Analysis of the polymorphisms and haplotypes of cyclin-dependent kinase inhibitor 2B antisense RNA 1 gene in patients with ulcerative colitis / 中华消化杂志

Objective:To investigate the relationship between polymorphisms and haplotypes of cyclin-dependent kinase inhibitor 2B antisense RNA 1 ( CDKN2 B- AS1) gene and the risk of ulcerative colitis (UC). Methods:From January 2012 to January 2021, a total of 534 UC patients diagnosed at the Department of Gastroenterology, the Second Affiliated Hospital of Wenzhou Medical University (Yuying Children′s Hospital) and during the same period 560 gender- and age-matched healthy controls were selected. Genotypes of CDKN2 B- AS1 (rs1063192, rs10757274, rs10757278, rs1333048, rs2383207) in venous blood were determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry technique. Unconditional logistic regression was used to analyze the difference in the distribution of CDKN2 B- AS1 gene polymorphisms between UC patients and healthy controls, as well as the influence on the clinicopathologic characteristics of UC patients. Software Haploview 4.2 was used to analyze the linkage disequilibrium and haplotype. Chi-square test was used for statistical analysis. Results:The frequencies of variant genotype (AG+ GG) and variant allele (G) of rs1063192 in UC patients were higher than those in healthy controls (32.4%, 173/534 vs. 24.8%, 139/560; 18.1%, 193/1 068 vs. 13.7%, 153/1 120), and the differences were statistically significant ( OR=1.45 and 1.40, 95% confidence interval(95% CI) 1.12 to 1.89 and 1.11 to 1.77, P=0.006 and 0.004, corrected P=0.030 and 0.020). The frequency of variant allele (G) of rs10757274 in UC patients was lower than that in healthy controls (34.7%, 371/1 068 vs. 39.5%, 442/1 120), and the difference was statistically significant ( OR=0.82, 95% CI 0.69 to 0.98, P=0.025). However, the difference was not significant after Bonferroni correction (corrected P>0.05). According to the Montreal classification, the frequency of homozygous variant genotype (GG) of rs1063192 in the patients with extensive colitis was higher than that in patients with proctitis plus left-sided colitis (6.6%, 14/211 vs. 1.9%, 6/323), and the difference was statistically significant ( OR=3.92, 95% CI 1.47 to 10.42, P=0.006, corrected P=0.030). There was linkage disequilibrium among rs10757274, rs2383207, rs10757278 and rs1333048 of CDKN2 B- AS1 gene. The frequency of haplotype GGGC in UC patients was lower than that in healthy controls (33.3%, 355.5/1 068 vs. 37.8%, 423.4/1 120), and the frequency of haplotype AGGC in UC patients was higher than that in healthy controls (6.7%, 71.7/1 068 vs. 3.6%, 40.3/1 120), and the differences were statistically significant ( χ2=4.81 and 11.16, P=0.028 and<0.001). Conclusions:The variation of rs1063192 in CDKN2 B- AS1 gene may increase the risk of UC. The risk of extensive colitis in patients carrying homozygous variant genotype (GG) of rs1063192 may rise. Among the haplotypes composed of rs10757274, rs2383207, rs10757278 and rs1333048, the risk of UC may decrease in the individuals carrying haplotype GGGC. However, the risk of UC may increase in the individuals carrying haplotype AGGC. The correlation between the variation of 10757274 and the risk of UC still needs to be further verified by expanding the sample size.

Cdkn2a Tracer Mice: A Novel Model to Study Cellular Senescence in Post-traumatic Osteoarthritis / 中国生物化学与分子生物学报

