Molecular alterations of p73 in human esophageal squamous cell carcinomas: loss of heterozygosity occurs frequently; loss of imprinting and elevation of p73 expression may be related to defective p53

Yuyang Christine Cai, Guang-yu Yang, Yan Nie, Li-Dong Wang1, Xin Zhao, Yun-long Song, Darren N. Seril, Jie Liao, Eric Poe Xing and Chung S. Yang2

Laboratory for Cancer Research, College of Pharmacy, Rutgers–The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA and
1 Laboratory for Cancer Research, Henan Medical University, Zhengzhou, Henan 450052, China


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
p73 is structurally and functionally related to p53 and is possibly a tumor suppressor gene. Using 15 surgically resected frozen esophageal specimens containing both squamous cell carcinomas (ESCC) and neighboring normal epithelia, we studied p73 gene alterations and mRNA expression. Loss of heterozygosity of the p73 loci was found in nine of 14 informative cases (64%). A polymorphism at codon 173 (Thr) of p73 was identified (eight samples had ACC and seven had ACT), but mutation was not detected in tumor samples. Nine of the 15 ESCC samples (60%) displayed significantly elevated expression of p73 over the neighboring normal epithelium; of these nine samples, four displayed loss of imprinting (LOI) and one switched the expressed allele. Hypermethylation of exon 1 of the p73 gene was not detected, using the bisulfite modification method, in normal or tumor samples. Twelve of the 15 (80%) ESCC samples contained p53 defects, including missense mutation, non-frameshift small deletion or insertion, non-detectable transcripts and protein accumulation. The ESCC samples with p53 defects were significantly correlated with those which had elevated expression of p73 (Fisher's exact test, P < 0.05). The results suggest that increased expression of p73, including that by LOI, could be a partial compensatory mechanism for defective p53.

Abbreviations: EAC, esophageal adenocarcinoma; ESCC, esophageal squamous cell carcinomas; IHC, immunohistochemistry; LOH, loss of heterozygosity; LOI, loss of imprinting; SSCP, single strand conformation polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The p53 gene product is a critical tumor suppressor, which induces cell cycle arrest or apoptosis in response to cellular stresses such as DNA damage and oxygen starvation (1,2). The loss or inactivation of p53 is thought to contribute to the development of ~50% of all human cancers (3). Two structural homologs of the p53 genes, p73 and p63/p51/KET, have recently been identified (46). The p73 gene is located on human chromosome 1p36.33, a region found to be frequently deleted in neuroblastoma and other tumors (4). Like p53, the p73 protein localizes in the nucleus. It exhibits sequence homology to the p53 transcriptional activation (29%), DNA-binding (63%) and oligomerization (38%) domains as well as functional similarity to p53. In the SK-N-AS cell line, expression of wild-type p73 by transfection can elevate the level of p21waf1 protein, which is comparable to activation of wild-type p53 (4). It has been found that p73 could induce apoptosis in the cancer cell line when the p53 pathway is shut down (7) and that p73 is a target of the non-receptor tyrosine kinase c-Abl in response to DNA damage (810).

The differences between p53 and p73 are also obvious. For example, point mutations in the p53 gene are among the most common genetic abnormalities documented in human cancers and usually localized to `hot-spots' within the DNA-binding domain, exons 5–8 (11). However, mutations in p73 have rarely been identified in human tumors (1219). Whereas p53 null mice are highly prone to tumors (20), homozygous p73 knock-out mice exhibit a striking lack of tumor susceptibility. These mice demonstrate no cancer phenotype after almost 3 years, but suffer severe developmental abnormalities. The functional relationship between p73 and p53 is not clear.

