A scalable and reproducible preparation for the antitumor protein TLC, a human-derived telomerase inhibitor.

Telomerase, which is overexpressed in approximately 90% of liver cancer cells, is an ideal target for anti-liver cancer therapy. LPTS, a putative liver tumor suppressor, is the only human-derived protein that can bind telomerase directly and inhibit the extension of telomere activity. Our previous studies demonstrated that TAT-LPTS-LC (TLC), a recombinant protein fused by the C-terminal 133-328 fragment of LPTS and TAT peptides, could be delivered into cells to inhibit telomerase-positive hepatoma cell growth in vitro and in vivo with very low toxicity. In the present study, E. coli strains which expressed TLC in abundance were screened and cultured in a laboratory bioreactor. A reproducible protein separation process was built, and this process was suitable for industrial amplification. The yields of TLC protein were up to 184 mg in one batch with a purity of approximately 95%. The purified TLC protein had a similar inhibitory effect on telomerase activity in vitro compared with those purified by Ni-affinity chromatography. Furthermore, TLC protein could be delivered into the cell nucleus to increase the doubling time of the cell and suppress cell growth in telomerase-positive liver cancer cell lines. Cell growth inhibition was negatively correlated with telomere length, suggesting that TLC is a highly targeted telomerase-telomere anticancer agent. These results will contribute to future preclinical studies of the TLC protein.

The LIS1/NDE1 Complex Is Essential for FGF Signaling by Regulating FGF Receptor Intracellular Trafficking.

Intracellular transport of membranous organelles and protein complexes to various destinations is fundamental to signaling transduction and cellular function. The cytoplasmic dynein motor and its regulatory proteins LIS1 and NDE1 are required for transporting a variety of cellular cargos along the microtubule network. In this study, we show that deletion of Lis1 in developing lung endoderm and limb mesenchymal cells causes agenesis of the lungs and limbs. In both mutants, there is increased cell death and decreased fibroblast growth factor (FGF) signaling activity. Mechanistically, LIS1 and its interacting protein NDE1/NDEL1 are associated with FGF receptor-containing vesicles and regulate FGF receptor intracellular trafficking and degradation. Notably, FGF signaling promotes NDE1 tyrosine phosphorylation, which leads to dissociation of LIS1/NDE1 complex. Thus, our studies identify the LIS1/NDE1 complex as an important FGF signaling regulator and provide insights into the bidirectional regulation of cell signaling and transport machinery for endocytosis.

Hepatic loss of Lissencephaly 1 (Lis1) induces fatty liver and accelerates liver tumorigenesis in mice.

The liver is a major organ in lipid metabolism, and its malfunction leads to various diseases. Nonalcoholic fatty liver disease, the most common chronic liver disorder in developed countries, is characterized by the abnormal retention of excess lipid within hepatocytes and predisposes individuals to liver cancer. We previously reported that the levels of Lissencephaly 1 (LIS1, also known as PAFAH1B1) are down-regulated in human hepatocellular carcinoma. Following up on this observation, we found that genetic deletion of Lis1 in the mouse liver increases lipid accumulation and inflammation in this organ. Further analysis revealed that loss of Lis1 triggers endoplasmic reticulum (ER) stress and reduces triglyceride secretion. Attenuation of ER stress by addition of tauroursodeoxycholic acid (TUDCA) diminished lipid accumulation in the Lis1-deficient hepatocytes. Moreover, the Golgi stacks were disorganized in Lis1-deficient liver cells. Of note, the Lis1 liver-knockout mice exhibited increased hepatocyte ploidy and accelerated development of liver cancer after exposure to the liver carcinogen diethylnitrosamine (DEN). Taken together, these findings suggest that reduced Lis1 levels can spur the development of liver diseases from steatosis to liver cancer and provide a useful model for delineating the molecular pathways that lead to these diseases.

Dysfunction of PLA2G6 and CYP2C44-associated network signals imminent carcinogenesis from chronic inflammation to hepatocellular carcinoma.

