Genome-wide Identification and Analysis of DNA Methyltransferases in Grape

DNA methylation is an important epigenetic regulation mechanism, which is catalyzed by DNA methyltransferases. In this study, eight DNA methyltransferase genes were identified in grape genome to analyze the selective pressure, gene expression and codon usage bias. The results showed grape DNA methyltransferase MET subfamily underwent relatively strong purifying selection during evolution, while chromomethylase CMT subfamily underwent positive selection during evolution. Under different abiotic(heat, drought or cold) stresses, the expression level of many grape DNA methyltransferase genes changed significantly. The expression level of these genes might be related with cis-regulatory elements of their promoters. The results of codon usage bias analysis showed that synonymous codon bias existed in grape DNA methyltransferase gene family, which might be affected by mutation pressure. These results laid a solid foundation for in-depth study of DNA methyltransferases in grape.

Regulation of neuronal survival by DNA methyltransferases

The limited regenerative capacity of neuronal cells requires tight orchestration of cell death and survival regulation in the context of longevity, age-associated diseases as well as during the development of the nervous system. Subordinate to genetic networks epigenetic mechanisms like DNA methylation and histone modifications are involved in the regulation of neuronal development, function and aging. DNA methylation by DNA methyltransferases （DNMTs）, mostly correlated with gene silencing, is a dynamic and reversible process. In addition to their canonical actions performing cytosine methylation, DNMTs influence gene expression by interactions with histone modifying enzymes or complexes increasing the complexity of epigenetic transcriptional networks. DNMTs are expressed in neuronal progenitors, post-mi- totic as well as adult neurons. In this review, we discuss the role and mode of actions of DNMTs including downstream networks in the regulation of neuronal survival in the developing and aging nervous system and its relevance for associated disorders.

DNA methyltransferases in hematological malignancies

DNA methyltransferases(DNMTs)are an evolutionarily conserved family of DNA methylases,transferring a methyl group onto the fifth carbon of a cytosine residue.The mammalian DNMT family includes three major members that have functional methylation activities,termed DNMT1,DNMT3A,and DNMT3B.DNMT3A and DNMT3B are responsible for methylation establishment,whereas DNMT1 maintains methylation during DNA replication.Accumulating evidence demonstrates that regulation of DNAmethylation by DNMTs is critical for normal hematopoiesis.Aberrant DNA methylation due to DNMT dysregulation and mutations is known as an important molecular event of hematological malignancies,such as DNMT3A mutations in acute myeloid leukemia.In this reviewwe first describe the basic methylation mechanisms of DNMTs and their functions in lymphocyte maturation and differentiation,We then discuss the current understanding of DNA methylation heterogeneity in leukemia and lymphoma to highlight the importance of studying DNA methylation targets.We also discuss DNMT mu-tations and pathogenic roles in human leukemia and lymphoma.We summarize the recent understanding of how DNMTs interact with transcription factors or cofactors to repress the expression of tumor suppressor genes.Firnally.we highlight current clinical studies using DNMT inhibitors for the treatment of these hematological malignancies.

Single nucleotide polymorphism in DNA methyltransferase 3B promoter and its association with gastric cardiac adenocarcinoma in North China

AIM:To investigate the association between single nucleotide polymorphism (SNP) in promoter of the DNA methyltrans-ferase 3B(DNMT3B) gene and risk for development and lymphatic metastasis of gastric cardiac adenocarcinoma (GCA). METHODS: The hospital based case-control study included 212 GCA patients and 294 control subjects without overt cancer. The DNMT3B SNP was genotyped by PCR and restriction fragment length polymorphism (RFLP) analysis. RESULTS: The C/C genotype was not detected in both GCA patients and controls. In control subjects, the frequency of T/T and C/T genotypes was 94.9% and 5.1% respectively, and that of T and C alleles was 97.4% and 2.6%, respectively. The genotype and allelotype distribution in the GCA patients was not significantly different from that in controls (P=0.34 and 0.33, respectively). When stratified by smoking status and family history of upper gastrointestinal cancer, significant difference in the genotype distribution was not observed between GCA patients and controls. The distribution of DNMT3B genotypes in GCA patents with or without lymphatic metastasis did not show significant difference (P= 0.42). CONCLUSION: The distribution of DNMT3B SNP in North China is distinct from that in Caucasians. Although this SNP has been associated with susceptibility to lung, head, neck and breast cancer, it may not be used as a stratification marker to predict susceptibility and lymphatic metastasis of GCA, at least in the population of North China.

Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.

Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

AIM： To explore the relationship between DNA methyltransferase 1 （DNMT1） and hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） and its biological significance in primary HCC. METHODS： We carried out an immunohistochemical examination of DNMT1 in both HCC and paired nonneoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC celt line SMMC-7721. RESULTS： DNMT1 protein expression was increased in HCCs compared to histologically normal nonneoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation （P = 0.014）. There were more cases with DNMT1 overexpression in HCC with HBV （42.85%） than in HCC without HBV （28.57%）. However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen （PCNA）, even in HCC cell lines where DNMT1 was stably decreased. CONCLUSION： The findings implied that DNMT1 plays a key role in HBV-retated hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.

DNA methyltransferase3a expression is an independent poor prognostic indicator in gastric cancer

AIM: To explore the alteration of DNA methyltransferase expression in gastric cancer and to assess its prognostic value.

mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees(Apis mellifera)

In honeybee （Apis mellifera） colonies, queens and workers are altemative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR （target of rapamycin） and Dnmt3 （DNA methyltransferase 3） genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K （similar to RPS6-p70-protein kinase） gene linked closely to organismal growth and development in queen and worker larvae （1-day and 3-day old）. Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.

DNA methyltransferase 3B promoter polymorphism and its susceptibility to primary hepatocellular carcinoma in the Chinese Han nationality population:A case-control study

AIM： To investigate the correlation between C/T single nucleotide polymorphism （SNP） in the promoter of the DNA methyltransferase 3B （D/VMT3B） gene and risk for development and progression of primary hepatocellular carcinoma （HCC）. METHODS： One hundred case subjects were selected consecutively from Tongji Hospital （Wuhan, China）. from March to November 2006. They did not receive radiotherapy or chemotherapy for newly diagnosed and histopathologically confirmed HCC. One hundred and forty control subjects having no history of cancerous or genetic diseases were healthy volunteers to Wuhan Blood Center in the same period. Frequency was matched for sex, age, alcohol consumption and cigarette smoking status of the case subjects. C/T polymorphism of the DNMT3B promoter was analyzed using polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） and sequencing analysis. The association between genotypes of DNMT3B and clinicopathological parameters among cases was also studied. RESULTS： The CC genotype HCC patients and controls. was not detected in both In control subjects, the frequency of TT and CT genotypes was 99.3% and 0.7% respectively, and that of T and C alleles was 99.6% and 0.4% respectively. The frequency of CT genotype was higher in HCC （3.0%）. The frequency of T and C alleles was 98.5% and 1.5% respectively. However, the genotype and allelotype distribution in HCC patients was not significantly different from that in controls. CONCLUSION： C/T polymorphism is not associated with the increased risk of HCC. DNMT3B genetic polymorphism is variable in different races, ethnic groups or geographic areas. Further study is needed to clarify the role of DNMT3B SNP in the development of HCCamong other populations.

Aberrant DNA methyltransferase 1 expression in clear cell renal cell carcinoma development and progression

Objective： To better understand the contribution of dysregulated DNA methyltransferase 1 （DNMT1） expression to the progression and biology of clear cell renal cell carcinoma （ccRCC）. Methods： We examined the differences in the expression of DNMT1 in 89 ecRCC and 22 normal tissue samples by immunohistochemistry. In addition, changes in cell viability, apoptosis, colony formation and invading ability of ccRCC cell lines （786-0 and Caki-1） were assessed after transfection with DNMT1 siRNA. Results： We found DNMT1 protein was significantly higher expressed in ccRCC than that of in no-tumor tissues （56.2% and 27.3%, respectively, P=0.018）. The expression of DNMT1 was strongly associated with ccRCC tumor size, tumor pathology stage, histological grading, lymph node metastasis, vascular invasion, recurrence and prognosis. Moreover, knockdown of DNMT1 expression significantly inhibited ccRCC cell viability, induced apoptosis, decreased colony formation and invading ability. Conclusions： Expression of DNMTI protein is increased in ccRCC tissues, and DNMT1 expression is associated with poor prognosis of patients. Experiments in vitro further showed DNMT1 played an essential role in proliferation and invasion of renal cancer cells. Moreover, targeting this enzyme could be a promising strategy for treating ccRCC, as evidenced by inhibited cell viability, increased apoptosis, decreased colony formation and invading ability.

Influence of DNA methyltransferase 3b on FHIT expression and DNA methylation of the FHIT promoter region in hepatoma SMMC-7721 cells

BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression and on DNA methylation of the FHIT promoter region in the hepatoma cell line SMMC-7721. METHODS: DNMT3b siRNA was used to down-regulate DNMT3b expression. DNMT3b and FHIT proteins were determined by Western blotting. Methylation-specific PCR was used to analyze the methylation status of the FHIT gene. RESULTS: After DNMT3b siRNA transfection, the expression of DNMT3b was inhibited in SMMC-7721 cells, and the expression of FHIT was significantly higher than that in the control group. There was no significant difference in methylation status between the DNMT3b siRNA transfected cells and control cells. CONCLUSION: DNMT3b may play an important role in regulation of FHIT expression in hepatoma SMMC-7721 cells, but not through methylation of the FHIT promoter. (Hepatobiliary Pancreat Dis Int 2009; 8: 273-277)

cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.

Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

Amplification and function analysis of N6-adenine-specific DNA methyltransferase gene in Nilaparvata lugens

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase（N6AMT） is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens（Nlu-N6AMT） and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenine Mlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding ds RNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

Cryopreservation by Vitrification of Vitis vinifera cv. ＂Red Globe＂ Zygotic Embryos and Effect on the Expression of DNA Methyltransferase Genes

Mexico is a large producer of table grape （Vitis vinifera L.） and therefore it is important to develop protocols to store the grape varieties germplasm. The objective of the present work was to design a protocol for the cryopreservation by vitrification of zygotic embryos of V. vinifera cv. ＂Red Globe＂ and evaluate possible epigenetics changes. The plant vitrification solution 2 （PVS2） was utilized before the utilization of liquid nitrogen （LN）. The effect of this protocol on embryo viability was tested by the triphenyl-tetrazolium chloride solution, as well as by the in vitro development of grape embryos into plantlet. A cDNA expression library of grape zygotic embryos was created to isolate expressed sequence tags of several DNA methyltrasferases. Gene expression of domains rearranged methyltransferase type 1 （DMR1） and DNA （cytosine-5）-methyltransferase 1 （MET1-2） isozymes was analyzed by quantitative reverse transcriptase PCR. The optimal conditions for vitrification were 10 min in 50% PVS2, followed by I0 min in 100% PVS2. Under these conditions, about 30% of plantlet was obtained from embryos after cryopreservation. It was recorded a reduction in the MET1-2 gene expression, which plays a role in the maintenance of DNA methylation. It is possible to cryopreserve viable grape zygotic embryos, although the treatment seems to induce alterations in the normal DNA methylation pattern of the zygotic embryo genome.

Synergy of DNA methylation and histone deacetylase inhibitors in the re-expression of RASSF1A and P16 genes silenced in QBC cells

Objective: To investigate the effects of DNA methylation and histone deacetylase inhibitors in the re-expression of P16 and RASSIF1A of QBC939. Methods: The QBC939 cells were treated with hydralazine and valproate either alone or combined, and the control group was added with RPIM-1640 culture medium. After 48 h, the expression of P16 and RASSF1A genes were evaluated by reverse transcription-PCR, Western blot, and the methylation status of the two genes were detected with MSP (methylation specific PCR). Results: Hydralazine and valproate could induce demethylation of the promoter region of the two genes, and could make them re-active. The expressions of P16 and RASSF1A of cells treated with both drugs were higher than that of the cells treated with either hydralazine or valproate (P < 0.01). There was no RASSF1A gene, and few P16 gene expressing in the control group. The demethylation effect could be found in the groups treated with hydralazine or both drugs, whereas no demethylation effect happened in the valproate group. Conclusion: The two drugs could synergistically re-express P16 and RASSF1A genes silenced in QBC939, and they exerted a great anti-tumour effect on QBC cells.

A High-Affinity Methyl-CpG-Binding Protein for Endonuclease-Free and Label-Free DNA Methyltransferase Activity Detection

DNA methyltransferase(MTase)activity detection has received increasing attention as a promising biomarker and therapeutic target.However,most of these detection methods rely on endonuclease digestion and signal groups labeling.Herein,we present a novel platform for sensing DNA MTase activity that overcomes these limitations.Our approach is both endonuclease-free and label-free,utilizing a combination of a high-affinity streptavidin-methyl-CpG-binding domain(SA-MBD)protein and surface plasmon resonance(SPR)technology.The SA-MBD protein specifically recognizes a hairpin probe containing methylated CpG sites,which is treated with M.SssI MTase.This recognition event generates a corresponding SPR response signal.The limit of detection is as low as 0.016 U/mL,owing to the high-affinity of the SA-MBD protein.Notably,we have demonstrated the feasibility of our method for M.SssI MTase activity analysis in serum and inhibitor screening,which implies the potential prospects for biomedical research.