Osteoarthritis (OA) is one of the most common geriatric motor system diseases in the clinical practice. Aging, whose hallmark is cellular senescence, is an important factor leading to the occurrence and development of OA, but its exact role in the pathological development of OA is not completely clear. Studies have proved that targeting senescence can effectively treat aging-related diseases. In this study, the heterozygous mouse model of Cdkn2a-e (Luc-2A-tdTomato-2a-CreerT2-Wpre-PA)1 was established by the CRISPR/ Cas9 technique. The expression of Cdkn2a (p16, p16INK4a), a classical marker of senescence, can be traced in vivo in mice. We then utilized anterior cruciate ligament transection (ACLT) to induce OA in Cdkn2a mice, and we hope to verify the relationship between aging and the occurrence and development of OA and visualize aging changes during OA development through the mice model. In this study, 10-12 weeks old Cdkn2a mice were randomly divided into no surgery control group, sham operation group and ACLT group. OA model was constructed in mice by ACLT operation. After surgery for 4 weeks, animals were collected for fluorescence imaging detection in vivo, which showed that local fluorescence expression of Cdkn2a increased in knee joints of mice in the ACLT group four weeks after surgery (P < 0. 05) . The Safranin O-Fast Green Staining of mouse knee tissue sections showed degeneration of the knee cartilage in the ACLT group at 4 weeks after surgery (P < 0. 05) . Immunohistochemical staining of Cdkn2a was performed on the knee tissue of mice. Compared with the other two groups, Cdkn2a staining on the cartilage surface of the knee tissue of mice in the ACLT group was deeper. The results showed that the OA model induced by surgery showed local aging, which further verified the relationship between aging and OA. At the same time, the Cdkn2a tracer mouse model can reflect the aging progress of mice in vivo, with combination of imaging examinations, so that the occurrence and progress of the relationship between aging and OA can be observed in real time. This heterozygous mouse model of Cdkn2a-e(Luc-2A-tdTomato-2a-CreerT2-Wpre-PA) 1 is not only useful for mechanism research of aging and OA diseases, but also beneficial for finding more potential therapy targets to OA and aging.

Effects of lncRNA CDKN2B-AS1 targeting miR-98-5p on proliferation and invasion of lung cancer A549 cells / 中华内分泌外科杂志

Objective:To investigate the effects of long non-coding RNA (lncRNA) CDKN2B-AS1 targeting miR-98-5p on proliferation and invasion of lung cancer A549 cells.Methods:A549 cells cultured in vitro were divided into control group, pcDNA group (transfected with pcDNA) , CDKN2B-AS1 group (transfected with pcDNA CDKN2B-AS1) and double transfection group (transfected with pcDNA CDKN2B-AS1 and pcDNA miR-98-5p) . The expression of lncRNA CDKN2B-AS1, miR-98-5p and the protein expression of PCNA, MMP-9 in A549 cells were detected. The activity, clone number, cloning efficiency, and the number of invasive cells of A549 cells were detected.Results:Compared with pcDNA group, the expression level of lncRNA CDKN2B-AS1 [ (2.14±0.14) vs (1.03±0.10) ], OD value in each time points, clone number [ (314.60±18.13) vs (220.08±12.46) ], cloning efficiency [ (85.81±3.06) % vs (60.03±2.85) %], invasive cell number [ (233.30±18.98) vs (140.84±12.30) ], expression levels of PCNA [ (0.78±0.08) vs (0.48±0.07) ] and MMP-9 [ (0.75±0.06) vs (0.38±0.06) ] proteins in A549 cells in CDKN2B-AS1 group were significantly increased ( P<0.05) ; the expression level of miR-98-5p [ (0.23±0.03) vs (0.99±0.09) ] was significantly decreased ( P<0.05) ; compared with CDKN2B-AS1 group, there was no significant difference in the expression level of lncRNA CDKN2B-AS1 in A549 cells in double transfection group ( P>0.05) , while the expression level of miR-98-5p in A549 cells was significantly increased ( P<0.05) . The OD value in each time points, clone number, cloning efficiency, invasive cell number, expression levels of PCNA and MMP-9 proteins were significantly decreased ( P<0.05) . Conclusion:LncRNA CDKN2B-AS1 can promote the proliferation and invasion of lung cancer A549 cells by targetingly inhibiting the expression of miR-98-5p.

