Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esophageal cancer in Linxian, China

Esophageal transit scintigraphy seems to be a valid methodology to assess impaired esophageal motility in early stages of disease. The purpose of this study was to discriminate patients with primary Raynaud's phenomenon (RP) and patients with systemic sclerosis (SSc) from healthy subjects by esophageal scintigraphy with a semisolid meal. METHODS: We studied 32 patients with primary RP, 18 with SSc and 13 healthy subjects. Dysphagia, acid regurgitation and heartburn were scored. After an overnight fast, all subjects underwent esophageal scintigraphy, using a semisolid orally ingested bolus (10 mL apple puree) labeled with 99mTc-sulfur colloid. Esophageal transit and emptying time and integral value were evaluated with the subjects in the upright (sitting) and supine positions. Transit time was defined as the time from the entry of 50% of radioactivity into the upper esophagus until the clearance of 50% of the bolus from the whole esophagus. Emptying time was defined as the time from the entry of 50% of radioactivity into the upper esophagus, until the clearance of 100% of the bolus from the whole esophagus. Integral value was defined as the total counts under the time-activity curve normalized to the maximum. RESULTS: Esophageal transit and emptying time and integral value, evaluated in both positions, were significantly higher in patients with SSc than in healthy subjects and than in patients with RP. Moreover, patients with RP had all three parameters, assessed in supine position, significantly longer compared to healthy subjects. Clinical scores regarding dysphagia, acid regurgitation and heartburn were not significantly different between patients with RP and SSc. CONCLUSION: Esophageal transit and emptying time and integral value appear to be able to discriminate patients with primary RP from patients with SSc and patients with RP from healthy subjects, suggesting an early mild esophageal dysfunction in RP.     

Inactivation of the p15INK4B and p16INK4 genes in hematologic malignancies

The recently discovered p15INK4B and p16INK4 genes encoding cell cycle regulating proteins, map to a region on chromosome 9p21 that is commonly deleted in a variety of malignant diseases. The p16INK4 gene has now been shown to be a tumor suppressor gene. It is frequently inactivated in cancer and is possibly the second most often mutated gene in human malignant disease after p53. The role of the p15INK4B and p16INK4 genes in hematologic malignancies has been the subject of intense investigation since their discovery. In this review we address the function and possible role in tumorigenesis of the p15INK4B and p16INK4 genes and discuss their significance as prognostic markers in hematologic malignancies.     

Lack of ETV6 (TEL) gene rearrangements or p16INK4A/p15INK4B homozygous gene deletions in infant acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) occurring in infants less than 1 year of age differs clinically and biologically from that observed in older children. Cytogenetically, 11q23 translocations are detected in approximately 50% of infant ALLs and fuse the 11q23 gene HRX with a variety of partner chromosomal loci. Overall, HRX rearrangements are detected molecularly in 70-80% of infant ALLs as compared to 5-7% of ALLs arising in older children. Two recently described molecular abnormalities in childhood ALL are ETV6 gene rearrangements and homozygous deletions of p16(INK4A) and/or p15(INK4B). Each of these abnormalities occurs in 15-20% of all childhood ALLs, and neither can be accurately identified by routine cytogenetic analyses. The incidence of these genetic abnormalities and their potential relationship to HRX gene status in infant ALL is unknown. Using Southern blot analyses, we determined ETV6 and p16(INK4A)/p15(INK4B) gene status in a cohort of infant ALLs. No ETV6 rearrangements or homozygous deletions (n=69) or homozygous p16(INK4A) and/or p15(INK4B) gene deletions (n=54) were detected in any of the infant ALLs. Therefore, ETV6 and p16(INK4A)/p15(INK4B) do not play a significant role in the pathogenesis of infant ALL, further emphasizing the distinctive biology of this subset of leukemias.     

