Multifaceted behavior of PEST sequence enriched nuclear proteins in cancer biology and role in gene therapy.

The amino acid sequence enriched with proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) is a signal-transducing agent providing unique features to its substrate nuclear proteins (PEST-NPs). The PEST motif is responsible for particular posttranslational modifications (PTMs). These PTMs impart distinct properties to PEST-NPs that are responsible for their activation/inhibition, intracellular localization, and stability/degradation. PEST-NPs participate in cancer metabolism, immunity, and protein transcription as oncogenes or as tumor suppressors. Gene-based therapeutics are getting the attention of researchers because of their cell specificity. PEST-NPs are good targets to explore as cancer therapeutics. Insights into PTMs of PEST-NPs demonstrate that these proteins not only interact with each other but also recruit other proteins to/from their active site to promote/inhibit tumors. Thus, the role of PEST-NPs in cancer biology is multivariate. It is hard to obtain therapeutic objectives with single gene therapy. An especially designed combination gene therapy might be a promising strategy in cancer treatment. This review highlights the multifaceted behavior of PEST-NPs in cancer biology. We have summarized a number of studies to address the influence of structure and PEST-mediated PTMs on activation, localization, stability, and protein-protein interactions of PEST-NPs. We also recommend researchers to adopt a pragmatic approach in gene-based cancer therapy.

Destrin Contributes to Lung Adenocarcinoma Progression by Activating Wnt/ß-Catenin Signaling Pathway.

Lung cancer, especially lung adenocarcinoma, is one of the most common neoplasms worldwide. However, the mechanisms underlying its initiation, development, and metastasis are still poorly understood. Destrin (DSTN) is a member of ADF/cofilin family. Its detailed biological function remains unknown, although it is reported that DSTN is involved in cytoskeleton remodeling and regulation of actin filament turnover. Recent evidence has shown that high expression of cofilin-1 is associated with invasion and poor prognosis of several types of human tumors, but the detailed mechanism is still entirely unclear, particularly in lung cancer tumorigenesis and malignancy. Here, we report that DSTN was highly expressed in a mouse lung cancer model induced by urethane and in clinical lung adenocarcinoma tissue samples. Its expression level was positively correlated with cancer development, as well as metastasis to the liver and lymph nodes. Consistently, it was directly associated with the poor prognosis of lung adenocarcinoma patients. Furthermore, we also found that DSTN promotes cell proliferation, invasion, and migration in vitro, and facilitates subcutaneous tumor formation and lung metastasis via intravenous injection in vivo. Mechanically, DSTN associates with and facilitates nuclear translocation of ß-catenin, which promotes epithelial-to-mesenchymal transition (EMT). Taken together, our results indicated that DSTN enhances lung cancer malignancy through facilitating ß-catenin nuclear translocation and inducing EMT. Combined with multivariate analyses, DSTN might potentially serve as a therapeutic target and an independent prognostic marker of lung adenocarcinoma. IMPLICATIONS: This finding indicates that DSTN facilitates ß-catenin nuclear translocation and promotes malignancy in lung adenocarcinoma.

Taking a holistic view of PEST-containing nuclear protein (PCNP) in cancer biology.

Polypeptide sequences enriched with proline (P), glutamic acid (E), aspartic acid (D) and serine (S)/ threonine (T) (PEST) have been reported to be the most abundant and frequently distributed at the cellular level. There is growing evidence that PEST sequences act as proteolytic recognition signals for degradation of residual proteins which is critical for activation or deactivation of regulatory proteins involved in cellular signaling pathways of cell growth, differentiation, stress responses and physiological death. A PEST containing nuclear protein (PCNP) was demonstrated as a tumor suppressor in a neuroblastoma cancer model and tumor promoter in lung adenocarcinoma cancer model. Its unique properties like ubiquitination by NIRF, co-localization with NIRF in nucleus and tumor progression attract the attention of researchers. PCNP was reported to be ubiquitinated by ring finger protein NIRF in E3 ligase manner and as modulator of MAPK and PI3K/AKT/mTOR signaling pathways. In this review, we summarize PCNP linked DNA damage response, Post translational modifications, and transportation to address initiation, prognosis, and resistance of tumor cells in terms of cell cycle regulation, transcription and apoptosis. Hence, we demonstrate PCNP as a novel target in cancer diagnosis and treatment.

[Inhibitory Effect of Histone Deacetylase Inhibitor SAHA on Proliferation of Mouse Multiple Myeloma Cell Line SP2/0 in vitro and in vivo].

