p53 gene mutations in colorectal tumors from patients with familial polyposis coli.

The p53 gene has been elucidated as a tumor suppressor gene, and inactivation of this gene caused by deletion or point mutations may play a crucial role in the development of human malignancies. In colorectal carcinomas with an allelic deletion of the p53 gene, the remaining p53 gene was mutated with considerable frequency. It is most difficult to detect point mutations or small deletions of the gene because the mutations occur in diverse regions, although four hot spots have been observed [J.M. Nigro et al., Nature (Lond.), 342: 705-708, 1989]. The polymerase chain reaction and denaturing gradient gel electrophoresis facilitate detection of mutations in the hot spots of the p53 gene. Using these methods, we detected mutations in three adenomatous polyps and one carcinoma from familial polyposis coli patients and three carcinomas of sporadic cases. The DNA sequence analysis confirmed mutations of the p53 gene in 2 adenomas (13 base-pair deletions in one and a point mutation in the other) and 1 carcinoma (point mutation) from familial polyposis coli patients. These results suggest that the p53 gene mutations may be involved in the formation not only of carcinomas but also of adenomas which occur in familial polyposis coli patients.     

p53蛋白三维空间结构改变与甲状腺乳头状瘤p21^WAF1/CIP1蛋白定量表达关系的研究

目的：从结构分子生物学深度探讨p53蛋白三维空间结构改变与甲状腺乳头状癌（PTC）及甲状腺滤泡性腺瘤（TFA）中p21^WAF1/CIP1。蛋白定量表达的关系。方法：采用PCR-SSCP、DNA序列分析、计算机重构p53蛋白三维空间结构和有关分析软件、免疫组织化学和图像分析等技术进行研究。根据p53基因Exon5—8突变与否把PTC划分为Ⅰ组（p53基因突变组）和Ⅱ组（无p53基因突变组），再根据突变型p53蛋白三维构象改变的空间位点不同，把Ⅰ组分为ⅠA和ⅠB组。结果：Ⅱ组和ⅠA组的阳性细胞的平均积分光密度值（MOD）差异无统计学意义，两者均数差1．26，P〉0．05。Ⅲ组（TFA组）和Ⅱ组、Ⅲ组和ⅠB组、Ⅲ组和ⅠA组之间，尤其是ⅠB组和ⅠA组之间的MOD的差异有统计学意义，均数差分别为12．57、25．80、13．84和-1．96，P〈0．01。结论：p53蛋白空间构象的改变与p21^WAF1/CIP1蛋白定量表达的变化之间存在着质变与量变的关系。     

p53 Gene Mutations Associated with Anaplastic Transformation of Human Thyroid Carcinomas

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4–9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.     

p53 gene mutations associated with anaplastic transformation of human thyroid carcinomas.

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4-9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.     

Frequent alterations of the p14(ARF) and p16(INK4a) genes in primary central nervous system lymphomas

To elucidate the role of p53/p16(INK4a)/RB1 pathways in the tumorigenesis of primary central nervous system lymphomas (PCNSLs), we have analyzed p14(ARF), p16(INK4a), RB1, p21(Waf1), and p27(Kip1) status in a series of their 18 sporadic cases of diffuse large B-cell lymphoma, using methylation-specific PCR, differential PCR, and immunohistochemistry. Homozygous deletion or methylation of p14(ARF) was detected in 10 (56%) PCNSLs, and they were almost entirely deletions (except 1 case). A total of 11 (61%) PCNSLs demonstrated homozygous deletion (6 cases) or methylation (5 cases) of p16(INK4a). Six tumors showed both p14(ARF) and p16(INK4a) homozygous deletions. Hypermethylation of the RB1 and the p27(Kip1) promoter region was detected in 2 (11%) cases, whereas p21(Waf1) methylation was not detected in any. Immunohistochemistry revealed loss of p14(ARF) and p16(INK4a) expression in 10 (56%) samples, correlating with the gene status. Four cases showed independent negative immunoreactivity for pRB and p27(Kip1), and nearly one-half of cases (8 of 18; 44%) were characterized by lack of p21(Waf1) expression. These results indicate that inactivation of p14(ARF) and p16(INK4a) by either homozygous deletion or promoter hypermethylation represents an important molecular pathogenesis in PCNSLs. Hypermethylation of RB1, p21(Waf1), and p27(Kip1) appears to be of minor significance, these genes being independently methylated in PCNSLs.     

