Molecular Analysis of Oncogenes, ras Family Genes (N-ras, K-ras, H-ras), myc Family Genes (c-myc, N-myc) and mdm2 in Natural Killer Cell Neoplasms

Natural killer (NK) cell neoplasms are rare diseases. Frequent abnormalities of the tumor suppressor genes Rb, p53, p151NK4B, p161NK4A and p14ARF have been reported. However, no oncogenes associated with tumorigenesis of NK cell neoplasms have been reported so far. We analyzed the status of oncogenes including N-ras, K-ras, H-ras, c-myc, N-myc and mdm2 by Southern blot, PCR-SSCP, western blot analysis and immunohistochemical staining. We analyzed four cell lines derived from NK cell neoplasms and 31 clinical samples with five subclasses of NK cell neoplasms. We found no point mutations of the ras family genes. We detected no mutations in the c-myc and N-myc genes. No overexpression of c-Myc protein was detected by western blot analysis. Although we found neither amplification nor rearrangement of the mdm2 gene, we found high expression of MDM2 protein in some cases by western blot analysis. Immunohistochemical staining confirmed the overexpression of MDM2 protein. We found 14 cases with overexpression of MDM2 protein out of 15 cases (93%) with four subclasses of NK cell neoplasms except chronic NK lymphocytosis. Our previous and these results suggested that the expression level of MDM2 protein is independent of the status of the p14ARF, p53, Rb genes. MDM2 protein might independently contribute to car-cinogenesis of NK cell neoplasms. Although the number of the cases we analyzed was not large, alterations of ras and myc family genes may rarely contribute to tumorigenesis in NK cell neoplasms. In contrast, overexpression of MDM2 might be associated with tumorigenesis of NK cell neoplasms, especially aggressive subclasses.     

Molecular analysis of tumor suppressor genes, Rb, p53, p16INK4A, p15INK4B and p14ARF in natural killer cell neoplasms.

Natural killer (NK) cell neoplasms, which are derived from mature or precursor NK cells, are rare diseases and are observed predominantly in Asian countries. We analyzed the status of the Rb, p53, p15INK4B, p16INK4A and p14ARF genes in these diseases by Southern blot, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and western blot analysis. We used 31 NK cell neoplasms, including four cell lines derived from NK cell neoplasms, 3 myeloid / NK cell precursor acute leukemias, 4 blastic NK cell lymphoma / leukemias, 4 aggressive NK cell leukemia / lymphomas, 4 nasal NK cell lymphomas, and 12 chronic NK lymphocytosis. We found gene amplification of the p53 gene in one nasal NK cell lymphoma, and point mutations of the p53 gene in one blastic NK cell lymphoma / leukemia and one chronic NK lymphocytosis. In addition, homozygous deletions of p15, p16 and p14 genes in 5 out of 31 samples were detected; 3 were from nasal NK cell lymphoma and 2 from blastic NK cell lymphoma / leukemia. Also hemizygous deletion of the Rb gene in one blastic NK cell lymphoma was detected. Rb proteins were highly expressed in one cell line as well as two myeloid / NK cell precursor acute leukemias. In other cell lines, complete loss and an aberrant migration pattern of Rb protein expression were observed. Comparative genomic hybridization suggested that the homozygous deletions of the p15, p16 and p14 were subtle chromosomal deletions and could not be identified by standard karyotyping in some cases. Although the number of cases we analyzed was not large, alterations identified in the Rb, p53, p16, p15 and p14 genes are of significance and might be associated with tumorigenesis in NK cell neoplasms.     