Genetic and epigenetic alterations of the cyclin-dependent kinase inhibitors p15INK4b and p16INK4a in human thyroid carcinoma cell lines and primary thyroid carcinomas

BACKGROUND: D-type cyclins, in association with the cyclin-dependent kinases CDK4 and CDK6, promote progression through the G1 phase of the cell cycle. CDK activity is modulated by inhibitors such as p15INK4b and p16INK4a. Loss of function of p15INK4b and p16INK4a (multiple tumor suppressor-I and CDK4 inhibitor) determines impairment in the control of the cell cycle and contributes to the transformation of several cell types. METHODS: The authors examined 20 thyroid neoplasms (12 papillary carcinomas and 8 follicular adenomas) and 4 human thyroid carcinoma cell lines for gene mutations and epigenetic modifications of the p15INK4b and p16INK4a genes by Southern blot analysis, single strand conformation polymorphism, and a polymerase chain reaction-based methylation assay. RESULTS: Abnormalities of p16 were found in the four cell lines studied. In follicular carcinoma (WRO) cells, both the p15 and p16 genes were homozygously deleted. Undifferentiated carcinoma (FRO) cells had a nonsense point mutation at codon 72 (CGA-TGA, Arg-Stop) of p16, whereas the poorly differentiated papillary carcinoma (NPA) line harbored a point mutation at the exon 1-intron 1 boundary that altered the donor splicing site and caused an aberrantly spliced form of p16INK4a. Furthermore, p16 allelic loss was evident in the DNA of both FRO and NPA cells. Finally, p16 expression was absent in the ARO cell line, likely due to a de novo methylation of exon 1 of p16INK4a. Regarding the primary thyroid tumors, a missense point mutation at codon 91 was found in 1 of 12 papillary thyroid carcinomas (GCC-GTC, Ala-Val). No mutations were found in follicular adenomas. However, in 6 of 20 primary tumors there was hypermethylation at exon 1 of p16. CONCLUSIONS: The high prevalence of p15 and p16 mutations in the cell lines described suggests involvement of these genes in immortalization in vitro. The p16 defects may have preexisted in a small subclone of the primary tumor that were selected for in vitro. Alternatively, p16 mutations may have arisen de novo during cell culture. Mutations of p15INK4b and p16INK4a do not appear to be critical events in the development of follicular adenomas or papillary carcinomas. However, de novo methylation of the 5' CpG island of p16 is common in primary tumors, indicating that the function of this gene may be lost as an epigenetic event during disease progression.     

Mutational effects on the p16INK4a tumor suppressor protein

Several point mutations of p16INK4a were studied by site-specific mutagenesis and functional analysis to assess the effects of these mutations on the function of the protein. These mutations were reported in several malignancies. Three deletional mutants of p16INK4a were also analyzed to reveal the relationship between p16INK4a and p15INK4b and to test the importance of the ankyrin repeats observed in both proteins. We studied the activity of these mutants using the yeast two-hybrid system and an in vitro kinase assay. Our results suggest that point mutations in the conserved ankyrin consensus affect the activity of p16INK4a. However, not all of the point mutations observed in tumors have a detectable effect on the activity. The COOH-terminal region of p16INK4a is not required for the protein to bind and to inhibit CDK4, but the deletion of the 4th ankyrin repeat abolished the activity completely.     

Rb and p16INK4a expression in resected non-small cell lung tumors

Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.     

Loss of p16/INK4A protein expression in non-Hodgkin's lymphomas is a frequent finding associated with tumor progression

The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.     

Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.     

Hypermethylation of the p15INK4B gene in myelodysplastic syndromes

Previous studies have shown that the cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and that they are candidate tumor-suppressor genes. Although genetic alterations in these genes may be limited to lymphoid malignancies, it has been reported that their inactivation by aberrant methylation of 5' CpG islands may be involved in various hematologic malignancies. In this study, we investigated the p15INK4B and p16INK4A genes to clarify their roles in the pathogenesis of myelodysplastic syndrome (MDS). Southern blotting analysis showed no gross genetic alterations in either of these genes. However, hypermethylation of the 5' CpG island of the p15INK4B gene occurred frequently in patients with MDS (16/32 [50%]). Interestingly, the p15INK4B gene was frequently methylated in patients with high-risk MDS (refractory anemia with excess blasts [RAEB], RAEB in transformation [RAEB-t], and overt leukemia evolved from MDS; 14/18 [78%]) compared with patients with low-risk MDS (refractory anemia [RA] and refractory anemia with ring sideroblast [RARS]; 1/12 [8%]). Furthermore, methylation status of the p15INK4B gene was progressed with the development of MDS in most patients examined. In contrast, none of the MDS patients showed apparent hypermethylation of the p16INK4A gene. These results suggest that hypermethylation of the p15INK4B gene is involved in the pathogenesis of MDS and is one of the important late events during the development of MDS.     

