NBR2 promotes the proliferation of glioma cells via inhibiting p15 expression.

PURPOSE: The purpose of this study was to investigate whether NBR2 can affect the proliferation of glioma cells by inhibiting the expression of p15, so as to promote the occurrence and development of glioma. METHODS: The expression of NBR2 in 44 glioma tissue specimens was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of NBR2 on cell viability, cell colony formation as well as cell migration and invasion abilities were examined by cell counting kit-8 (CCK-8) assay, plate cloning assay and Transwell assay. p15 protein was detected using Western blot. After simultaneous knockdown of NBR2 and p15, qRT-PCR, CCK-8, and plate cloning experiments were adopted to analyze p15 gene level, cell viability and proliferation ability, respectively. RESULTS: NBR2 was highly expressed in glioma tissues, and the level in stage III/IV glioma tissues was conspicuously higher than that in stage I/II. The overall survival rate of glioma patients with high NBR2 level was conspicuously lower than those with low NBR2 expression. Clinical data analysis revealed that NBR2 expression was correlated with the WHO stage of clinical patients. After knockdown of NBR2, it was found that NBR2 level, cell viability, cell proliferation ability as well as migration and invasion abilities were all conspicuously reduced. In addition, the protein level of p15 was significantly increased after NBR2 was inhibited. Meanwhile, knockout of p15 could reverse the inhibitory effect of NBR2 on glioma cell proliferation. CONCLUSIONS: The highly-expressed NBR2 inhibits the expression of p15, thus promoting the proliferation of glioma cells.

MXD/MIZ1 transcription regulatory complexes activate the expression of MYC-repressed genes.

MXDs are transcription repressors that antagonize MYC-mediated gene activation. MYC, when associated with MIZ1, acts also as a repressor of a subset of genes, including p15 and p21. A role for MXDs in regulation of MYC-repressed genes is not known. We report that MXDs activate transcription of p15 and p21 in U2OS cells. This activation required DNA binding by MXDs and their interaction with MIZ1. MXD mutants deficient in MIZ1 binding interacted with the MYC-binding partner MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA-binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of target genes.

CDKN2B gene expression is affected by 9p21.3 rs10757278 in CAD patients, six months after the MI.

BACKGROUND: Chromosomal region 9p21.3 is most robustly associated with coronary artery disease (CAD) in western European populations. However, heterogeneity in CAD phenotypes leads to uncertainty whether 9p21.3 is associated with stable and/or acute clinical presentations of CAD. 9p21.3 is rich in regulatory elements, but the underlying mechanisms of its actions in CAD remain unclear. We investigate the association of 9p21.3 two haplotype blocks lead variants (rs10757278 and rs518394) with first-ever non-fatal myocardial infarction (MI) in CAD patients and their association with CDKN2B mRNA expression in peripheral blood mononuclear cells 6 months after the event. METHODS: We included CAD patients with sustained first MI (n = 523) and controls (n = 583). Gene expression was assessed in 72 patients 6 months after MI and 43 healthy controls. TaqMan® technology was used for the gene expression and genotyping analysis. RESULTS: CDKN2B mRNA was significantly lower in MI patients compared with the controls (p = 0.002) and in patients carrying the rs10757278 G risk allele versus AA homozygotes (p = 0.012) 6 months after the event. While we confirmed the association of rs10757278 with CDKN2B expression in MI patients, we failed to find an association between the investigated variants and MI or disease burden. CONCLUSIONS: We suggest a dysregulation of gene expression in the 9p21.3 region six months after acute MI, which is affected by a genetic variant in patients. The rs10757278 rare allele is one factor that might lead to prolonged risk for proatherogenic complications.

CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells.