Molecular Analysis of Tumor Suppressor Genes, Rb, p53,pl6INK4A, pl5INK4B and p14ARF in Natural Killer Cell Neoplasms

Natural killer (NK) cell neoplasms, which are derived from mature or precursor NK cells, are rare diseases and are observed predominantly in Asian countries. We analyzed the status of the Rb, p53, p15INK4B, p16INK4A and p14ARF genes in these diseases by Southern blot, polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) and western blot analysis. We used 31 NK cell neoplasms, including four cell lines derived from NK cell neoplasms, 3 myeloid/ NK cell precursor acute leukemias, 4 blastic NK cell lymphoma/leukemias, 4 aggressive NK cell leukemia/lymphomas, 4 nasal NK cell lymphomas, and 12 chronic NK lymphocytosis. We found gene amplification of the p53 gene in one nasal NK cell lymphoma, and point mutations of the p53 gene in one blastic NK cell lymphoma/leukemia and one chronic NK lymphocytosis. In addition, homozygous deletions of pl5,p16 and p14 genes in 5 out of 31 samples were detected; 3 were from nasal NK cell lymphoma and 2 from blastic NK cell lymphoma/leukemia. Also hemizygous deletion of the Kb gene in one blastic NK cell lymphoma was detected. Rb proteins were highly expressed in one cell line as well as two myeloid/NK cell precursor acute leukemias. In other cell lines, complete loss and an aberrant migration pattern of Rb protein expression were observed. Comparative genomic hybridization suggested that the homozygous deletions of the p15, p16 and p14 were subtle chromosomal deletions and could not be identified by standard karyotyping in some cases. Although the number of cases we analyzed was not large, alterations identified in the Rb, p53, p16, p15 and p14 genes are of significance and might be associated with tumorigenesis in NK cell neoplasms     

Expression of the p14ARF tumor suppressor predicts survival in acute myeloid leukemia.

Cell cycle aberrations are associated with therapy outcome in many types of cancer. We analyzed mRNA expression levels of 18 cell cycle-related genes in bone marrow samples from 78 acute myeloid leukemia (AML) patients and six controls using high-throughput quantitative RT-PCR. Samples of AML patients contained significantly increased mRNA expression levels of the mdm2 and c-myc oncogenes. Also, the average expression levels of p14ARF and p16INK4A were higher in patient samples compared to controls. Leukemic blasts and control bone marrow samples did not differ significantly in the expression levels of proliferation-associated genes such as cyclin A2 and pcna. When single genes were analyzed for prognostic significance in Kaplan-Meier and Cox regression analyses, a low p14ARF level emerged as a strong and independent predictor for poor survival (P=0.04 and 0.029). Subsequently, p14ARF mRNA levels were analyzed in a second, independent patient population (n=57). Again, low p14ARF levels were associated with a worse outcome. Finally, immunohistochemistry analysis of AML tissue arrays confirmed the widespread expression of c-myc and p14ARF in AML on the protein level. Taken together, the expression of the p53 regulators mdm2 and p14ARF are altered in AML, and low p14ARF levels indicate a poor prognosis.     

p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2

The INK4a/ARF locus encodes two distinct tumor suppressors, p16INK4a and p14ARF. Although the contribution of p16INK4a to human tumorigenesis through point mutation, deletion, and hypermethylation has been widely documented, little is known about specific p14ARF lesions and their consequences. Recent data indicate that p14ARF suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14ARF prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14ARF epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14ARF promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14ARF hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2'-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14ARF promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14ARF was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14ARF promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14ARF methylation. Taken together, these data support that epigenetic silencing of p14ARF by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.     

CDK4 and MDM2 gene alterations mainly occur in highly proliferative and aggressive mantle cell lymphomas with wild-type INK4a/ARF locus

Amplification of 12q13 locus occurs in some mantle cell lymphomas (MCL), potentially involving CDK4 and MDM2 genes. To determine the role of these genes in MCL, we have examined their gene status and expression and their relationship to INK4a/ARF and p53 gene aberrations in 69 tumors. Increased CDK4 gene copy number was detected in 4 of 19 (21%) highly proliferative blastoid variants and was associated with mRNA and protein overexpression. Three additional cases showed mRNA overexpression with no structural alterations of the gene. MDM2 gene overexpression was detected in three blastoid tumors (16%) with no relationship to gene copy gains. INK4a/ARF and p53 aberrations were observed in 13 and 12 tumors, respectively. Four of the seven lymphomas with CDK4 aberrations had concurrent inactivation of p53 gene, whereas only one case had a concomitant homozygous deletion of INK4a/ARF. No other gene alterations were found in the three cases with MDM2 overexpression. Patients with INK4a/ARF deletions or simultaneous aberrations of p53 and CDK4 had a significantly shorter median survival (17 months) than patients with isolated alterations of p53, MDM2, or CDK4 (32 months) and patients with no alterations in any of these genes (77 months). The prognostic impact of the concomitant oncogenic alterations of the p14ARF/p53 and p16INK4a/CDK4 pathways was independent of the proliferation of the tumors. These findings indicate that CDK4 and MDM2 gene alterations mainly occur in MCL with a wild-type INK4a/ARF locus and may contribute to the higher proliferation and more aggressive behavior of the tumors.     