Characteristics of DNMT3A mutations in acute myeloid leukemia

BACKGROUND: DNMT3A mutations occur in approximately 20% of AML cases and are associated with changes in DNA methylation. CDKN2B plays an important role in the regulation of hematopoietic progenitor cells and DNMT3A mutation is associated with CDKN2B promoter methylation. We analyzed the characteristics of DNMT3A mutations including their clinical significance in AML and their influence on promoter methylation and CDKN2B expression.METHODS: A total of 142 adults, recently diagnosed with de novo AML, were enrolled in the study. Mutations in DNMT3A, CEBPA, and NPM1 were analyzed by bidirectional Sanger sequencing. We evaluated CDKN2B promoter methylation and expression using pyrosequencing and RT-qPCR.RESULTS: We identified DNMT3A mutations in 19.7% (N=28) of enrolled patients with AML, which increased to 29.5% when analysis was restricted to cytogenetically normal-AML. Mutations were located on exons from 8–23, and the majority, including R882, were found to be present on exon 23. We also identified a novel frameshift mutation, c.1590delC, in AML with biallelic mutation of CEBPA. There was no significant difference in CDKN2B promoter methylation according to the presence or type of DNMT3A mutations. CDKN2B expression inversely correlated with CDKN2B promoter methylation and was significantly higher in AML with R882H mutation in DNMT3A. We demonstrated that DNMT3A mutation was associated with poor AML outcomes, especially in cytogenetically normal-AML. The DNMT3A mutation remained as the independent unfavorable prognostic factor after multivariate analysis.CONCLUSION: We characterized DNMT3A mutations in AML and revealed the association between the DNMT3A mutation and CDKN2B expression and clinical outcome.

Correlation between methylation level of and genes and aging in healthy individuals / 南方医科大学学报

OBJECTIVE@#To analyze the relationship between and gene methylation with aging in the general population.@*METHODS@#We collected peripheral blood samples from 284 male and 246 female healthy subjects for detection of methylation levels of and genes using quantitative methylation-specific PCR (qMSP). The relationship between the methylation levels of and genes and aging was analyzed using Spearman or Pearson correlation test.@*RESULTS@#We found a significant positive correlation between the methylation levels of the two genes in these subjects ( < 0.05). In the overall population as well in the female subjects, methylation was found to be inversely correlated with age ( < 0.05). The methylation levels of and genes were inversely correlated with TG, ApoE, Lp(a) and AST in the overall population ( < 0.05). In both the female and male subjects, the methylation levels of the two genes were inversely correlated with Lp(a) ( < 0.05). In the male subjects, methylation was inversely correlated with AST ( < 0.05), while methylation was inversely correlated with HDL and ApoE ( < 0.05). In the female subjects, methylation was positively correlated with LDL and inversely correlated with ApoE and AST ( < 0.05).@*CONCLUSIONS@#The methylation levels of and are closely related to age and the levels of multiple proteins in healthy subjects.

Effect of long-chain non-coding CDKN2B on miR-19 in chronic myeloid leukemia / 中国免疫学杂志

Objective:To investigate the effect of long-chain non-coding CDKN2B targeting miR-19 on the biological behavior of chronic myeloid leukemia cells and its mechanism.Methods: The expression of CDKN2B in different leukemia cells were detected by qPCR.Double luciferase reporter gene was used to detect the interaction between CDKN2B and miR-19.MTT proliferation assay and flow cytometry were used to detect the effect of CDKN2B on the proliferation and apoptosis of HL-60 cells.The changes of migration ability of leukemia HL-60 cells after overexpress of CDKN2B were detected by scratch test.The changes of invasion ability of leukemia HL-60 cells after silencing CDKN2B were detected by Transwell invasion assay.Scaling healing experiment and Transwell invasion assay were used to detect the effect of miR-19 on the migration and invasion of leukemia cells after silencing CDKN2B.The morphological changes of cytoskeleton microfilament microtubules after silencing CDKN2B were detected by phalloidin staining.Western blot was used to detect the expression of PI3K/AKT signaling pathway after silencing CDKN2B.Results: The expression level of CDKN2B was the lowest in leukemia cell HL-60.CDKN2B binds specifically to the 3′UTR of miR-19;overexpression of CDKN2B could inhibit the proliferation and enhance the apoptosis of leukemia HL-60 cells.Overexpression of CDKN2B can inhibit the invasion and migration of leukemia HL-60 cells.After overexpressed of CDKN2B,the cytoskeleton showed decreased pseudopodia and decreased exercise capacity.The expression of actin was down-regulated.The expression of PI3K/AKT pathway protein was down-regulated after overexpressed of CDKN2B.Conclusion: CDKN2B can target the regulation of miR-19 to regulate the biological behavior of leukemia cells.

