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Mutational Analysis of the p63/p73L/p51/p40/CUSP/KET Gene in Human Cancer Cell Lines Using Intronic Primers

Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
After the identification of p73, a second homologue of the human p53 tumor suppressor gene has been reported and named p63/p73L/p51/p40/CUSP/KET. We have investigated the hypotheses that: (a) p63 is mutated in diverse types of human cancers; and (b) p63 functions in the same pathway as p53 and p73 in the process of carcinogenesis; therefore, mutations in these three genes would be mutually exclusive. We have analyzed the genomic structure of the p63 gene and have performed mutational analyses on 54 human cell lines using intronic primers flanking each exon. We have confirmed that the human p63 open reading frame encodes the same length of protein as murine p63 that was initially reported to be 39 amino acids longer than human p63. By mutational analysis, we have shown that DLD1 and SKOV3 cells have either heterozygous mutations or polymorphisms in the putative DNA binding domain of p63. In these cell lines, p63 is biallelically expressed. We conclude that mutations in the p63 gene are rare in human cell lines. The fact that DLD1 is abnormal for both p63 and p53 genes suggests that they may not be involved in the same tumor suppressor pathway.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
The existence of a p53 homologue had long been considered unlikely. However, emerging evidence indicates that there is a p53 gene family (1) . After the identification of the p73 gene (2) , the second homologue of p53 was reported from several investigators under the names p73L (3) , p51 (4) , p40 (5) , CUSP,² KET (6) , and p63 (7) . Although the amino acid sequences and the molecular weights were reported to be different, they have proven to be isotypes derived from a single gene with two promoters, two 3'-end exons, and at least three alternative splicing patterns (summarized in Table 1 ). In this report, the name p63 will be used hereafter. p53, p63, and p73 have much sequence homology throughout their length and share domain structures that include a transactivation domain, a DNA binding domain, and an oligomerization domain from the NH₂ terminus to the COOH terminus. Because p63 and p73 share more homology than p53, they are considered to be evolutionarily more closely related. A prominent feature of p63 is its lack of the transactivation domain (Np63s: shown in Table 1 ) in some isotypes transcribed from the second promoter (7) . p63 isotypes having the transcriptional transactivation domain transactivate p53 target sequences such as the p21^WAF-1 promoter (4) and the minimal p53 binding sequence, PG-13 (7) . Overexpression of p63 induces apoptosis, although this ability differs among isotypes (4 , 7) . The expression of p63 is tissue specific, and many tissues have dominantly expressed isotypes (3, 4, 5 , 7) . By immunohistochemical analysis, high p63 expression was observed in the basal cells of various epithelial tissues (7) . Mice lacking p63 show developmental defects in organs of ectodermal origin (8 , 9) ; mice lacking p53 have exencephaly and other developmental defects in about 25% of the embryos (10, 11, 12) . This suggests that p63 and p53 activate different target genes. Because p63 has a high homology with p53 and p73, we tested two hypotheses: (a) that p63 is mutated in diverse types of human cancers; and (b) that p63 is functioning in the same pathway as p53 and p73 in the process of carcinogenesis so that mutations would be exclusive among the three genes. Detailed mutational analysis is necessary to investigate these hypotheses. In addition to the six isotypes shown in Table 1 , p63 has even more minor isotypes produced by additional alternative splicing patterns (4 , 8 , 13) . This makes mutation detection by RT-PCR-SSCP³ analysis difficult because the differently spliced mRNAs exhibit additional bands on the gel (4) . In this report, we studied the p63 genomic sequence, designed PCR primers in the introns flanking each exon, and then performed the mutation screening using genomic DNA. This enables accurate p63 mutational analysis without being affected by the presence of multiple isotypic mRNAs produced by alternative splicing.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