Intragenic mutations of the p16(INK4), p15(INK4B) and p18 genes in primary non-small-cell lung cancers

The p15(INK4B), p16(INK4) and p18 genes are members of the gene family coding for inhibitors of cyclin-dependent kinases 4 and 6. p15(INK4B) and p16(INK4) are located at 9p21, a chromosomal region frequently deleted in many human neoplasms. To examine the role of these 3 genes in lung carcinogenesis, somatic mutations within the genes were analyzed by single-strand conformation polymorphism and DNA sequencing in 71 non-small-cell lung cancer (NSCLC) samples. Six somatic mutations in the p16(INK4) gene and 3 cases with a polymorphic allele were observed. Loss of heterozygosity in the p18 gene was found in 1 sample. We did not find any intragenic mutations in the p15(INK4B) or p18 genes. We conclude that p16(INK4) mutations play a role in the formation of some NSCLCs, whereas the involvement of p15(INK4B) and p18 is uncommon.     

Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia

Measures of arterial elasticity have been proposed as surrogate markers for asymptomatic atherosclerosis. We investigated the relations of serum lipoproteins, oxidized low-density lipoprotein (ox-LDL), and familial hypercholesterolemia (FH) to arterial elasticity among young men. As a marker of arterial elasticity we measured compliance in the thoracic aorta by using magnetic resonance imaging and in the common carotid artery by using ultrasound. LDL diene conjugation was used as a marker of ox-LDL. In study I, 25 healthy men (aged 29 to 39) were classified into 2 extreme groups according to previously measured high-density lipoprotein cholesterol to total cholesterol ratio (HDL-C/TC ratio). In study II, the healthy men were used as controls for 10 age matched asymptomatic patients with FH. In healthy men, the group with low HDL-C/TC ratio had decreased carotid artery compliance (2. 3+/-0.4% versus 1.9+/-0.5%/10 mm Hg, P=0.034). In univariate analysis, the compliance of the carotid artery associated with ox-LDL (r =-0.49, P=0.016) and HDL-C/TC ratio (r=0.41, P=0.040). In multivariate regression analyses, ox-LDL was the only independent determinant for compliance of the carotid artery (P=0.016). Aortic elasticity was not related to standard lipid variables, but the compliance of the ascending aorta associated with ox-LDL (r=-0.44, P=0.030). In FH patients, arterial elasticity was similar to that in controls. We conclude that elasticity of the common carotid artery is affected by serum lipid profile in young men. The current study demonstrates for the first time an in vivo association between ox-LDL and arterial elasticity suggesting that oxidative modification of LDL may play a role in the alteration of arterial wall elastic properties.     

p16INK4 gene mutations are relatively frequent in ampullary carcinomas

A high incidence of gene mutations or deletions of p16INK4, a cell cycle regulator which inhibits the activity of cyclin-dependent kinase 4/cyclin D complex and blocks the G1-to-S transition, has been reported in pancreato-biliary tract cancers. In order to investigate p16INK4 gene alterations in sporadic ampullary carcinomas, 17 sporadic ampullary carcinomas were examined. After histological diagnosis, DNA samples extracted separately from both cancerous and normal paraffin-embedded tissues were investigated. Loss of heterozygosity (LOH) was investigated utilizing 3 microsatellite markers on 9p21-22, and a mutational analysis was performed by cloning and sequencing. LOH was observed in 3 cases (17.6%) and somatic mutations with retention of heterozygosity were found in 7 cases (41.2%). Of note was that two mutations resulted in truncated incomplete proteins and one was a point mutation at the consensus site in the conserved ankyrin repeats, which would be crucial for function. Although two-hit inactivation was not evident in any of the mutation cases and further investigation would be needed to elucidate the role of altered p16INK4, these results suggest that the p16INK4 gene mutations are relatively frequent and its inactivation might be important in ampullary carcinogenesis.     

