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Background: The limited radiosensitivity of osteosarcoma poses a challenge in applying radiotherapy, necessitating the search for effective radiosensitizing targets., Methods: The lentiviral vectors were employed to establish CDKN2C-overexpressing (CDKN2C-OE) and CDKN2C-negative control (CDKN2C-NC) HOS and U2OS osteosarcoma cells. Cells were treated with or without irradiation (IR) to assess radiosensitization via viability, proliferation, apoptosis, and cell cycle analysis. A mouse model with subcutaneous tumors from CDKN2C-OE and CDKN2C-NC HOS cells evaluated tumor growth post-IR. Immunohistochemical staining and Western blot analysis were conducted to confirm model establishment and explore mechanisms., Results: CDKN2C-OE combined with IR inhibited cell viability and proliferation, promoting apoptosis in vitro and inhibiting tumor growth in vivo. CDKN2C-OE inhibited G1 phase progression post-IR by suppressing Cyclin-dependent kinase 4 (CDK4) expression and Thr172 phosphorylation, reducing retinoblastoma protein (RB) phosphorylation at Ser807/811. CDKN2C-OE did not primarily impact the cell cycle by regulating the expression of CDK6 and Cyclin D1. Furthermore, when CDKN2C-OE was combined with IR, the expression of BAX, Caspase-3, and its active cleavage product, cleaved Caspase-3, was upregulated., Conclusions: Our research results indicate that overexpression of CDKN2C enhances radiosensitivity in osteosarcoma through the induction of G1 phase arrest and subsequent apoptosis. G1 phase arrest is mediated by the suppression of CDK4 expression and Thr172 phosphorylation, which consequently affects the expression of phosphorylated RB at the Ser807/811 sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: Carriers of CDKN2A mutations have high risks of melanoma and certain other cancers. In this study we examined the occurrence of tumors among CDKN2A wild type (wt) members of melanoma-prone families with CDKN2A mutations., Methods: Swedish and US melanoma-prone families with CDKN2A mutations were included. Data was collected on tumors diagnosed among family members. Among the CDKN2A mutated families, members with CDKN2A wt status who were diagnosed with melanoma were designated phenocopies., Results: Of patients with melanoma in the CDKN2A mutated families (n = 266), 7.1%, were seen among members with CDKN2A wt status (phenocopy rate). Among the CDKN2A wt family members of the CDKN2A mutated families (n = 256), 7.4% were diagnosed with melanoma. The prospective relative risk for melanomas was significantly higher among the CDKN2A wt subjects compared with population-based controls (7.4 (95% confidence interval 1.7-33.2)), while no elevated risks of nonmelanoma cancers were seen and their offspring did not have significantly elevated risks of melanoma or other cancers., Conclusion: Members of CDKN2A mutation carrying families who test negative for their family's mutation have moderately increased risk for melanoma and should, in addition to being considered for continuing dermatologic surveillance, be encouraged to follow sun safety recommendations and practice skin self-exams.