Much research has been conducted on p73 imprinting and its regulation. p73 was reported to be imprinted in cell lines (4) and human normal tissues such as normal lung cells, renal cells and other cell types (21,22). Loss of imprinting (LOI) of p73 has been reported in lung cancer (21). However, biallelic expression of p73 has been observed in normal tissues such as the neuroblast and non-cancerous cell lines (12,15). In addition, there is significant variation of p73 expression among normal tissues taken from the same patient (23). For example, one allele of the p73 gene was preferentially expressed in lung and liver, the other in stomach and both in the small intestine, spleen and kidney. Hypermethylation of the CpG island of exon 1 in cancer cell lines has been reported to be associated with p73 gene silencing and low expression levels (24,25). However, many authors have reported increased p73 expression levels in lung cancer (21), renal cell carcinomas (22), colorectal carcinomas (26), brain tumors (27), bladder cancer (13), neuroblastoma (12), prostate cancer (16), esophageal carcinoma (18), breast cancer (28) and cancerous cell lines (14). To better understand p73 status and the relationship between p53 and p73, we studied these two genes in human esophageal cancer.

Esophageal cancer is the sixth most common cancer world wide. The uneven geographic distribution of this disease (29,30) suggests that certain dietary and cultural practices are important risk factors in the high incidence regions. The mortality rates of esophageal cancer are very similar to the incidence rates, due to the relatively late stage of diagnosis and the poor efficiency of treatment; the 5 year survival rate is <10%. In previous studies using resected esophageal tumors from a high incidence area of Linzhou (formerly Linxian) in Henan, China, we found frequent p53 alterations in human esophageal squamous cell carcinomas (ESCC), with >50% of the samples containing mutations and an even larger number of samples with p53 protein accumulation (31,32). In the present study, we have characterized the molecular changes in p73 together with those of p53 in the frozen resected esophageal samples from Linzhou. The present communication reports the high frequency of loss of heterozygosity (LOH) and LOI of p73 as well as a strong correlation between elevated p73 mRNA content and defective p53 in human ESCC. We suspect that increased expression of p73 by LOI and other mechanisms could be a compensatory mechanism for the loss of p53 functions.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Esophageal tissue specimens
Fifteen human esophageal tissue specimens containing ESCC and two gastric cardia specimens containing esophageal adenocarcinomas (EAC) were obtained at the time of surgery from patients with carcinomas from Linzhou, Henan, China. The samples were stored in liquid N2 or at –80°C until use. Parts of the tumor tissue and adjacent normal epithelium tissue were cut at –20°C and kept at –80°C until use.

DNA/RNA preparation
The areas with tumor or normal epithelium were scraped from hematoxylin and eosin stained glass slides. DNA was prepared with a QIAamp Tissue Kit (Qiagen, Valencia, CA) and RNA was prepared with a Rneasy Mini Kit (Qiagen).

LOH and allelic expression
Genomic DNA of normal/tumor samples was amplified with p73 primers 5'-CAGGAGGACAGAGCACGAA and 5'-CGAAGGTGGCTGAGGCTAG (21). cDNA was amplified with primers A-1 (5'-GGGCTGCGACGGCTGCAGAGC, located in the 5'-untranslated region) and A-2 (5'-GAGAGCTCCAGAGGTGCTC, located in exon 1) (21). The PCR product was digested with StyI overnight and analyzed on a 3% agarose gel.

RT-Multiplex-PCR AdvantageTM
An RT-for-PCR kit (catalogue no. K1402-1; Clontech Laboratories, Palo Alto, CA) was used to reverse transcribe mRNA to cDNA. Then, p73, p53, p21 and an internal control were amplified simultaneously. Primers for p73 were 5'-CTCCCCGCTCTTGAAGAAAC (located in exon 3) and 5'-GTTGAAGTCCCTCCCGAGC (located in exon 4); primers for p21 were 5'-CGACTTTGTCACCGAGACAC and 5'-CGTTTTCGACCCTGAGAGT; primers for p53 were 5'-CTGAGGTTGGCTCTGACTGTACCACCATCC and 5'-CTCATTCAGCTCTCGGAACATCTCGAAGCG; primers for the internal control (human 23 kDa highly basic protein) were 5'-TAAACAGGTACTGCTGGGCCGGAAGGTG and 5'-CACGTTCTTCTCGGCCTGTTTCCGTAGC (Clontech Laboratories). The cycle number was optimized for each DNA sample to ensure that amplification was in the linear range and the results were quantitative. The multiplex PCR product (5 µl), run in duplicate, was analyzed and separated on a 20% polycrylamide gel with a Thermoflow system (Novex, San Diego, CA) by electrophoresis at 20°C for 50 min. An Image-Pro Plus 1.3 System was used for quantification.