Homozygous Deletion of INK4a/ARF Genes and Overexpression of Bcl-2 in Relation with Poor Prognosis in Immunocompetent Patients with Primary Central Nervous System Lymphoma of the Diffuse Large B-cell Type

Only a few reports have been published on molecular genetic alterations in primary central nervous system lymphomas (PCNSLs) of the diffuse large B-cell type and no reports have addressed the correlation between the genetic alterations and clinical course of the patients with this neoplasm. Thus, the molecular background of the PCNSL and its importance for the clinical course of the patients are still unclear. We investigated a series of 14 patients with PCNSL to determine structural alterations of the INK4a/ARF, MDM2, and TP53 genes, the status of bcl-2 and bcl-6 protein expression, and the clinical course of the patients (i.e. their survival time after diagnosis). No structural alterations of MDM2 and TP53 genes were found. Only INK4a/ARF genes whose expression affects both the p16INK4a–Rb and p14ARF–mdm2–p53 pathways in the regulation for cell cycle and apoptosis, showed an alteration of the homozygous deletions at a high frequency (nine of 14 patients: 64%). This specific alteration was not related with the bcl-6 expression, but a relation was shown with overexpression of the bcl-2 anti-apoptotic protein (p = 0.036, chi-square test), as well as a shorter patient survival (p = 0.044, Wilcoxon test). There was only a tendency, not a significant correlation, in which the patients with bcl-2 overexpression resulted in poor prognosis (p = 0.149). The present study is the first to suggest that the INK4a/ARF gene homozygous deletions and overexpression of the bcl-2 protein may be correlated with each other and together serve as important predictors for the prognosis of patients with PCNSL.     

Aberrations of the p14(ARF) and p16(INK4a) genes in renal cell carcinomas.

The INK4a / ARF locus on chromosome 9p21, which encodes two distinct genes, p14(ARF) and p16(INK4a), is frequently altered in human neoplasms. To investigate the potential roles of p14(ARF) and p16(INK4a) genes in human renal cell carcinomas (RCCs), we analyzed 6 human RCC cell lines and 91 primary RCCs for homozygous deletion, promoter hypermethylation and expression of the p14(ARF) and p16(INK4a) gene products using differential PCR, methylation-specific PCR, and immunohistochemistry, respectively. Five cell lines showed homozygous co-deletion of both genes and one demonstrated promoter hypermethylation of the p16(INK4a) gene only. Eight of 91 RCCs showed aberrations of p14(ARF) or p16(INK4a) status and six of these featured gross extension into the renal vein. The results suggest that p14(ARF) and p16(INK4a) aberrations may play roles in the relatively late stage of renal tumorigenesis associated with tumor progression.     

p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53

The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.     

ARF-mdm2-p53的相互作用途径

细胞周期素依赖性激酶4（cyclin-dependent kinase4,CDK4)抑制蛋白基因p16INK4α及其可变读框基因（alterative reading frame,ARF)-INK4α-ARF基因位于人染色体9p21的CDKN2A位点,该位点编码两个重叠的基因;p16INK4α和ARF基因,这两个基因的阅读框架不同,因此,p16和ARF基因的氨基酸顺序是完全不同的,ARF主要通过mdm2参与mdm2-p53通路的调节。目前研究表明ARF基因在肿瘤发生过程中可有起一定的作用,ARF基因功能的失活可能主要通过该基因启动子高度甲基化。本文就ARF与mdm2及p53相互作用途径做一综述。     

Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells

BACKGROUND: The p14(ARF) protein encoded by the INK4a/ARF locus promotes degradation of the MDM2 protein and thus prevents the MDM2-mediated inhibition of p53. Homozygous deletion of the INK4a/ARF locus is common in human mesothelioma and may result in the loss of p14(ARF) and the inactivation of p53. We designed this study to evaluate the biologic and potential therapeutic roles of p14(ARF) expression in mesothelioma cells. Methods and Results: We constructed Adp14, an adenoviral vector carrying human p14(ARF) complementary DNA, and used it to transfect human mesothelioma cell lines H28, H513, H2052, and MSTO-211H. Overexpression of p14(ARF) led to increased amounts of p53 and the p21(WAF) proteins and dephosphorylation of the retinoblastoma protein. The growth rate of mesothelioma cells was inhibited markedly by infection with Adp14 compared with mock infection or infection with a control adenovirus vector, AdCtrl. Overexpression of p14(ARF) induced G(1)-phase cell cycle arrest and apoptotic cell death. Cytotoxicity assays showed that Adp14 had a statistically significantly (P =.002) greater effect on colon cancer (HCT116) cell lines containing two copies of the wild-type p53 gene than on p53-null cells, suggesting that functional p53 is a critical determinant of p14(ARF)-mediated cytotoxicity. CONCLUSIONS: The transfection of p14(ARF) into mesothelioma cells led to the overexpression of p14(ARF), which resulted in G(1)-phase arrest and apoptotic cell death. These results suggest that this gene therapy-based approach may be of use in the treatment of mesothelioma.     

H-, K- and N-Ras inhibit myeloid leukemia cell proliferation by a p21WAF1-dependent mechanism

Mutated ras genes are frequently found in human cancer. However, it has been shown that oncogenic ras inhibits growth of primary cells, through pathways involving p53 and the cell cycle inhibitors p16INK4a and p19ARF. We have analysed the effect of the ectopic expression of the three mammalian ras genes on the proliferation of K562 leukemia cells, which are deficient for p53, p16INK4a, p15INK4b and p19ARF genes. We have found that high expression levels of both wild-type and oncogenic H-, K- and N-ras inhibit the clonogenic growth of K562 cells. Induction of H-rasV12 expression in K562 transfectants retards growth and this effect is accompanied with an increase of p21WAF1 mRNA and protein levels. Furthermore, p21WAF1 promoter is activated potently by oncogenic ras and less pronounced by wild-type ras. This induction is p53-independent since a p21WAF1 promoter devoid of the p53 responsive elements is still activated by Ras. Finally, inhibition of p21WAF1 expression by an antisense construct partially overcomes the growth inhibitory action of oncogenic H-ras. Altogether, these results indicate that the antiproliferative effect of ras in myeloid leukemia cells is associated to the induction of p21WAF1 expression and suggest the existence of p19ARF and p16INK4a-independent pathways for ras-mediated growth inhibition.     

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.     

Clearing up the p16INK4a-p14/p19ARF imbroglio?]

Since its discovery, the CDKN2/MTS1 locus has been considered as an important site for the understanding of cell cycle deregulations that are involved in cancer cell generation. A comprehensive approach of the respective roles played by the two p16INK4a and p14/p19ARF (ARF) proteins encoded by this locus was not yet achieved because of the structural intrication of their genes. Inactivation of the only p16INK4a gene in mouse allowed to get better insight into this puzzle. In vivo results presented by de Pinho's group showed that inactivation of both p16INK4a alleles generated a panel of various types of tumors from the 28th week following birth. Bern's group dit not confirm this result but showed that the presence of only one ARF functional copy increases sensitivity of p16-/- mice to tumor occurrence indicating that insufficient dosage of ARF protein may facilitate tumorigenesis. It seems now established that, at least in mouse, ARF controls senescence in vitro, immortalisation and transformation by oncogenic ras. p16INK4a inactivation appears to be crucial for the induction of carcinogens-induced tumors.     

