CDKN2 Gene Research Articles

Genetic Predisposition to Type II Diabetes in the Hispanic Population of Orange County in Relation to SNP rs1333051 on the CDKN2 Gene

Thirty‐four million individuals cope with diabetes within the United States, 95 percent of which have Type II Diabetes (T2D). Hispanics are particularly affected by diabetes and being the largest minority in the U.S., it is a significant public health issue. Since there is a diverse genetic variation between different ethnicities, researchers study genes through single nucleotide polymorphism (SNPs) to determine an individual's risk of developing T2D. The SNP rs1333051 located on the CDKN2 gene correlates with T2D. Evidence shows a nucleotide change of A allele by T allele. The allele A is also reported to be associated with coronary heart disease in T2D patients. The project’s primary goal is to find the prevalence of SNP rs133051 in 24 controls and 23 diabetic patients in the Hispanic population of Orange County. DNA was extracted, taken from a buccal swab sample, and subjected to a polymerase chain reaction (PCR), followed by gel electrophoresis to confirm PCR products. Enzymatic digestion was done using Bsrl to detect the alleles based on restriction fragment length patterns. The alleles A and T were identified in both groups. However, the allele T was predominant in the diabetic group. External factors possibly play a larger role than genetic factors. To know more about the association of the T allele, increasing the number of samples and applying statistical analysis is necessary. The overall knowledge in the identification of SNPs associated with T2D allows for preventative measures to be taken within the Hispanic population at risk for this disease.

Whole-Genome Sequencing of Vero E6 (VERO C1008) and Comparative Analysis of Four Vero Cell Sublines.

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of Vero sublines have been developed and can be classified into four major cell lineages. In this study, we determined the whole-genome sequence of Vero E6 (VERO C1008), which is one of the most widely used cell lines for the proliferation and isolation of severe acute respiratory syndrome coronaviruses (SARS-CoVs), and performed comparative analysis among Vero JCRB0111, Vero CCL-81, Vero 76, and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that these four sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoVs, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 harbored only the allele with isoleucine, resulting from the loss of one of the X chromosomes.

Transformation of Chronic Lymphocytic Leukemia Towards Richter´s Syndrome Is Induced By AKT Activation

Richter's syndrome (RS) is an aggressive transformation of Chronic Lymphocytic Leukemia (CLL) to Diffuse Large B Cell Lymphoma (DLBCL) refractory to current therapies with dismal prognosis. Richter Syndrome arises from CLL cells independent of common DLBCL mutations. Frequently, mutations in p53, CDKN2 or cMyc genes are involved, but a significant proportion displays no specifically acquired driver mutation.We could observe activation of AKT in 6 out 48 Richter syndrome biopsies by positive staining for active phosphorylated AKT while in CLL lymph nodes, DLBCL and Burkitt´s Lymphoma no phospho-AKT by IHC could be observed.However in primary patient CLL cases we could detect varying levels of pAKT by Western blot, elevated levels were identified predominantly in patients harboring high-risk mutations such p53, ATM, NOTCH1 and XPO1. Furthermore, B-cell receptor mediated stimulation of the PI3K/AKT axis provided protection towards genotoxic stress induced apoptosis via post-translation stabilization of MCL1. This provides subsequently a synergistic induction of apoptosis by combining idelalisib and bendamustin.Thus we analyzed the functional impact of AKT signaling using a conditional constitutive allele for AKT (AKT-C) specifically activated using CD19-Cre and Cγ1-Cre fro post-GC-activation. AKT activation alone could not induce a malignant phenotype, however we could demonstrate that Eµ-Tcl-1 mice with AKT-C develop Richter Syndrome. Both in EµTCL1:CD19-CreAKT-C (TCA) and EµTCL1:Cγ1- CreAKT-C (TCγ1A) mice developed a high-grade lymphoma phenotype leading to decreased survival. Transformed cells displayed blastoid characteristics with significantly increased cellular size and the histomorphological features of DLBCL. Large transformed cells show high percentage of KI67-positive staining (>90%) and frequent mitotic figures.Here, AKT-mediated GSK-3b inhibition and subsequent cMyc and Mcl-1 stabilization might transform CLL to RS cells and combinatory treatments with DNA-damaging and PI3K-inhibiting compounds revealed promising therapeutic results. Collectively, we have identified AKT signaling as an oncogenic signaling pathway in progression of CLL towards Richter´s syndrome and generated the first murine Richter Syndrome model (TCA and TCγ1A) providing novel mechanistic insights into the molecular understanding of Richter’s transformation that is amenable to model therapeutic strategies and to address the efficacy of synergistic treatment combinations. DisclosuresKlapper:Roche, Novartis, Amgen, Takeda: Research Funding. Hallek:Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.