p73 mutation analysis
Exons 4–7 of the p73 gene were amplified from the cDNA and the PCR products were run on cold single strand conformation polymorphism (SSCP) gels at 10°C (20% TBE gel in a Thermoflow system). Direct sequencing was also applied for p73 mutation analysis. PCR products for DNA sequencing were purified with QIAquick Gel Extraction Kits (catalogue no. 28706; Qiagen).

p53 mutation analysis
A newly established multiplex PCR–SSCP system was used for p53 mutation analysis. Exons 5–8 of p53 with partial intron sequence were amplified simultaneously by multiplex PCR in three steps: (1) genomic DNA was amplified in the first step PCR with primers 5-R, 6-R, 7-R and 8-R, which contain only specific sequence for the targets, and long primers 5-L-F, 6-L-F, 7-L-F and 8-L-F, which contain both specific sequence for the targets and an artificial universal tail 1 (T1); (2) PCR products were amplified with 5-L-F, 6-L-F, 7-L-F and 8-L-F as well as 5-L-R, 6-L-R, 7-L-R and 8-L-R, which contained both specific sequence for targets and an artificial universal tail 2 (T2); (3) PCR products were amplified with 33P-labeled primers T1 and T2. All the related primers are listed in Table IGo. The multiplex PCR products were mixed with 15 µl of denaturing buffer (TBE buffer containing methylmercury hydroxide and dyes) and denatured at 95°C for 10 min. Then, the sample was loaded onto a 6% polyacrylamide gel containing 5% glycerol. The running conditions were 40 W, 4–5 h at room temperature. Shift bands were cut, eluted with TE buffer at 95°C for 15 min and reamplified by PCR.


View this table:
[in this window]
[in a new window]
 
Table I. Primers for multiplex PCR–SSCP in p53 mutation analysis
 
Methylation detection
Genomic DNA from tumor and normal tissues was modified by bisulfite reaction as previously described (33). Methylation-specific PCR was performed using the primers and PCR conditions taken from Corn et al. (25). The PCR products were resolved on a 3% agarose gel and methylation was determined by the presence of methylated-specific PCR products.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Alterations of the p73 gene in esophageal carcinomas
Using the restriction fragment length polymorphism marker in exon 2 (GC/AT polymorphism) (21), we found that nine of the 14 informative ESCC cases (64%) had LOH. In these nine tumor samples, two patterns of LOH were observed (Figure 1Go): seven had lost AT alleles and two had lost GC alleles in the tumor samples (Table IIGo). Homozygous deletion of the p73 gene was not detected. In previous reports, the LOH frequency for the p73 gene varied from 5.3% (2/38, marker in intron 9) in prostatic carcinomas (34) to 42% (11/29, marker in exon 1) in lung cancers (21). In lung squamous cell carcinoma, an even higher frequency (60%, six of 10 informative cases) of LOH was observed (21). The difference could be due to several factors, including differences in cancer types, populations involved, carcinogen exposure, markers used and the small sample size used in the study (4,17,21,26,34). In the two EAC samples analyzed, both lost the AT alleles (Table IIGo).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. Patterns of LOH of the tumor samples (T) and normal epithelia (N). After StyI digestion, the GC allele had one fragment (229 bp) and the AT allele had two fragments (157 and 72 bp) on a 3% agarose gel. Lanes A, non-informative case; lanes B, informative case without LOH; lanes C, LOH, loss of AT allele in tumor samples; lanes D, LOH, loss of GC allele in tumor samples. M, 100 bp DNA ladder as molecular marker.

 

View this table:
[in this window]
[in a new window]
 
Table II. Summary of p73 and p53 status of the tumor samples
 
No p73 mutations were found in exons 4–7. This region has the highest homology to the p53 DNA-binding domain where most of the p53 mutations occur. This observation is consistent with previous reports (4,34,35). A polymorphism at codon 173 (Thr) was identified: ACC was observed in eight ESCC and one EAC case and ACT was observed in seven ESCC and one EAC case (Table IIGo). This type of polymorphism has been reported in oligodendrogliomas, however, the reported frequency of ACC was 10% (36), much lower than that in our samples.