Background: Gene copy number variations (CNVs) have been reported to be frequent in renal cell carcinoma (RCC), with potential prognostic value for some. However, their clinical utility, especially to guide treatment of metastatic disease remains to be established. Our objectives were to assess CNVs on a panel of selected genes and determine their clinical relevance in patients who underwent treatment of metastatic RCC., Patients and Methods: The genetic assessment was performed on frozen tissue samples of clear cell metastatic RCC using quantitative multiplex polymerase chain reaction of short fluorescent fragment method to detect CNVs on a panel of 14 genes of interest. The comparison of the electropherogram obtained from both tumor and normal renal adjacent tissue allowed for CNV identification. The clinical, biologic, and survival characteristics were assessed for their associations with the most frequent CNVs., Results: Fifty patients with clear cell metastatic RCC were included. The CNV rate was 21.4%. The loss of CDKN2A and PLG was associated with a higher tumor stage (P < .05). The loss of PLG and ALDOB was associated with a higher Fuhrman grade (P < .05). The loss of ALDOB was also associated with a worse Heng prognostic score (95% vs. 66%; P = .029) and lower 24-month survival rate (18% vs. 58%; P = .012). The loss of both ALDOB and PLG was frequent (32%) and was associated with a higher tumor stage and grade (P < .05)., Conclusion: As expected, we showed that several CNVs were associated with clinical relevance, especially those located on CDKN2A, PLG, and ALDOB, in a homogeneous cohort of patients with clear cell metastatic RCC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC), embryonal carcinoma cells, and 3T3 fibroblasts ectopically expressing Dppa4. We find a core set of Dppa4 binding sites shared across cell types, and also a substantial number of sites unique to each cell type. Across cell types Dppa4 shows a preference for binding to regions with active chromatin signatures, and can influence chromatin modifications at target genes. In 3T3 fibroblasts with enforced Dppa4 expression, Dppa4 represses the cell cycle inhibitor Cdkn2c and activates Ets family transcription factor Etv4, leading to alterations in the cell cycle that likely contribute to the oncogenic phenotype. Dppa4 also directly regulates Etv4 in mESC but represses it in this context, and binds with Oct4 to a set of shared targets that are largely independent of Sox2 and Nanog, indicating that Dppa4 functions independently of the core pluripotency network in stem cells. Together these data provide novel insights into Dppa4 function in both pluripotent and oncogenic contexts., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: A recent study on the comprehensive genomic profile of advanced urothelial bladder cancer (UBC) showed cyclin-dependent kinase inhibitor 2A (CDKN2A) and fibroblast growth factor receptor 3 (FGFR3) as the most often clinically relevant genomic alterations. Therefore, the prognostic role of FGFR3 and CDKN2A/p16 for pT1 UBC was studied., Patients and Methods: Clinical data and formalin-fixed paraffin-embedded tissues of pT1 UBC treated with an organ-preserving approach was analyzed retrospectively. Total RNA was isolated using commercial RNA extraction kits and mRNA expression of CDKN2A/p16 and FGFR3 was measured using single step reverse transcription quantitative real time polymerase chain reaction using RNA-specific TaqMan assays., Results: Data from 296 patients (79.4% male; median age: 72 years) could be used for the final evaluation. Spearman correlation revealed a statistically significant negative correlation between mRNA expression of CDKN2A/p16 and FGFR3. There was a positive correlation between CDKN2A/p16 and G3 tumors (ρ = 0.1875; P = .0012) and associated carcinoma in situ (ρ = 0.1703, P = .0033) and a negative correlation between FGFR3 and these factors (ρ = -0.2791, P < .0001 and ρ = -0.2182, P = .0002). High CDKN2A/p16 expression (≥38.04) and low FGFR3 expression (<39.14) were statistically significantly associated with worse progression-free survival (PFS; P = .0194 and P = .0089). Multivariate Cox regression analysis could identify patients with low FGFR3 and high CDKN2A/p16 expression (log rank (LR) χ 2  = 10.69; P = .0048) as well as tumor size ≥3 cm (LR χ 2  = 6.03; P = .0141) as independent predictors for PFS., Conclusion: High expression of CDKN2A/p16 and low expression of FGFR3 show a correlation with established prognostic features for non-muscle-invasive bladder cancer and can predict progression of stage pT1 UBC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Developments in high-throughput genotyping technology have driven discovery of genomic regions associated with an increased risk of endometriosis. In all, 16 genomic regions have been associated with risk of endometriosis in one or more populations. The latest meta-analysis including 17,045 endometriosis cases identified 14 genomic regions supported by results from multiple studies. No independent associations were identified from direct genotyping of common and low-frequency protein-coding variants. This suggests that the most common genetic factors that contribute to endometriosis risk are located in regulatory DNA sequences and alter the regulation of gene transcription. Evidence from different methods is essential to identify the target genes and transcripts that contribute to altered disease risk. Potential target genes in three chromosome regions showing altered gene regulation include LINC00339 and CDC42 on chromosome 1, CDKN2A-AS1 on chromosome 9, and VEZT on chromosome 12. Further functional studies are needed to confirm the causal genes in these and other regions to understand pathways that increase endometriosis risk and help identify novel targets for interventions to improve diagnosis and treatment., (Copyright © 2018. Published by Elsevier Ltd.)