Association of CDKN2B-AS1 rs1333049 with Brain Diseases: A Case-control Study and a Meta-analysis

OBJECTIVE: CDKN2B-AS1 polymorphisms were shown to associate with the risk of stroke in European. The goal of this study was to evaluate the contribution of CDKN2B-AS1 rs1333049 to the risk of hemorrhagic stroke (HS) and brain tumor (BT) in Han Chinese. METHODS: A total of 142 HSs, 115 BTs, and 494 controls were included in the current association study. The genotyping test was performed using the melting temperature shift method. RESULTS: We failed to validate the association of CDKN2B-AS1 rs1333049 with the risk of brain disease. Significantly higher levels of low-density lipoprotein cholesterol (LDL-C) (p=0.027), high-density lipoprotein cholesterol (HDL-C) (p0.05). The meta-analysis of 10 studies among 133,993 individuals concluded that rs1333049 of CDKN2B-AS1 gene was likely to increase a 16% incidence rate of cerebrovascular disease (CD) among various populations (odds ratio 1.16, 95% confidence interval 1.08–1.25; p<0.0001, random-effect method). CONCLUSION: Our case-control study identified rs1333049 genotypes showed different association with the concentration of the LDL-C, HDL-C and TC in the HS patients. Meta-analysis supported the association between rs1333049 and CD risk in various populations, although we were unable to observe association between rs1333049 and the risk of HSs in Han Chinese.

Research progress in the long non-coding RNA ANRIL / 基础医学与临床

Long non-coding RNAs are important regulators of gene expression.ANRIL which was coded on the Chr9p21.3 loci participates in the pathogenesis of tumor, coronary artery disease, type 2 diabetes mellitus and oth-er diseases.Multiple ANRIL isoforms are tissue-specific.ANRIL mainly functions through Polycomb proteins, while there are also other downstream targets.The mechanism of each isoform and the downstream pathways are hotspots incurrent researches.

Prognostic value of CDKN2A mRNA level in glioblastoma / 肿瘤研究与临床

Objective To investigate the prognostic value of CDKN2A mRNA in glioblastoma (GBM).Methods CDKN2A gene mRNA data were obtained from three different GBM database online (TCGA,REMBARNDT and GSE16011).The correlations between overall survival (OS) and CDKN2A expression were analyzed by Kaplan-Meier method and multivariate Cox regression analysis.Results In the TCGA database (n =358),patients with high CDKN2A mRNA level got longer OS than those with low expression level [median OS:18.0 months (95 ％ CI 15.0-21.0 months) vs 13.9 months (95 ％ CI 12.4-15.4 months),P =0.001].In another two validation datasets,patients with high CDKN2A mRNA level had longer OS than those with low expression level [median OS in REMBRANT:16.6 months (95 ％ CI 13.3-19.8 months) vs 11.8 months (95 ％ CI 7.3-16.4 months),P =0.019; in GSE16011:11.9 months (95 ％ CI 8.3-15.6 months) vs 8.4 months (95 ％ CI 6.2-10.5 months),P =0.005].CDKN2A mRNA level was an independent prognostic factor for GBM.The combination of CDKN2A mRNA expression with MGMT promoter methylation status or G-CIMP status/IDH1 mutations provided an optimized prognostic factor in GBM patients.Conclusion The CDKN2A mRNA has prognostic value in GBM patients,which provided an optimized stratification strategy based on multiple biomarkers.