Little is known about how chronic inflammation contributes to the progression of hepatocellular carcinoma (HCC), especially the initiation of cancer. To uncover the critical transition from chronic inflammation to HCC and the molecular mechanisms at a network level, we analyzed the time-series proteomic data of woodchuck hepatitis virus/c-myc mice and age-matched wt-C57BL/6 mice using our dynamical network biomarker (DNB) model. DNB analysis indicated that the 5th month after birth of transgenic mice was the critical period of cancer initiation, just before the critical transition, which is consistent with clinical symptoms. Meanwhile, the DNB-associated network showed a drastic inversion of protein expression and coexpression levels before and after the critical transition. Two members of DNB, PLA2G6 and CYP2C44, along with their associated differentially expressed proteins, were found to induce dysfunction of arachidonic acid metabolism, further activate inflammatory responses through inflammatory mediator regulation of transient receptor potential channels, and finally lead to impairments of liver detoxification and malignant transition to cancer. As a c-Myc target, PLA2G6 positively correlated with c-Myc in expression, showing a trend from decreasing to increasing during carcinogenesis, with the minimal point at the critical transition or tipping point. Such trend of homologous PLA2G6 and c-Myc was also observed during human hepatocarcinogenesis, with the minimal point at high-grade dysplastic nodules (a stage just before the carcinogenesis). Our study implies that PLA2G6 might function as an oncogene like famous c-Myc during hepatocarcinogenesis, while downregulation of PLA2G6 and c-Myc could be a warning signal indicating imminent carcinogenesis.

Adenovirus-mediated delivery of siRNA targeting TM4SF4 attenuated liver cancer cell growth in vitro and in vivo.

Gene targeting using short interfering RNA (siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. The human transmembrane 4 superfamily member 4 (TM4SF4) was originally identified in intestine and liver as a cell proliferation-related gene. Recently, it showed an increased expression in the hepatocellular carcinoma (HCC) tissues. In this study, we developed an adenoviral vector harboring an effective siRNA targeting TM4SF4 (AdSiTM4SF4) and identified its function in suppression of tumor cell growth. It was confirmed that TM4SF4 was overexpressed in HCC tissues compared with its paired non-tumor tissues by western blot analysis and immunohistochemistry. Remarkably, it was more abundant on the cell surface of HCC cells. The signals of ectopically expressed TM4SF4 in four cell lines dramatically localized in the plasma membrane, slightly in the cytoplasm, and absent in the nucleus, demonstrating that TM4SF4 is a membrane protein. Targeting TM4SF4 by AdSiTM4SF4 successfully exerted a gene knockdown effect. The QGY-7701 and SMMC-7721 HCC cells infected with AdSiTM4SF4 displayed remarkably attenuated growth potential. Moreover, intratumoral injection of AdSiTM4SF4 significantly suppressed tumor growth in a xenograft mouse model using SMMC-7721 hepatoma cells. Our results indicated that targeting TM4SF4 might be a promising modality for inhibition of HCC.

Microspherule protein 2 associates with ASK1 and acts as a negative regulator of stress-induced ASK1 activation.

Microspherule protein 2 (MCRS2) has been reported to associate with the cellular function of telomerase inhibition, transcriptional regulation and cellular transformation. Here, we report a novel function of MCRS2 in ASK1 pathway. We found that MCRS2 directly binds to ASK1 in vivo and co-localises with ASK1 in the cytoplasm. Overexpression of MCRS2 inhibited oxidative stress (H(2)O(2))-induced ASK1 activation. Knockdown of MCRS2 expression accelerated p38 and JNK phosphorylation and promoted apoptosis in response to H(2)O(2). Finally, H(2)O(2) treatment induced proteasomal degradation of MCRS2, which was further enhanced by activated ASK1. Our results clearly demonstrate that MCRS2 plays a negative role in stress-induced ASK1 activation.

Human tetraspanin transmembrane 4 superfamily member 4 or intestinal and liver tetraspan membrane protein is overexpressed in hepatocellular carcinoma and accelerates tumor cell growth.