Tumorigenicity is a well-documented risk to overcome for pluripotent or multipotent cell applications in regenerative medicine. To address the emerging demand for safe cell sources in tissue regeneration, we established a novel, protein-based reprogramming method that does not require genome integration or oncogene activation to yield multipotent fibromodulin (FMOD)-reprogrammed (FReP) cells from dermal fibroblasts. When compared with induced pluripotent stem cells (iPSCs), FReP cells exhibited a superior capability for bone and skeletal muscle regeneration with markedly less tumorigenic risk. Moreover, we showed that the decreased tumorigenicity of FReP cells was directly related to an upregulation of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression during the FMOD reprogramming process. Indeed, sustained suppression of CDKN2B resulted in tumorigenic, pluripotent FReP cells that formed teratomas in vivo that were indistinguishable from iPSC-derived teratomas. These results highlight the pivotal role of CDKN2B in cell fate determination and tumorigenic regulation and reveal an alternative pluripotent/multipotent cell reprogramming strategy that solely uses FMOD protein.

Linear and circular CDKN2B-AS1 expression is associated with Inflammatory Bowel Disease and participates in intestinal barrier formation.

AIMS: The role of long non-coding RNA's (lncRNA) in the biology of ulcerative colitis (UC) is not well understood. We have previously detected changes in lncRNA's associated with UC. This study aims to characterize one specific lncRNA, CDKN2B-AS1 whose expression was downregulated in UC patients. MAIN METHODS: UC biopsies were used to determine the levels of linear and circular CDKN2B-AS1 relative to healthy controls. In situ hybridization was used to determine the localization of CKDN2B-AS1 in the colon. The intestinal epithelial cell line, Caco-2, was used to study the effects of shRNA mediated loss of CDKN2B-AS1. Transepithelial electrical resistance was used to measure barrier function. An RT-PCR array, immunoblots and immunohistochemistry were used to determine tight junction proteins that CDKN2B-AS1 regulates. KEY FINDINGS: CDKN2B-AS1 is transcribed into not only linear transcripts but also as circular RNA through back-splicing and both forms are decreased in IBD. CDKN2B-AS1 is expressed mainly in colonic epithelial cells. Cells with down-regulated CDKN2B-AS1 exhibited increased proliferation and no alterations in apoptosis. Targeting both the linear and circular transcripts of CDKN2B-AS1 with short hairpin RNAs enhanced barrier function. We subsequently determined that Claudin-2, a "leaky Claudin" known to decrease barrier function, was decreased in CDKN2B-AS1 knockdown cells. SIGNIFICANCE: This study identifies a novel lncRNA with both linear and circular transcripts affecting UC biology.

Expression of p15 in a spectrum of spitzoid melanocytic neoplasms.

BACKGROUND: Accurate classification of spitzoid melanocytic lesions is difficult due to overlapping clinical and histopathologic features between Spitz nevi, atypical Spitz tumors (ASTs), and spitzoid melanomas. Expression of p16 (CDKN2A) has been used as a marker of spitzoid lesions. However, its expression may be variable. p15 is a tumor suppressor encoded by CDKN2B, loss of which has been recently shown to promote transition from nevus to melanoma. We sought to determine whether p15 is a useful immunohistochemical marker to distinguish Spitz nevi from spitzoid melanomas and to compare p15 and p16 staining in this population. METHODS: Immunohistochemistry for p15 and p16 was performed on Spitz nevi (n = 19), ASTs (n = 41), and spitzoid melanomas (n = 17). Immunoexpression was categorized by a four-tiered system: 0 (negative), 1+ (weak), 2+ (moderate), 3+ (strong). RESULTS: 3+/strong p15 staining was observed in 68.4% of Spitz nevi, 34.2% of ASTs, and 17.7% of spitzoid melanomas. By contrast, we observed 3+ p16 staining in roughly equivalent percentages of Spitz nevi (57.9%), ASTs (56.1%), and spitzoid melanomas (58.8%). CONCLUSION: These data illustrate that p15 may be more useful than p16 as a biomarker to help distinguish benign from malignant spitzoid lesions.

