TPP1 Expression Research Articles

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

Abstract LB096: Telomere length as a predictor of therapy response and survival in patients diagnosed with ovarian carcinoma

Abstract Impaired telomere length (TL) maintenance in ovarian tissue may play a pivotal role in epithelial ovarian carcinoma (OvC) onset. TL in either target or surrogate tissue (blood) is currently being investigated as a predictor in anti-OvC therapy or as a biomarker of the disease progression, respectively. There is presently an urgent need for an appropriate approach to chemotherapy response prediction. We performed a monochrome multiplex qPCR measurement of TL in peripheral blood and tumor tissues of 209 OvC patients. Methylation status and gene expression of shelterin complex and telomerase catalytic subunit (hTERT) were determined within tumor tissues by High-Throughput DNA methylation profiling and RNA sequencing (RNA-Seq) analysis, respectively. The patients sensitive to cancer treatment (n=46) had shorter telomeres in peripheral blood cells compared to those treatment-resistant (n=93; P=0.037). Cancer tissue TL lower than the median predisposed to better overall survival (P=0.002). Gene expression of TPP1, which recruits telomerase to telomeres, was positively associated with TL in tumor tissue (P=0.026). TL measured in peripheral blood cells could serve as a marker of platinum therapy response in OvC patients. Additionally, TL determined in tumor tissue provides information on OvC patients' overall survival. The study was financed by the project NPO (LX22NPO05102), the Czech Health Research Council (NU21-03-00145), and the Czech Science Foundation (21-27902S). Citation Format: Kristyna Tomasova, Karolina Seborova, Michal Kroupa, Josef Horak, Ludmila Vodickova, Lukas Rob, Martin Hruda, Marcela Mrhalova, Alena Bartakova, Jiri Bouda, Thomas Fleischer, Vessela Kristensen, Pavel Vodicka, Radka Vaclavikova. Telomere length as a predictor of therapy response and survival in patients diagnosed with ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB096.

TPP1 promoter mutations cooperate with TERT promoter mutations to lengthen telomeres in melanoma.

Overcoming replicative senescence is an essential step during oncogenesis, and the reactivation of <i>TERT</i> through promoter mutations is a common mechanism. <i>TERT</i> promoter mutations are acquired in about 75% of melanomas but are not sufficient to maintain telomeres, suggesting that additional mutations are required. We identified a cluster of variants in the promoter of <i>ACD</i> encoding the shelterin component TPP1. <i>ACD</i> promoter variants are present in about 5% of cutaneous melanoma and co-occur with <i>TERT</i> promoter mutations. The two most common somatic variants create or modify binding sites for E-twenty-six (ETS) transcription factors, similar to mutations in the <i>TERT</i> promoter. The variants increase the expression of <i>TPP1</i> and function together with TERT to synergistically lengthen telomeres. Our findings suggest that <i>TPP1</i> promoter variants collaborate with <i>TERT</i> activation to enhance telomere maintenance and immortalization in melanoma.

Dihydroartemisinin inhibits HepG2.2.15 proliferation by inducing cellular senescence and autophagy

Dihydroartemisinin (DHA) has been reported to possess anti-cancer activity against many cancers. However, the pharmacologic effect of DHA on HBV-positive hepatocellular carcinoma (HCC) remains unknown. Thus, the objective of the present study was to determine whether DHA could inhibit the proliferation of HepG2.2.15 cells and uncover the underlying mechanisms involved in the effect of DHA on HepG2.2.15 cells. We found that DHA effectively inhibited HepG2.2.15 HCC cell proliferation both in vivo and in vitro. DHA also reduced the migration and tumorigenicity capacity of HepG2.2.15 cells. Regarding the underlying mechanisms, results showed that DHA induced cellular senescence by up-regulating expression levels of proteins such as p-ATM, p-ATR, γ-H2AX, P53, and P21 involved in DNA damage response. DHA also induced autophagy (green LC3 puncta gathered together and LC3II/LC3I ratio increased through AKT-mTOR pathway suppression). Results also revealed that DHA-induced autophagy was not linked to senescence or cell death. TPP1 (telomere shelterin) overexpression could not rescue DHA-induced anticancer activity (cell proliferation). Moreover, DHA down-regulated TPP1 expression. Gene knockdown of TPP1 caused similar phenotypes and mechanisms as DHA induced phenotypes and mechanisms in HepG2.2.15 cells. These results demonstrate that DHA might inhibit HepG2.2.15 cells proliferation through inducing cellular senescence and autophagy.

Global Brain Transcriptome Analysis of a Tpp1 Neuronal Ceroid Lipofuscinoses Mouse Model.

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.