Objective: To establish a high throughput, low cost, and simple nanotechnology-based method for the detection of single nucleotide polymorphism (SNP) loci in type 2 diabetes mellitus (T2DM)., Methods: Multiplex ligase detection reaction (LDR) amplification was performed using fluorescently labeled magnetic nanosphere-bound upstream LDR probes and downstream probes labeled with a unique fluorescent group for each SNP locus. The amplified LDR products were separated by magnetic nanospheres and then scanned by fluorescence spectroscopy. Four SNP loci associated with T2DM were detected, including the rs13866634 locus in SLC30A8, rs10811661in CDKN2A/2B, rs1111875 in the HHEX gene, and rs7903146 in the TCF7L2 gene. The SNP genotype was also determined by DNA sequencing as a control., Results: The SNP genotypes of the four gene loci determined by the nanosphere-based multiplex LDR method were consistent with the DNA sequencing results. The accuracy rate was 100%., Conclusion: A method based on multiplex PCR and LDR was established for simultaneous detection of four SNP loci of T2DM susceptibility genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Germline mutations in CDKN2A are frequently identified among melanoma kindreds and are associated with increased atypical nevus counts. However, a clear relationship between pathogenic CDKN2A mutation carriage and other nevus phenotypes including counts of common acquired nevi has not yet been established. Using data from GenoMEL, we investigated the relationships between CDKN2A mutation carriage and 2-mm, 5-mm, and atypical nevus counts among blood-related members of melanoma families. Compared with individuals without a pathogenic mutation, those who carried one had an overall higher prevalence of atypical (odds ratio = 1.64; 95% confidence interval = 1.18-2.28) nevi but not 2-mm nevi (odds ratio = 1.06; 95% confidence interval = 0.92-1.21) or 5-mm nevi (odds ratio = 1.26; 95% confidence interval = 0.94-1.70). Stratification by case status showed more pronounced positive associations among non-case family members, who were nearly three times (odds ratio = 2.91; 95% confidence interval = 1.75-4.82) as likely to exhibit nevus counts at or above the median in all three nevus categories simultaneously when harboring a pathogenic mutation (vs. not harboring one). Our results support the hypothesis that unidentified nevogenic genes are co-inherited with CDKN2A and may influence carcinogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

ARID1A as a subunit of SWI/SNF chromatin complexes is frequently mutated in human pancreatic cancer, however its exact role in pancreatic tumorigenesis remain unclear. In this study, we investigated the effects of ARID1A loss on human pancreatic epithelial cell lines HPNE, BxPC-3 with KRAS mutant (KRAS G12D ) expression. We found that ARID1A knockdown promoted cell proliferation and colony formation in cooperation with active mutant KRAS G12D . Function assay revealed that ARID1A knockdown accelerated cell cycle progression, and repressed KRAS G12D -induced cell senescence. Transcriptome analysis revealed ARID1A knockdown led to miR-503 upregulation. CDKN2A was identified as a target of miR-503, which contributes to cell senescence. Thus, our data suggests that ARID1A deficiency promote KRAS G12D -driven pancreatic tumorigenesis through miR-503/CDKN2A-mediated senescence., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Inherited genetic variations offer a possible explanation for the observed peculiarities