Gemcitabine/Cisplatin Treatment Induces Concomitant SERTAD1, CDKN2B and GADD45A Modulation and Cellular Changes in Bladder Cancer Cells Regardless of the Site of TP53 Mutation.

Simultaneous use of cisplatin (CIS) and gemcitabine (GEN) for treating bladder cancer has increased because of their complementary effects. However, the molecular mechanisms underlying the activities of these two antineoplastic drugs are not fully known. Here, molecular biology techniques and microscopy were used to investigate transcriptomic and morphological changes in low and high-grade urinary bladder transitional carcinoma cell lines [RT4 - wild type TP53; 5637 - two TP53 mutations, one in codon 72 (Arg-Pro) and other in codon 280 (Arg-Thr) and T24 - in-frame deletion of tyrosine 126 in the TP53 allele] simultaneously treated with CIS/GEN. Gene expression profile was evaluated by PCR arrays; cell morphology by scanning and transmission electron microscopy, and apoptosis was analyzed using fluorescent dye. Results showed concomitantly upregulation of CDKN2B (G1/S transition), GADD45A (DNA repair and apoptosis) and SERTAD1 (regulation of transcription) gene, increased number of nuclear chamfers and apoptotic cells, and reduced number of microfilaments, organelles and in the size of the nucleus in 5637 and T24 cells after simultaneous treatment with CIS/GEN. In conclusion, independently of the TP53 mutation status and tumor grade, CIS/GEN induced gene modulation accompanied by changes in cell morphologies, which confirm the antiproliferative activity of the treatment protocol. These findings help to understand the pathways modulated by these antineoplastic agents and may provide insights for anti-cancer chemotherapy.

Upregulation of MicroRNA-15a Contributes to Pathogenesis of Abdominal Aortic Aneurysm (AAA) by Modulating the Expression of Cyclin-Dependent Kinase Inhibitor 2B (CDKN2B).

BACKGROUND The objective of the present study was to identify the association between miR-15a-5p and CDKN2B, and their roles in regulating the development of abdominal aortic aneurysm (AAA). MATERIAL AND METHODS We searched the miRNA database online (www.mirdb.org) and used a luciferase reporter assay system to study the regulatory relationship between miR-15a-5p and CDKN2B. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CDKN2B among different patient groups (participants with abdominal aortic aneurysm (AAA) and normal controls) or cells treated with scramble control, miR-15a-5p mimics, CDKN2B siRNA, and miR-15a-5p inhibitors. RESULTS We found that CDKN2B was a virtual target of miR-15a-5p with potential binding sites in the 3'UTR of CDKN2B (77-83 bp). We also showed that miR-15a-5p could bind to the CDKN2B 3'UTR, resulting in a significant decrease in luciferase activity compared with the scramble control. Furthermore, we found that the cells isolated from AAA participants showed an over-expression of miR-15a-5p compared to the normal controls, while the CDKN2B mRNA and protein expression level of the AAA group were much lower than the normal control group. Additionally, the expression of CDKN2B mRNA and the protein of the cells transfected with miR-15a-5p mimics and CDKN2B siRNA was downregulated, while the cells showed upregulated expression subsequent to transfection with miR-15a-5p inhibitors compared to the scramble control. CONCLUSIONS The data revealed a negative regulatory role of miR-15a-5p in the apoptosis of smooth muscle cells via targeting CDKN2B, and showed that miR-15a-5p could be a novel therapeutic target of AAA.

Long non-coding RNA IRAIN suppresses apoptosis and promotes proliferation by binding to LSD1 and EZH2 in pancreatic cancer.

