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.

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.

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.

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.

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.

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.

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.

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.

Dual promoters control the cell-specific expression of the human cell death-inducing DFF45-like effector B gene.

CIDE-B [cell death-inducing DFF45 (DNA fragmentation factor 45)-like effector B] is a member of the CIDE family of apoptosis-inducing factors. The highly restricted pattern of expression of CIDE-B in the liver and spleen suggests that a mechanism exists for the tissue- and cell-specific regulation of transcription of this gene. We have analysed the promoters of the human CIDE-B gene, particularly the mechanism of cell-specific transcription. Expression of CIDE-B is driven by two promoters which are responsible for the synthesis of two types of transcript, and Sp1 and Sp3 are key regulators of basal transcription from both the upstream and the internal promoter, as indicated by EMSAs (electrophoretic mobility-shift assays) and site-directed mutagenesis. Bisulphite sequencing analysis demonstrated that the upstream promoter was hypermethylated in cells that did not express the long transcript of CIDE-B, but was hypomethylated in cells that expressed this transcript. Furthermore, methylation of this region in vitro reduced the promoter activity to approximately 5% of the control. Thus methylation at CpG sites in the upstream promoter region appeared to be important for cell-specific synthesis of the long transcript. By contrast, HNF4alpha (hepatocyte nuclear factor-4alpha) bound to the internal promoter and enhanced its activity. Moreover, the short transcript of CIDE-B gene was expressed in cells which do not normally express this transcript upon introduction of exogenous HNF4alpha, demonstrating the involvement of HNF4alpha in the cell-specific synthesis of the short transcript. Thus our analysis revealed a novel mechanism for the cell-specific transcription of the human CIDE-B gene, which involves epigenetic and genetic control at separate respective promoters.

Cloning and characterization of the promoter region of human LPTS/PinX1 gene.

The gene for LPTS/PinX1 encodes a potent telomerase inhibitor and suppresses tumor cell growth. In order to investigate the transcriptional regulation of this gene, we isolated its 5'-flanking region from the human genomic BAC clone and identified a major transcriptional initiation site. The sequence of the 5'-flanking region is GC-rich, lacks canonical TATA box, but contains potential binding sites for a variety of transcription factors. The deletion analysis indicated that the proximal 100 bp (from nt -66 to +34) is essential for minimal promoter activity and the regions of promoter from nt -1272 to -573 and nt -330 to -66 are required for maximal expression of the LPTS/PinX1 gene. Four DNase I hypersensitive sites (DHS1-4) mapping to the regions of transcription initiation and promoter in LPTS/Pinx1 gene were also revealed.

p73beta inhibits transcriptional activities of enhancer I and X promoter in hepatitis B virus more efficiently than p73alpha.

AIM: p73, as a novel member of a family of p53-related transcription factors, shares redundant functions with p53, such as the abilities of inducing apoptosis and suppressing growth. It is well known that p53 can repress HBV expression and transcription efficiently. The aim of this paper is to investigate the transcriptional effect of p73alpha and p73beta on hepatitis B virus (HBV) and to understand the correlation between HBV and p73. METHODS: To construct an x-gene inactivated HBV plasmid which was cotransfected with p73alpha or p73beta expression vectors into HepG2 cells. After transiently transfection, HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) were detected by ELISA. Viral transcripts synthesized by HBV were evaluated by Northern blotting analysis. The activities of HBV regulatory elements, including enhancer I/X promoter (ENI/Xp) and enhancer II/core promoter (ENII/Cp) were monitored by luciferase assays. RESULTS: Both p73alpha and p73beta could repress HBsAg and HBeAg expression by downregulating the ENI/Xp and ENII/Cp activities. But p73beta exerted stronger inhibition on the activity of ENI/Xp than p73alpha, resulting in much lower level of viral transcripts and the antigens expression. CONCLUSION: p73beta as a novel member of p53 family can efficiently inhibit HBV transcription mainly through downregulating the activities of the HBV ENI/Xp regulatory elements.

Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma.

AIM: To find the point mutations meaningful for inactivation of liver-related putative tumor suppressor gene (LPTS) gene, a human novel liver-related putative tumor suppressor gene and telomerase inhibitor in hepatocellular carcinoma. METHODS: The entire coding sequence of LPTS gene was examined for mutations by single strand conformation polymorphism (SSCP) assay and PCR products direct sequencing in 56 liver cancer cell lines, 7 ovarian cancer and 7 head neck tumor cell lines and 70 pairs of HCC tissues samples. The cDNA fragment coding for the most frequent mutant protein was subcloned into GST fusion expression vector. The product was expressed in E.coli and purified by glutathione-agarose column. Telomeric repeat amplification protocol (TRAP) assays were performed to study the effect of point mutation to telomerase inhibitory activity. RESULTS: SSCP gels showed the abnormal shifting bands and DNA sequencing found that there were 5 different mutations and/or polymorphisms in 12 tumor cell lines located at exon2, exon5 and exon7. The main alterations were A(778)A/G and A(880)T in exon7. The change in site of 778 could not be found in HCC tissue samples, while the mutation in position 880 was seen in 7 (10 %) cases. The mutation in the site of 880 had no effect on telomerase inhibitory activity. CONCLUSION: Alterations identified in this study are polymorphisms of LPTS gene. LPTS mutations occur in HCC but are infrequent and of little effect on the telomerase inhibitory function of the protein. Epigenetics, such as methylation, acetylation, may play the key role in inactivation of LPTS.

