P53 and Rb tumor suppressor gene alterations in gastric cancer

Alterações dos genes supressores tumorais p53 e Rb no câncer gástrico

ORIGINAL RESEARCH

P53 and Rb tumor suppressor gene alterations in gastric cancer

Alterações dos genes supressores tumorais p53 e Rb no câncer gástrico

Rejane Mattar; Suely Nonogaki; Cleonice Silva; Venancio Alves; Joaquim J. Gama-Rodrigues

From the Department of Gastroenterology, Hospital das Clínicas, Faculty of Medicine, University of São Paulo and Division of Pathology, Adolfo Lutz Institute - São Paulo/SP, Brazil. E-mail: [e-mail]

ABSTRACT

Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma.

METHOD: Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using ³⁵S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone.

RESULTS AND DISCUSSION: No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P = .90). There was no correlation of gene alterations with the disease severity.

CONCLUSIONS: The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

Key words: Gastric cancer. APC. DCC. Rb. p53.

RESUMO

A inativação de genes supressores tumorais tem sido freqüentemente observada na carcinogênese gástrica. O nosso objetivo foi estudar o envolvimento dos genes p53, APC, DCC e Rb no câncer gástrico.

MÉTODO: Vinte e dois casos de câncer gástrico foram estudados por PCR-LOH (reação de polimerase em cadeia- perda de alelo heterozigoto) dos genes p53, APC, DCC e Rb; e por PCR-SSCP (reação de polimerase em cadeia- polimorfismo de conformação de cadeia única) dos exons 5-6 e exons 7-8 do gene p53, empregando ³⁵S-dATP e expressão de p53 por imunoperoxidase com monoclonal anti-p53.

RESULTADOS E DISCUSSÃO: Perda de alelo heterozigoto não foi detectada nos genes estudados; deleção homozigótica foi observada no gene Rb em 23% (3/13) dos casos de câncer gástrico do tipo intestinal. Desvio de motilidade de banda nos exons 5-6 e/ou exons 7-8, indicando mutação do gene p53 foi encontrada em 18 casos (81.8%). A expressão de p53 foi positiva nas células de câncer gástrico em 14 casos (63.6%). A mucosa gástrica normal não corou com anti-p53, portanto, a reatividade imune deve representar formas mutantes. A correlação de desvio de motilidade de banda e expressão imune de p53 não foi significante (p=0.90). Não houve correlação entre as alterações genéticas e a extensão da doença.

CONCLUSÃO: A inativação dos genes p53 e Rb tem papel na carcinogênese gástrica no nosso meio. A perda do gene Rb observada apenas no câncer gástrico do tipo intestinal deve ser avaliada posteriormente em associação com infecção pelo Helicobacter pylori. O gene p53 estava afetado em ambos os tipos histopatológicos.

Unitermos: Câncer gástrico. p53. APC. DCC. Rb.

Gastric cancer is a heterogeneous pathology, being classified by Laurén (1965)¹ into 2 general subtypes: intestinal (differentiated) and diffuse (undifferentiated). Intestinal-type gastric cancer may or may not be preceded by preneoplastic lesions; it is more prevalent in older individuals and in certain geographical areas where there is a high incidence of gastric carcinoma, such as in Japan.²

The differences between these two histological subtypes occur also at the molecular level, suggesting different genetic pathways. The intestinal-type gastric carcinoma presents tumor suppressor gene alterations similar to colorectal tumors and distinct from diffuse-type gastric cancer.³

An accumulation of multiple genetic and epigenetic alterations of oncogenes, tumor suppressor genes, DNA repair genes, cell cycle regulators, cell adhesion molecules, and growth factor/receptor systems are involved throughout the course of the multistep conversion of normal epithelial cells to clinical gastric cancer.⁴

In gastric cancer, p53 gene alterations have been observed in both histological subtypes,⁵ being correlated with node-positive cancer,^6,7 depth of tumor invasion,⁸ and poor survival.^9,10 The p53 protein is a transcriptional factor that arrests the cell cycle in the G1 phase when DNA is damaged¹¹ by inducing the expression of the p21 protein, an inhibitor of Cdk kinase and PCNA.^12,13 Thus, damaged DNA can not replicate, allowing time for the repair system to act.¹¹ If this system fails, p53 induces apoptosis by transactivation of the bax gene.¹⁴