Correlation between deletion of the CDKN2 gene and tyrosine kinase inhibitor resistance in adult Philadelphia chromosome-positive acute lymphoblastic leukemia.

BackgroundFrequency relapses are common in Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukemia (ALL) following tyrosine kinase inhibitors (TKIs). CDKN2A/B is believed to contribute to this chemotherapy resistance.MethodsTo further investigate the association between CDKN2 status and TKI resistance, the prevalence of CDKN2 deletions and its correlation with a variety of clinical features was assessed in 135 Ph-positive ALL patients using interphase fluorescence in situ hybridization (I-FISH).ResultsResults showed that no difference occurred between patients with CDKN2 deletion (44/135) and wild-type patients in sex, age, and complete remission (CR) rate following induction chemotherapy combined with tyrosine kinase inhibitors (TKIs). However, CDKN2 deletion carriers demonstrated higher white blood cell (WBC) count, enhanced rates of hepatosplenomegaly (P = 0.006), and upregulation of CD20 expression (P = 0.001). Moreover, deletions of CDKN2 resulted in lower rates of complete molecular response (undetectable BCR/ABL), increased cumulative incidence of relapse, short overall survival (OS), and disease-free survival (DFS) time (P < 0.05) even though these patients received chemotherapy plus TKIs followed by allogenic hematopoietic stem cell transplantation (Allo-HSCT). In the case of 44 patients who presented with CDKN2 deletion, 18 patients were treated with dasatinib treatment, and another 26 patients were treated with imatinib therapy, and our study found that there were no differences associated with OS (P = 0.508) and DFS (P = 0.555) between the two groups.ConclusionsCDKN2 deletion is frequently acquired during Ph-positive ALL progression and serves as a poor prognostic marker of long-term outcome in Ph-positive ALL patients with CDKN2 deletion even after the second-generation tyrosine kinase inhibitor treatment.

CDKN2 Gene Deletion As Poor Prognosis Predictor Involving in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients

Background: Genes CDKN2A (MTS1) and CDKN2B (MTS2), which encode for p16INK4a/p14ARF and p15INK4b respectively from the 9p21.3 locus, are thought to be an important growth suppressor gene. INK4a/ARF regulates p53 tumor suppressor function by interacting with MDM2. P15/INK4b works as a CDK inhibitor, which inhibits phosphorylation of Rb by inactivating cyclin/ CDK-4/6 complexes and thereby controls cell-cycle G1 progression. Deletion of CDKN2A/B was well known in many hematologic malignancies, but only few report investigated this deletion effect on clinical prognosis.Methods: This study performed analysis of the CDKN2 deletion in 215 adult B-ALL patients, and relation with cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; MLL rearrangement; MYC, IGH translocation).Results: The prevalence of CDKN2 deletions in all study population was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells(WBC) count, BM blast percentage,extra infiltration and induction complete remission(CR) rate. Analysis at relapse patients revealed increased rate of CDKN2 loss (44.6%) compared with the rate in all patients (28.4%) (p=0.006). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (p<0.001), lower disease free-survival (DFS) (p<0.001), and increased cumulative incidence of relapse (p=0.002); Also, CDKN2 deletions were strongly associated with IGH translocation (p=0.021); and had an adverse effect on patients who with BCR-ABL fusion gene or MLL rearranged. Among of patients with the CDKN2 deletions , the subgroup of received Allo-HSCT treatment had a better OS and DFS compared with the subgroup received chemotherapy only (OS: p<0.000; DFS: p<0.000).Conclusion: Patients with CDKN2 gene deletion were benefited from allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was a possible poor prognostic marker in long-term outcomes. DisclosuresNo relevant conflicts of interest to declare.