Long non-coding RNA (lncRNA) modulates gene expression, while lncRNA dysregulation is associated with human cancer. Furthermore, while recent studies have shown that lncRNA IRAIN plays an important role in other malignancies, the role of IRAIN in pancreatic cancer (PC) progression remains unclear. In this study, we found that upregulation of lncRNA IRAIN was significantly correlated with tumor size, TNM stage, and lymph node metastasis in a cohort of 37 PC patients. In vitro experiments showed that knockdown of IRAIN by small interfering RNA (siRNA) significantly induced cell apoptosis and inhibited cell proliferation in both BxPC-3 and PANC-1 cells. Further mechanism study showed that, by binding to histone demethylase lysine-specific demethylase 1 (LSD1), an enhancer of zeste homolog 2 (EZH2), IRAIN reduced PC tumor cell apoptosis and induced growth arrest by silencing the expression of Kruppel-like factor 2 (KLF2) and P15. Moreover, IRAIN expression was inversely correlated with that of KLF2 and P15 in PC tissues. To our knowledge, this is the first report elucidating the role and mechanism of IRAIN in PC progression.

Inhibition of miR-154 Protects Against Cardiac Dysfunction and Fibrosis in a Mouse Model of Pressure Overload.

Expression of miR-154 is upregulated in the diseased heart and was previously shown to be upregulated in the lungs of patients with pulmonary fibrosis. However, the role of miR-154 in a model of sustained pressure overload-induced cardiac hypertrophy and fibrosis had not been assessed. To examine the role of miR-154 in the diseased heart, adult male mice were subjected to transverse aortic constriction for four weeks, and echocardiography was performed to confirm left ventricular hypertrophy and cardiac dysfunction. Mice were then subcutaneously administered a locked nucleic acid antimiR-154 or control over three consecutive days (25 mg/kg/day) and cardiac function was assessed 8 weeks later. Here, we demonstrate that therapeutic inhibition of miR-154 in mice with pathological hypertrophy was able to protect against cardiac dysfunction and attenuate adverse cardiac remodelling. The improved cardiac phenotype was associated with attenuation of heart and cardiomyocyte size, less cardiac fibrosis, lower expression of atrial and B-type natriuretic peptide genes, attenuation of profibrotic markers, and increased expression of p15 (a miR-154 target and cell cycle inhibitor). In summary, this study suggests that miR-154 may represent a novel target for the treatment of cardiac pathologies associated with cardiac fibrosis, hypertrophy and dysfunction.

Rapamycin restores p14, p15 and p57 expression and inhibits the mTOR/p70S6K pathway in acute lymphoblastic leukemia cells.

The aim of the present study was to investigate the effects of rapamycin and its underlying mechanisms on acute lymphoblastic leukemia (ALL) cells. We found that the p14, p15, and p57 genes were not expressed in ALL cell lines (Molt-4 and Nalm-6) and adult ALL patients, whereas mTOR, 4E-BP1, and p70S6K were highly expressed. In Molt-4 and Nalm-6 cells exposed to rapamycin, cell viability decreased and the cell cycle was arrested at the G1/S phase. Rapamycin restored p14, p15, and p57 gene expression through demethylation of the promoters of these genes. As expected, rapamycin also increased p14 and p15 protein expression in both Molt-4 and Nalm-6 cells, as well as p57 protein expression in Nalm-6 cells. Rapamycin additionally decreased mTOR and p70S6K mRNA levels, as well as p70S6K and p-p70S6K protein levels. However, depletion of mTOR by siRNA did not alter the expression and promoter methylation states of p14, p15, and p57. These results indicate that the inhibitory effect of rapamycin may be due mainly to increased p14, p15, and p57 expression via promoter demethylation and decreased mTOR and p70S6K expression in ALL cell lines. These results suggest a potential role for rapamycin in the treatment of adult ALL.

Curcumin-Mediated Reversal of p15 Gene Promoter Methylation: Implication in Anti-Neoplastic Action against Acute Lymphoid Leukaemia Cell Line.