LOH and mutation are the two most common mechanisms in shutting down tumor suppressors, and play important roles in carcinogenesis. Typical examples are the high frequency of LOH in Rb and mutations in p53. Although frequent LOH of the p73 gene is observed in our samples, its functional importance is not clear. Therefore the p73 expression level was studied.

Changes in the expression of p73
In our 16 informative normal human esophageal epithelia, only monoallelic expression of p73 was observed (Figure 2Go): 14 expressed the GC allele and the other two expressed the AT allele (Table IIGo). The data suggest that p73 is imprinted in the normal esophagus. In the five informative ESCC cases without LOH, four tumor samples had biallelic expression of p73 (Table IIGo). We believe those were due to LOI, namely activation of the normally silent alleles in the tumor samples. The remaining one informative ESCC case without LOH had only the GC allele expressed in both the normal epithelium and tumor samples; interestingly, there was no detectable alteration of p53 in this sample. LOI was not observed in the two EAC cases, which lost the AT alleles.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. Patterns of allelic expression of the tumor samples (T) and normal epithelia (N). After StyI digestion, the AT allele had a fragment of 84 bp and the GC allele had a fragment of 116 bp on a 3% agarose gel. Lanes A, the AT allele was silent in both normal and tumor samples; lanes B, the normally silent GC allele was activated in the tumor sample; lanes C, the normally silent AT allele was activated in the tumor sample; lanes D, the GC allele was silent in both normal and tumor samples; lanes E, the GC allele was lost in the tumor sample and the AT allele was activated. M, 100 bp DNA ladder as molecular markers.

 
Of the 11 ESCC samples with LOH, 10 had the same allelic expression pattern in both the normal and tumor tissues. The remaining one displayed switching from the GC allele in the normal epithelium to the AT allele in the tumor. Apparently the GC allele was lost in this tumor and the silent allele was activated. Combining this sample and the four ESCC samples with biallelic expression, we have observed five cases with activation of the normally silent alleles.

During the study we also found biallelic expression of p73 in the `normal' cells of one sample. After we re-examined the tissue, a precancerous lesion was identified and was screened out from our study. It appears that alteration of p73 can occur before histopathological changes are manifest.

Because of the limited sample source, we used a sensitive multiplex RT–PCR procedure to determine the mRNA levels of p73, p53 and p21waf1 together with an internal control. Efforts were made to run the reaction under conditions in which the signals could be quantified. We first tested the interaction of the primers for a control gene, p53, p73 and p21waf1 in placental DNA and picked the four-pair combination with the least interference and most similar magnitude of amplification (Figure 3AGo). Subsequently, the linear range of amplification with these primers was studied simultaneously at different cycle numbers. Cycle 27 was picked as the standard (Figure 3B and CGo). DNA extracted from human normal/tumor samples in pairs were amplified side by side with the four pairs of primers. In nine of 15 ESCC samples the levels of p73 mRNA in tumors were significantly higher than in the normal tissues (3- to 13-fold, average 6-fold) (Figure 4Go and Table IIGo): four samples exhibited LOI, one switched allele expression. In the two EAC cases, the p73 mRNA levels were also elevated; both displayed LOH but no LOI. It appears that the level of p73 expression is independent of LOH. We believe that there might be an important gene other than p73 in the loci which plays the role of a tumor suppressor. Combined with a rare mutation in p73, if there is any, p73 shut-down does not seem to play the role of a typical tumor suppressor.