Loss of p21Waf1 expression is a strong predictor of reduced survival in primary superficial bladder cancers.

p21Waf1 is a downstream effector of p53 and belongs to the Cip1/Kip1 family of cyclin-dependent kinase inhibitors. Thus, it is a potential tumor suppressor gene and likely plays an important role in tumor development. Moreover, reduced expression of p21Waf1 has been reported to have prognostic value in several human malignancies. In this study, we evaluated the prognostic value of p21Waf1 in bladder cancer compared with other clinicopathological features and with p27Kip1 and p53 expression. A total of 96 superficial (pTa-1) human bladder carcinomas were immunohistochemically stained for p21Waf1 protein expression. Positive p21Waf1 staining (> or =5% positive nuclei) was observed in 68 of the 96 (71%) tumors. p21Waf1 expression was neither associated with tumor stage (P = 0.9) nor with tumor grade (P = 0.18) but was significantly associated with both p53 protein expression (> or =20% positive nuclei; P = 0.007) and with p53 gene mutations (P = 0.017). A significant correlation was also observed between positivity for p21Waf1 and high (>50% positive cells) p27Kip1 expression (P = 0.04). With regard to prognosis, patients whose tumors showed absence of p21Waf1 staining displayed a significantly shorter overall survival (P = 0.01 by log-rank test). However, p21Waf1 expression did not correlate with disease-free survival (P = 0.15 by log-rank test). On a multivariate analysis that also included p53 and p27Kip1 expression, negative p21Waf1 staining was an independent predictor of reduced overall survival (P = 0.004; relative risk, 5.32), stronger than age and tumor stage. These data indicate that expression of p21Waf1 protein strongly correlates with survival and might represent a useful prognostic marker in primary superficial bladder carcinomas.     

p53 mutations in childhood thyroid tumors from Belarus and in theyroid tumors without radiation history

Mutations in the p53 tumour-suppressor gene (exons 5–8) were investigated in 31 Belarussian childhood thyroid tumours (24 cases of papillary thyroid carcinoma, 3 benign tumours and 2 cases each of thyroiditis and goiter); 33 thyroid tumours from juveniles and adults without radiation exposures (25 carcinomas of various histological types, including 11 papillary carcinomas and 8 adenomas) and 6 tumours from adults (4 papillary carcinomas, 1 adenoma, 1 goiter) served as controls. The mutational spectrum of p53 differed greatly between the childhood thyroid carcinomas from Belarus and the control groups. In the control groups of 29 malignant thyroid tumours, 7 different mutations were detected on exons 5–8, none of which occurred among the 15 papillary carcinomas in this group. Five mutations were found in tissue samples of the 24 childhood papillary carcinomas, and they were all the same p53 point mutation (CGA |iO CGG) on codon 213 of exon 6. To determine whether this mutation is simply a polymorphism or whether it is specific to the tumour cells, laser-assisted microdissection was applied to collect various areas of tumorous and non-tumorous cells (10–20 cells per sample) from each paraffin-embedded tissue section of 8 of the papillary thyroid carcinomas. Using PCR-SSCP and sequence analysis on these cells, the very same p53 mutation on codon 213 was detected in various microdissected tumour samples of 2 cases, but it was not found in any microdissected non-tumorous sample. The exclusive occurrence of this p53 mutation in selective microdissected samples of tumour cells, even as homozygous mutation in 1 case, reflects a distinct tumour heterogeneity within papillary childhood thyroid carcinomas. Int. J. Cancer 73:802–807, 1997. © 1997 Wiley-Liss, Inc.     

Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after gamma-irradiation.

The p53/p21Cip1/Waf1-dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo fibroblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent trans-activation of the target p21cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21Cip1/Waf1 and p27KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21Cip1/Waf1 and p27KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21Cip1/Waf1-associated kinase activity is increased upon gamma-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-specific antibodies. During a temporary G2/M delay induced by gamma-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21Cip1/Waf1, contributes to formation of polyploid cells which then die due to apoptosis.     

Association between polymorphism in p21(Waf1/Cip1) cyclin-dependent kinase inhibitor gene and human oral cancer.