Curcumin has been documented to exert anticancer effects by interacting with altered proliferative and apoptotic pathways in cancer models. In this study, we evaluated the potential of curcumin to reverse promoter methylation of the p15 gene in Raji cells and its ability to induce apoptosis and genomic instability. Anti-neoplastic action of curcumin showed an augmentation in reactive oxygen species (ROS) and cell cycle arrest in G1 phase. Subsequently, curcumin- exposed Raji cells showed structural abnormalities in chromosomes. These observations suggest that curcumin also causes ROS-mediated apoptosis and genomic instability. The treatment of Raji cell line with 10 µM curcumin caused hypomethylation of the p15 promoter after six days. Hypomethylation of p15 was further found to be favoured by downregulation of DNA methyltransferase 1 after 10 µM curcumin treatment for six days. Methylation-specific PCR suggested demethylation of the p15 promoter. Demethylation was further validated by DNA sequencing. Reverse-transcription PCR demonstrated that treatment with curcumin (10 µM) for six days led to the up-regulation of p15 and down-regulation of DNA methyltransferase 1. Furthermore, curcumin- mediated reversal of p15 promoter methylation might be potentiated by down-regulation of DNA methyltransferase 1 expression, which was supported by cell cycle analysis. Furthermore, curcumin acts as a double-pronged agent, as it caused apoptosis and promoter hypomethylation in Raji cells.

Opposing growth regulatory roles of protein kinase D isoforms in human keratinocytes.

PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15(INK4B) and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis.

INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform.

The naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15(INK4b), p16(INK4a), and ARF (alternate reading frame). Although p15(INK4b) has its own ORF, p16(INK4a) and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15(INK4b) exon 1 joined to p16(INK4a) exons 2 and 3. We have named this isoform pALT(INK4a/b) (for alternative splicing). We show that pALT(INK4a/b) is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALT(INK4a/b) expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALT(INK4a/b) has stronger ability to induce cell-cycle arrest than either p15(INK4b) or p16(INK4a). We hypothesize that the presence of the fourth product, pALT(INK4a/b) of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species.

Role of heme oxygenase-1 in demethylating effects on SKM-1 cells induced by decitabine.

We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.

A phase I and pharmacodynamic study of the histone deacetylase inhibitor belinostat plus azacitidine in advanced myeloid neoplasia.

Background We hypothesized that targeting two mechanisms of epigenetic silencing would be additive or synergistic with regard to expression of specific target genes. The primary objective of the study was to establish the maximum tolerated dose (MTD) of belinostat in combination with a fixed dose of azacitidine (AZA). Methods In Part A of the study, patients received a fixed dose of AZA, with escalating doses of belinostat given on the same days 1-5, in a 28 day cycle. Part B was designed to evaluate the relative contribution of belinostat to the combination based on analysis of pharmacodynamic markers, and incorporated a design in which patients were randomized during cycle 1 to AZA alone, or the combination, at the maximally tolerated dose of belinostat. Results 56 patients with myeloid neoplasia were enrolled. Dose escalation was feasible in part A, up to 1000 mg/m(2) dose level of belinostat. In Part B, 18 patients were assessable for quantitative analysis of specific target genes. At day 5 of therapy, MDR1 was significantly up-regulated in the belinostat/AZA arm compared with AZA alone arm (p = 0.0023). There were 18 responses among the 56 patients. Conclusions The combination of belinostat and AZA is feasible and associated with clinical activity. The recommended phase II dose is 1000 mg/m(2) of belinostat plus 75 mg/m(2) of AZA on days 1-5, every 28 days. Upregulation in MDR1 was observed in the combination arm at day 5 compared with the AZA alone arm, suggesting a relative biologic contribution of belinostat to the combination.

Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

Anticancer bioactive peptide (ACBP) inhibits gastric cancer cells by upregulating growth arrest and DNA damage-inducible gene 45A (GADD45A).