Lack of germ-line mutations of CDK4, p16(INK4A), and p15(INK4B) in families with glioma

Gliomas are tumors of the central nervous system that may be inherited in some patients. The gene(s) responsible for the clustering of gliomas in families have not yet been identified. Molecular studies of sporadic high-grade gliomas have revealed mutations or deletions of the genes encoding the protein kinase inhibitors p16(INK4A) and p15(INK4B) in a large proportion of tumors. Moreover, those tumors without deletions frequently display gene amplification and/or over-expression of mRNA encoding the protein kinase cdk4. We hypothesized that germ-line mutations in the p16(INK4A), p15(INK4B), or CDK4 genes might contribute to some cases of familial gliomas. To address this issue, we analyzed 36 kindreds with a predisposition to glial tumors. Genomic DNA from index members of these families was screened by PCR-single-strand conformational polymorphism analysis. We did not detect any functional mutations in the p16(INK4A), p15(INK4B), or CDK4 genes, although two individuals did have a previously described A140T polymorphism in p16(INK4A). Thus, despite the association between the sporadic forms of high-grade glioma and abnormalities of p16(INK4A), p15(INK4B), or CDK4, we found no evidence that germ-line mutations in the coding region of these three genes predispose to inherited glial tumors.     

Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations

The gross and microscopic anatomy of the rat larynx was studied with particular attention to myology and neuromuscular structures to further validate it as a model to evaluate morphologic and functional changes induced by botulinum injection. A laryngeal alar cartilage (LAIC), alar cricoarytenoid (ACA) muscle, and a superior cricoarytenoid muscle (SCA) were identified as anatomic structures not previously described. Two portions (medial and lateral) of the thyroarytenoid muscle (TA) were distinguished. The function of the ACA was suggested to be similar to the aryepiglottis muscle in humans and the function of the SCA was suggested to be similar to the human interarytenoid muscle. The predominant pattern of motor endplate (MEP) distribution in rat laryngeal muscles (posterior cricoarytenoid, lateral cricoarytenoid, cricothyroid, and SCA) was to have MEPs concentrated mostly at the midbelly of muscle where they were distributed throughout the cross-sectional area of the midbelly. The TA and ACA differed from this pattern. The lateral TA had MEPs concentrated at the anterior third of its belly and those of the medial TA were located at the midbelly. Motor endplates in the ACA were located mostly at the posterior portion of muscle. Muscle fiber-typing showed subtle differences between the intrinsic laryngeal muscles. Fast fibers were predominant in the rat laryngeal muscles. This study supports the expanded use of rats in studies of laryngeal neuromuscular function and disease in humans.     

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.     

High frequency of p16INK4A gene alterations in hepatocellular carcinoma

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR-SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5' CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5' CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.     

A high prevalence of functional inactivation by methylation modification of p16INK4A/CDKN2/MTS1 gene in primary urothelial cancers

We analyzed the genetic and epigenetic alterations of p16INK4A/CDKN2/MTS1 gene (MTS1 gene) in 38 primary urothelial cancers. Genetic alterations of the MTS1 gene consisted of one base substitution mutation in exon 2 (2.6%) and 6 homozygous deletions (16.2%). Hypermethylation of the 5' CpG island in exon 1 of the MTS1 gene was observed in 12 tumors (37.5%). Consequently, 19 of 38 tumors (50%) showed genetic alterations or epigenetic hypermethylation of the MTS1 gene. Retention of hypermethylated MTS1 gene(s) in 36% of the tumors showing loss of heterozygosity at the critical region indicates that the methylation modification could be an initial event followed by genomic rearrangements associated with total loss of MTS1 gene function. Immunohistochemical analysis of MTS1 expression revealed that all the tumors with genetic alterations of the MTS1 gene and 9 of 12 highly methylated tumors displayed an absence of MTS1 nuclear antigen. Genetic and epigenetic changes of the MTS1 gene were not correlated with the grade and stage of tumors, indicating that these alterations are early events in urothelial carcinogenesis, in which functional inactivation by hypermethylation is a predominant mechanism.