Genética molecular del melanoma familiar: revisión del tema y aplicación clínica / Genetics of familial cutaneous melanoma

El melanoma maligno cutáneo (MMC) representa el 4 por ciento del total de los tumores malignos de piel, dando cuenta del 80 por ciento de las muertes producidas por cáncer cutáneo. La sobrevida a cinco años de individuos portadores de enfermedad metastásica se estima en 14 por ciento. Las formas de presentación incluyen una variante esporádica y otra familiar o hereditaria. En ambas el papel de diferentes mutaciones genéticas que otorgan susceptibilidad al desarrollo de MMC es indiscutido, así como la interacción entre las características genéticas del individuo y eventos ambientales. En el MMC familiar se han establecido dos genes de alta susceptibilidad con diferente penetrancia y frecuencia: CDK4 y CDKN2A. CDKN2A (Cyclin-dependent kinase inhibitor 2A) es el más importante gen de susceptibilidad a MMC, cuyas mutaciones se han identificado en aproximadamente un 40 por ciento de familias que presentan tres o más casos de MMC. Las características clínicas asociadas a la mutación de CDKN2A son número elevado de individuos afectados por MMC dentro de una familia, MMC primario múltiple y presencia conjunta de MMC y cáncer de páncreas dentro de una familia. En Chile de desconoce la frecuencia y tipos de mutaciones que afectan a CDKN2A en familias con predisposición a MMC familiar.

Cutaneous malignant melanoma (CMM) represents 4 percent of all malignant skin tumors and accounts for 80 percent of deaths related to cutaneous cancer. 5-year survival rates in individuals with metastatic disease is around 14 percent. An hereditary or familial variant of CMM has been identified. It is related to different mutations of so-called susceptibility genes as well as to interactions between genetic characteristics and environmental factors. Familial CMM is related to two genes of elevated susceptibility, penetrance, and frequency: CDK4 and CDKN2A (Cyclin-dependent kinase inhibitor 2A). CDKN2A is the most important susceptibility gene and its mutations have been identified in approximately 40 percent of the families bearing three or more members with CNM. Clinical features associated to CDKN2A mutations are elevated number of family members with CMN, multiple primary CMM, and the presence of both CMN and pancreatic cancer in the same family. In Chile, the incidence and mutation variants of CDKN2A in families with CMM is unknown.

Genes de predisposición al melanoma. Melanoma familiar / Melanoma predisposition genes: familial melanoma

El melanoma es un tumor que ha crecido en frecuencia y para el cual todavía no existe un tratamiento efectivo. En la génesis del melanoma participan factores externos, como la radiación ultravioleta (RUV), y factores genéticos. Si bien el estudio de los genes involucrados en el melanoma aun está en su inicio, se conocen en la actualidad múltiples mutaciones genéticas transmisibles que confieren a su portador mayor riesgo de desarrollar melanoma, tales como las del gen CDK-N2A, del receptor de la melanocortina 1 (MCR1) y las de los genes de la vía de las MAP kinasas, entre otras. Muchos de estos genes determinan la predisposición al melanoma familiar o al melanoma múltiple y aumentan la susceptibilidad a otros tumores no cutáneos. El conocimiento cada vez más exacto y complejo de la acción de cada gen en la génesis y en la progresión del melanoma permitirá en un futuro próximo la creación de tratamientos más eficaces y más específicos para cada paciente.

Cutaneous melanoma is an increasingly frequent tumor that, as yet,does not have a satisfactory treatment. Environmental risk factors such as ultraviolet radiation (UVR) and genetic factors as well, participate in its genesis. Although studies of the genes involved in melanoma are scarce, it is well known that multiple transmissible genetic mutations, such as CDKN2A gene, the melanocortin-receptor 1(MCR1) gene and the MAPK signaling pathway genes, among others,confer to their carrier greater risk of developing melanoma. Many of these genes determine the predisposition to familial or multiple melanoma and increase the susceptibility to noncutaneous tumors. The precise and more complex knowledge of the role of each gene in the genesis and progression of melanoma, will allow the development of more successful and specific treatments for each patient in thenear future.