CDKN2 Gene Deletion Impair Prognosis in Adult Ph+ Acute Lymphoblastic Leukemia Even in Tyrosine Kinase Inhibitors Era

Background: Philadelphia chromosome-positive (BCR-ABL1+) ALLs benefit from the addition of a specific tyrosine kinase inhibitors (TKIs) to chemotherapy, However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The 9p21 locus, encoding important tumor suppressors (CDKN2A/B), is a major target of inactivation in the pathogenesis of many human tumors, but only few report investigated this deletion effect on clinical prognosis in Ph+ ALL.Purpose: Many studies found that deletion of CDKN2 was associated with poor prognosis in ALL, and CDKN2 deletion also as a frequent cytogenetic aberration in Ph+ ALL patients. Here we study about the prognostic significance of the CDKN2 in Ph+ ALL in TKIs era.Patients and Methods: To explore the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive ALL and to investigate their prognostic value, 135 patients (98 denovo and 37 relapsed cases) were analyzed by Paired diagnosis-relapse samples were interphase fluorescence in situ hybridization(I-FISH).Results: The prevalence of CDKN2 deletions in all study population was 27.4% (37/135). There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells(WBC) count, BM blast percentage, and induction complete remission(CR) rate. Compared with patients with wild-type CDKN2, the patients with CDKN2 deletion had higher rates of hepatosplenomegaly, CD20 expression (p<0.05). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (P<0.001), lower disease free-survival (DFS) (P<0.001), and increased cumulative incidence of relapse (P=0.003). In case of 37 patients with CDKN2 deletion, 20 patients treated with chemotherapy and Dasatinib followed by allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and another 13 patients treated with chemotherapy and Imatinb followed by Allo-HSCT,there were no difference associated with OS(P=0.813) and DFS(p=0.513) between the above groups.Conclusions: Deletion of CDKN2 by I-FISH is a frequent event in Ph-positive ALL, and frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes even though treated by adding dasatinib. We also found CDKN2 deletion patients had higher CD20 expression, this group patients may be benefit from rituximab. DisclosuresNo relevant conflicts of interest to declare.

CDKN2 Gene Deletion as Poor Prognosis Predictor Involved in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients.

Deletion of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) is well known in many hematologic malignancies, but only few reports have investigated this deletion effect on clinical prognosis. This study performed analysis of the CDKN2 deletion in 215 adult B- lineage acute lymphoblastic leukemia (B-ALL) patients, and related cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; Mixed Lineage Leukemia (MLL) rearrangement; MYC, Immunoglobulin heavy locus (IGH) translocation). The prevalence of CDKN2 deletions in all study populations was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells (WBC) count, BM blast percentage, extra infiltration and induction complete remission (CR) rate. Analysis in relapse patients revealed that the distribution of CDKN2 deletion is higher in relapse patients (44.6%) than all patients (28.4%, P=0.006). Deletion of CDKN2 was significantly associated with poor outcomes including decreased overall survival (OS) (P<0.001), lower disease free-survival (DFS) (P<0.001), and increased cumulative incidence of relapse (P=0.002); Also, CDKN2 deletion was strongly associated with IGH translocation (P=0.021); and had an adverse effect on patients with BCR-ABL fusion gene or with MLL rearrangement. Patients with CDKN2 gene deletion benefited from allogenic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was poor prognostic marker in long-term outcomes.