of stroke in sub - Saharan African populations. Interleukin-6 polymorphisms have been previously associated with ischemic stroke in some non-African populations., Aim: Herein we investigated, for the first time, the association of genetic polymorphisms of IL-6, CDKN2A- CDKN2B and other genes with ischemic stroke among indigenous West African participants in the Stroke Investigative Research and Education Network (SIREN) Study., Methods: Twenty-three previously identified single nucleotide polymorphisms (SNPs) in 14 genes of relevance to the neurobiology of ischemic stroke were investigated. Logistic regression models adjusting for known cardiovascular disease risk factors were constructed to assess the associations of the 23 SNPs in rigorously phenotyped cases (N=429) of ischemic stroke (Men=198; Women=231) and stroke- free (N=483) controls (Men=236; Women=247)., Results: Interleukin-6 (IL6) rs1800796 (C minor allele; frequency: West Africans=8.6%) was significantly associated with ischemic stroke in men (OR=2.006, 95% CI=[1.065, 3.777], p=0.031) with hypertension in the model but not in women. In addition, rs2383207 in CDKN2A/CDKN2B (minor allele A with frequency: West Africans=1.7%) was also associated with ischemic stroke in men (OR=2.550, 95% CI=[1.027, 6.331], p=0.044) with primary covariates in the model, but not in women. Polymorphisms in other genes did not show significant association with ischemic stroke., Conclusion: Polymorphisms rs1800796 in IL6 gene and rs2383207 in CDKN2A/CDKN2B gene have significant associations with ischemic stroke in indigenous West African men. CDKN2A/CDKN2B SNP rs2383207 is independently associated with ischemic stroke in indigenous West African men. Further research should focus on the contributions of inflammatory genes and other genetic polymorphisms, as well as the influence of sex on the neurobiology of stroke in people of African ancestry., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The definition of biological donor organ age rather than chronological age seems obvious for the establishment of a valid pre-transplant risk assessment. Therefore, we studied gene expression for candidate markers in 60 zero-hour kidney biopsies. Compared with 29 younger donors under age 55, 31 elderly donors age 55 and older had significant mRNA expression for immunoproteasome subunits (PSMB8, PSMB9 and PSMB10), HLA-DRB, and transcripts of the activating cytotoxicity receptor NKG2D. Gene expression was validated in an independent donor cohort consisting of 37 kidneys from donors 30 years and under (Group I), 75 kidneys from donors age 31-54 years (Group II) and 75 kidneys from donors age 55 and older (Group III). Significant gene induction was confirmed in kidneys from Group III for PSMB9 and PSMB10. Strikingly, transcripts of NKG2D had the significantly highest gene induction in Group III versus Group II and Group I. Similar results were obtained for CDKN2A, but not for telomere length. Both NKG2D and CDKN2A mRNA expression were significantly correlated with creatinine levels at 24 months after transplantation. Univariate regression analysis showed significant predictive power regarding graft function at 6 and 12 months for NKG2D and CDKN2A. However, only NKG2D remained significantly predictive in the multivariate model at 12 months. Thus, our results reveal novel candidate markers in aged renal allografts, which could be helpful in the assessment of organ quality., (Copyright © 2017 International Society of Nephrology. All rights reserved.)

