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Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the estrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal, and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P = 0.0002). The molecular apocrine group is androgen receptor-positive (AR+) and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is more common in the molecular apocrine group than the other groups. Genes that best split the three groups were identified by the Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8–14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER-AR-) and molecular apocrine (ER- AR+).

In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants. Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GCAT transitions represented 73% of all single base pair substitutions. In 70% of the cases the mutated G was preceded 5' by a purine. The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P0.0003) and CCNU induced only GCAT transitions at those positions. The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion. One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations. This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.

We have used chromatin immunoprecipitation (ChIP) to measure p53-dependent histone acetylation at the p21, MDM2, and PUMA promoters. The pattern of histone acetylation was different at each promoter. H3 and H4 acetylation increased at both the p21 and PUMA promoters in response to p53 activation, whereas there was only a minimal increase in H4 acetylation and no increase in H3 acetylation at the MDM2 promoter. The high p53 occupancy of the p21, MDM2 and PUMA promoters has been attributed to the presence of two p53 binding sites in these promoters, but mutation of the p53 binding sites in integrated p21 promoter constructs showed that the two sites in the p21 promoter do not cooperate to stabilize p53 binding. Despite 10-fold higher p53 binding to the proximal than the distal site in the p21 promoter, both sites showed similar patterns of H3 and H4 acetylation. Mutation of the binding sites showed that acetylation of the proximal, low-affinity site requires p53 binding to that site but not to the distal, high-affinity site. Since low-affinity p53 binding sites can confer strong acetylation, the DNA binding affinity in vitro is an unreliable guide to the likely importance of p53 in regulating candidate target genes in vivo.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P

Recent studies have suggested that a rare class of p53 mutants found in tumours has a subtle transcriptional defect affecting bax induction but not p21 induction. We have therefore developed simple functional assays in yeast which can be used to identify these mutants. Analysis of 51 different mutations observed in human tumours showed that all mutants tested scored as mutant with the bax reporter strain but nine scored as wild-type with the p21 reporter strain. These results, which can be explained by the lower affinity of the p53 protein for the bax site, may suggest that p21 is not the key target of p53 mutations in tumours. Since p21 status has recently been shown to modulate the chemotherapeutic and radiotherapeutic sensitivities of cancerous cells, the functional assays described here may have important clinical implications.

p53 triple mutants (120N/121G/277H, 120H/121G/ 277H, 120S/121G/277H and 120H/121G/277Y) have altered sequence specificity in bandshift assays in vitro and transcription assays in vivo. These mutants activate transcription from the site TTT CATG AAA but not from wild type sites. The triple mutants activate more strongly than p53 with a single 277Y mutation. The TTT site matches the wild type p53 consensus at only 4/10 positions and is not recognised by wild type p53. 277Y mutations have been described in human tumours, and Ewing tumour cells expressing this mutant from the endogenous p53 locus selectively activate transcription from transfected luciferase reporters regulated by TTT-mutant p53 binding sites. p53 mutants with altered sequence specificity have potential advantages for cancer gene therapy: if used to activate transcription of conditionally toxic genes they would allow tumour-targeting by p53, which acts as a sensor for the malignant state, but place control over cell killing in the hands of the clinician. Rare tumours expressing such mutants from the endogenous p53 locus could be targeted directly with p53-regulated suicide vectors, but for most tumours both the p53 mutant and the reporter would need to be encoded by the virus.

Field cancerisation of the aerodigestive tract is caused by chronic exposure to alcohol and tobacco, but the nature of the genetic alterations preceding overt malignancy is unknown. To identify potential field changes we have used a functional assay which tests the transcriptional competence of human p53 expressed in yeast. To increase the sensitivity and reliability of the technique for samples containing under 20% mutant p53, the 5′ and 3′-ends of the p53 cDNA were examined separately. With this split form of the assay the tissue p53 mRNA acts as its own control for RNA quality. Mutations were detected in 87% (46/53) of tumours, reflecting the high sensitivity of the technique. Multiple biopsies of histologically normal tissue from the upper aero-digestive tract were tested and clonal p53 mutations were identified in 76% (38/50) of biopsies from patients presenting with multiple tumours compared with 32% (38/117) of biopsies from patients presenting with single tumours (P