The cyclin-dependent kinase inhibitor gene p21(Waf1/Cip1) plays a central role in inducing cellular growth arrest, terminal differentiation, and apoptosis. Alterations in this gene may adversely affect regulation of these processes and increase susceptibility for cancer. We have recently reported a novel polymorphism in the p21(Waf1/Cip1) gene in the Indian population and its association with esophageal cancer. An A-->G transition at codon 149 resulted in amino acid substitution from aspartate to glycine in the proliferating cell nuclear antigen binding COOH-terminal domain of p21(Waf1/Cip1) that may affect PCNA-p21(Waf1/Cip1) interactions, thereby affecting regulation of cellular proliferation, and may increase susceptibility for development of cancer. In a parallel study in our laboratory, we searched for putative p21(Waf1/Cip1) mutations in oral premalignant and malignant lesions. No somatic mutation was detected in exon 2 of p21(Waf1/Cip1). Interestingly, a codon 149 polymorphism variant (A-->G) was identified in 11 of 30 (37%) premalignant lesions (7 of 19 hyperplastic lesions and 4 of 11 dysplastic lesions) and 11 of 30 (37%) squamous cell carcinomas (SCCs). This codon 149 variant was also identified in paired lymphocytes of all of the patients with premalignant lesions and SCCs harboring the variant allele, suggesting the occurrence of a polymorphism. Lymphocyte DNA isolated from 50 unrelated age- and gender-matched healthy subjects was screened for this polymorphism. Seven of 50 (14%) normal controls harbored the A-->G codon 149 variant allele. Immunohistochemical analysis of p21(Waf1/Cip1) protein expression showed immunoreactivity in 19 of these 30 (63%) oral premalignant lesions and 16 of 30 (53%) SCCs. The most intriguing features of the study were: (a) the significant increase in frequency of this polymorphism not only in patients with oral SCCs (P = 0.038), but also in patients with premalignant lesions (P = 0.038), compared with normal controls; and (b) the significantly higher frequency of p21(Waf1/Cip1) variants (codon 149) in oral premalignant lesions (10 of 11 cases) and SCCs (11 of 11 cases) with wild-type p53 (P = 0.045) than in lesions with p53 mutations, suggesting that this polymorphism affects the p53 pathway and may play a vital role in oral tumorigenesis. Furthermore, overexpression of p21 protein in oral lesions harboring missense mutations in the p53 gene suggest a p53-independent role for p21 in the pathogenesis of oral cancer.     

Disrupted p53 function as predictor of treatment failure and poor prognosis in B- and T-cell non-Hodgkin's lymphoma.

Mutation of the p53 gene has been associated with treatment failure and poor outcome in various malignancies. It has been suggested that immunohistochemical analysis of p53 and p21Waf1, a downstream target, can be used to screen for p53 gene mutations. We determined the value of immunohistochemical screening for p53 gene mutations as a prognostic marker in a population-based group of B- and T-cell non-Hodgkin's lymphomas (NHLs). On the basis of p53 gene mutation status and immunohistochemically detected p53 and p21Waf1 expression in 34 lymphomas, we established an immunophenotype (delta p53) correlating with p53 gene mutation. The immunohistochemical analysis was extended to encompass 199 lymphomas from a population-based registry and was correlated with clinical parameters. Delta p53 showed 100% concordance with p53 gene mutation and was detected in 42 cases (21%). Multivariate analysis of advanced stage lymphomas showed that delta p53 was independently associated with treatment failure (relative risk, 3.8; P = 0.001). Delta p53 predicted poor survival when analyzing all patients (P = 0.0001), as well as B-cell (P = 0.04) and T-cell NHL (P = 0.000002). In multivariate analysis, delta p53 (relative risk, 2.2; P = 0.001) maintained prognostic significance. The impact on prognosis of delta p53 was highly significant in the low-intermediate-risk group (P = 0.00002). Comparing survival of the aggressive lymphoma patients in this group showed that the 8 delta p53 patients died within 1 year, whereas the median survival of the 28 non-delta p53 patients was 36 months. These results suggest that immunohistochemically assessed p53 status may predict treatment response and outcome in B- and T-cell NHL patients.     

甲状腺肿瘤组织中p14ARF基因纯合性缺失及其产物表达状况的研究

目的检测甲状腺肿瘤组织中p14ARF基因变异及其产物的表达,探讨此基因与甲状腺肿瘤发生发展的关系.方法应用双重PCR和免疫组化SP法分别检测20例甲状腺腺瘤和28例甲状腺癌组织中p14ARF基因纯合性缺失及其蛋白表达状况.28例甲状腺癌包括11例甲状腺滤泡癌,12例乳头状癌,4例髓样癌以及1例未分化癌.结果1)p14ARF基因在甲状腺腺瘤及癌中的纯合性缺失率分别为5.0%(1/20)和42.9%(12/28),两者差异有统计学意义,P=0.0036.2)p14ARF蛋白在甲状腺腺瘤及甲状腺癌中的阳性率分别为90.0%(18/20)和35.7%(10/28),两者差异有统计学意义,P=0.0002.结论甲状腺肿瘤中p14ARF基因的纯合性缺失是其蛋白表达失活的重要机制之一,与甲状腺肿瘤的发生发展密切相关.p14ARF可能是甲状腺肿瘤的分子标志物之一,可作为鉴别甲状腺腺瘤与滤泡癌的辅助指标.     