OBJECTIVE: To explore the anti-myeloma effect of suberoylanilide hydroxamic acid (SAHA) and on mouse myeloma cell line SP2/0 in vitro and in vivo and its mechanism. METHODS: The inhibitory effect of SAHA on SP2/0 cells was measured by CCK-8 assay,and the apoptosis and cell cycle were analyzed by flow cytometry FACS. The protein expression of Caspase-3 and p53 of SP2/0 cells treated with SAHA were examined by Western blot. Annexin V/7-AAD double staining was performed to detect the apoptosis of SP2/0 induced by SAHA in vitro. SP2/0 cells (1×106) resuspended in 200 µl PBS were inoculated subcutaneously and intravenously into BALB/c mice, so as to establish aggressive or non-aggressive myeloma-bearing mouse models respectively. On day 3 after modeling, mice received SAHA or vehicle control treatment by intraperitoneal injection. The dose of SAHA was 60 mg/kg·d, 5 times a week for 3 weeks. RESULTS: In SAHA-treated SP2/0 cells, the proliferation inhibition rate and apoptotic cells increased in a dose dependent manner. Also, SAHA significantly increased the ratio of cells in G2 phase and decreased in S phase. Molecular mechanisms of apoptosis and cell cycle arrest of SP2/0 induced by SAHA partly correlated with up-regulating the expression level of Caspase-3 and p53. In the non-aggressive myeloma-bearing mice, SP2/0 cells disappeared in peripheral blood after SAHA treatment. In the aggressive myeloma-bearing mice, inhibition of tumor growth and prolongation of the cell survival were observed after SAHA treatment. CONCLUSION: SAHA inhibited SP2/0 cell proliferation, this effect associates with inducing apoptosis and cell cycle arrest, the mechanism of SAHA ralates partly with activating Caspase-3 and p53 pathway.

Immunohistochemical detection of Ndrg2 in the mouse nervous system.

NDRG2, a member of the N-myc downstream-regulated gene (NDRG) family, is involved in cell differentiation and development. However, the distribution and function of Ndrg2 in the central nervous system remains unclear. Here, we analyzed the expression and distribution of Ndrg2 in the mouse brain and explored the potential physiological functions of Ndrg2. Ndrg2 was expressed in different regions of the brain, including the cerebral cortex, olfactory bulb, midbrain, hippocampus, and thalamus, with high levels in the midbrain and thalamus. Immunohistochemistry assay revealed that Ndrg2-positive cells distributed widely in the adult mouse brain and some of them showed nuclear staining. Indirect immunofluorescence and confocal microscopy studies showed that Ndrg2 protein colocalized with glial fibrillary acidic protein, indicating that Ndrg2 is expressed in astrocytes. Furthermore, Ndrg2 expression increased in glioma cells that were differentiating into astrocytes. Taken together, these findings suggest that Ndrg2 is possibly associated with glial cell proliferation and differentiation based on its immunolocalization in this study.

Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse.

The widespread distribution of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the adult brain suggests its role in a broad range of brain functions. Here we show evidence supporting a physical interaction of PTEN with a region in the third intracellular loop (3L4F) of the serotonin 5-HT2C receptor (5-HT2cR, formerly 5-HT1c receptor) in cell cultures. PTEN limits agonist-induced phosphorylation of 5-HT2cR through its protein phosphatase activity. We showed the probable existence of PTEN:5-HT2cR complexes in putative dopaminergic neurons in the rat ventral tegmental area (VTA), a brain region in which virtually all abused drugs exert rewarding effects by activating its dopamine neurons. We synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Systemic application of Tat-3L4F or the 5-HT2cR agonist Ro600175 suppressed the increased firing rate of VTA dopaminergic neurons induced by delta9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Using behavioral tests, we found that Tat-3L4F or Ro600175 blocks conditioned place preference of THC or nicotine, and that Ro600175, but not Tat-3L4F, produces anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggest a potential strategy for treating drug addiction with the Tat-3L4F peptide.

Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects.