The human transmembrane 4 superfamily member 4 or intestinal and liver tetraspan membrane protein (TM4SF4/il-TMP) was originally cloned as an intestinal and liver tetraspan membrane protein and mediates density-dependent cell proliferation. The rat homolog of TM4SF4 was found to be up-regulated in regenerating liver after two-thirds hepatectomy and overexpression of TM4SF4 could enhance liver injury induced by CCl(4). However, the expression and significance of TM4SF4/il-TMP in liver cancer remain unknown. Here, we report that TM4SF4/il-TMP is frequently and significantly overexpressed in hepatocellular carcinoma (HCC). Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that TM4SF4/il-TMP mRNA and protein levels were up-regulated in ∼80% of HCC tissues. Immunohistochemical analysis of a 75 paired HCC tissue microarray revealed that TM4SF4/il-TMP was significantly overexpressed in HCC tissues (P< 0.001), and high immunointensity of TM4SF4/il-TMP tended to be in well-to-moderately differentiated HCC compared with poorly differentiated tumors. Functional studies showed that overexpression of TM4SF4/il-TMP in QGY-7701 and BEL-7404 HCC cell lines through stable transfection of TM4SF4 expression plasmid significantly promoted both cell growth and colony formation of HCC cells. Reduction of TM4SF4/il-TMP expression in QGY-7701 and BEL-7404 cells by stably transfecting TM4SF4 antisense plasmid caused great inhibition of cell proliferation. Our findings suggest that TM4SF4/il-TMP has the potential to be biomarker in HCC and plays a crucial role in promotion of cancer cell proliferation.

Hepatitis B virus X protein reduces starvation-induced cell death through activation of autophagy and inhibition of mitochondrial apoptotic pathway.

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.

Human cell-death-inducing DFF45-like effector C induces apoptosis via caspase-8.

Human cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector C (CIDEC) is a potent apoptotic inducer. Previous studies have indicated that the Fat-specific protein 27 (Fsp27), a mouse homolog of CIDEC, induces apoptosis via caspase-3, -7, and -9 and triggers the release of cytochrome c from mitochondria, which implies that the mitochondrial pathway is involved in Fsp27-induced apoptosis. In the current study, we found that CIDEC-induced apoptosis was mediated by caspase-8. The caspase inhibitor assay showed that CIDEC-induced apoptosis was dramatically reduced in the presence of the general caspase inhibitor, the caspase-3 inhibitor, and the caspase-8 inhibitor, whereas the caspase-9 inhibitor only weakly inhibited CIDEC-induced apoptosis. These results confirmed that the activation of caspase-3 and caspase-8 were involved in CIDEC-induced apoptosis. Moreover, in caspase-3- or caspase-8-deficient cells, CIDEC-induced apoptosis were dramatically decreased, which demonstrated that CIDEC-induced apoptosis might require the activation of caspase-3 and caspase-8. Because caspase-8 in general is a key effecter of death-receptor pathway and activated by Fas-Associated protein with Death Domain (FADD), we examined whether FADD was involved in CIDEC-induced apoptosis. Our results demonstrated that CIDEC-induced apoptosis was independent of FADD, suggesting that CIDEC-induced apoptosis might be in a death-receptor-independent, caspase-8-dependent manner. It was also found that the region of amino acid 168-200 in carboxyl domain of CIDEC was critical for its crucial pro-apoptotic function.

The human LIS1 is downregulated in hepatocellular carcinoma and plays a tumor suppressor function.

The human lissencephaly-1 gene (LIS1) is a disease gene responsible for Miller-Dieker lissencephaly syndrome (MDL). LIS1 gene is located in the region of chromosome 17p13.3 that is frequency deleted in MDL patients and in human liver cancer cells. However, the expression and significance of LIS1 in liver cancer remain unknown. Here, we investigated the expression of LIS1 in hepatocellular carcinoma (HCC) tissues by real-time PCR, Western blot, and immunohistochemistry. The results indicated that the mRNA and protein levels of LIS1 were downregulated in about 70% of HCC tissues, and this downregulation was significantly associated with tumor progression. Functional studies showed that the reduction of LIS1 expression in the normal human liver cell line QSG7701 or the mouse fibroblast cell line NIH3T3 by shRNA resulted in colony formation in soft agar and xenograft tumor formation in nude mice, demonstrating that a decrease in the LIS1 level can promote the oncogenic transformation of cells. We also observed that the phenotypes of LIS1-knockdown cells displayed various defective mitotic structures, suggesting that the mechanism by which reduced LIS1 levels results in tumorigenesis is associated with its role in mitosis. Furthermore, we demonstrated that ectopic expression of LIS1 could significantly inhibit HCC cell proliferation and colony formation. Our results suggest that LIS1 plays a potential tumor suppressor role in the development and progression of HCC.

HIV-Tat-mediated delivery of an LPTS functional fragment inhibits telomerase activity and tumorigenicity of hepatoma cells.