The N-myc and c-myc downstream pathways include the chromosome 17q genes nm23-H1 and nm23-H2

Gain of chromosome 17q material is the most frequent genetic abnormality in neuroblastomas. The common region of gain is at least 375 cR large, which has precluded the identification of genes with a role in neuroblastoma pathogenesis. Neuroblastoma also frequently show amplification of the N-myc oncogene, which correlates closely with 17q gain. Both events are strong predictors of unfavorable prognosis. To identify genes that are part of the N-myc downstream pathway, we constructed SAGE libraries of an N-myc transfected and a control cell line. This identified the chromosome 17q genes nm23-H1 and nm23-H2 as being 6-10 times induced in the N-myc expressing cells. Northern and Western blot analysis confirmed this up-regulation. Time-course experiment shows that both genes are induced within 4 h after N-myc is switched on. Furthermore, we demonstrate also that c-myc can up-regulate nm23-H1 and nm23-H2 expression. Neuroblastoma tumor and cell line panels reveal a striking correlation between N-myc amplification and mRNA and protein expression of both nm23 genes. We show that the nm23 genes are located at the edge of the common region of chromosome 17q gain previously described in neuroblastoma cell lines. Our findings suggest that nm23-H1 and nm23-H2 expression is increased by 17q gain in neuroblastoma and can be further up-regulated by myc overexpression. These observations suggest a major role for nm23-H1 and nm23-H2 in tumorigenesis of unfavorable neuroblastomas.     

Alterations of the p16(INK4) locus in human malignant mesothelial tumors

The INK4 locus has two promoters and encodes two unique proteins that share exons in different reading frames, p16(INK4a) and p14(ARF). The p16(INK4a) protein, by inhibiting cyclin-dependent kinase, down regulates Rb-E2F and leads to cell cycle arrest in the G1 phase. The p14(ARF) protein interacts with the MDM2 protein, neutralizing MDM2-mediated degradation of p53. Since p53/Rb genes are not altered in malignant mesothelioma, additional components of these pathways, such as p16(INK4a) and p14(ARF), are candidates for inactivation. In this study, we have examined p16(INK4a) and p14(ARF) alterations (gene deletion, mutation and promoter methylation) in 45 primary malignant mesothelioma specimens. Fourteen patients (31%) had altered p16; four tumors had a methylated promoter region (8.8%), 10 tumors showed p16 to be deleted (22.2%), and one tumor had a point mutation (2%). We did not find any instances of methylation in the p14(ARF) 5'-CpG island. Patients whose tumors had p16 deletion were significantly younger than those with methylation, and, in the patients whose lungs were studied for the prevalence of asbestos fibers, those with any p16 alteration had lower fiber counts than those with no p16 alteration. Hence, p16 gene alteration is relatively common in malignant mesothelioma, while p14(ARF) is rarely, if ever, methylated. Our data suggest that deletion of p16 occurs in a relatively susceptible subset of the population.     

Alterations of p14ARF and p16INK4a genes in salivary gland carcinomas

The p14ARF and p16INK4a genes are localized to 9p21, where genetic alterations have been reported to be frequent in various human neoplasms. To elucidate their status in salivary gland tumorigenesis, we analyzed a series of 36 salivary gland carcinomas (SGCs) using methylation-specific PCR, differential PCR and immunohistochemistry. Homozygous deletion (3 cases) or methylation (7 cases) of p14ARF was detected in 10 (28%) SGCs, one and three showing co-deletion and co-methylation of both p14ARF and p16INK4a genes, respectively. A total of 5 (14%) SGCs demonstrated homozygous deletion (1 case) or methylation (4 cases) of p16INK4a, all but one being adenoid cystic carcinomas. Immunohistochemical study revealed loss of p14ARF and p16INK4a expression in 11 samples (31%), correlating with the gene status. These results indicate that inactivation of p14ARF and p16INK4a genes by either homozygous deletion or promoter hypermethylation may be important for the molecular pathogenesis of salivary malignant tumors, and provide clear evidence that epigenetic changes like methylation are related to salivary gland carcinogenesis.