The loss of heterozygosity (LOH) and the loss of expression of the DCC gene have been more frequently found in the intestinal-type gastric cancer^15-17 and have often been encountered (35.3%) in gastric cancer in stage III and IV. Thus, in gastric cancer, LOH of the DCC gene was a late event associated with malignant progression.^15,18 The DCC gene, located on the long arm of chromosome 18,¹⁹ encodes a netrin-1 receptor component with functions in cell migration,²⁰ G2/M cell cycle arrest, and apoptosis.²¹

The mutated APC germline gene on chromosome 5q21 is responsible for the inheritance of familial adenomatous polyposis; in addition, it was somatically altered in sporadic colorectal cancer patients.²² In gastric cancer, the incidence of allelic deletions of APC was significantly higher in the intestinal phenotypes than in the diffuse phenotypes.²³ Furthermore, APC down-regulates the proto-oncogene b-catenin that is critical for intercellular adhesion and has linked colorectal carcinogenesis to the Wnt-signal transduction pathway.²⁴ Increased b-catenin mRNA levels were significantly more frequent in intestinal-type gastric cancers as compared with the diffuse-type gastric cancers. APC gene mutations found in these cases of intestinal-type gastric cancer were associated with the increase of b-catenin mRNA levels.²⁵

Loss of the retinoblastoma (Rb) gene has been associated with esophageal tumorigenesis,²⁶ and at a lower rate to gastric cancer development.¹⁷ However, more recent reports have shown that Helicobacter pylori (H. pylori) infection generated gastric cancer through p53-Rb tumor-suppressor system mutation and telomerase reactivation.²⁷ The Rb gene encodes a nuclear protein that acts as a cell cycle control checkpoint at the G1 phase.²⁸

The purpose of our study was to further analyze the involvement of p53, APC, DCC, and Rb tumor suppressor genes in gastric carcinoma cases.

MATERIALS AND METHOD

Gastric cancer tissues and corresponding leukocytes were obtained from 22 patients after surgical treatment during the period of 1996 to 1997 at the Hospital of Clinics, Department of Gastroenterology, and were immediately frozen in liquid nitrogen and stored prior to DNA extraction. The average age of the patients (18 men, 4 women) was 63.4 + 14.3 years. The tumors were classified as intestinal and diffuse types according to Láuren1; 13 cases were of the intestinal type and 9 cases were of the diffuse type. The TNM stage grouping was performed according to the criteria of the Japanese Classification of Gastric Carcinoma.²⁹

DNA extraction

DNA was extracted from the thawed cancer tissue and peripheral leucocytes using a phenol-chloroform method³⁰ and stored at 4 ºC until use.

Polymerase Chain Reaction (PCR)

One microgram or 300 ng of genomic DNA was used as a template in a reaction volume of 50 uL, containing 50 pmol of each primer (Table 1), 200 uM of each dNTP and 2.5 U of Taq DNA polymerase (Gibco BRL, Gaithersburg, MD, USA). The PCR was performed in a 2400 GeneAmp PCR system (Perkin Elmer, Branchburg, NJ, USA). Amplification was performed for 35 cycles at an annealing temperature of 68 ºC for APC,³¹ 62 ºC for p53,³² 55 ºC for DCC,³¹ and 57 ºC for Rb-D13S270.³³ The amplification conditions for Rb intron1 were 40 cycles in 2 steps (94 ºC for 1 min and 50 ºC for 1 min), followed by 1 extension step at 72 ºC for 5 min; inclusion of 10% dimethyl sulfoxide (DMSO) was necessary for generating the 180 bp fragment.³⁴ A second primer pair that spans the same locus was used in the cases where the PCR product was absent from the carcinoma DNA template when compared with a strong product from the paired constitutional DNA template.

Restriction fragment length polymorphism analysis

Products of the PCR (~45 uL) were digested with 60 U of MspI (DCC) (Stratagene, La Jolla, CA, USA) and RsaI (APC) (Stratagene, La Jolla, CA, USA), 15 U of BamHI (Rb) (Stratagene, La Jolla, CA, USA), and 8 U of AccII (p53) (Amersham Life Science, Cleveland, OH, USA) at 37 ºC overnight. The DNA fragments were separated by electrophoresis on 4% low-melting-point agarose gels. For VNTR, Rb (D13S270), and after MspI digestion, PCR products were separated on 3% low-melting-point agarose gels.