BACKGROUND & AIMS: Human liver-related putative tumor suppressor (LPTS) is a gene that encodes a telomerase inhibitory protein that is similar to human Pin2/TRF1-interacting protein. The LPTS protein binds directly to the telomerase catalytic subunit (human telomerase reverse transcriptase) and suppresses telomerase activity. Telomere maintenance and telomerase activity are required for long-term proliferation of cancer cells, so LPTS might be used in anticancer strategies. METHODS: The carboxy-terminal (functional) fragment of LPTS was fused to the transactivator of transcription of human immunodeficiency virus (Tat)-an 11-amino acid peptide that translocates across the cell membrane; the TAT-fused C-terminal of LPTS (TAT-LPTS-LC) was purified and transduced into cells. Telomerase activity was identified by using the telomeric repeat amplification protocol. The effects of the TAT-LPTS-LC protein on cell proliferation and death were evaluated by colorimetric tetrazolium salt and flow cytometry analyses. Tumor growth was analyzed in nude mice. RESULTS: The purified TAT-LPTS-LC protein was efficiently delivered into the cells, where it suppressed telomerase activity and shortened telomere length. TAT-LPTS-LC inhibited proliferation of telomerase-positive hepatocellular carcinoma BEL-7404 and hepatoblastoma HepG2cells and induced their death; however, it had no effect on telomerase-negative liver cell line L02 and osteosarcoma cell line Saos-2. In mice, tumor formations by BEL-7404 cells were suppressed by TAT-LPTS-LC treatments. CONCLUSIONS: Transduction of hepatoma cells with a fusion protein that contains the C-terminal, functional fragment of LPTS and human immunodeficiency virus Tat (TAT-LPTS-LC) causes telomere shortening, limits proliferation, and inhibits growth of tumors from these cells in mice. TAT-LPTS-LC inhibits telomerase activity and might be developed as an anticancer agent.

Proteomic mining in the dysplastic liver of WHV/c-myc mice--insights and indicators for early hepatocarcinogenesis.

Because of the asymptomatic process of carcinogenesis, the early detection of cancers such as hepatocellular carcinoma (HCC) is very challenging. Tumor-prone transgenic mouse models of oncogenesis can provide a stable and powerful tool for the analysis of cancer initiation, and are therefore promising for the discovery of early putative biomarkers of HCC. Using a label-free proteomic quantification strategy, we comprehensively investigated the protein expression profile in the livers of three 2-month-old WHV/c-myc mice at the dysplastic stage, with age-matched wt-C57 mice as controls. We identified 2781 proteins, 540 of which were differentially expressed. These proteins successfully characterized certain precancerous biological processes and alterations in transcriptional regulators before tumor onset. Two candidates, FK506-binding protein 4 (FKBP52) and ferritin heavy chain, were taken as examples for a search from the mouse model to clinical human tissues. Their levels in serum samples were determined by western blotting, and showed a noteworthy ability to distinguish between HCC and control cases. Immunohistochemical analysis with tissue microarrays confirmed the differential expression of FKBP52 between HCC and the paired controls (P < 0.001). The regulation of FKBP52 was also discovered to be relevant to HCC staging, with a dramatic decline at stage III (P < 0.05). The potentials of the candidate pool in this study were discussed in terms of delineating c-myc-induced hepatocarcinogenesis and facilitating biomarker discovery for early HCC diagnosis.

C-terminal amino acids 290-328 of LPTS/PinX1 confer telomerase inhibition.

LPTS/PinX1, a telomerase inhibitor composed of 328 amino acids, binds to the telomere associated protein Pin2/TRF1 and to the telomerase catalytic subunit hTERT. However, the mechanism by which LPTS/PinX1 regulates telomerase activity remains unclear. Here we show, for the first time, that LPTS/PinX1 uses different domains to interact with Pin2/TRF1 and hTERT. The LPTS/PinX1(254-289) fragment specifically binds to Pin2/TRF1, and LPTS/PinX1(290-328) can associate with hTERT. Compared with the full-length LPTS/PinX1 protein, LPTS/PinX1(290-328) shows stronger in vitro telomerase inhibitory activity. Moreover, the LPTS/PinX1 protein was recruited to telomeres for binding to Pin2/TRF1. Overexpression of LPTS/PinX1(290-328), which contains a nucleolus localization signal, in cells resulted in telomere shortening and progressive cell death. Conversely, telomere elongation was induced by expression of the dominant-negative LPTS/PinX1(1-289). Our results suggest that the C-terminal fragment of LPTS/PinX1 (LPTS/PinX1(290-328)) contains a telomerase inhibitory domain that is required for the inhibition of telomere elongation and the induction of cell crisis. Our studies also provide evidence that LPTS/PinX1 interaction with Pin2/TRF1 may play a role in the stabilization of telomeres.