Extraskeletal Myxoid Chondrosarcoma of the Vulva With PLAG1 Gene Activation

Extraskeletal myxoid chondrosarcoma (EMC) is a rare mesenchymal neoplasm, rarely reported in the genitourinary tract with only 5 cases reported in the vulva. We investigated 2 cases of vulvar sarcomas whose morphologic appearance and immunohistochemical profiles were consistent with EMC using fluorescence in situ hybridization (FISH), reverse-transcription polymerase chain reaction, and a whole genome expression array. FISH and reverse-transcription polymerase chain reaction assays showed no EWSR1 and NR4A3 loci rearrangements. Microarray-based analysis also revealed no changes in NR4A3 and EWSR1 gene transcription levels. Microarray data showed a significant downregulation of the muscle-related genes (eg, myosin heavy chain family, actins, myoglobin, desmin, creatine kinase, troponins) and cytokeratins (KRT6A, 6B, 13, 14, and 78), upregulation of several neuron-specific genes [neural cell adhesion molecule 1 (NCAM-1/CD56), neurofilament (NEFH)], along with some well-characterized tumor biomarkers [carbonic anhydrase IX (CA-9), topoisomerase IIα (TOP2A), matrix metalloproteinases (MMP-7, MMP-9), CDKN2 gene (p16-INK4a), checkpoint homolog 2 (CHEK2)]. Notably, both tumors showed upregulation of the pleomorphic adenoma gene 1 (PLAG1), and in 1 case PLAG1 gene rearrangement was detected by break-apart FISH. Some vulvar tumors with morphologic and immunohistochemical characteristics of EMC may represent a molecular genetic entity separate from EMCs arising in other locations. PLAG1 gene activation appears to be involved in the development of these neoplasms.

A review of epigenetic and gene expression alterations associated with intracranial meningiomas

A more comprehensive understanding of the epigenetic abnormalities associated with meningioma tumorigenesis, growth, and invasion may provide useful targets for molecular classification and development of targeted therapies for meningiomas. The authors performed a review of the current literature to identify the epigenetic modifications associated with the formation and/or progression of meningiomas. Several epigenomic alterations, mainly pertaining to DNA methylation, have been associated with meningiomas. Hypermethylation of TIMP3 inactivates its tumor suppression activity while CDKN2 (p14[ARF]) and TP73 gene hypermethylation and HIST1H1c upregulation interact with the p53 regulation of cell cycle control. Other factors such as HOX, IGF, WNK2, and TGF-β epigenetic modifications allow either upregulation or downregulation of critical pathways for meningioma development, progression, and recurrence. Genome-wide methylation profiling demonstrated that global hypomethylation correlates with tumor grades and severity. Identification of additional epigenetic changes, such as histone modification and higher-order chromosomal structure, may allow for a more thorough understanding of tumorigenesis and enable future individualized treatment strategies for meningiomas.

Abstract 3953: Senescence induced of smooth muscel protein 22-alpha (SM22) and Radiation patients change human-link genes expression level

Abstract P53 and RB protein is famous for its capacity to suppress tumor senescence pathway member. Senescence is a leading cause of cancer-related death, considerable uncertainty still remains about their effectiveness. However, each marker in an increased risk that experience genomic data with a cancer-specific mutations. So, senescence marker that techniques can't develop patient-specific treatment for particular about experimental study-conditions and result-analysis. This study was undertaken to evaluate the sm22 (smooth muscel protein 22-alpha) protein relation of senescence marker and changes condition of the radiation at human-link senescence marker genes. We were determined the expression of senescence-associated β-galactosidase and western-blot assay. It was performed in colon cancers that were 17 patients each of normal and adenoma, 16 patients each of normal and adenocarcinoma. To conformed enzyme efficiency is β-galactosidase, senescence pathway using of p16, p21 and p53. On the other hand, sm22 use of senescence marker in study. we have decided to micro-array analysis that 3 patients each of normal and adenoma, 2 patients each of normal and adenocarcinoma, 2 patients each of normal and irradiation-adenocarcinoma (2 weeks after) available miRNA sequences on the Agilent Human whole genome 8x60K array chip. Gene expression levels were calculated with Feature Extraction v10.7.3.1 (Agilent technologies, CA) Relative signal intensities for each gene were generated using the Robust Multi-Array Average algorithm. Senescence induced p16 expression in dependent β-galactosidase assay results, sm22 protein suppressed cellular senescence levels as well as p16 protein. The expression of p16 and sm22 was analyzed that not involved each normals, adenomas and adenocarcinomas tissue by western blot analysis. In addition, it found that these results were similar to those of micro array. The three groups (adenoma, adeno-carcinoma and irradiation-adeno-carcinoma) can be compared in terms of total 2-fold changed gene (least 200% of controls for up-regulated and lower than 50% of controls for down-regulated genes). We found this integrative data analysis tool to the LCN2, LYPD5 and LOC100132051 genes as well as CDKN2 (p16) and TAGLN2 (sm22) gene expression. Irradiation groups was much less than the adenoma (45.3 %) and adenocarcinoma (71.1 %) 2-fold changed gene expression. The LCN2, LYPD5 and LOC100132051 genes lower than adeno-carcinoma group level. Our observations suggest that shared gene and protein expression profiles may contribute to sm22 and p16 of senescence marker, irradiation induces cellular senescence of colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3953. doi:1538-7445.AM2012-3953