The hippocampal dentate gyrus in the adult mammalian brain contains neural stem/progenitor cells (NS/PCs) capable of generating new neurons, i.e., neurogenesis. Most drugs of abuse examined to date decrease adult hippocampal neurogenesis, but the effects of cannabis (marijuana or cannabinoids) on hippocampal neurogenesis remain unknown. This study aimed at investigating the potential regulatory capacity of the potent synthetic cannabinoid HU210 on hippocampal neurogenesis and its possible correlation with behavioral change. We show that both embryonic and adult rat hippocampal NS/PCs are immunoreactive for CB1 cannabinoid receptors, indicating that cannabinoids could act on CB1 receptors to regulate neurogenesis. This hypothesis is supported by further findings that HU210 promotes proliferation, but not differentiation, of cultured embryonic hippocampal NS/PCs likely via a sequential activation of CB1 receptors, G(i/o) proteins, and ERK signaling. Chronic, but not acute, HU210 treatment promoted neurogenesis in the hippocampal dentate gyrus of adult rats and exerted anxiolytic- and antidepressant-like effects. X-irradiation of the hippocampus blocked both the neurogenic and behavioral effects of chronic HU210 treatment, suggesting that chronic HU210 treatment produces anxiolytic- and antidepressant-like effects likely via promotion of hippocampal neurogenesis.

High expression of bcl-x(L) in K562 cells and its role in the low sensitivity of K562 to realgar-induced apoptosis.

Arsenic compounds (As(2)O(3 )or()As(4)S(4)) have been used successfully for the treatment of acute promyelocytic leukemia (APL) for quite a long time. It has been noticed that the sensitivity to apoptosis induced by As(2)O(3 )varies among various leukemia cells. It was reported by several groups that As(2)O(3) could induce apoptosis in APL-derived NB4 cells at concentrations of 0.5-1 mumol/l, whereas in other leukemia cells like K562, As(2)O(3) has no effects at the same concentration. K562 cells undergo apoptosis only when the concentration of As(2)O(3 )is greater than 2 mumol/l. Another arsenic compound, realgar (As(4)S(4)), a traditional Chinese mineral medicine, has been used to treat APL effectively and demonstrated to have lower toxicity than As(2)O(3). It would be interesting to know whether NB4 and K562 cells will show different sensitivity to realgar as well and if there is a difference, what is the cellular mechanism of it. In our present study, K562 cells were much less sensitive than NB4 cells to apoptosis induced by realgar. We confirm that the expression of bcl-x(L) is significantly higher in K562 cells than that in NB4 cells and is not downregulated upon realgar treatment. K562 cells become sensitive to realgar at clinically acceptable concentrations when bcl-x(L) expression level is downregulated by transfecting bcl-x(L) antisense RNA vector into the cells. Our results suggest that the increased bcl-x(L) expression in K562 cells contributes to its insensitivity to realgar-induced apoptosis.

[Cloning tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction-based subtractive hybridization].

BACKGROUND & OBJECTIVE: Glioma is a common tumor in central nervous system with no specific clinical therapy. Its pathogenesis is unclear. This study was to clone tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction (PCR)-based subtractive hybridization, and to explore the molecular biological mechanism of tumorigenesis of glioma. METHODS: mRNA was isolated from a sample of human glioma, and reversely transcribed into cDNA. PCR-based subtractive hybridization was used to clone tumor-related genes and tumor suppressor genes from it. RESULTS: In tumor-related candidate gene group, phospho-protein enriched in astrocytes of 15 (PEA15) and homology of acid fibroblast growth factor (aFGF) were picked up. Whereas, in tumor suppressor gene group, interferon-induced protein 17 and ndr2 were picked up. ndr2 was widely expressed in normal brain tissue, but absent in glioma tissue. CONCLUSION: ndr2 gene is a candidate tumor suppressor gene, and may play a role in tumorigenesis of glioma.

Involvement of extracellular regulated kinase and p38 kinase in hippocampal seizure tolerance.

The mechanisms underlying brain seizure tolerance, a phenomenon in which brief periods of seizures protect brain against the lethal effects of subsequent sustained seizures, are poorly understood. Because brain seizure tolerance and brain ischemia tolerance likely share certain common mechanisms, the recent evidence that activation of extracellular regulated kinase (ERK) and p38 kinase pathways plays a critical role in ischemic preconditioning suggests that a similar mechanism may underlie brain seizure tolerance. We investigated the hypothesis in a rat kainic acid preparation of seizure preconditioning and tolerance, which was established by induction of one episode of priming epileptic status lasting for 20 min on the first day and another episode of sustained epileptic status lasting for 2 hr on the second day. We observed that acute seizures lead to a rapid activation of ERK and p38 in the hippocampal CA3 area, the brain region most susceptible to the lethal effects of epileptic status. Pretreatment with the ERK inhibitor PD98059 and the p38 inhibitor SB203580 selectively reduces seizure-elicited activation of ERK and p38, respectively, and significantly reduces priming seizure-induced protection of CA3 neurons. These findings indicate that, similar to brain ischemia tolerance, brain seizure tolerance also involves the ERK and p38 signaling pathways.