View larger version (166K):
[in this window]
[in a new window]
 
Fig. 3. Multiplex RT–PCR and expression analysis. (A) Test of primers. The four pairs of primers used showed little interference and had similar magnitudes of amplification. Lane 1, amplification of the control gene (human 23 kDa highly basic protein); lane 2, amplification of both the control gene and p53; lane 3, amplification of the control gene, p53 and p73 simultaneously; lane 4, amplification of the control gene, p53, p73 and p21 simultaneously. M-15 marker was used as a standard for molecular weight and the sizes of the PCR products for the four genes were approximately 480, 370, 290 and 180 bp. (B). The control, p53, p21 and p73 genes were amplified simultaneously for different cycle numbers and loaded on a 20% polyacrylamide gel. M-15 marker (M) was used as a standard for molecular weight and the sizes of the PCR products for the four genes were approximately 480, 370, 290 and 180 bp. (C) Linear range study, which showed that cycle number 27 was a good basis for RT–PCR in expression analysis.

 


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 4. Alterations at the mRNA level. The tumor and neighboring normal epithelium were analyzed by multiplex RT–PCR in parallel under the optimized conditions and loaded on a 20% polyacrylamide gel. M-15 marker (M) was used as a standard for molecular weight. The relative expression of different genes was quantified as the ratio to the internal control.

 
There have been reports on the down-regulation of p73 expression corresponding to hypermethylation of the CpG island in exon 1 (24,25). We checked this sequence in exon 1 (GenBank accession no. AF077616) and found that the 290 bp sequence contained six CpG dinucleotides and 29 GpC dinucleotides. The CpG:GpC ratio was 0.21, lower than the normal ratio of >0.6 for CpG islands. Seventeen pairs of tumor and normal samples were screened with methylation-specific primers for p73 methylation according to the reported method (25). None of the samples showed methylation in exon 1.

Correlation between elevated p73 expression and p53 alterations
p53 mutation analysis was performed on exons 5–8. Four point mutations, one small insertion and two deletions were identified in 15 ESCC samples (Figures 5 and 6GoGo). Only one ESCC case had two genetic alterations, one point mutation and one deletion (Table IIGo). Three mutations occurred at codon 273, which is one of the hot-spots. Since we only analyzed exons 5–8, mutations in other exons or introns might have been missed. p53 protein accumulation is also regarded as a good indicator for alterations in p53. Thirteen tumor samples displayed p53 immunohistochemistry (IHC) positive staining, representing an abnormal p53 protein level (Figure 7Go). p53 mRNA remained unchanged in most tumors, as measured by semi-quantitative RT–PCR. This is reasonable, since p53 is primarily regulated at the post-transcription level. However, we did observe two ESCC samples with increased p53 transcripts (up to 9-fold); one ESCC sample showed transcripts at 15% of the level in the paired normal sample and one had no detectable transcript. In total, 14 of 17 tumor samples had defective p53, including missense mutation, non-frameshift deletion or insertion, non-detectable transcripts and protein accumulation based on IHC (Table IIGo). Among the 11 tumor samples with elevated expression of p73, one had no detectable p53 transcript and 10 showed p53 protein accumulation based on IHC. Of these, eight displayed p53 missense mutations or non-frameshift deletions or insertions. Interestingly, all displayed concomitant elevated or normal expression of p21waf1. In the three cases without any detectable p53 defects, no changes in p73 and p21waf1 expression were detected. The association between elevated expression of p73 and p53 defects was statistically significant as determined by Fisher's exact test (P < 0.05) (Table IIIGo). If only p53 mutations, deletions and insertions were considered as genetic alterations, the association was still significant (Fisher's exact test, P < 0.05). In the three tumor samples without elevation of p73 expression but abnormal p53 protein, overexpression of MDM-2 was observed.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 5. Multiplex PCR–SSCP for p53 mutation analysis. Four shift bands were identified and labeled 1–4.

 


View larger version (88K):
[in this window]
[in a new window]
 
Fig. 6. Sequencing was used to confirm the p53 mutations after SSCP. (A) Two-direction (a and b) sequencing results which confirmed the point mutation at exon 8 (shift band 2, CGT->TGT ). (B) Two-direction (a and b) sequencing results which confirmed the non-frameshift deletion at exon 7 (shift band 4).

 


View larger version (117K):
[in this window]
[in a new window]
 
Fig. 7. IHC study of p53 protein accumulation with Ab-6. The dark stains show significant accumulation of p53 protein.