PCR Analysis of Single-Strand Conformation Polymorphism

Genomic DNA (300 - 500 ng) was used as a template in a reaction volume of 25 µL containing 50 pmol of each primer (Table 1), 200 µM of deoxynucleotide triphosphate (dNTP), ³⁵S-dATP (0.5 µL), and 2.5 U of Taq DNA polymerase (Amersham Biosciences). Exons 5-6 and 7-8 of the p53 gene were amplified in 35 cycles according to the following schedule: 94 ºC for 30 s, 63 ºC for 50 s, and 72 ºC for 1 min. The elongation step was done at 72 ºC for 10 min. Amplification products were diluted 5-fold in 0.1% SDS, 10 mM EDTA and were rediluted 1:2 in sequencing stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol); they were then heated at 90 ºC for 5 min, chilled on ice, and loaded onto a nondenaturing polyacrylamide gel (6% acrylamide, 10% glycerol, 1x TBE). Electrophoresis was carried out at 8 W at room temperature. After 4 hours of migration for exons 5-6 and 7 to 9 hours of migration for exons 7-8, the gels were dried and subjected to autoradiography using Kodak T-Mat G/RA film at —80 ºC with an intensifying screen. One sample of amplification product from blood was run together with tumor DNA amplification products. The conditions have been previously described, with modifications.^35,36 The primers that were used for PCR were those according to Tohdo et al. 1993.³⁷

LSAB-immunoperoxidase

Sequential sections of 3 µm from formalin-fixed, paraffin-embedded samples were placed on slides previously treated with 3-aminopropyltriethoxy-silane (Sigma, A-3648, USA). After deparaffinization in xylene and rehydration in alcohol, antigen retrieval was performed with 10 mM citric acid pH 6.0 in a pressure cooker for 4 min. Endogenous peroxidase activity was blocked with 6% H₂O_2. Incubation with 1:100 monoclonal antibody anti-p53 clone DO-7 (Dako, M7001, Denmark) in 1% bovine serum albumin-phosphate buffered solution, was performed for 16 h at 4 ºC. The slides were then incubated for 30 min at 37 ºC with secondary biotinylated goat anti-mouse/rabbit Ig, followed by incubation for 30 min at 37 ºC with the complex, streptavidin and biotinylated peroxidase (Dako, K492, Denmark). Slides were developed using 3,3'-diaminobenzidine tetrahydrochloride (Sigma, D-5637, USA), 6% H₂O₂, and dimethyl sulphoxide and counterstained with Harris' hematoxylin. The samples were observed with optic microscope by 2 observers (SN and VAFA) and were scored as previously described by Harvey³⁸; estimated proportion and intensity scores were added to obtain a total score that ranged from 0.2 to 8. A score greater than 2 was used to define p53 positivity.

Statistics

The statistic analysis was performed with the chi-square and Fisher exact tests using the SPSS 7.5 for Windows, student version. A P value of <.05 was considered statistically significant.

RESULTS

The results of the analysis of PCR products for loss of heterozygosity (LOH) at the Rb, APC, DCC, and p53 loci on 22 gastric cancer tissues (13 of intestinal type and 9 of diffuse type) matched with corresponding peripheral leukocytes are listed in Table 2. Heterozygosity was found for APC in 13 of 21 (61.9%) cases, for DCC in 13 of 22 (59%) cases, for p53 in 11of 21 (52.4%) cases, and for Rb in 7 of 19 (36.8%) cases. Those cases that had no amplification in leukocyte and tumor DNA for unknown reasons were excluded from this analysis. No LOH was observed in these tumor suppressor genes. Nonetheless, no PCR product was obtained for the tumor DNA when compared to the normal DNA at the DCC gene in 1 patient (CA.6) and at the Rb gene in 3 patients (CA.6, CA.11, CA.22), suggesting homozygous deletion.

To confirm homozygous deletion at the DCC and Rb genes, a second pair of primers that span the same locus was used, a variable number of the tandem repeat markers (VNTR) was used for the DCC gene, and a microsatellite marker (D13S270) was used for the Rb gene. Homozygous deletion was confirmed only at the Rb gene (Figure 1). These 3 cases of the intestinal-type gastric carcinomas (3/13—23%) with homozygous deletion at Rb gene were classified as stage Ia, stage II, and stage IIIb, respectively.