Caracterização do lócus CDKN2A e dos genes CDK4 e MC1R em pacientes com critérios clínicos para o diagnóstico da síndrome do melanoma familial e melanoma múltiplo esporádico / Caracterization of the CDKN2A, CDK4 and MC1R genes in patients with clinical criteria for the diagnosis of Familial Melanoma Syndrome and Multiple Sporadic Melanoma

A Síndrome do Melanoma Familial (SMF) é uma síndrome rara de predisposição ao câncer associada preponderantemente ao lócus CDKN2A. Algumas poucas famílias no mundo apresentam mutações no gene CDK4, relacionado a mesma via molecular da pRb. Fatores relacionados ao fenótipo e polimorfismos do gene MC1R modulam de desenvolvimento de melanoma nestes pacientes. Os objetivos deste trabalho são pesquisar as mutações germinativas do lócus CDKN2A e do gene CDK4, além de caracterizar os polimorfismos dos gene MC1R em famílias brasileiras com diagnóstico clínico de Síndrome do Melanoma Familial (SMF) e em pacientes com Melanoma Múltiplo Esporádico (MME) e descrever os fatores de risco pessoais e ambientais de melanoma em probandos e familiares. Foram incluídos 50 probandos que preencheram os critérios clínicos, sendo 32 para SMF e 18 para MME. A metodologia incluiu o seqüenciamento direto dos 4 éxons do lócus CDKN2A, do éxon 2 do gene CDK4 e do éxon único do gene MC1R. Foram encontradas 8 mutações germinativas e uma alteração no lócus CDKN2A. Nenhuma mutação foi detectada no gene CDK4. Uma ampla variedade de polimorfismos do MC1R foi encontrada em 87% dos pacientes pesquisados. As mutações patogênicas p.Pro48Thr e c.-34G>T foram as mais freqüentes, sendo detectadas em 3 probandos cada uma. Cada um dos demais probandos apresentaram as mutações p.Gly101Trp e c.IVS-105A>G. A alteração p.Val84Met nunca descrita na literatura foi detectada em um dos probandos e tem potencial patogênico incerto. Estudos adicionais de segregação e funcionais são necessários. Este estudo é o quarto estudo realizado na América Latina a respeito da definição das mutações dos genes de predisposição ao Melanoma Familial e Melanoma Múltiplo Esporádico, sendo o único com critérios de seleção que seguem o padrão dos outros estudos descritos na literatura.

Methylation status of ANAPC1, CDKN2A and TP53 promoter genes in individuals with gastric cancer

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80°C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4 percent of gastric cancer samples, with 35 percent methylation in diffuse-type and 26.9 percent in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30 percent diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.

DNA methylation analysis of the tumor suppressor gene CDKN2B in Brazilian leukemia patients

The aim of this work was to evaluate the methylation profile of the p15 (CDKN2B) gene in Brazilian patients with leukemia and to correlate the CDKN2B gene expression with the percentage of methylated CpG dinucleotides in its promoter region. Thirty-one samples from six patients with acute lymphocytic leukemia (ALL), four with chronic myeloid leukemia (CML), and 21 with acute myeloid leukemia (AML) were evaluated by MSP (Methylation-Specific PCR). The CDKN2B gene was found to be methylated in four (67 percent) of the six ALL samples and in 16 (76 percent) of the 21 AML samples, but in none of the four CML samples analyzed. We observed a correlation between the CDKN2B mRNA expression (RT-PCR) and the percentage of methylated CpG dinucleotides. Therefore, this study in Brazilian patients confirms that the CDKN2B gene is methylated in the majority of leukemia patients.