 

View this table:
[in this window]
[in a new window]
 
Table III. Correlation between elevated expression of p73 and p53 alterations
 
LOI is one mechanism by which the expression of a gene can be elevated (3739). Of the five ESCC informative cases without LOH (Table IIGo), four showed LOI as well as p53 defects, and the one case without LOI had no detectable p53 defect. The association between p73 LOI and p53 alteration is also significant by Fisher's exact test (P < 0.05). Apparently, LOI is one reason for elevated expression of p73, but LOI by itself is not sufficient.

It has been suggested that p73 is primarily involved in development and active in response to certain DNA damages (20). On the other hand, p73 is up-regulated (elevated mRNA) and maintains its functional status (no mutation) in many tumors (12,16,18,26,28). The elevated expression of p73 and its correlation with the defective p53 suggests that p73 overexpression may be a partial compensatory mechanism for loss of p53 function in the esophagus. Apparently, the compensation is not enough to prevent cancer formation over the long term since carcinomas developed in our samples.


    Acknowledgments
 
We wish to thank Dr Honghai Li for help in establishing the multiplex PCR–SSCP methodology in our laboratory and Dr S.K.Chhabra for help in the preparation of the manuscript. Supported by NIH Grant CA65871 and facilities from NIEHS Center Grant ES05022, Cancer Center Support Grant CA72720 and the China National Natural Science Foundation (39770296).


    Notes
 
2 To who correspondence should be addressed Email: csyang{at}rci.rutgers.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