Biological Evaluation of a Novel 2’-Fluoro Derivative 5-Azacytidine as a Potent DNA Methyltransferase Inhibitor

Background: DNA methyltransferases (DNMTs) are key epigenetic regulatory enzymes involved in the expression of many genes and are considered as an attractive target for cancer treatment, especially hematological malignancies. Therefore, promising DNMT inhibitors characterized by low toxicity, target activity and high selectivity are crucial for the development of new cancer therapy and research on the inhibitory mechanism. We had previously demonstrated that the novel 2’-fluoro-2’-deoxy-arabinofuranosyl 5-azacytosine nucleoside (2’F-araAC) showed high antiproliferative activity in vitro and increased hydrolytic stability compared to the known agents like azacitidine and decitabine. Objective: The objective of the present study was to investigate the effect of novel 2’F-araAC as potent anti-leukemia agent and DNMTs inhibitor on nuclear extract of the HCT-116 human colorectal cell line and P388 and L1210 mouse leukemia cell lines. Methods: The DNMTs activity was evaluated using the fluorometric DNMT Activity Quantification Kit (Abcam) and were reported as the percentage of control. Nuclear proteins were extracted from HCT-116 cell line using the Nuclear Extraction Kit (Abcam). To explore the mechanism of anti-leukemic activity of 2’F-araAC, cell cycle and apoptosis analyses were performed on P388 and L1210 cell lines. Results: It has been shown that the DNMTs activity was significantly reduced at 1 and 10 µM of 2’F-araAC compared to controls. Moreover, 2’F-araAC can induce G2/M cell cycle arrest and apoptosis in P388 and L1210 mouse leukemia cell lines as shown by flow cytometry method. Apoptosis was 54.53% and 43.35% for 2’F-araAC vs. 2.88% and 5.25% for the control P388 and L1210 cell lines, respectively. Conclusions: Thus, our study presents a new and promising compound to further develop new epigenetic regulators to be used as antitumor agents.

Reduced non-CpG methylation is a potential epigenetic target after spinal cord injury

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury.To investigate the role of DNA methylation in spinal cord injury,we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points(day 0-42)after spinal cord injury in mice.Global DNA methylation levels,specifically non-CpG(CHG and CHH)methylation levels,decreased modestly following spinal cord injury.Stages post-spinal cord injury were classified as early(day 0-3),intermediate(day7-14),and late(day 28-42)based on similarity and hie rarchical cluste ring of global DNA methylation patterns.The non-CpG methylation level,which included CHG and CHH methylation levels,was markedly reduced despite accounting for a minor proportion of total methylation abundance.At multiple genomic sites,including the 5’untranslated regions,promoter,exon,intron,and 3’untranslated regions,the non-CpG methylation level was markedly decreased following spinal cord injury,whereas the CpG methylation level remained unchanged at these locations.Approximately one-half of the differentially methylated regions were located in intergenic areas;the other differentially methylated regions in both CpG and non-CpG regions were cluste red in intron regions,where the DNA methylation level was highest.The function of genes associated with differentially methylated regions in promoter regions was also investigated.From Gene Ontology analysis results,DNA methylation was implicated in a number of essential functional responses to spinal cord injury,including neuronal synaptic connection creation and axon regeneration.Notably,neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells.In summary,our work elucidated the dynamic pattern of DNA methylation in the spinal co rd following injury and identified reduced nonCpG methylation as an epigenetic target after spinal cord injury in mice.

Molecular mechanism underlying the functional loss of cyclindependent kinase inhibitors p16 and p27 in hepatocellular carcinoma

Hepatocellular carcinoma （HCC） is one of the most common human cancers, and its incidence is still increasing in many countries. The prognosis of HCC patients remains poor, and identification of useful molecular prognostic markers is required. Many recent studies have shown that functional alterations of cellcycle regulators can be observed in HCC. Among the various types of cell-cycle regulators, p16 and p27 are frequently inactivated in HCC and are considered to be potent tumor suppressors, p16, a G1-specific cell-cycle inhibitor that prevents the association of cyclindependent kinase （CDK） 4 and CDK6 with cyclin DI, is frequently inactivated in HCC via CpG methylation of its promoter region, p16 may be involved in the early steps of hepatocarcinogenesis, since p16 gene methylation has been detected in subsets of pre-neoplastic liver cirrhosis patients, p27, a negative regulator of the G1-S phase transition through inhibition of the kinase activities of Cdk2/cyclin A and Cdk2/cyclin E complexes, is now considered to be an adverse prognostic factor in HCC. In some cases of HCC with increased cell proliferation, p27 is overexpressed but inactivated by sequestration into cyclin D1-CDK4-containing complexes. Since loss of p16 is closely related to functional inactivation of p27 in HCC, investigating both p16 and p27 may be useful for precise prognostic predictions in individuals with HCC.