Nonalcoholic steatohepatitis (NASH) is characterized by excess accumulation of lipids in liver, accompanied with hepatocyte injury, cell death and inflammation. Although p16 is known as tumor suppressor in multiple cancer types, it remains unclear whether p16 plays a critical role in NASH. To determine whether p16 could play a role in the pathogenesis of NASH, wild-type mice and p16 -/- mice were fed on a methionine and choline-deficient (MCD) diet for 3 weeks, and liver steatosis, fibrosis, and inflammation were evaluated. Our data show that p16 -/- mice fed with MCD diet displayed more significant hepatic steatosis, hepatocyte damage, increased oxidative stress and inflammatory cell infiltration compared to MCD-fed WT mice. It was also clear that the increased ROS and the accumulation of lipid in BEL-7402 cells occurred when p16 expression was depleted with siRNA. These findings indicate that p16 may play a critical role in the development of NASH by reining in ROS production and by inhabiting inflammatory response., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In cancer treatment, specifically targeting cancer cells is important for optimal therapeutic efficacy. One strategy is to utilize a cancer specific promoter to express a cytotoxic gene or a viral gene required for replication. In this approach, the therapeutic window is dependent on the relative promoter activity in cancer cells versus normal cells. Therefore, a promoter with optimal cancer cell-specificity should be used. The tumor suppressor ARF promoter, which specifically responds to deregulated E2F activity, is a potent candidate. Defects in the RB pathway resulting in deregulated E2F activity are observed in almost all cancers. Furthermore, the ARF promoter exhibits greater cancer cell specificity than the E2F1 promoter and consequently, adenovirus expressing HSV-TK under the control of the ARF promoter (Ad-ARF-TK) has more selective cytotoxicity in cancer cells than the analogous E2F1 construct. Ideally, cancer specific gene expression driven by the ARF promoter could be enhanced for optimal therapeutic efficacy, with minimal side effects. We show here that ectopic expression of the CDK inhibitor p21 Cip1 enhanced deregulated E2F activity and pro-apoptotic E2F target gene expression in cancer cells. Moreover, ectopic expression of p21 Cip1 augmented cancer specific cytotoxicity of Ad-ARF-TK, and apoptosis induced by p21 Cip1 was dependent on deregulated E2F activity. These results suggest that p21 Cip1 specifically enhances deregulated E2F activity and that a combination of the CDK inhibitor with Ad-ARF-TK could be effectively employed for cancer therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT + CD20 - precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: CDKN2A is the main high-risk melanoma-susceptibility gene, but it has been poorly assessed in Latin America. We sought to analyze CDKN2A and MC1R in patients from Latin America with familial and sporadic multiple primary melanoma (SMP) and compare the data with those for patients from Spain to establish bases for melanoma genetic counseling in Latin America., Methods: CDKN2A and MC1R were sequenced in 186 Latin American patients from Argentina, Brazil, Chile, Mexico, and Uruguay, and in 904 Spanish patients. Clinical and phenotypic data were obtained., Results: Overall, 24 and 14% of melanoma-prone families in Latin America and Spain, respectively, had mutations in CDKN2A. Latin American families had CDKN2A mutations more frequently (P = 0.014) than Spanish ones. Of patients with SMP, 10% of those from Latin America and 8.5% of those from Spain had mutations in CDKN2A (P = 0.623). The most recurrent CDKN2A mutations were c.-34G>T and p.G101W. Latin American patients had fairer hair (P = 0.016) and skin (P < 0.001) and a higher prevalence of MC1R variants (P = 0.003) compared with Spanish patients., Conclusion: The inclusion criteria for genetic counseling of melanoma in Latin America may be the same criteria used in Spain, as suggested in areas with low to medium incidence, SMP with at least two melanomas, or families with at least two cases among first- or second-degree relatives.Genet Med 18 7, 727-736.

Background: Squamous cell cancers of the anal canal (ASCC) are increasing in frequency and lack effective therapies for advanced disease. Although an association with human papillomavirus (HPV) has been established, little is known about the molecular characterization of ASCC. A comprehensive genomic analysis of ASCC was undertaken to identify novel genomic alterations (GAs) that will inform therapeutic choices for patients with advanced disease., Patients and Methods: Hybrid-capture-based next-generation sequencing of exons from 236 cancer-related genes and intronic regions from 19 genes commonly rearranged in cancer was performed on 70 patients with ASCC. HPV status was assessed by aligning tumor sequencing reads to HPV viral genomes. GAs were identified using an established algorithm and correlated with HPV status., Results: Sixty-one samples (87%) were HPV-positive. A mean of 3.5 GAs per sample was identified. Recurrent alterations in phosphoinositol-3-kinase pathway (PI3K/AKT/mTOR) genes including amplifications and homozygous deletions were present in 63% of cases. Clinically relevant GAs in genes involved in DNA repair, chromatin remodeling, or receptor tyrosine kinase signaling were observed in 30% of cases. Loss-of-function mutations in TP53 and CDKN2A were significantly enhanced in HPV-negative cases (P < 0.0001)., Conclusions: This is the first comprehensive genomic analysis of ASCC, and the results suggest new therapeutic approaches. Differing genomic profiles between HPV-associated and HPV-negative ASCC warrants further investigation and may require novel therapeutic and preventive strategies., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Competing Interests: The authors state no conflict of interest.