  1. Livingstone,L.R., White,A., Sprouse,J., Livanos,E., Jacks,T. and Tlsty,T.D. (1992) Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell, 70, 923–935.[ISI][Medline]
  2. Lowe,S.W., Schmitt,E.M., Smith,S.W., Osborne,B.A. and Jacks,T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes [see comments]. Nature, 362, 847–849.[ISI][Medline]
  3. Levine,A.J. (1997) p53, the cellular gatekeeper for growth and division. Cell, 88, 323–331.[ISI][Medline]
  4. Kaghad,M., Bonnet,H., Yang,A., Creancier,L., Biscan,J., Valent,A., Minty,A., Chalon,P., Lelias,J., Dumont,X., Ferrara,P., McKeon,F. and Caput,D. (1997) Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell, 90, 809–819.[ISI][Medline]
  5. Yang,A., Kaghad,M., Wang,Y., Gillett,E., Fleming,M.D., Dotsch,V. andrews,N.C., Caput,D. and McKeon,F. (1998) p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing and dominant-negative activities. Mol. Cell, 2, 305–316.[ISI][Medline]
  6. Osada,M., Ohba,M., Kawahara,C., Ishioka,C., Kanamaru,R., Katoh,I., Ikawa,Y., Nimura,Y., Nakagawara,A., Obinata,M. and Ikawa,S. (1998) Cloning and functional analysis of human p51, which structurally and functionally resembles p53 [see comments]. Nature Med., 4, 839–843. [Erratum, Nature Med. (1998), 4, 982.][ISI][Medline]
  7. Prabhu,N.S., Somasundaram,K., Satyamoorthy,K., Herlyn,M. and El-Deiry,W.S. (1998) p73beta, unlike p53, suppresses growth and induces apoptosis of human papillomavirus E6-expressing cancer cells. Int. J. Oncol., 13, 5–9.[ISI][Medline]
  8. Yuan,Z.M., Shioya,H., Ishiko,T., Sun,X., Gu,J., Huang,Y.Y., Lu,H., Kharbanda,S., Weichselbaum,R. and Kufe,D. (1999) p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature, 399, 814–817.[ISI][Medline]
  9. Agami,R., Blandino,G., Oren,M. and Shaul,Y. (1999) Interaction of c-Abl and p73alpha and their collaboration to induce apoptosis [see comments]. Nature, 399, 809–813.[ISI][Medline]
  10. Gong,J.G., Costanzo,A., Yang,H.Q., Melino,G., Kaelin,W.G.Jr, Levrero,M. and Wang,J.Y. (1999) The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage [see comments]. Nature, 399, 806–809.[ISI][Medline]
  11. Hollstein,M., Shomer,B., Greenblatt,M., Soussi,T., Hovig,E., Montesano,R. and Harris,C.C. (1996) Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acids Res., 24, 141–146.[Abstract/Free Full Text]
  12. Kovalev,S., Marchenko,N., Swendeman,S., LaQuaglia,M. and Moll,U.M. (1998) Expression level, allelic origin and mutation analysis of the p73 gene in neuroblastoma tumors and cell lines. Cell Growth Differ., 9, 897–903.[Abstract]
  13. Yokomizo,A., Mai,M., Tindall,D.J., Cheng,L., Bostwick,D.G., Naito,S., Smith,D.I. and Liu,W. (1999) Overexpression of the wild type p73 gene in human bladder cancer. Oncogene, 18, 1629–1633.[ISI][Medline]
  14. Han,S., Semba,S., Abe,T., Makino,N., Furukawa,T., Fukushige,S., Takahashi,H., Sakurada,A., Sato,M., Shiiba,K., Matsuno,S., Nimura,Y., Nakagawara,A. and Horii,A. (1999) Infrequent somatic mutations of the p73 gene in various human cancers. Eur. J. Surg. Oncol., 25, 194–198.[ISI][Medline]
  15. Tsao,H., Zhang,X., Majewski,P. and Haluska,F.G. (1999) Mutational and expression analysis of the p73 gene in melanoma cell lines. Cancer Res., 59, 172–174.[Abstract/Free Full Text]
  16. Yokomizo,A., Mai,M., Bostwick,D.G., Tindall,D.J., Qian,J., Cheng,L., Jenkins,R.B., Smith,D.I. and Liu,W. (1999) Mutation and expression analysis of the p73 gene in prostate cancer. Prostate, 39, 94–100.[ISI][Medline]
  17. Ichimiya,S., Nimura,Y., Kageyama,H., Takada,N., Sunahara,M., Shishikura,T., Nakamura,Y., Sakiyama,S., Seki,N., Ohira,M., Kaneko,Y., McKeon,F., Caput,D. and Nakagawara,A. (1999) p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent. Oncogene, 18, 1061–1066.[ISI][Medline]
  18. Nimura,Y., Mihara,M., Ichimiya,S., Sakiyama,S., Seki,N., Ohira,M., Nomura,N., Fujimori,M., Adachi,W., Amano,J., He,M., Ping,Y.M. and Nakagawara,A. (1998) p73, a gene related to p53, is not mutated in esophageal carcinomas. Int. J. Cancer, 78, 437–440.[ISI][Medline]
  19. Kroiss,M.M., Bosserhoff,A.K., Vogt,T., Buettner,R., Bogenrieder,T., Landthaler,M. and Stolz,W. (1998) Loss of expression or mutations in the p73 tumour suppressor gene are not involved in the pathogenesis of malignant melanomas. Melanoma Res., 8, 504–509.[ISI][Medline]