Next Generation Sequencing of Prostate Cancer from a Patient Identifies a Deficiency of Methylthioadenosine Phosphorylase, an Exploitable Tumor Target

Castrate-resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumor sequencing program, a patient tumor was analyzed using Illumina genome sequencing and a matched renal capsule tumor xenograft was generated. Both tumor and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2, and ARF genes. It is rare for this deletion to occur in primary prostate tumors, yet approximately 10% express decreased levels of methylthioadenosine phosphorylase (MTAP) mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it seems that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient-derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers as deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumors at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology.

P16 INK4a/CDKN2 expression and its relationship with oral squamous cell carcinoma is our current knowledge enough?

Oral squamous cell carcinomas (OSCC) are the most common malignancy of the oral cavity and their multistep development requires the accumulation of multiple genetic and epigenetic alterations. Inactivation of p16 INK4a, encoded by the CDKN2 gene has been widely associated with this type of tumors. The purpose of this review is to elucidate the relationship between p16 INK4a expression and the different clinical and pathological aspects of OSCC, analyze the variation in results between studies, detailing the described genetic/epigenetic alterations that result in gene silencing and the relationship between p16 INK4a and HPV infection.

Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells.

Despite the predominance of ultraviolet A (UVA) relative to UVB in terrestrial sunlight, solar mutagenesis in humans and rodents is characterized by mutations specific for UVB. We have investigated the kinetics of repair of UVA- and UVB-induced DNA lesions in relation to mutagenicity in transgenic mouse fibroblasts irradiated with equilethal doses of UVA and UVB in comparison to simulated-sunlight UV (SSL). We have also analyzed mutagenesis-derived carcinogenesis in sunlight-associated human skin cancers by compiling the published data on mutation types found in crucial genes in nonmelanoma and melanoma skin cancers. Here, we demonstrate a resistance to repair of UVB-induced cis-syn cyclobutane pyrimidine-dimers (CPDs) together with rapid removal of UVA-induced oxidized purines in the genome overall and in the cII transgene of SSL-irradiated cells. The spectra of mutation induced by both UVB and SSL irradiation in this experimental system are characterized by significant increases in relative frequency of C-->T transitions at dipyrimidines, which are the established signature mutation of CPDs. This type of mutation is also the predominant mutation found in human nonmelanoma and melanoma tumor samples in the TP53, CDKN2, PTCH, and protein kinase genes. The prevailing role of UVB over UVA in solar mutagenesis in our test system can be ascribed to different kinetics of repair for lesions induced by the respective UV irradiation.

P16INK4A tumor suppressor gene expression and CD3ϵ deficiency but not pre-TCR deficiency inhibit TAL1-linked T-lineage leukemogenesis

Inactivation of the CDKN2 genes that encode the p16(INK4A) and p14(ARF) proteins occurs in the majority of human T-cell acute lymphoblastic leukemias (T-ALLs). Ectopic expression of TAL1 and LMO1 genes is linked to the development of T-ALL in humans. In TAL1xLMO1 mice, leukemia develops in 100% of mice at 5 months. To identify the molecular events crucial to leukemic transformation, we produced several mouse models. We report here that expression of P16(INK4A) in developing TAL1xLMO1 thymocytes blocks leukemogenesis in the majority of the mice, and the leukemias that eventually develop show P16(INK4A) loss of expression. Events related to the T-cell receptor beta selection process are thought to be important for leukemic transformation. We show here that the absence of the pTalpha chain only slightly delays the appearance of TAL1xLMO1-induced T-ALL, which indicates a minor role of the pTalpha chain. We also show that the CD3epsilon-mediated signal transduction pathway is essential for this transformation process, since the TAL1xLMO1xCD3epsilon-deficient mice do not develop T-ALL for up to 1 year.

