The p53 tumor suppressor gene, which is induced by DNA damage and/or stress stimuli, causes cells to undergo G1-arrest or apoptotic death; thus it plays an essential role in human carcinogenesis. We have searched for p53-related genes by using degenerate PCR, and have identified two cDNA fragments similar to but distinct from p53: one previously reported, p73, and the other new. We cloned two major splicing variants of the latter gene and named these p51A and p51B (a human homologue of rat Ket). The p51A gene encodes a 448-amino-acid protein with a molecular weight of 50.9 kDa; and p51B, a 641-amino-acid protein with a molecular weight of 71.9 kDa. In contrast with the ubiquitous expression of p53, expression of p51 mRNA was found in a limited number of tissues, including skeletal muscle, placenta, mammary gland, prostate, trachea, thymus, salivary gland, uterus, heart and lung. In p53-deficient cells, p51A induced growth-suppression and apoptosis, and upregulated p21waf-1 through p53 regulatory elements. Mutations in p51 were found in some human epidermal tumors.

We performed a comprehensive survey of commonly inactivated tumor suppressor genes in esophageal squamous cell carcinoma (ESCC) based on functional reactivation of epigenetically silenced tumor suppressor genes by 5-aza-2'-deoxycytidine and trichostatin A using microarrays containing 12599 genes. Among 58 genes identified by this approach, 44 (76%) harbored dense CpG islands in the promoter regions. Thirteen of twenty-two tested gene promoters were methylated in cell lines, and ten in primary ESCC accompanied by silencing at the mRNA level. Potent growth suppressive activity of three genes including CRIP-1, Apolipoprotein D, and Neuromedin U in ESCC cells was demonstrated by colony focus assays. Pharmacologic reversal of epigenetic silencing is a powerful approach for comprehensive identification of tumor suppressor genes in human cancers.

p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63 ؉/؉ mouse, it is undetectable in these tissues in the p63 ؊/؊ mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.p63 is a member of the p53 tumor suppressor gene family. Similar to p53, p63 is a transcription factor that activates target genes through sequence-specific DNA binding (35,41,43,52,56). It has been shown that expression of p21 waf-1 , MDM2, and BAX are induced by TAp63s through binding to p53 response elements (p53-REs) (45). In spite of their structural similarities, p63 functions differ greatly from those of p53. The most striking difference is the apparent involvement of p63 in skin and limb development. The p63 knockout mouse exhibits skin and limb defects as well as craniofacial abnormalities (29, 57). On the other hand, the p53 knockout mouse develops normally but is prone to suffering from various cancers from an early age (7). Heterozygous p63 germ line mutations cause several skin and other developmental disorders (1,3,17,28,53). On the other hand, germ line mutations of p53 cause Li-Fraumeni syndrome, in which affected individuals are exceptionally prone to developing cancer (26). p63 complements p53-dependent apoptosis induced by DNA damage. However, p63 itself induces apoptosis to a lesser extent than p53 (12, 42).These differences may be due to the differential regulation of target genes by p53 and p63. The p53 and p63 proteins can bind to two or more tandem repeats of RRRCWWGYYY (p53-RE) or some other motifs and subsequently activate target gene expression (5, 9, 54, 56). In the case of the 14-3-3 promoter, p53 and p63 differentially bind to two distinct response elements (55). Until now, a number of genes have been reported to be targets of p63 and its close relative, p73, such as JAG1, JAG2, IL4R, ⌬Np73, AQP3, and REDD1 (11,30,39,40,59). However, p63-specific response elements (p63-REs) have not yet been defined. Thus, the specific mechanism of gene activation exhibited by p63 and its distinction from that exhibited by p53 remain unclear.In order...

Aberrant promoter hypermethylation of tumor suppressor genes is proposed to be a common feature of primary cancer cells. We recently developed a pharmacological unmasking microarray approach to screen unknown tumor suppressor gene candidates epigenetically silenced in human cancers. In this study, we applied this method to identify such genes in head and neck squamous cell carcinoma (HNSCC). We identified 12 novel methylated genes in HNSCC cell lines, including PGP9.5, cyclin A1, G0S2, bone-morphogenetic protein 2A, MT1G, and neuromedin U, which showed frequent promoter hypermethylation in primary HNSCC (60%, 45%, 35%, 25%, 25%, and 20%, respectively). Moreover, we discovered that cyclin A1 methylation was inversely related to p53 mutational status in primary tumors (P ‫؍‬ 0.015), and forced expression of cyclin A1 resulted in robust induction of wild-type p53 in HNSCC cell lines. Pharmacological unmasking followed by microarray analysis is a powerful tool to identify key methylated tumor suppressor genes and relevant pathways.

Promoter hypermethylation accompanied by gene silencing is a common feature of human cancers. We identified previously several new tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of reexpression on microarray analysis. In this study, we modified the selection of candidates from our previous microarray data by excluding genes that showed basal expression in cancer cell lines. With the new method, we found novel methylated genes with 90% accuracy. Among these 33 novel methylated genes that we identified in esophageal squamous cell carcinoma (ESCC) cell lines, N-methyl-D-aspartate receptor type 2B (NMDAR2B) was of particular interest. NMDAR2B was methylated in 95% of primary human ESCC tissue specimens and 12 ESCC cell lines by sequence analysis. NMDAR2B expression was silenced in all 12 ESCC cell lines and was reactivated by the demethylating agent 5-aza-2V-deoxycytidine. Moreover, reintroduction of the gene was accompanied by marked Ca 2+ -independent apoptosis in ESCC cell lines, suggesting that NMDAR2B can suppress tumor growth. Thus, NMDAR2B promoter methylation is common in ESCC, abrogating gene transcription and leading to cellular resistance to apoptosis.