Determination of the Intronic Sequences Flanking Each Exon of the Human p63 Gene.
The intronic sequences that flank each exon of the human p63 gene were determined by the "Long Distance Sequencer" method (14, 15, 16) from YAC clone yhCEPH913D2. In brief, YAC DNA was digested by HincII, RsaI, PvuII, NlaIV, HaeIII, or Cac8I (New England Biolabs) and ligated with a vectorette unit. PCR was then performed using a gene-specific primer and the 224 M13 primer (14) . A total of 69 gene-specific primers were designed from the reported cDNA sequences (3, 4, 5, 6 , 7) to determine all of the exon/intron boundaries. Two to four amplified fragments were selected and directly sequenced on the fluorescent DNA sequencer 370A (Perkin-Elmer) using the -21 M13 primer (Amersham) and the ThermoSequenase dye terminator sequencing kit (Amersham). Our genomic sequences were compared with the available cDNA sequences (3, 4, 5, 6 , 7) , and the exon/intron boundaries were assigned by the GT/AG rule (17) . The nucleotide sequences of the 5' untranslated regions of mRNAs with transactivation domains (TAp63s in Table 1 ) or without transactivation domains (Np63s in Table 1 ) had been reported in GenBank (accession numbers AB016072 and AF091627, respectively). We determined the genomic sequences corresponding to these untranslated regions; then the first translation start codons (ATG) of the open reading frames were assigned. The presence of the upstream in-frame stop codons, which defines the open reading frames of both TAp63s and Np63s, was also searched.

Preparation of DNA.
Fifty-four human cell lines were grown in the recommended medium. Cell lines used were as follows: 11 colon cancers (HCT116, DLD1, SW620, HT-29, SW480, COLO320DM, SW48, WiDr, LS174T, RKO, and SW403), 11 non-small cell lung cancers (866 MT, A2182, NCI-H292, Calu6, A427, Calu1, NCI-H358, NCI-H1155, NCI-H157, NCI-H596, and A549), 6 small cell lung cancers (NCI-N417, DMS92, NCI-H446, NCI-H146, NCI-H82, and NCI-H526), 5 breast cancers (MDA-MB-468, T-47D, MCF7, Hs578T, and ZR-75–1), 5 hepatocellular carcinomas (HA22T/VGH, HUH4, HEP3B, HUH7, and SK-HEP-1), 1 hepatoblastoma (HepG2), 1 hepatoblastoma transfected by hepatitis B virus (HB611), 1 SV40 immortalized liver cell line (THLE-5B), 3 pancreatic cancers (MIAPaCa-2, Capan-2, and AsPC-1), 2 oral cancers (FaDu and SSC-4), 2 mesotheliomas (M9K and M24), 1 T-cell lymphoblastic leukemia (CCRF CEM), 1 T-cell lymphoma (H9), 1 ovarian cancer (SKOV3), 1 cervical cancer (CaSki), 1 esophageal cancer (HCE7), and 1 glioblastoma (U118 MG). DNA was extracted using the Nucleon I DNA Extraction kit (Scotlab) and dissolved in 10 mM Tris-HCl (pH 8.0)/1 mM EDTA to a final concentration of 50 ng/µl.

PCR-SSCP Analysis.
Sixteen primer sets were designed from the intronic sequences to amplify all coding exons and the splicing junctions of the p63 gene. The primers and the lengths of the PCR fragments are shown in Table 2 . PCR was performed in a 25-µl reaction containing 1x XL buffer II (Perkin-Elmer), 1.1 mM Mg(OAc)₂, 200 µM deoxynucleotide triphosphates, 300 nM of each primer, and 2 units of rTth DNA polymerase, XL (Perkin-Elmer) using 40 cycles of 94°C for 40 s, 55°C for 30 s, and 68°C for 2 min. After confirming the amplification by agarose gel electrophoresis, the PCR fragments were labeled by five additional cycles of 94°C for 40 s, 55°C for 30 s, and 68°C for 2 min in a 5-µl labeling solution containing 1x XL buffer II, 1.1 mM Mg(OAc)₂, 50 µM deoxynucleotide triphosphates, 300 nM of each primer, 0.2 µCi of [-³³P]dATP (DuPont), and 0.1 unit of rTth DNA polymerase, XL. For fragments longer than 300 bp, the labeled fragment was then digested by an appropriate restriction enzyme (see Table 2 ) to divide it into two shorter fragments of <300 bp. Labeled fragments were denatured by heating at 70°C after adding an equal amount of denaturing solution containing 98% formamide, 0.025% xylene cyanol, and 0.025% bromphenol blue and were then run on a 0.5x MDE gel (FMC Bioproducts) at 20°C. The gel was dried and exposed to Kodak XAR film overnight.