Genes encoding Pir51, Beclin 1, RbAp48 and aldolase b are up or down-regulated in human primary hepatocellular carcinoma.

AIM: To reveal new tumor markers and target genes from differentially expressed genes of primary tumor samples using cDNA microarray. METHODS: The (33 )P labeled cDNAs were synthesized by reverse transcription of message RNA from the liver cancerous tissue and adjacent non-cancerous liver tissue from the same patient and used to hybridize to LifeGrid 1.0 cDNA microarray blot containing 8400 known and unique human cDNA gene targets, and an expression profile of genes was produced in one paired human liver tumor tissue. After a global analysis of gene expression of 8400 genes, we selected some genes to confirm the differential expression using Northern blot and RT-PCR. RESULTS: Parallel analysis of the hybridized signals enabled us to get an expression profile of genes in which about 500 genes were differentially expressed in the paired liver tumor tissues. We identified 4 genes, the expression of three (Beclin 1, RbAp48 and Pir51) were increased and one (aldolase b) was decreased in liver tumor tissues. In addition, the expression of these genes in 6 hepatoma cell lines was also showed by RT-PCR analysis. CONCLUSION: cDNA microarray permits a high throughput identification of changes in gene expression. The genes encoding Beclin 1, RbAp48, Pir51 and aldolase b are first reported that may be related with hepatocarcinoma.

Complete sequence of the 10.3 kb silkworm Attacus ricini rDNA repeat, determination of the transcriptional initiation site and functional analysis of the intergenic spacer.

A complete sequence of the 10.3 kb silkworm rDNA repeat in Attacus ricini (also called as Samia cynthia ricini) was first determined here, and sequences coding for 18S, 5.8S and 28S were also mapped to rDNA unit. The precise transcription initiation site was determined by primer extension experiment. Intergenic spacer (IGS) in the length of 2670 bp in which transcription of 45s rRNA precursor starts was found to contain three distinct AT-rich (A + T > 75%) regions. The AT-richest region (from -320 to -3, A + T > 83%) is just in front of the transcription initiation site. Many short sequence homologies were also found to exist in the transcription initiation region. It clearly demonstrates that the nucleotide sequence of initiation region of rRNA shows great diversity among different species, but the general functional architectures show some conservation. Analyzing the IGS region of A. ricini may provide new insights into the mechanisms of the expression regulation of RNA polymerase I contributed genes.

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.

Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family.

DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40 kDa caspase-activated nuclease (DFF40) and a 45 kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3alpha, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3alpha exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3alpha isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

HCRP1, a novel gene that is downregulated in hepatocellular carcinoma, encodes a growth-inhibitory protein.

One of the most frequent allelic deletions in hepatocellular carcinoma (HCC) has been found at chromosome 8p21-23. We reported here the identification and characterization of a novel gene for a hepatocellular carcinoma related protein 1 (HCRP1) localized at 8p22, which was isolated by positional candidate cloning. The expression of the gene for HCRP1 was most abundant in normal human liver tissue and significantly reduced or undetected in HCC tissues. The analysis of subcellular distribution showed that HCRP1 diffused in the cytoplasm with a significant fraction accumulated in the nuclei. After introduction of the sense and antisense cDNA of HCRP1 into HCC cell line SMMC-7721, we observed that the overexpression of HCRP1 significantly inhibited both anchorage-dependent and anchorage-independent cell growth in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of HCRP1 gene in the other HCC cell line BEL-7404 resulted in the cell growth greatly enhanced. Moreover, reduction of the HCRP1 gene expression could also elevate the invasive ability of BEL-7404 cells. Our results strongly suggest that HCRP1 might be a growth inhibitory protein and associated with decreasing the invasion of HCC cells.

Human MCRS2, a cell-cycle-dependent protein, associates with LPTS/PinX1 and reduces the telomere length.

Human LPTS/PinX1 is a telomerase-inhibitory protein, which binds to the telomere protein Pin2/TRF1 and the catalytic subunit hTERT of telomerase. To explore the proteins that might be involved in the telomerase pathway, we performed a yeast two-hybrid screening with LPTS/PinX1 as the bait. A novel gene, MCRS2, encoding for an isoform of MCRS1/p78 and MSP58 was isolated. The expression of MCRS2 protein is cell-cycle dependent, accumulating in the very early S phase. MCRS2 interacts with LPTS/PinX1 in vitro, in vivo and colocalizes with LPTS/PinX1 in cells. MCRS2 and its amino terminus inhibit telomerase activity in vitro and long-term overexpression of MCRS2 in SMMC-7721 cells results in a gradual and progressive shortening of telomeres. Our findings suggest that MCRS2 might be a linker between telomere maintenance and cell-cycle regulation.

High-level expression and secretion of recombinant mouse endostatin by Escherichia coli.

The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase.