Inactivation of the p14(ARF), p15(INK4B) and p16(INK4A) genes is a frequent event in human oral squamous cell carcinomas.

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.     

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.     

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.     

Ratio of expression of p16INK4a to p14ARF correlates with the progression of non-small cell lung cancer.

The CDKN2 gene is located on the short arm of chromosome 9p and encodes two unrelated proteins, p16(INK4a) and p14(ARF), through the use of independent first exons and shared exons 2 and 3. p16(INK4a) is a cyclin-dependent kinase inhibitor, whereas p14(ARF) regulates the cell cycle through a p53 and MDM2-dependent pathway. We have examined the expression of p16(INK4a) and p14(ARF) using competitive RT-PCR in 60 non-small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16(INK4a) and p14(ARF) were nearly equal or the intensity of the p16(INK4a) band slightly exceeded that of p14(ARF) in the normal lung tissues (n = 60). In 38 tumors the intensity of the p16(INK4a) band was similar to or slightly weaker than that of p14(ARF). In 6 tumors the intensity of the p16(INK4a) band was weaker than that of p14(ARF). In 15 tumors the intensity of the p14(ARF) band was very strong and the p16(INK4a) band was barely visible. In only one tumor was the intensity of the p16(INK4a) band very strong, while the band of p14(ARF) was barely visible. The ratio of the intensity of p16(INK4a) to p14(ARF) had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II - IV tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the p stage I tumors (P = 0.036). The T2 - 4 tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the T1 tumors (P = 0.005). The N1 - 3 tumors had significantly lower p16(INK4a) to p14(ARF) ratios than the N0 tumors (P = 0.014). Our results suggest that the ratio of expression of p16(INK4a) to p14(ARF) tends to decrease during the progression of NSCLC.     

Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.     

Individuals with presumably hereditary uveal melanoma do not harbour germline mutations in the coding regions of either the P16INK4A, P14ARF or cdk4 genes

In familial cutaneous malignant melanoma (CMM), disruption of the retinoblastoma (pRB) pathway frequently occurs through inactivating mutations in the p16 (p16INK4A/CDKN2A/MTS1) gene or activating mutations in the G1-specific cyclin dependent kinase 4 gene (CDK4). Uveal malignant melanoma (UMM) also occurs in a familial setting, or sometimes in association with familial or sporadic CMM. Molecular studies of sporadic UMM have revealed somatic deletions covering the INK4A-ARF locus (encoding P16INK4A and P14ARF) in a large proportion of tumours. We hypothesized that germline mutations in the p16INK4A, p14ARF or CDK4 genes might contribute to some cases of familial UMM, or to some cases of UMM associated with another melanoma. Out of 155 patients treated at the Institut Curie for UMM between 1994 and 1997, and interviewed about their personal and familial history of melanoma, we identified seven patients with a relative affected with UMM (n = 6) or CMM (n = 1), and two patients who have had, in addition to UMM, a personal history of second melanoma, UMM (n = 1), or CMM (n = 1). We screened by polymerase chain reaction single-strand conformation polymorphism the entire coding sequence of the INK4A-ARF locus (exon 1alpha from p16INK4A, exon 1beta from p14ARF, and exons 2 and 3, common to both genes), as well as the exons 2, 5 and 8 of the CDK4 gene, coding for the functional domains involved in p16 and/or cyclin D1 binding. A previously reported polymorphism in exon 3 of the INK4A-ARF locus was found in one patient affected with bilateral UMM, but no germline mutations were detected, either in the p16INK4A, p14ARF or CDK4 genes. Our data support the involvement of other genes in predisposition to uveal melanoma.     

INK4a／ARF抑癌基因与食管癌的研究进展

  INK4a／ARF基因位于染色体9p21的CDKN2A位点，它编码两种蛋白p16INK4a和p14ARF，这两个蛋白均为细胞周期调控因子，分别通过p16INK4a／pRB途径和p14ARF／p53途径履行调控细胞周期的职责，对INK4a／ARF位点p16INK4a和p14ARF的结构功能以及与食管癌的关系进行综述。     

The prognostic significance of p16INK4a/p14ARF and p15INK4b deletions in adult acute lymphoblastic leukemia.