RT-PCR using total RNA from DLD1 and SKOV3 cells was done as follows. Total RNA was isolated using the RNA Extraction kit (Stratagene). One µg of total RNA was reverse transcribed using SuperScript II reverse transcriptase (Life Technologies, Inc.) and then amplified by rTth DNA polymerase, XL using -21 M13 or -28 M13 reverse sequence-tagged primer pairs Ex5M13 (TGTAAAACGACGGCCAGTCCATGAGCTGAGCCGTGA-ATTC) and Ex6rev (AGGAAACAGCTATGACCATCAGCACACTCTG-TCTTCCTGTGAT) to check exon 6 and Ex12M13 (TGTAAAACGACGGCCAGTTATCCCACAGATTGCAGCATTGT) and Ex13rev (AGGAAACA-GCTATGACCATATCCATGGAGTAATGCTCAATCTG) to check exon 13. Each primer was designed on different exons to span the intervening intron and thus avoid amplification from contaminating genomic DNA. The nucleotide sequence was determined as described above. The correct exon/exon boundary sequences were confirmed to guarantee the mRNA-derived sequences.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Screening of the human YAC, PAC, and BAC genomic libraries using the p63-specific primer pairs gave five clones shown in Fig. 1A . To find a clone that contained the entire p63 gene, we first screened 270,000 PAC and BAC clones using primer pairs CF1-CB1 and CF51-CB68. Each PAC or BAC clone had an average insert size of 100 kb; thus, 270,000 clones were enough to cover the entire human genome nine times. Even so, we were not able to find a single clone that was positive for both primer pairs, suggesting that p63 is so large that it does not fit into a single PAC or BAC clone. We then screened a human YAC library. Most of these clones have from 500-kb to 2-Mb inserts. We found clones yhCEPH809A5, yhCEPH913D2, and yhCEPH914F12 that contain the entire p63 gene. These three clones are reported to have the STS marker D3S1288 that had been mapped to chromosome 3q27.⁴ This is consistent with the reported location of the p63 gene at 3q27–29 (3 , 4 , 6 , 7) . The intronic sequences flanking each exon were determined from yhCEPH913D2 by the Long Distance Sequencer method (14) . The sequences were deposited in GenBank under accession numbers AF124528–AF124540. PAC clone 71B1 and BAC clone 243L1, obtained using the primer pair CF1-CB1, were also mapped on the p63 genomic structure (Fig. 1A) . Each clone was found to contain only three exons. This suggests that the 5' exons of the p63 gene are widely dispersed in the genome, as is the case for the p53 and p73 genes (18) .

View larger version (48K): [in this window] [in a new window]   Fig. 1. Analysis of the human p63 gene. A, location of the human genomic clones (YAC, PAC, and BAC). YAC yhCEPH913D2 containing the entire p63 gene was used in this study. The extent of the insert of each clone was determined by mapping individual exons on the clone. The distances between exons have not yet been determined. B, comparison of the NH2 termini of the human and murine p63, human and murine p53, and human p73 proteins, with the human nucleotide sequence shown above. p63 has a longer open reading frame than either p53 or p73 in the NH2 terminus. The translation start codon is located in exon 1. The nucleotide sequence of exon 2 is boxed. Dashes (-) were inserted to maximize the alignment. Left, amino acid numbers for each protein. Amino acids common to human and murine are shown in reverse text, as well as any in p53 or p73 common to conserved amino acids in p63. ***, in-frame upstream stop codon. +++, "ATG" codon initially reported as the translation start codon.

We designed PCR primers in the introns to amplify all of the coding exons individually from the genomic DNA. This enables the mutational analysis to be independent of either the level of expression of each allele or of the presence of the alternative splicing patterns. We searched for p63 mutations in 54 human cell lines that we had previously analyzed for mutations in the p73 and p53 genes (19) . Of these 54 cell lines, only DLD1 and SKOV3 showed single nucleotide changes in one allele each. DLD1 was changed in exon 6, and SKOV3 was changed in exon 13. As shown in Fig. 2 , each cell line showed an abnormal band on the SSCP gel in addition to the normal band, indicating that each cell line is heterozygous for the altered exon. Direct sequencing analysis showed that the nucleotide change in exon 6 of DLD1 causes an amino acid change from ²⁷⁹Pro to ²⁷⁹His. The change in exon 13 of SKOV3 causes an amino acid change from ⁵⁶⁰Ser to ⁵⁶⁰Ala. Both changes are located in the putative DNA binding domain of the p63 protein. p73 has been reported to be monoallelically expressed in neuroblastoma cells (2) . To determine the pattern of allelic expression in p63, RT-PCR direct sequencing analysis was performed using primers spanning an intron to avoid amplification of contaminating genomic DNA. As shown in Fig. 2C , biallelic expression was found in both DLD1 and SKOV3.