Richter's syndrome (RS) is the development of an aggressive lymphoma in patients with a previous or concomitant diagnosis of chronic lymphocytic leukemia (CLL). The incidence rate for RS is ∼0.5% per year of observation. In the presence of clinical suspicious of RS, diagnosis of transformation and choice of the site of biopsy may take advantage of 18 FDG PET/CT. Molecular lesions of tumor suppression regulators (TP53), cell cycle (CDKN2A) and cell proliferation (NOTCH1, MYC) overall account for ∼90% of RS and may be responsible for its aggressive clinical phenotype. The prognosis of RS is generally highly unfavorable. However, the pattern of survival is not homogeneous and the clonal relationship between the CLL and the aggressive lymphoma clones is the most important prognostic factor. Rituximab-containing polychemotherapy represents the back-bone for induction treatment in RS. Younger patients who respond to induction therapy should be offered stem cell transplant to prolong survival., (Copyright © 2016. Published by Elsevier Ltd.)

Increase of fat cells (FCs) in adipose tissue is attributed to proliferation of preadipocytes or immature adipocytes in the early stage, as well as adipogenic differentiation in the later stage of adipose development. Although both events are involved in the FC increase, they are contrary to each other, because the former requires cell cycle activity, whereas the latter requires cell cycle withdrawal. Therefore, appropriate regulation of cell cycle inhibition is critical to adipogenesis. In order to explore the important cell cycle inhibitors and study their expression in adipogenesis, we adopted a strategy combining the Gene Expression Omnibus (GEO) database available on the NCBI website and the results of quantitative real-time PCR (qPCR) data in porcine adipose tissue. Three cell cycle inhibitors - cyclin G2 (CCNG2), cyclin-dependent kinase inhibitor 2C (CDKN2C) and peripheral myelin protein (PMP22) - were selected for study because they are relatively highly expressed in adipose tissue compared with muscle, heart, lung, liver and kidney in humans and mice based on two GEO DataSets (GDS596 and GDS3142). In the latter analysis, they were found to be more highly expressed in differentiating/ed preadipocytes than in undifferentiated preadipocytes in human and mice as shown respectively by GDS2366 and GDS2743. In addition, GDS2659 also suggested increasing expression of the three cell cycle inhibitors during differentiation of 3T3-L1 cells. Further study with qPCR in Landrace pigs did not confirm the high expression of these genes in adipose tissue compared with other tissues in market-age pigs, but confirmed higher expression of these genes in FCs than in the stromal vascular fraction, as well as increasing expression of these genes during in vitro adipogenic differentiation and in vivo development of adipose tissue. Moreover, the relatively high expression of CCNG2 in adipose tissue of market-age pigs and increasing expression during development of adipose tissue was also confirmed at the protein level by western blot analysis. Based on the analysis of the GEO DataSets and results of qPCR and Western blotting we conclude that all three cell cycle inhibitors may inhibit adipocyte proliferation, but promote adipocyte differentiation and hold a differentiated state by inducing and maintaining cell cycle inhibition. Therefore, their expression in adipose tissue is positively correlated with age and mature FC number. By regulating the expression of these genes, we may be able to control FC number, and, thus, reduce excessive fat tissue in animals and humans.

