 inactivation may involve cellular environmental factors. Indeed, we recently demonstrated that wild-type p53 activity in mammary epithelial cells was controlled by estrogens (Guillot et al., in press). In vitro, the absence of estrogenic activity leads to a dramatic decrease of p53 protein levels and a loss of transcriptional function in response to genotoxic treatment. Most of breast cancers develop in post-menopausal women. The inhibition of p53 functions due to the lack of estrogenic activity, may explain why most of breast cancers fail to select for TP53 mutations, that would lead to a de®nitive loss of p53 tumour suppressing functions.


Materials and methods Cell lines Characteristics of human breast adenocarcinoma cell lines used in this study are described in Table 1. Hepatoma cell lines were previously described (Puisieux et al., 1993). Cell lines (American Type Culture Collection) were maintained in D-MEM [Dulbecco's modi®ed Eagle's medium] supplemented with 10% FCS [fetal calf serum]. Cells were seeded 18 h before drug or radiation treatment and were 50 to 70% con¯uent at the time of treatment. Cells were treated with a chemotherapeutic agent doxorubicin (Adriamycin) at a concentration of 0.2 mg/ml or exposed to X-ray irradiation at 6 Gy. Tumour short term cultures Breast tumour sample was processed for culture on the day of surgery. It was freed from adipose tissue, minced and ground with a special blade. The micro-fragments were suspended in D-MEM/F12 medium containing 1% FCS and 0.25 U/ml of collagenase (Boehringer Mannheim), incubated in a water-bath at 378C under constant gentle magnetic stirring for a period which dependend on the rapidity of disappearance of the fragments in suspension (from 6 h to overnight). Then, the suspension was gently pipetted to break up the cell clumps, and centrifuged at 1500 r.p.m. for 5 min at room temperature. The cell pellet was resuspended in culture medium D-MEM/F12 containing 10% FCS. The cell suspensions was seeded on coverslips, in 12 well-plates. After 3 days, cultures were exposed for 18 h with 0.2 mg/ml Adriamycin and then processed for immunodetection of p53 and p21WAF1/CIP1. Western blot analysis Cell protein extracts were prepared by lysing cells into a buer containing 50 mM Tris-HCl, 0.25 M NaCl, 1 mM CaCl2, 0.1% triton X-100, 50 mM NaF and a cocktail of protease inhibitors (0.2 mg/ml leupeptin, 0.2 mg/ml aprotinin, 2 mg/ml tosylphenylalanine chloromethyl ketone, 10 mg/ml phenylmethyl-sulfonyl ¯uoride and 2 mg/ml soyabean trypsin inhibitor). Lysates were centrifuged 15 min at 13 000 r.p.m. and supernatants were recovered. Protein concentration was estimated using the Biorad protein assay. 100 mg of total protein was then separated on 10% SDS-polyacrylamide gel in a Tris-Glycine buer and transferred to PVDF Immunobilon membranes (Millipore). Filters were blocked for 2 h at room temperature in TBS-T [0.05% Tween in Tris-buered saline] containing 5% non-fat dry milk and incubated with primary antibodies overnight at +48C. The blots were then rinsed in TBS-T and incubated with a peroxidaseconjugated goat anti-mouse IgG antibody (P260, Dako) for 1 h at room temperature. After washing in TBS-T, antibody-antigen complexes were detected by ECL chemiluminescence (Amersham) according to the manufacturer's instructions. Anti-p53 monoclonal antibody PAb1801 (Ab-2; Oncogene Science) and anti-21WAF1/CIP1 (Ab-1; Oncogene Science) were used in TBS-T containing 2% non-fat dry milk. Immunocytochemical detection of p53 and p21WAF1/CIP1 in cell lines and primary cultures Cultured cell lines or short term cultures of tumours grown on coverslips were ®xed in 4% paraformaldehyde for

15 min, rinsed for 15 min with PBS, incubated 15 min in 1% Triton X-100 in PBS and rinsed for 10 min with PBS at room temperature. Then, cells were incubated for 30 min at room temperature with blocking solution (3% BSA in PBS) and incubated with primary antibodies over night at +48C. After two washes in PBS, cells were incubated with a murine secondary antibody and with the Vectastain ABC kit (Vector Laboratories). Streptavidin-coupled horseradish peroxidase/diaminobenzidine system was used for detection. Anti-p53 monoclonal antibodies PAb1801 (Ab-2; Oncogene Science), PAb240, (Ab-3; Oncogene Science), HR231 (kindly provided by T Soussi; Legros et al., 1993), PAb122 (Gurney et al., 1980) and anti-p21 monoclonal antibody (Ab-1; Oncogene Science) were used. In primary tumours, epithelial cells and ®broblasts were detected by immunochemical staining using, respectively, a cytokeratin antibody and a vimentin antibody (Boehringer Mannheim), and alkaline phosphatase/fast red system (Boehringer Mannheim). Epithelial cells were detected in association with p21WAF1/CIP1 immunostaining and ®broblasts with p53 immunostaining. Immunohistochemistry of paran embedded tissue Sections were deparanized in toluene, transferred to 100% and 95% ethanol, and air dried. They were then treated with 5% hydrogen peroxide for 20 min to exhaust endogenous peroxidase. Tissue sections were placed in sodium citrate solution (0.01 M , pH 6.0) and then incubated three times for 5 min in an 800 W microwave oven. After preincubation in 1% BSA in PBS, sections were incubated 90 min at 378C with the mouse monoclonal p53 anti-serum DO7 diluted 1 : 50 (Dako), washed and reacted with streptavadin-biotin-peroxidase reagents (Dako) and diaminobenzidine (DAB) chromogen. Sections were slightly counterstained with hematoxylin. Sequence-based analysis of TP53 The method was essentially as described by SjoÈgren et al. (1996). RNA was prepared from the frozen tumours under stringent conditions to avoid degradation and contamination. This was followed by an enzymatic conversion of the RNA to cDNA. TP53 was ampli®ed from the tumour cDNA by the polymerase chain reaction using four overlapping primer pairs covering the complete coding region of the TP53 gene. Biotin-labeled PCR products were generated with one of the primers (in each pair) being modi®ed with a biotin molecule, which facilitates solid-phase sequencing. Solid-phase sequencing was carried out using AutoLoadTm Solid Phase Sequencing Combs and T7 DNA polymerase (Pharmacia Biotech). The sequencing products generated were analysed using an automated laser ¯uorescence, ALFTm DNA sequencer (Pharmacia Biotech).

Acknowledgements The authors are grateful to Pharmacia Biotech, Sweden, for supplying reagents and equipment for sequencing. This work was supported by grants from la Ligue de Lutte contre le Cancer (ComiteÂ DeÂpartmental de l'Ain). CG was supported by doctoral fellowships from the Association pour la Recherche sur le cancer (ARC).

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