Cytogenetic/molecular abnormalities significantly influence the prognosis of patients with acute leukemia. Recently, two genes, p16INK4a and p15INK4b, encoding two cyclin-dependent kinase inhibitor proteins of the INK4 family of Mr 15,000 and 16,000, respectively, have been localized to 9p21. Remarkably, the p16INK4a locus has been found to encode a second protein, p14ARF, known as p19ARF in mice, with a distinct reading frame. Like p16INK4a, p14ARF is involved in cell cycle regulation, blocking cells at the G1 restriction point through the activity of MDM-2 and p53. We studied bone marrow samples of 42 newly diagnosed and untreated patients with acute lymphoblastic leukemia for the incidence of deletions of p16INK4a/p14ARF and p15INK4b using Southern blot analysis and determined the clinical outcome with regard to complete remission (CR) duration, event-free survival, and overall survival. We found deletions of p16INK4a/p14ARF in 17 of 42 patients (40%), with homozygous deletions in 11 of 42 patients (26%) and hemizygous deletions in 6 of 42 patients (14%). The gene for p15INK4b was codeleted in most, but not all, cases and was never deleted without deletion of p16INK4a/ p14ARF. No correlation was observed between molecular studies and karyotype abnormalities as determined by conventional cytogenetics. Furthermore, no difference was found in the CR rate, CR duration, event-free survival, and overall survival in patients with homozygous gene deletions compared to patients with no deletions or loss of only one allele.     

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.     

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.     

Loss of p16(INK4a) expression correlates with decreased survival in pediatric osteosarcomas

Abnormalities of the G1 cell-cycle checkpoint are commonly reported in cancers at various anatomic sites. pRB, p16(INK4a) and cyclin D1 are critical G1-checkpoint proteins responsible for maintaining the balance of cellular proliferation. We examined a series of 38 pediatric osteosarcomas for abnormal expression of pRB, p16(INK4a) and cyclin D1 by immunohistochemical analysis of archival biopsy specimens. Overall, 17/38 (45%) osteosarcomas showed evidence of G1-checkpoint abrogation, including 11/38 (29%) with loss of pRB expression and 6/38 (16%) with loss of p16(INK4a) expression. Cyclin D1 over-expression was not detected. There was an inverse correlation between loss of pRB and p16(INK4a) expression (p = 0.07). pRB and p16(INK4a) abnormalities were independent of site of disease, presence of metastasis at diagnosis and percentage of tumor necrosis in the resection specimen. Clinical follow-up was available on all patients (median 31.6 months, range 5.9-116 months). Absence of p16(INK4a) expression significantly correlated with decreased survival in univariate analysis (p = 0.03), while loss of pRB expression did not affect survival. Immunohistochemical analysis of p16(INK4a) expression in pediatric osteosarcomas may be a useful adjunctive marker of prognosis.     

Alterations of p16(INK4a) and p14(ARF) in patients with severe oral epithelial dysplasia

A number of genetic aberrations have been reported in end-stage squamous cell carcinoma of the head and neck, including p16(INK4a) and p14(ARF) (INK4a/ARF) inactivation rates of 70-85%. Still, the cell cycle-regulatory genes p16(INK4a) and p14(ARF) remain poorly understood in oral cavity premalignant lesions. This study evaluated INK4a/ARF locus alterations in 26 patients (28 samples) deemed to be at increased risk for malignant transformation to squamous cell carcinoma due to the diagnosis of severe oral epithelial dysplasia. Microscopically confirmed dysplastic oral epithelium and matching normal tissue were laser capture-microdissected from paraffin sections, DNA was isolated, and molecular techniques were used to evaluate p16(INK4a) and p14(ARF) gene deletion, mutation, loss of heterozygosity (LOH), and hypermethylation events. Deletion of exon 1beta, 1alpha, or 2 was detected in 3.8%, 11.5%, and 7.7% of patients, respectively. INK4a and ARF mutations were detected in 15.4% and 11.5% of patients with severe dysplasia of the oral epithelium. All identified mutations occurred in the INK4a/ARF conserved exon 2. Allelic imbalance was assessed using three markers previously reported to show high LOH rates in head and neck tumors. LOH was found in 42.1%, 35.0%, and 82.4% of patients for the markers IFNalpha, D9S1748, and D9S171, respectively. Hypermethylation of p16(INK4a) and p14(ARF) was detected in 57.7% and 3.8% of patients, respectively, using nested, two-stage methylation-specific PCR. The highest rates of p16(INK4a) hypermethylation occurred in lesions of the tongue and floor of the mouth. In addition, p16(INK4a) hypermethylation was significantly linked to LOH in two or more markers. These data support that INK4a/ARF locus alterations are frequent events preceding the development of oral cancer and that p16(INK4a) inactivation occurs to a greater extent in oral dysplasia than does p14(ARF) inactivation.     

Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer

The CDKN2A locus on human chromosome 9p21 encodes two tumor suppressors, p14(ARF) and p16(INK4A), which enhance the growth-suppressive functions of the retinoblastoma (Rb) and the p53 proteins, respectively. Conversely, the E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPVs) causally associated with carcinogenesis of the uterine cervix contributes to tumor development by inactivating p53 and Rb. Nevertheless, a correlation between expression of p14(ARF)/p16(INK4A) and HPV infection in uterine cervix is less clear. To clarify this, we examined 25 cervical cancers and 11 normal uterine cervixes. HPV was detected in 21 of 25 cervical cancers (84%) and their subtype was determined by PCR-RFLP. Quantitative real-time RT-PCR assays showed overexpression of p14(ARF) mRNA in all 21 HPV-positive cases (100%). p16(INK4A) mRNA was overexpressed in 17 cases of the HPV-positive cases (81%). In four HPV-negative cancers, reduced expression of p14(ARF) mRNA was detected in two cases (50%) and reduced p16(INK4A) mRNA in three cases (75%). Our data indicate that the overexpression of p14(ARF) and p16(INK4A) strongly associates with HPV-positive cervical cancers and that reduced expression of p14(ARF) and p16(INK4A) correlates with HPV-negative cervical cancers. These findings may indicate that impaired p14(ARF) and p16(INK4A) mRNA expression contribute to tumor development in HPV-negative cervical cancers by failure to support p53 and Rb instead of their inactivation by HPV E6 and E7.     

Aberrations of p16INK4A,p14ARF and p15INK4B genes in pediatric solid tumors

Expression of the p16INK4A (p16), p15INK4B (p15), and p14ARF genes, located at 9p21, was examined in pediatric neuroblastoma (NB), Ewing's sarcoma (ES), and rhabdomyosarcoma (RMS). p16 expression was absent in 4 of 5 ESs, and 2 of these 4 cases died. p16 expression was reduced or absent in 10 of 12 RMSs, and 4 of these 10 cases died. These results suggested the possibility that p16 expression was associated with the progression of ES and RMS. There has been no previous report on p14ARF in NB. Our investigation might indicate that abnormal expression of the p16 and p14ARF was associated with a poor prognosis in NB, although in some cases of NB normal p16 and abnormal p14ARF expression was seen. These findings suggest an important role of p14ARF gene in the tumorigenesis of NB. The different incidence of expression of the p16, p15, and p14ARF genes in these 3 tumor types may reflect differences of the molecular process through which the 3 tumors develop. Our results suggest that abnormal expression of the p16 and/or p14ARF may be associated with a poor prognosis in these 3 tumors.     

Role of the alternative INK4A proteins in human keratinocyte senescence: evidence for the specific inactivation of p16INK4A upon immortalization.

The INK4A locus on human chromosome 9p21 encodes two genes that have been implicated in replicative senescence and tumor suppression, p16INK4A and p14ARF. In contrast to p16INK4A, which is up-regulated to high levels, we were unable to detect p14ARF protein in senescent human keratinocytes. Also, p53, an established target of p14ARF, did not increase, suggesting that p14ARF is not instrumental in human keratinocyte senescence. In neoplastic keratinocyte cultures, p16INK4A inactivation was invariably associated with the immortal phenotype, and there was evidence for the inactivation of p16INK4A, independent of p14ARF, in 6 of 10 lines that lacked large homozygous deletions. In contrast, we failed to detect exon 1beta mutations or p16INK4A-independent deletions. These results emphasize the previously proposed role for p16INK4A in human keratinocyte senescence but do not rule out a supporting role for p14ARF inactivation.     

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.