[Inhibition of hepatitis C virus gene expression by antisense nucleotide in vitro].

OBJECTIVE: To study the mechanism of hepatitis C virus (HCV) gene regulation and the inhibitory effect of antisense RNA on HCV gene expression in vitro. METHODS: The hepatoblastoma cell line (HepG2) was co-transfected by recombinant plasmid of antisense RNA complementary to HCV 5' untranslated region (5'UTR)and HCV 5' UTR Directed luciferase (luc) gene expression recombinant plasmid. Meanwhile a reversed HCV 5'UTR recombinant plasmid which can not transcribe as antisense RNA in the cell and a recombinant plasmid in which the luc was regulated by simian virus 40 (sv40) 5'UTR were used as controls respectively. The level of luc gene expression was determined by an enzymatic assay. RESULTS: The antisense RNA which directed to HCV 5'UTRcould obviously knock down the level of luc gene expression and the close-dependent inhibition of antisense RNA was observed at the same time. However the above inhibition was not shown in the cells co-transfected by reversed HCV 5'UTR recombinant plasmid and HCV 5'UTR directed luc gene expression recombinant plasmid. No reduction was observed in luc gene expression level in the cell co-transfected by both antisense RNA recombinant plasmid and SV40 5'UTR directed luc gene expression recombinant plasmid. CONCLUSION: HCV 5'UTR plays an important role in regulation of viral gene expression. The antisense RNA complementary to HCV 5'UTR could effectively inhibit the gene expression regulated by HCV 5'UTR in vitro.

[Cloning, sequencing and bioinformatics analysis of a new tumor suppressor gene ndr2 from mouse].

BACKGROUND & OBJECTIVE: Ndr2 (N-myc down stream regulator) gene in human is a new gene cloned with the human adult whole brain cDNA as template in 1999, which accession number is AF159092 in GenBank. Locating backward position of the N-myc gene in human chromosome, this gene was named Ndr2 gene. The previous experimental results showed Ndr2 gene probably is a tumor suppressor gene. To research the function of Ndr2 gene, the authors cloned the genomic sequence of ndr2 from mouse. METHODS: To clone Ndr2 genomic sequence by reverse transcription-polymerase chain reaction(RT-PCR) with the mouse genome library as template; automatic sequencing was performed using 310 Genetic Analyzer; homogeneous analysis was made using GenBank BLAST; open reading fragment(ORF) analysis was made using PC Gene and ORF Finder; domain analysis was made using ProDom system. RESULTS: A fragment (about 3310bp,identified by agarose gel electrophoresis) was obtained using RT-PCR with the mouse genome library as template. The fragment was cloned in pMD18-T vector. BLAST analysis showed that the sequence was highly homogeneous (with the homogeneity rate of 91.4%) with Ndr2 gene in human and non-homogeneous with genomic sequence database in mouse. ORF analysis showed that there was a complete coding region in it, which including 8 extrons and 7 introns; it can interpret a protein containing about 200 amino acid residuals. ProDom analysis showed there was a domain like acyl carrier protein(ACP) in it. CONCLUSION: The authors cloned Ndr2 gene in mouse and proved that the sequence is a new genome sequence in mouse genomic sequence database. At present, the genome sequence has been submitted to GenBank(the accession number: AY151387).

Binding of Insulin Receptor Substrate 1 PH Domain to Protein Kinase C.

To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total RNAs were isolated and purified from fresh liver and mRNAs were reversely transcribed into cDNAs. After PCR the fragments of the DNA were cloned into vector pUC19. The sequence of the fusion gene was confirmed by sequencing, and the gene was correctly expressed in E.coli as fusion protein with glutathione S- transferase(GST). The fusion protein was purified by glutathione agarose beads, then was incubated with the lysate of Jurkat cells. After SDS-PAGE, the proteins were transferred to PVDF membrane, and an anti-PKC antibody was used to detect binding between the PH domain with PKC. The sequence of the gene encoding for the PH domains was confirmed to be correct, and the PH domain was successfully expressed in E.coli JM 109 in soluble form. Western blots confirmed the binding of the PH domain with PKC in vitro. In conclusion, purified IRS-1 PH domain GST fusion protein was obtained and its biological activity was confirmed.