View larger version (59K): [in this window] [in a new window]   Fig. 2. SSCP-sequencing analysis of the p63 gene in DLD1 and SKOV3 cells. A, SSCP analysis in exon 6 and exon 13 of the p63 gene. DLD1(left column, arrow) and SKOV3 (right column, arrow) each show an abnormal band in addition to the common bands, suggesting that both cell lines have an abnormal allele in addition to a normal allele. B, direct genomic sequencing of p63 exon 6 in DLD1 and p63 exon 13 in SKOV3 cells using the PCR product amplified by the intronic primers for each exon. In both cell lines, both normal and abnormal sequences were seen to overlap. The abnormal allele encodes 279His instead of 279Pro in DLD1 and 560Ala instead of 560Ser in SKOV3. C, direct sequencing of the RT-PCR products of p63 exon 6 in DLD1 and p63 exon 13 in SKOV3 cells. PCR primer sets spanning the intervening introns allow only cDNA-derived sequences to be amplified. In both cell lines, both normal and abnormal alleles are biallelically expressed.

In the literature, one p63 somatic mutation (¹⁸⁷Ala to ¹⁸⁷Pro) was found in 66 primary human tumors, and two mutations (¹⁸⁴Ser to ¹⁸⁴Leu in Ho-1-U-1 cells and ²⁰⁴Gln to ²⁰⁴Leu in SKG-III cells) were observed in 67 human cell lines evaluated by RT-PCR-SSCP analysis. The authors did not define the mutations as homozygous or heterozygous (4) . Another group (13) found no mutations in 45 primary lung cancers but found a heterozygous frameshift mutation in one cell line (EBC1) of the 44 lung cancer cell lines examined. In EBC1, the abnormal allele has an insertion of an adenine after the ²³³Lys codon. All of these mutations, including ours, are located in the putative DNA binding domain of the p63 protein. We conclude that mutations in p63 are not frequent in the human tumors or in the human cell lines examined. The high percentage of homology among the oligomerization domains of p53, p63, and p73 suggests that they may form heterotetramers. Indeed, it has been reported that p53 and p73 interact in the yeast two-hybrid system (8) . Hypothetically, for the precise regulation of cell proliferation, p53, p63, and p73 may all need to be functionally and physically intact. If this were the case, the mutations would be expected to show a mutually exclusive pattern in cancer cells. In the 54 cell lines that we investigated, 36 of these have mutations in p53, whereas 18 are wild-type. Two (A427 and NCI-H1155) have mutations in p73, whereas 52 are wild-type (19) . A427 has a mutation in p73, but has wild-type p53 and p63 genes. NCI-H1155 and DMS92 have mutations in both p73 and p53 genes but are wild-type for p63. DLD1 has a mutation in p53 and p63 (heterozygous) but is wild-type for p73. SKOV3 has a mutation in p63 (heterozygous) but is wild-type for p73 and p53. Other cell lines have wild-type p73 and p63, with or without p53 mutations. Therefore, mutations in p53 and p63, as well as in p53 and p73, are not mutually exclusive. The relationship between p63 and p73 mutations is difficult to evaluate because of the low mutation rates in both genes.

We detected one single nucleotide polymorphism in intron 10, as shown in Table 3 . This polymorphism can be detected by SSCP using primer pair exon 10-F and exon 10-B (Table 2) and is thus useful for detecting a loss of heterozygosity in the p63 locus.

	ACKNOWLEDGMENTS

 
We thank Dorothea Dudek for editorial assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, National Cancer Institute, Building 37, Room 2C01, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-2048; Fax: (301) 496-0497;

² Genbank accession number AF091627.

³ The abbreviations used are: RT-PCR, reverse transcriptase-PCR; SSCP, single strand conformation polymorphism; YAC, yeast artificial chromosome; PAC, P1 artificial chromosome; BAC, bacterial artificial chromosome.

⁴ Internet address: http://kiwi.imgen.bcm.tmc.edu:8088/bio/access_yac.html.

Received 5/ 6/99. Accepted 7/19/99.

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