QSG-7701 human hepatocytes form polarized acini in three-dimensional culture.

Hepatocytes are polarized and fulfill a variety of liver-specific functions in vivo; but the polarized tissue structure and many of these functions are lost when the cells are cultured on plastic. To recapitulate the polarized structure and tissue-specific function of liver cells in culture, we established a three-dimensional (3D) culture assay with the human hepatocyte line QSG-7701. In 3D Matrigel culture, QSG-7701 cells formed polarized spheroids with a center lumen, which is reminiscent of bile canaliculi in the liver. Immunofluoresence analysis showed that F-actin bundles and radixin were mainly located at the apical membrane and that alpha6 and beta1 integrins were localized basally in 3D culture. Lumen formation was associated with the selective apoptosis of centrally located cells and was accompanied by proliferative suppression during acinar development. Compared to QSG-7701 cells in 2D or agarose gel cultures, the cells in 3D Matrigel culture maintained a given direction of biliary excretion and acquired higher levels of cytochrome P450 and albumin expression. Our study shows that the immortal human hepatocytes, QSG-7701, in 3D Matrigel culture reacquire cardinal features of glandular epithelium in vivo, providing an ex vivo model to study liver-specific function and tumorigenesis.

Expression and function on embryonic development of lissencephaly-1 genes in zebrafish.

Lissencephaly is a severe disease characterized by brain malformation. The main causative gene of lissencephaly is LIS1. Mutation or deletion of LIS1 leads to proliferation and migration deficiency of neurons in brain development. However, little is known about its biological function in embryonic development. In this article, we identified the expression patterns of zebrafish LIS1 gene and investigated its function in embryonic development. We demonstrated that zebrafish consisted of two LIS1 genes, LIS1a and LIS1b. Bioinformatics analysis revealed that LIS1 genes were conserved in evolution both in protein sequences and genomic structures. The expression patterns of zebrafish LIS1a and LIS1b showed that both transcripts were ubiquitously expressed at all embryonic developmental stages and in adult tissues examined. At the protein level, the LIS1 products mainly exist in brain tissue and in embryos at early stages as shown by western blotting analysis. The whole-mount immunostaining data showed that LIS1 proteins were distributed all over the embryos from 1-cell stage to 5 day post-fertilization. Knockdown of LIS1 protein expression through morpholino antisense oligonucleotides resulted in many developmental deficiencies in zebrafish, including brain malformation, circulation abnormality, and body curl. Taken together, our study suggested that zebrafish LIS1 plays a very important role in embryonic development.

Hepatitis B virus X protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression.

UNLABELLED: Human beclin 1 is the first identified mammalian gene to induce autophagy. It is commonly expressed at reduced levels in breast tumors; however, it is overexpressed in hepatitis B virus (HBV)-infected cancerous liver tissues. To expose the possible mechanism and biological significance of this up-regulation of beclin 1, we investigated the regulation of beclin 1 expression by HBV x protein (HBx) in hepatic or hepatoma cell lines. Here, we showed that enforced expression of HBx by transfection technology results in the up-regulation of the endogenous messenger RNA (mRNA) and protein levels of Beclin 1 in the tested cells. Using a luciferase- reporter assay, we demonstrated that HBx transactivates beclin 1 promoter activity in a dose-dependent manner. The promoter region of the beclin 1 gene identified in this study is located at nt -277/+197 and has the maximum transcriptional activity. HBx-mediated up-regulation of beclin 1 expression might be direct, that is, via its promoter. Furthermore, the cells that transiently or stably expressed HBx showed an enhanced accumulation of vacuoles carrying the autophagy marker LC3 as compared with the control cells, which was induced by nutrient starvation, indicating HBx-enhanced autophagy. Moreover, this enhanced autophagy occurred in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA, suggesting that HBV infection also causes increased levels of autophagy under starvation conditions. Treatment of cells with beclin 1 small interfering RNA (siRNA) blocked HBx-enhanced autophagy, demonstrating that the function of HBx in influencing autophagy is Beclin 1 dependent. CONCLUSION: Our findings suggest a novel function of HBx in increasing autophagy through the up-regulation of beclin1 expression, and this may provide an important mechanism in HBV-infected hepatocytes growing under nutrient-deficient conditions.