MicroRNA-423 promotes cell growth and regulates G(1)/S transition by targeting p21Cip1/Waf1 in hepatocellular carcinoma

MicroRNAs (miRNAs) are small non-coding RNA molecules that are often located in genomic breakpoint regions and can act as oncogenes or tumor suppressor genes in human cancer. Our previous study showed that microRNA-423 (miR-423), which localized to the frequently amplified region of chromosome 17q11, was upregulated in hepatocellular carcinoma (HCC). However, the potential functions and exact mechanistic roles of miR-423 in hepatic carcinogenesis remain unknown. Here, we demonstrated that miR-423 significantly promotes cell growth and cell cycle progression at the G(1)/S transition in HCC cells. In particular, we found that miR-423-3p contributes to these effects, whereas miR-423-5p does not. Further studies revealed that p21Cip1/Waf1 is a downstream target of miR-423 in HCC cells, as miR-423 bound directly to its 3' untranslated region and reduced both the messenger RNA and protein levels of p21Cip1/Waf1. Moreover, enforced expression of p21Cip1/Waf1 abrogated miR-423-induced effects on HCC cell proliferation and cell cycle progression. These findings indicate that miR-423 exerts growth-promoting effects in hepatic carcinogenesis through the suppression of tumor suppressor p21Cip1/Waf1 expression. The results of this study define miR-423 as a new oncogenic miRNA in HCC.     

Loss of Heterozygosity on Chromosome 1p in Thyroid Adenoma and Medullary Carcinoma, but not in Papillary Carcinoma

We analyzed 53 loci on 21 chromosomes other than chromosome 4 to detect possible loss of heterozygosity in 31 thyroid tumors using polymorphic DNA markers that detect allelic deletions at specific chromosomal loci. Loss of heterozygosity on chromosomes 1, 7 and 12 was detected in one follicular thyroid adenoma, and on chromosome 1 in two medullary thyroid carcinomas. However, no loss of heterozygosity was detected at any of the loci examined in papillary thyroid carcinomas. These results suggest that chromosomal loss detected in thyroid adenoma is one of the signals for risk of premalignant transformation, and that inactivation of unknown genes on chromosome 1p contributes to tumorigenesis of medullary thyroid carcinoma. Some genetic changes other than chromosomal losses may participate in the tumorigenesis of papillary thyroid carcinoma.     

High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines

The RAS-RAF-MEK-ERK-MAP kinase pathway mediates the cellular response to extracellular signals that regulate cell proliferation, differentiation, and apoptosis. Mutation of the RAS proto-oncogene occurs in various thyroid neoplasms such as papillary thyroid carcinomas (PTCs), follicular thyroid adenomas and carcinomas. A second genetic alteration frequently involved in PTC is RET/PTC rearrangements. Recent studies have shown that BRAF, which is a downstream signaling molecule of RET and RAS, is frequently mutated in melanomas. This study tests whether BRAF is also mutated in thyroid tumors and cell lines. We analyzed BRAF gene mutation at codon 599 in thyroid tumors using mutant-allele-specific PCR and in 10 thyroid tumor cell lines by DNA sequencing of the PCR-amplified exon 15. We found that BRAF was mutated in 8 of 10 thyroid tumor cell lines, including 2 of 2 papillary carcinoma cell lines, 4 of 5 anaplastic carcinoma cell lines, 1 of 2 follicular carcinoma cell lines, and 1 follicular adenoma cell line. BRAF mutation at codon 599 was detected in 21 of 56 PTC (38%) but not in 18 follicular adenomas and 6 goiters. BRAF mutation occurred in PTC at a significantly higher frequency in male patients than in female patients. To test whether BRAF mutation may cooperate with RET/PTC rearrangements in the oncogenesis of PTC, we tested whether BRAF-mutated PTCs were also positive for RET/PTC rearrangements. Immunohistochemical staining was conducted to evaluate RET/PTC rearrangements by using two different anti-RET antibodies. Surprisingly, we found that a large number of BRAF-mutated PTCs (8 of 21) also expressed RET, indicating that the RET proto-oncogene is rearranged in these BRAF-mutated PTCs. These observations suggest that mutated BRAF gene may cooperate with RET/PTC to induce the oncogenesis of PTC.