Hematopoietic stem cells (HSCs) are protected in a metabolically dormant state within the bone marrow stem cell niche. Inflammation has been shown to disrupt HSC dormancy and cause multiple functional changes. Here, we investigated whether HSC functions were altered in systemic lupus erythematosus (SLE)-prone mice and whether this contributed to clinical manifestations of SLE. We found that HSCs were significantly expanded in lupus mice. The increase in HSC cellularity was caused by both genetic lupus risk factors and inflammatory cytokines in lupus mice. In addition, the inflammatory conditions of lupus led to HSC mobilization and lineage-biased hematopoiesis. Strikingly, these functionally altered HSCs possessed robust self-renewal capacity and exhibited repopulating advantages over wild-type HSCs. A single-nucleotide polymorphism in the cdkn2c gene encoding p18(INK4c) within a SLE susceptibility locus was found to account for reduced p18(INK4c) expression and the increase in HSC self-renewal capacity in lupus mice. Lupus HSCs with enhanced self-renewal capacity and resistance to stress may compete out transplanted healthy HSCs, thereby leading to relapses after HSC transplantation.

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) associated with poor prognosis. Because the drug-resistance of leukemia cells is partly dependent on cell quiescence in the bone marrow niche, EVI1 may be involved in cell cycle regulation in leukemia cells. As a candidate regulator of the cell cycle in leukemia cells with high EVI1 expression (EVI1(high)), we analyzed angiopoietin1 (Ang1), which is a down-regulated gene in EVI1-deficient mice and is involved in the quiescence of hematopoietic stem cells. The results of real-time PCR analyses showed that Ang1 is highly expressed in leukemia cell lines and primary AML cells with EVI1(high) expression. Introduction of shRNA against EVI1 into EVI1(high) leukemia cells down-regulated Ang1 expression. Moreover, knockdown of Ang1 in EVI1(high) leukemia cells promoted cell cycle progression and down-regulated the CDK inhibitor p18 (INK4c). Treatment with a decoy Tie2/Fc protein also down-regulated the expression of p18. These results suggest that Ang1/Tie2 signaling may suppress cell cycle progression via maintenance of G0/G1 phase through up-regulation of p18 expression. This mechanism may help to maintain EVI1(high) leukemia cells in the bone marrow niche and promote resistance to anti-cancer drugs., (Copyright © 2011. Published by Elsevier Inc.)

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27(Kip1)/CDKN1B (p27) and p18(Ink4C)/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Inhibition of Cdk4/Cdk6 by p18(INK4c) (p18) is pivotal for generation of noncycling immunoglobulin (Ig)-secreting plasma cells (PCs). In the absence of p18, CD138(+) plasmacytoid cells continue to cycle and turnover rapidly, suggesting that p18 controls PC homeostasis. We now show that p18 selectively acts in a rare population of rapidly cycling CD138(hi)/B220(hi) intermediate PCs (iPCs). While retaining certain B-cell signatures, iPCs are poised to differentiate to end-stage PCs although the majority undergo apoptosis. p18 is dispensable for the development of the PC transcriptional circuitry, and Blimp-1 and Bcl-6 are expressed fully and mutually exclusively in individual iPCs. However, a minor proportion of iPCs express both, and they are preferentially protected by p18 or Bcl-xL overexpression, consistent with expansion of the iPC pool by Bcl-xL overexpression, or loss of proapoptotic Bim or Noxa. Expression of Noxa is induced during B-cell activation, peaks in iPCs, and selectively repressed by p18. It is required to promote apoptosis of cycling B cells, especially in the absence of p18. These findings define the first physiologic function for Noxa and suggest that by repressing Noxa, induction of G₁ arrest by p18 bypasses a homeostatic cell-cycle checkpoint in iPCs for PC differentiation.