Recently, we reported that anticancer bioactive peptide (ACBP), purified from goat spleens immunized with human gastric cancer extracts, significantly inhibited gastric cancer cells in vitro and gastric tumors in vivo via repressing cell growth and promoting apoptosis, making it a promising potential biological anticancer drug. However, it is not known what genes are functionally required for the ACBP effects. Here, we first found that two tumor suppressor genes, cyclin-dependent kinase inhibitor 2B (CDKN2B) and growth arrest and DNA damage-inducible alpha (GADD45A), were upregulated significantly in the cells with ACBP treatment by microarray screening and the findings were validated by real-time RT-PCR. Next, GADD45A mRNA and protein expressions were downregulated in the gastric cancer cells by lentivirus-mediated RNAi; then, cell viability, cell cycle, and apoptosis were assayed by MTT and flow cytometry. Interestingly, our results indicated that cell viability was not dependent on GADD45A without ACBP treatment; however, cell sensitivity to ACBP was significantly decreased in ACBP-treated gastric cancer cells with GADD45A downregulation. Therefore, we demonstrate that GADD45A was functionally required for ACBP to inhibit gastric cancer cells, suggesting that GADD45A may become a biomarker for ACBP sensitivity. Our findings have significant implications on the molecular mechanism understanding, biomarker development, and anticancer drug development of ACBP.

Study on the treatment of the p15 gene combined with Bcr-abl-specific siRNA and STI571 for chronic myeloid leukemia.

The aim of this study was to investigate the effect of the p15 gene combined with Bcr-abl-specific siRNA and STI571 on the proliferation, cell cycle and apoptosis of K562 chronic myeloid leukemia cells. Using the gene sequence results, we amplified the p15 gene from normal peripheral blood by RT-PCR, and constructed a p15-pcDNA3.1 vector. The K562 cell line with G418 resistance was screened, synthesized and transfected for bcr-abl gene fusion point for 21-nt siRNA. In p15-pcDNA3.1-K562 cells, the growth rate was slower than that of the control K562 cells, G0/G1-phase was increased and S-phase was decreased significantly. In the siRNA group, bcr-abl fusion gene expression was significantly decreased in K562 cells accompanied by the downregulation of BCL-xL protein expression and G1-phase arrest. Cell survival rate was significantly decreased compared with the sole p15-K562 cell group and the sole RNA interference-K562 cell group. In the combination of p15-pcDNA3.1-K562 cells with STI571, the proportion of apoptosis was significantly increased and the cell survival rate was significantly decreased compared with the p15-K562 cell group and STI571-K562 cell group. siRNA at 30 pM combined with 0.5 µM STI571 promoted apoptosis compared with sole application. The p15 gene combined with siRNA had a synergistic effect on the inhibition of proliferation and the induction of apoptosis in K562 cells. Exogenous p15 protein expression combined with STI571 appeared to have a synergistic effect on proliferation inhibition and apoptosis induction in K562 cells. The combination of low-dose RNA interference with STI571 showed a synergistic effect in inducing apoptosis.

ANRIL inhibits p15(INK4b) through the TGFß1 signaling pathway in human esophageal squamous cell carcinoma.

The INK4b-ARF-INK4a gene cluster encodes three tumor suppressors: p15(INK4b), p14(ARF), and p16(INK4a). Antisense non-coding RNA in the INK4 locus (ANRIL) is transcribed in the opposite direction from this gene cluster. Recent studies suggest that ANRIL represses the expression of p15(INK4b), p14(ARF), and p16(INK4a); however, the underlying mechanism is unclear. In this study, the expressions of ANRIL in human esophageal squamous cell carcinoma (ESCC) tissues and matched adjacent non-tumor tissues were examined by quantitative real-time polymerase chain reaction. Compared with matched adjacent non-tumor tissues, the expression levels of ANRIL in ESCC tissues were significantly increased. Furthermore, inhibition of ANRIL was found to increase the expression of p15(INK4b) and transforming growth factor ß1 (TGFß1) and depletion of ANRIL in ESCC cell lines may inhibit cellular proliferation. Thus, our findings suggest a significant role of ANRIL in the occurrence and development of ESCC through TGFß1 signaling pathways.