Identification of zebrafish LPTS: a gene with similarities to human LPTS/PinX1 that inhibits telomerase activity.

Human LPTS/PinX1 is a newly identified telomerase inhibitory protein. Overexpression of the LPTS/PinX1 gene suppresses telomerase activity, results in shortened telomeres. To investigate the role of the LPTS gene in zebrafish, we cloned the homologous gene, zLPTS, which encodes a protein of 355 amino acids. Sequence analysis revealed that, like human LPTS/PinX1, the zLPTS protein has a conserved G-patch domain at its N-terminus and a lysine-rich domain at its C-terminus. Bioinformatics analysis showed the evolutionary conservation of zLPTS. Using RT-PCR and northern blot, we found that zLPTS was expressed in all zebrafish tissues with higher level in ovary, and in all embryonic developmental stages examined. Whole mount in situ hybridization revealed that zLPTS was expressed in all regions of early developmental embryos. The subcellular localization of zLPTS protein was showed in the nucleolus and telomeres. We also cloned the gene for zebrafish Telomerase Reverse Transcriptase (zTERT), a catalytic subunit of telomerase, and demonstrated that zLPTS protein can interact with zTERT through the TR-binding domain of zTERT. Further, we verified that zLPTS could inhibit telomerase activities in zebrafish embryos and human cancer cell line by TRAP assay. Our results clearly demonstrate that zLPTS is ubiquitously expressed in tissues and embryos and plays a function of inhibiting telomerase activity. This study may provide a useful system for further investigating the mechanism of telomere length regulation.

CpG methylation plays a vital role in determining tissue- and cell-specific expression of the human cell-death-inducing DFF45-like effector A gene through the regulation of Sp1/Sp3 binding.

Cell-death-inducing DFF45-like effector A (CIDE-A) belongs to a family of proapoptotic proteins, the expression of which is highly restricted in human tissues and cells. Here, the core region of the human CIDE-A promoter was characterized. Surprisingly, two Sp1/Sp3-binding sites, rather than tissue-specific transcription factors, were found to be required for the promoter activity. Although the ubiquitously expressed Sp1 and Sp3 were crucial, they alone could not adequately regulate the specific expression of CIDE-A. We found that the expression of CIDE-A was further regulated by CpG methylation of the promoter region. By performing bisulfite sequencing, we observed dense CpG methylation of the promoter region in tissues and cells with low or no expression of CIDE-A but not in tissues with high level of CIDE-A expression. In vitro methylation of this region showed significantly reduced transcriptional activity. Treatment of CIDE-A-negative cells with 5-aza-2'-deoxycytidine demethylated the CpG sites; this opened the closed chromatin conformation and markedly enhanced the binding affinity of Sp1/Sp3 to the promoter in vivo, thereby restoring CIDE-A expression. These data indicated that CpG methylation plays a crucial role in establishing and maintaining tissue- and cell-specific transcription of the CIDE-A gene through the regulation of Sp1/Sp3 binding.

[Preparation of a novel telomerase inhibitory protein LPTS-L].

The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein, the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS. The LPTS-L protein was expressed in E. coli BL21 solublely, and purified by Ni Sepharose affinity chromatography which, however, is not fit for large scale protein purification. The gene of LITS-L was then PCR amplified to remove the 6 x His tag, and cloned into pET-24a. The non-fusion protein of LPTS-L was expressed in E. coli B21, and purified by phosphocellulose P11 chromatography. The purity of LPTS-L protein was about 55% after that procedure,and arrived at 80% after second purification by Sephadex G-100 chromatography. Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein. The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro. It was observed that the purified LPTS-L inhibited the activity of telomerase greatly, similarly with that of LPTS-L protein purified by Ni Sepharose 4B. Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein. Through this study, a technique for preparation of LPTS-L protein in a large scale is established.