Cyclin-dependent kinase inhibitors (CDKIs) are known targets to become deregulated in various tumour types, including endocrine tumours. Typically, these cell cycle regulators are somatically inactivated in sporadic endocrine tumours. Recently, it became known that certain CDKI genes cause inherited susceptibility to endocrine neoplasia. Multiple endocrine neoplasia type 4 (MEN4) emerged as a novel form of multiple endocrine neoplasia, caused by mutations in the CDKI gene CDKN1B/p27(Kip1). The MEN4 phenotype remains unclear, but all MEN4 patients identified thus far present with parathyroid involvement, and less typically with pituitary adenomas and other endocrine features. Moreover, the CDKI gene CDKN2C/p18(INK4C) has been also implicated in endocrine neoplasia susceptibility. This review presents the recent advances in these novel MEN-related states and summarises the current knowledge of how these CDKIs may be implicated in endocrine neoplasia. In addition, it briefly presents data from Cdkn1b/p27(Kip1) and Cdkn2c/p18(INK4C) murine models, which strongly support the protective role of these inhibitors against endocrine tumourigenesis., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Pancreatic endocrine tumors (PETs) arise sporadically or are associated with multiple endocrine neoplasia type 1 (MEN1) syndrome or von Hippel-Lindau syndrome. About 90% of patients with familial MEN1 display detectable MEN1 gene (menin) mutations. The cyclin-dependent kinase inhibitor p27 (CDKN1B) is a downstream target of menin and has been recently shown to be responsible for the multiple endocrine neoplasia-like syndrome in rats, where affected animals develop multiple tumors and hyperplasia in endocrine tissues, including the pancreatic islets of Langerhans. A germline nonsense truncation mutation of p27 has been recently described in a suspected MEN1 family without MEN1 mutation, raising the possibility that p27 mutation could be responsible for MEN1 phenotype. Somatic MEN1 mutations occur at low frequency in sporadic PETs; here, we subjected p27 to mutational analysis in 27 sporadic PETs. As an additional menin target, analysis of the p18 (CDKN2C) gene was included. In the p27 gene, one common polymorphism (V109G) and one novel polymorphism (g/a) in the noncoding part of exon 2 were identified. Three known polymorphisms were found in the p18 gene. These data suggest that p27 and p18 are unlikely to present classic tumor-suppressor genes in sporadic PETs.

Erythroid Kruppel-like factor (EKLF, KLF1) is an essential erythroid cell specific C(2)H(2) zinc finger transcription factor that binds CACC box elements in promoters and distant regulatory elements to activate transcription. Forced expression of EKLF arrests cell division. The cyclin dependent kinase (Cdk) inhibitor p18(INK4c) was identified as a potential novel EKLF target gene from an expression profiling study. The p18(INK4c) protein functions as an inhibitor of Cdk4 and Cdk6 activity during early G1 phase of the cell cycle, thus acting as a physiological brake on cell division. We confirmed p18(INK4c) is downregulated in EKLF null mice by real-time PCR and Western blotting, and identified three closely associated and highly conserved EKLF binding sites (CCNCNCCCN) approximately 1 kb upstream of the p18(INK4c) transcriptional start site. We showed that EKLF binds to one of these elements by gel shift assay and demonstrated this site is capable of driving EKLF dependent transcription. We also determined by chromatin immunoprecipitation (ChIP) that this region of the p18(INK4c) promoter is bound by EKLF in erythroid cells. Thus, EKLF is a direct regulator of p18(INK4c) gene expression, and much of EKLF's role in driving erythroid cell differentiation may occur via p18(INK4c).

Integrative genomic and gene-expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene-expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated 20 homozygous deletions at 7 chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms. Notably, the pattern of genetic and epigenetic inactivation differed among B-NHL subtypes. Thus, the P53-inducible PIG7/LITAF was silenced by homozygous deletion in primary mediastinal B-cell lymphoma and by promoter hypermethylation in germinal center lymphoma, the proapoptotic BIM gene presented homozygous deletion in mantle cell lymphoma and promoter hypermethylation in Burkitt lymphoma, the proapoptotic BH3-only NOXA was mutated and preferentially silenced in diffuse large B-cell lymphoma, and INK4c/P18 was silenced by biallelic mutation in mantle-cell lymphoma. Our microarray strategy has identified novel candidate tumor suppressor genes inactivated by genetic and epigenetic mechanisms that substantially vary among the B-NHL subtypes.

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.