Since p53 gene alterations are very frequent in gastric cancer and no allelic loss was detected, we decided to search for mutations using PCR analysis for single-strand conformation polymorphism (PCR-SSCP) and p53 protein overexpression using LSAB-immunoperoxidase. Eighteen (81.8%) cases of both histological types showed mobility shifts in exons 5-6 and/or 7-8 of the p53 gene (Figure 2). The p53 protein expression was positive in gastric cancer cells in 14 cases (63.6%). There was no significant correlation of band mobility shift and immunoreactivity to anti-p53 (P = .90). The staining of p53 was never observed in the normal gastric mucosa adjacent to the tumor tissue; thus, p53 protein immunoreactivity likely indicated mutant forms of the p53 gene. The correlation of band mobility shifts and immunoreactivity was not observed in 9 cases. In 7 cases, a mobility shift was detected in exons 7-8 with negative immunoreactivity to anti-p53, 4 were 0 + 0 = 0 and 3 cases were 1p + 1i = 2. The immunoreactivity to anti-p53 was positive in 2 cases (2p + 3i = 5; 2p + 1i = 3) with a negative band mobility shift.

The statistical analysis showed that there was no correlation of sex, age, histology, or severity of disease with mutation and with the immunoreactivity to anti-p53.

DISCUSSION

To understand the molecular events of gastric carcinogenesis in our environment, the DNA of 22 gastric tumors paired with corresponding DNA of peripheral leukocytes were studied for loss of heterogeneity (LOH) of the p53, APC, DCC, and Rb genes. These tumor suppressor genes were chosen for this study based on previous reports showing that their inactivations have a role in gastric carcinogenesis.

Loss of heterogeneity at the APC locus was detected in 87% of primary gastric carcinomas in both intestinal and diffuse types in both early and advanced stages.³⁹ Therefore, LOH at APC was considered one of the most prevalent genetic alterations in human gastric carcinoma, and it occurred at an early stage of the carcinogenesis and was not a prognostic factor. Moreover, Sano et al.⁴⁰ had observed LOH on chromosome 5q where the APC gene is located in 60% of early well-differentiated carcinoma, but not in poorly differentiated carcinoma.

In our study, no LOH was detected at the APC locus. Other authors^17,41 studying the same polymorphic site found a low incidence (27% - 30%) of LOH in gastric cancer. Also in Africa, where the frequency of gastric cancer is low as it is in Brazil, only 1 patient presented LOH in the 5q region.⁴² Furthermore, in gastric cancer patients from north-central Italy, no intragenic mutations were found in APC codons 686 through 1693, and allelic loss was detected in loci near APC.⁴³

These authors argued that epidemiologic studies have not observed a higher risk of gastric cancer in patients with inherited familial adenomatous polyposis. Fundic gland polyps are the most common gastric lesion in familial adenomatous polyposis and are generally believed to have little or no potential for malignant transformation in the population at large. The development of high-grade dysplasia or gastric adenocarcinoma associated with diffuse fundic gland polyposis was described in a few cases of familial adenomatous polyposis.⁴⁴ Gastric-type adenomas were less likely to show high-grade dysplasia and adenocarcinoma and were found in 10 patients with familial adenomatous polyposis.⁴⁵

High frequencies of allelic deletions affecting the DCC locus have been previously described to occur in 30%¹⁷ to 60%¹⁶ of cases of both types of gastric cancer¹⁵ and more often in advanced (50%) than in early (14.3%) disease. Only 1 case in the present study (CA.6) showed what seemed to be homozygous deletion at the DCC gene by RFLP; however, with another pair of primers, a region of the DCC gene was amplified. Thus, the possibility of homozygous deletion was excluded. Since the frequency of DCC loss is higher in advanced disease and more frequent in the intestinal type, we were expecting that at least in the 5 cases that had advanced intestinal-type gastric cancer and were informative for DCC gene, loss would be found.

Homozygous deletion at the Rb gene was detected in 3 (3/13—23%) cases of the intestinal-type gastric cancer, which seems to be an early event, since 2 of these patients had initial disease (stage Ia and stage II). The inactivation of the Rb gene by mutation or loss has been considered an important genetic alteration in esophageal carcinogenesis.^{26, 31} Our data suggest that Rb gene inactivation may be involved in the development and/or progression of intestinal-type gastric cancer.