Identification of oligodendroglioma specific chromosomal copy number changes in the glioblastoma MI-4 cell line by array-CGH and FISH analyses

Glioblastomas, the most frequent and malignant glial tumors, are known to be phenotypically heterogeneous. A low fraction of glioblastomas is associated with specific chromosomal losses at 1p and 19q, which are commonly found in oligodendrogliomas and are generally considered to be a primary event in the development of these tumors. Subsequent progression of oligodendroglial tumors appears to be triggered by additional molecular features underlying the transition to anaplastic oligodendroglioma and glioblastoma multiforme (GBM) such as deletions of 9p and 10q, and alterations of CDKN2A (p16), which is located at 9p21. These findings strengthen the view that GBM on rare occasions may develop from oligodendroglial differentiated cells. In the present study, we evaluated the newly established MI-4 glioblastoma cell line, which displays 1p and 19q specific alterations targeting preferential regions of allelic loss in glial neoplasms, by array-CGH and fluorescence in situ hybridization (FISH) analyses that were combined to obtain a high resolution map of targeted chromosome rearrangements and copy number changes throughout the genome. Genome-wide and chromosome 19 full coverage array-CGH analysis of the MI-4 cell line revealed that in this particular cell line, 1p-specific loss, including the CDKN2 (p18) gene, is not accompanied by loss of the previously described 19q13.3 tumor suppressor candidate region. Interestingly, the array-CGH (CGHa) profile showed an increase in copy number along most of 19q including the AKT2 oncogene and the KLKs gene family, which have previously been shown to be amplified in pancreatic carcinomas and upregulated in several tumors, respectively. The concomitant 1p partial loss and chromosome 19 alterations, with the +7 and −10-specific GBM markers associated with homozygous deletion of 9p21.3 including CDKN2A (p16), are distinct features of the glioblastoma MI-4 cell line, illustrating its origin from an olidodendroglial tumor. Based on these results, we conclude that the MI-4 glioblastoma cell line might function as a model system for investigations into the behavior of a defined oligodendroglioma subtype.

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

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.1) 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.

Structural organization, mapping, characterization and evolutionary relationships of CDKN2 gene family members in Xiphophorus fishes

Xiphophorus fishes and their hybrids are used as models for the study of melanoma and other diseases. The cyclin-dependent kinase inhibitor gene family in humans is comprised of four members, including CDKN2A (P16), and dysregulation of this gene is implicated in numerous neoplasms including melanomas. We have investigated the status of the gene family in the southern platyfish X. maculatus. Xiphophorus harbors at least two such loci, which we now term CDKN2A/B and CDKN2D. Both loci map to Xiphophorus linkage group 5, a genomic area that has long been known to harbor the DIFF tumor suppressor locus. Within this report, we report on the complete cloning, genomic exon/intron boundary delineation, linkage mapping and expressional characteristics of Xiphophorus CDKN2D. We also compare and contrast this expression to that of the previously isolated CDKN2AB locus in normal and neoplastic tissues derived from non-hybrid and hybrid fishes. The hypothetical evolutionary relationships of gene family members and their involvement in melanoma is evaluated. In comparison to CDKN2A/B, the RNA expression of Xiphophorus CDKN2D differs in normal tissues and is not associated with melanotic/pathologic tissues, confirming functional divergence between obvious homologues.