Activation and Abnormalities of Cell Cycle Regulating Factor in Head and Neck Squamous Cell Carcinoma Cell Lines: Abnormal Expression of CDKN2 Gene in Laryngeal Squamous Cell Carcinoma / 영남의대학술지

BACKGROUND: Cyclin-dependent kinase (CDK) inhibitors are family of molecules that regulate the cell cycle. The CDKN2, a CDK4 inhibitor, also called p16, has been implicated in human tumorigenesis. The CDKN2 inhibits the cyclin/CDK complexes which regulate the transition from G1 to S phase of cell cycle. There is a previous report that homozygous deletion of CDKN2 region on chromosome 9p21 was detected frequently in astrocytoma, glioma and osteosarcoma, less frequently in lung cancer, leukemia and ovarian cancer, but not detected in colon cancer and neuroblastoma. However, little is known about the relationship between CDKN2 and laryngeal cancer. Therefore this study was initiated to investigate the role of CDKN2 in human laryngeal squamous cell carcinoma development. MATERIALS AND METHODS: We used 5 human laryngeal carcinoma cell lines whether they have deletions or losses of CDKN2 gene expression by DNA-PCR or RT-PCR, respectively. We examined 8 fresh frozen human laryngeal cancer tissues to detect the loss of heterozygosity (LOH) of CDKN2. PCR was performed by using microsatellite markers of short arm of human chromosome 9 (D9S126, D9S144, D9S156, D9S161, D9S162, D9S166, D9S171, D9S200 and D9SIFNA). For informative cases, allelic loss was scored if the signal of one allele was significantly decreased in tumor DNA when compared to the same allele in normal DNA. RESULTS: The CDKN2 DNA deletion was observed in 3 cell lines. The CDKN2 mRNA expression was observed in only one cell line, which was very weak. LOH was detected in 7 cases (87.5%). CONCLUSION: These results suggest that CDKN2 plays a role in the carcinogenesis of human laryngeal squamous cell carcinoma.

Significance of the Methylation of Cyclin-Dependent Kinase Inhibitor 2A gene in the Prognosis of Osteosarcoma / 대한정형외과학회잡지

PURPOSE: The methylation status of the CpG promoter regions of the p16INK4A and p14ARF genes, mutations of 4 exons of the CDKN2A gene, and the expression of the corresponding proteins were examined. Prognostic implications were assessed in osteosarcoma. MATERIALS AND METHODS: Methylation-specific PCR, sequence analysis, and immunohistochemical staining were performed upon 32 frozen osteosarcoma tissues. RESULTS: Methylation of p16INK4A was found in 16%, and methylation of p14ARF in 47%. Metastasis and poor survival was statistically related to the methylation of p14ARF. The methylation of p14ARF correlated with the repression of the corresponding protein, and repression of p14ARF with the repression of p21 and of wild type of p53. No sequence alterations were found in the four exons of the CDKN2A gene. Methylation of p14 showed highest hazard ratio by multivariate survival analysis. CONCLUSION: Our data suggest that methylation of the CDKN2A gene seems to be the main mechanism of protein repression. For p14ARF, the methylation of its promoter region was related to the repression of p21 and wild type p53, distant metastasis and a poor prognosis. Further study regarding cell cycle regulatory factors should shed light on oncogenesis and the possibility of a new treatment strategy for osteosarcoma.

Relationship between exon deletion frequency of CDKN2/P16 and pathological type,metastasis,sex in osteosarcoma / 中国组织工程研究

Objective To study on the CDKN2/P16 gene in primary osteosarcoma.Method By using molecular biological methods that inclued genome DNA extraction from paraffined tissue and PCR SSCP analysis technique, we studied alternations of CDKN2/P16 gene in 25 primary osteosarcomas.Results (1)The deletions frequency in differentiation degree of osteosarcomas was① bone brood cell, 16.7% ;② cartilage brood cell,12.5% ;③ Fiber brood cell:20% ,(P >0.05).(2)The deletion frequency in male patients was 17.6% , female patients 12.5% ,(P >0.05).(3)In early metastatic osteosarcomas the deletion rate was 33.3% ,which was significantly higher than that of the control group with the rate of 10.5% (P< 0.05).(4)The deletion rate was 16% and the mutations were not found.Conclusion (1)The deletion rate was 16% and the mutations were not found.This suggests that the deletions of CDKN2/P16 gene were closely related to the genesis of primary osteosarcoma and that the main type of the alternation of CDKN2/P16 gene was deletion.(2)In early metastatic osteosaarcomas the deletion rate was 33.3% , which was significantly higher than that of the control group with the rate of 10.5% .This indicates to great extend that the deletions of CDKN2/P16 gene were closely related to the metastatic ability.(3)The deletions frequency had no significant relationship with differentiation degree of osteosarcomas, so was with the sex of the patient.