Homozygous deletions are thought to be the result of 2 events: the loss of a larger chromosomal region and the independent loss of a considerably smaller area.⁴⁶ In previous reports, homozygous deletion was described in a variety of cancers as a mechanism of total gene inactivation, and the presence of at least 1 tumor suppressor gene within the deleted region was suggested.^47-50

A higher prevalence (30%) of Rb-LOH in both histological types of gastric cancer was found by other authors.¹⁷ A more recent report⁵¹ showed that the mRNA levels of Rb and p53 in gastric cancer tissues were both significantly lower than were those in their noncancerous tissue samples. These studies indicate that the suppression of both Rb and p53 may be associated with the tumorigenesis of the stomach.

Loss of heterozygosity on chromosome 17p (p53 locus) and mutation of the p53 gene have been observed in more than 60% of gastric carcinomas, regardless of the histological type,⁴⁰ and have been correlated with short survival times.⁹ Surprising, no LOH at the p53 gene was observed in our cases. One possible explanation for the fact that we did not find LOH at p53 gene may be that this gene may be altered through another mechanism, such as point mutation. To search for p53 gene mutations, we decided to use PCR-SSCP and to correlate with immunoreactivity to anti-p53. The half-life of wild-type p53 is short, around 20 to 30 minutes; consequently, p53 protein expression usually is negative in normal tissues. Mutations render the p53 protein to be a more stable compound with a longer half-life (1.5 - 7 h).⁵² Thus, p53 protein overexpression is likely to represent mutant forms.⁵³

In the literature, the authors usually employ ³²P-dCTP for PCR-SSCP^35,36; however, ³²P is a high-energy b particle emitter, so we decided to try the low-energy b particle emitter, ³⁵S-dATP. The bands were easily visualized in dried gels exposed at —80^º C for at least 3 days. Thus, the use of ³⁵S-dATP was more advantageous than ³²P-dATP, since it lowered the risk for the personnel of the lab.

Following PCR-SSCP, band mobility shifts were observed mainly in exons 7-8 (86.36%) that encompasses codons 225 to 326. Other authors⁵⁴ studying p53 in gastric cancer also found a predominance of mutations in exons 7-8 (70%).

Non-missense mutations or frameshifts could explain negative immunoreactivity with band mobility shifts in 7 cases. Moreover, analyzing the Allred criteria only 4 were truly negative 0+0=0, and 3 cases were 1p+1i=2 (considered negative). Allred 2 should be better evaluated and, perhaps, considered as positive for p53 expression as determined by immunohistochemistry. Seta et al., 1998⁵⁴ found a p53 mutation in 2 cases of gastric cancer with less than 25% of cells positive (+) by immunohistochemistry. Assuming Allred 2 to be positive for p53, the agreement of immunohistochemistry and PCR-SSCP would be 72.7%.

In 2 cases that had immunoreactivity to anti-p53 and most probably harbored mutation in the p53 gene, PCR-SSCP was unable to detect mutation. A discrepancy between results by PCR-SSCP and DNA sequencing has been reported in the literature. In MKN-7 and MKN-28 gastric cancer cell lines, p53 gene mutations were not detected by PCR-SSCP³⁶; however, point mutations at codons 278 and 251, respectively, were found by cDNA sequencing.⁵³

In conclusion, the inactivation of p53 is involved in the development and/or progression of both diffuse and intestinal types of gastric cancer. However, loss of the Rb gene plays role only in the intestinal-type gastric cancer progression. It has been suggested that H. pylori infection initiates gastric cancer through p53-Rb tumor-suppressor system mutation and telomerase reactivation.²⁷ Even though the incidence of H. pylori in the normal population of Brazil is high (80%), the association of H. pylori infection and gastric cancer has been observed exclusively with the intestinal type (P = .008)⁵⁵. Further studies are needed to demonstrate the direct involvement of H. pylori in p53 and Rb gene inactivation in our cases of gastric cancer.

ACKNOWLEDGEMENTS

The authors thank Ana Maria Alexandrino for doing PCR-LOH of the cases.

Received for publication on November 05, 2003

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Publication Dates

• Publication in this collection
  09 Sept 2004
• Date of issue
  2004

History

• Received
  05 Nov 2003