Retrospective Analysis of Familial Clustering for Malignant Melanoma

Background Malignant Melanoma (MM) is familial in 5–7% of cases. Germline mutations of the CDKN2 and CDK4 genes are involved in the pathogenesis of hereditary MM, but the high frequency of familial clustering suggests other genes involved. MM seems to cluster with hereditary syndrome such as Hereditary Breast/Ovarian Cancer (HBOC). In this respect, some authors reported that carriers of BRCA 2 germline mutations have a significantly increased risk for MM (RR: 2.58). AIMS We investigate family history of MM patients to define the familial clustering for MM and history for other tumours. Methods We retrospectively analysed family history of 342 consecutive MM cases referred from 1994 to 2002 to the “Gruppo Melanoma”, Azienda Universitaria Policlinico, University “Federico II” of Naples. We divided the cases into four subsets: 1) multiple MM; 2) MM aggregated with other tumours in the proband; 3) MM with a family history of MM; and 4) MM with a family history of other tumours. We collected the pedigree, after informed consent, in all cases with a family history of MM or other tumours. Results Data analysis showed: multiple MM in 5/342 cases (1.46%); MM aggregated with other tumours in the proband in 10/342 cases (2.9%); MM with a family history of MM in 13/342 cases (3.8%) and MM with a family history of other tumours in 28/342 cases (8.2%). We propose pedigree construction to 41 subjects: we collected 23 pedigrees, 14 pedigree are ongoing, while 4 cases refused their consent. Conclusion Patient affected by Malignant Melanoma seems to be at increased risk to develop other tumours and healthy individuals belonging to families with a high clustering for malignant melanoma and other tumours could be at risk for cancers. Supported by MIUR-COFIN 2001.

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, p16INK4a and p14ARF, through the use of independent first exons and shared exons 2 and 3. p16INK4a is a cyclin‐dependent kinase inhibitor, whereas p14ARF regulates the cell cycle through a p53 and MDM2–dependent pathway. We have examined the expression of p16INK4a and p14ARF using competitive RT‐PCR in 60 non‐small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16INK4a and p14ARF were nearly equal or the intensity of the p16INK4a band slightly exceeded that of p14ARF in the normal lung tissues (n=60). In 38 tumors the intensity of the p16INK4a band was similar to or slightly weaker than that of p14ARF. In 6 tumors the intensity of the p16INK4a band was weaker than that of p14ARF. In 15 tumors the intensity of the p14ARF band was very strong and the p16INK4a band was barely visible. In only one tumor was the intensity of the p16INK4a band very strong, while the band of p14ARF was barely visible. The ratio of the intensity of p16INK4a to p14ARF had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II‐IV tumors had significantly lower p16INK4a to p14ARF ratios than the p stage I tumors (P=0.036). The T2–4 tumors had significantly lower p16INK4a to p14ARF ratios than the T1 tumors (P=0.005). The N1–3 tumors had significantly lower p16INK4a to p14ARF ratios than the NO tumors (P=0.014). Our results suggest that the ratio of expression of p16INK4a to p14ARFtends to decrease during the progression of NSCLC.

Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia.

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor, p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor, p14ARF, which blocks MDM2-induced p53 degradation resulting in an increase in p53 levels that leads to cell cycle arrest. Recent studies have revealed that expression of p16 and p14ARF is influenced markedly by the status of pRb and p53, and p16 overexpression has been demonstrated in cervical neoplasia because of functional inactivation of pRb by the human papillomavirus (HPV) E7 protein. To clarify the p14ARF status and the relationship between p16/p14ARF and other cell cycle molecules in cervical carcinogenesis, immunohistochemical analysis of p16, p14ARF, p53 and MDM2 was performed on 65 samples of cervical and genital condylomatous and neoplastic lesions, including nine HPV-negative tumors. In most cervical cancers and preneoplastic lesions with HPV infection of high and intermediate risk, a marked overexpression of p14ARF as well as the p16 protein (i.e. dotted nuclear immunostaining) was observed. All condyloma acuminata except one and low-grade dysplasia with HPV infection of low risk, such as HPV 6, immunohistochemically showed completely negative staining for p14ARF, also seen in non-neoplastic and mesenchymal cells. Our results clearly show that the mode of p14ARF overexpression in cervical neoplastic cells with HPV association differs from that in cancers of other organs without HPV association, and the p14ARF overexpression may be attributable to a negative feedback result in the functional inactivation of the pRb and p53 proteins by HPV oncoproteins.