Mutational Analysis of CDKN2 (p16-INK4A/MTS1) Gene in Childhood Acute Leukemia / 대한소아혈액종양학회지

PURPOSE: The human chromosome 9p21 region that is a frequent site of deletions and rearrangements in many tumor types including leukemias implied the existence of a tumor suppressor gene within 9p21 which is involved in tumor formation. CDKN2 (p16) gene is located in the same chromosomal region. The loss of CDKN2 function is probably one of the most common genetic alterations and is now thought to play a key role in leukemogenesis. We examined the frequency of the point mutation of CDKN2 gene by analyzing the DNA sequence and demonstrated the prognostic implication of mutations of CDKN2 gene in childhood acute leukemia. METHODS: We investigated the prevalence of the point mutation in thirty patients with 20 cases of acute lymphoblastic leukemia (ALL) and 10 cases of acute myeloid leukemia (AML). The point mutation of CDKN2 gene was analyzed in a PCR generated DNA sequencing technique. RESULTS: There was no point mutation in exon 1 of CDKN2 gene. A missense mutation (G--

The Study of CDKN2/p16INK4A Mutation in Human Breast Cancer

The p16 is a cyclin-dependent kinase inhibitor(CDKI) that inhibits cell cycle progression. In recent studies, homozygous deletions of p16 gene have been noted in some cancer cell lines, which implies the deletion or mutation of p16 gene may contribute to the malignant progression of cells in some ways. This study was to investigate the frequency of p16 gene mutation in breast cancer patients by using polymerase chain reaction-single stranded confromational polymorphysm(PCR-SSCP) analysis. Examination of 24 blood samples and corresponding 16 tissue samples from 24 breast cancer patients were performed by PCR-SSCP method. Four from 24 blood samples(16.7%) disclosed 3 abnormal bands and one band shifting. Among 13 tissue samples revealed three conformational changes(23.1%). In two cases, there were abnormal bands in both blood samples and cancer tissues. One case with no products by PCR in the tissue sample showed a band shifting in the blood sample. Three cases with no PCR products in tissue samples may considered as total allelic deletion of the p16. The cases of abnormal PCR-SSCP results show some abnormalities on direct sequencing by Sanger method as T base insertion, C/T and A/G bases substitution. The results may suggest some of breast cancer patients have germline mutations of the p16 gene and some have somatic mutations. In the carcinogenesis of some breast cancers, p16 gene mutation may dysregulates the cell cycle, that may play an important role in the unlimited tumor cell proliferations.

Alterations of CDKN2 (MTS1/p16INK4A) gene in paraffin-embedded tumor tissues of human stomach, lung, cervix and liver cancers

The CDKN2 (MTS1/p16INK4A) gene, encoding cyclin dependent kinase inhibitor, was found to be homozygously deleted at a high frequency in cell lines from many different types of cancer and some primary cancers. To determine the frequency of CDKN2 mutations in most common human cancers in Korea, PCR and PCR-SSCP analyses for the exon 2 of CDKN2 were performed on each set of 20 formalin-fixed and paraffin-embedded tumor tissues of stomach adenocarcinomas, lung cancers, cervix cancers and hepatocellular carcinomas. No mutations in exon 2 of CDKN2 were found in 20 stomach adenocarcinomas. In contrast to rare mutations in stomach adenocarcinomas, a high frequency of CDKN2 mutations was identified in other 3 cancers, 11 of 20 (55%) lung cancers (7 of 10 NSCLCs and 4 of 10 SCLCs), 14 of 20 (70%) cervix cancers and 11 of 20 (55%) hepatocellular carcinomas. These results suggest that mutations of the CDKN2 gene might be an important genetic change in NSCLCs, cervix cancers and hepatocellular carcinomas.