  20. White,E. and Prives,C. (1999) DNA damage enables p73 [news; comment]. Nature, 399, 734–735, 737.[ISI][Medline]
  21. Mai,M., Yokomizo,A., Qian,C., Yang,P., Tindall,D.J., Smith,D. and Liu,W. (1998) Activation of p73 silent allele in lung cancer. Cancer Res., 58, 2347–2349.[Abstract]
  22. Mai,M., Qian,C., Yokomizo,A., Tindall,D.J., Bostwick,D., Polychronakos,C., Smith,D.I. and Liu,W. (1998) Loss of imprinting and allele switching of p73 in renal cell carcinoma. Oncogene, 17, 1739–1741.[ISI][Medline]
  23. Nomoto,S., Haruki,N., Kondo,M., Konishi,H. and Takahashi,T. (1998) Search for mutations and examination of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers. Cancer Res., 58, 1380–1383.[Abstract]
  24. Kawano,S., Miller,C.W., Gombart,A.F., Bartram,C.R., Matsuo,Y., Asou,H., Sakashita,A., Said,J., Tatsumi,E. and Koeffler,H.P. (1999) Loss of p73 gene expression in leukemias/lymphomas due to hypermethylation. Blood, 94, 1113–1120.[Abstract/Free Full Text]
  25. Corn,P.G., Kuerbitz,S.J., van Noesel,M.M., Esteller,M., Compitello,N., Baylin,S.B. and Herman,J.G. (1999) Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5' CpG island methylation. Cancer Res., 59, 3352–3356.[Abstract/Free Full Text]
  26. Sunahara,M., Ichimiya,S., Nimura,Y., Takada,N., Sakiyama,S., Sato,Y., Todo,S., Adachi,W., Amano,J. and Nakagawara,A. (1998) Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas. Int. J. Oncol., 13, 319–323.[ISI][Medline]
  27. Loiseau,H., Arsaut,J. and Demotes-Mainard,J. (1999) p73 gene transcripts in human brain tumors: overexpression and altered splicing in ependymomas. Neurosci. Lett., 263, 173–176.[ISI][Medline]
  28. Zaika,A.I., Kovalev,S., Marchenko,N.D. and Moll,U.M. (1999) Overexpression of the wild type p73 gene in breast cancer tissues and cell lines. Cancer Res., 59, 3257–3263.[Abstract/Free Full Text]
  29. Cook-Mozaffari,P. (1979) The epidemiology of cancer of the esophagus. Nutr. Cancer, 1, 51–60.
  30. Warwick,G.P. (1973) Some aspects of the epidemiology and etiology of esophageal cancer with particular emphasis on the Transkei, South Africa. Adv. Cancer Res., 17, 81–229.
  31. Gao,H., Wang,L.D., Zhou,Q., Glodstein,S., Hong,J.Y., Shao,P., Qiu,S.L. and Yang,C.S. (1994) p53 tumor suppressor gene mutation in early esophageal precancerous lesions and carcinoma among high-risk populations in Henan, China. Cancer Res., 54, 4342–4346.[Abstract]
  32. Wang,L.D., Hong,J.Y., Qiu,S.L., Gao,H.K. and Yang,C.S. (1993) Accumulation of p53 protein in human esophageal precancerous lesions: a possible early marker for carcinogenesis. Cancer Res., 53, 1783–1787.[Abstract]
  33. Xing,E.P., Nie,Y., Song,Y., Yang,G.-Y., Cai,Y.C., Wang,L.D. and Yang,C.S. (1999) Mechanisms of inactivation of p14ARF, p15INK4b and p16INK4a genes in human esophageal squamous cell carcinoma. Clin. Cancer Res., 5, 2704–2713.[Abstract/Free Full Text]
  34. Takahashi,H., Ichimja,S., Nimura,Y., Watanabe,M., Furusato,M., Wakui,S., Yatani,R., Aizawa,S. and Nakagawara,A. (1998) Mutation, allelotyping and transcription analyses of the p73 gene in prostatic carcinoma. Cancer Res., 58, 2076–2077.[Abstract]
  35. Nomoto,S., Haruki,N., Kondo,M., Konishi,H., Takahashi,T., Takahashi,T. and Takahashi,T. (1998) Search for mutations and examination of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers. Cancer Res., 58, 1380–1383.[Abstract]
  36. Mai,M., Huang,H., Reed,C., Qian,C., Smith,J., Alderete,B., Jenkins,R., Smith,D. and Liu,W. (1998) Genomic organization and mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletion. Genomics, 51, 359–363.[ISI][Medline]
  37. Wilkins,R.J. (1998) Genomic imprinting and carcinogenesis. Lancet, 1, 329–331.
  38. Wu,M., Wang,H., Lin,C., Sheu,J., Shun,C., Lee,W. and Lin,J. (1997) Loss of imprinting and overexpression of IGF2 gene in gastric adenocarcinoma. Cancer Lett., 120, 9–14.[ISI][Medline]
  39. Feinberg,A. (1993) Genomic imprinting and gene activation. Nature Genet., 4, 110–113.[ISI][Medline]
Received June 1, 1999; revised November 2, 1999; accepted November 19, 1999.