Abstract 341: The role of telomere protective protein TPP1 in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a worldwide malignancy. To date, surgery is still the most effective treatment with curative potential, but only about 20% of patients with HCC are eligible for surgical intervention. Therefore exploring the pathogenesis of liver cancer, looking for molecules targeting therapy, is very important. Telomeres, the chromosome extremities, are protected by specific proteins, such as POT1, RAP1, TIN2, TRF1, TRF2,and TPP1. More and more findings have discovered that TPP1 plays an important role in the tumorigenesis. However, the mechanism of TPP1 in HCC remains unknown. The purpose of this study is to explore the role of TPP1 in HCC. Currently 42 cases of HCC patients were recruited in this study. First, the patients' tumor tissue, adjacent tissue and normal tissue were analyzed by Western blot. The results showed that TPP1 expression in patients' adjacent tissue and normal tissue was lower than that in tumor tissue, respectively (P&amp;lt;0.05; P&amp;lt;0.05). Furthermore, investigation on the protein expression levels of TPP1 with clinicopathologic characteristics discovered that the higher TPP1 in patients' tumor tissue was correlated with the tumor clinical stages, demonstrating that those patients who were in earlier stages (I + II) showed lower TPP1 protein levels than those in later or advanced stages (III + IV) (P&amp;lt;0.05). More importantly, the survival analysis showed that the overall survival was significantly shorter in patients with high TPP1 expression than in others (P&amp;lt;0.05). To further study the role of telomere protective protein TPP1 in HCC, we used lentiviral shRNA embedding TPP1 to knock down the expression of TPP1 in HepG2 and SK-Hep1 cells. The cell growth curve results showed that knockdown of TPP1 could inhibit the growth and proliferation of the above cells. Meanwhile we found that the migration and invasion abilities of the cells were significantly reduced by knocking down the expression of TPP1 compared with the cells transfected with the control lentiviral shRNA. Through scratch test and cell invasion transwell chamber test, TPP1 depletion dramatically suppressed the colony formation on soft agar, which indicated that TPP1 loss could reduce the transformation ability of HCC. More interestingly, we found that ATM pathway was activated after knockdown of TPP1, resulting in γ-H2AX and phosphorylated ATM(p-ATM) upregulation. On the other hand, removal of TPP1 initiated mTOR pathway activation, which resulted in phosphorylated mTOR (p-mTOR) augmentation, followed by the increase of p70S6K and 4EBP1 phosphorylation. Eventually the autophagy marker LC3 I/II conversion and p62 declined obviously by depletion of TPP1 in HepG2 and SK-Hep1 cells. Taken together, TPP1 could be a potential predictor of hepatocellular carcinoma staging and overall survival, and targeting TPP1 might be a therapeutic approach for HCC. Citation Format: Jiang Zou, Ru Sun, Jingruo Xia, Dan Xiao, Chang Liu, Hebin Liao, Lei Xu, Qiang Ma, Yan Cai, Jingdong Li, Xiaolan Guo. The role of telomere protective protein TPP1 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 341.

Abstract 5745: Study on telomere shelterin component TPP1 in esophageal squamous cell carcinoma

Abstract Esophageal cancer (EC) is one of the most aggressive cancer and ranks the sixth leading cause of cancer-related mortality worldwide, approximately 70% of global esophageal carcinoma cases occur in China and esophageal squamous cell carcinoma (ESCC) is the most frequent histological subtype of EC. Lacking of reliable early diagnostic markers and efficient medical and surgical treatment approaches, the five-year survival rate of ESCC is only about 4.4%.Therefore exploration of the genetic basis and the affiliated molecular mechanisms in ESCC could be beneficial to improve the diagnosis, therapy, and prevention of this disease. As an important telomere binding protein, TPP1 protects the ends of telomeres and maintains the genomic stability and integrity of chromosomes in eukaryotes. Previous investigation discovered that TPP1 might play an important role in the carcinogenesis and progression of some cancers. However, whether and/or how TPP1 participates in ESCC remains unclear. Here TPP1 was studied in 65 ESCC patients’ specimens including tumor tissue, the adjacent tissue, and the peripheral blood leukocytes, also the ESCC cell lines were employed to further explore the molecular mechanisms of TPP1 involved in the tumorigenesis of ESCC. In this study, TPP1 mRNA and protein were measured in the ESCC tissue specimens, peripheral blood leukocytes and also in ESCC cell lines using real time RT-PCR and Western Blot. The results showed that TPP1 mRNA level was significantly higher in ESCC tumor tissues compared with the adjacent tissues (P&amp;lt;0.05); the same trend was observed in ESCC cells (Eca109, Kyse150) vs human esophageal epithelial cells (HET-1A), respectively. However, in peripheral blood leukocytes, the expression of TPP1 did not show any difference between the cancer patients and the healthy controls. Among the 65 ESCC patients, TPP1 protein expression in 46 patients was obvious higher in tumor tissues than that in the matched adjacent tissues, although there were 19 patients in exception. Moreover, the TPP1 expression was correlated with the lymph nodes metastasis and CYF2, which is one of the potential tumor markers for diagnosis of esophageal carcinoma. To further explore the potential biological function of TPP1 in ESCC, stably- transfecting shRNA-TPP1 (sh-TPP1) and shRNA-Negative Control (sh-NC) expression constructs in ESCC cell lines were performed to study TPP1’s roles in ESCC. The colongenic assay showed that depletion of TPP1 decreased ESCC cells proliferation and cell growth, and the wound healing experiment and cell invasion transwell chamber test demonstrated that down-regulation of TPP1 suppressed the cell migration and invasion capabilities in vitro. Taken together, our findings revealed that TPP1 might play crucial roles in the pathogenesis of ESCC and targeting TPP1 could be a valuable potential for ESCC early diagnosis, intervention and therapeutics. Note: This abstract was not presented at the meeting. Citation Format: Lihua Yao, Jiang Zou, Ru Sun, Jiajing Cai, Guangcheng Huang, Qiang Ma, Lei Xu, Mayong Fu, Xiaolan Guo. Study on telomere shelterin component TPP1 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5745. doi:10.1158/1538-7445.AM2017-5745

Metabolic correlation between polyol and energy-storing carbohydrate under osmotic and oxidative stress condition in Moniliella megachiliensis

Moniliella megachiliensis, the osmo-tolerant basidiomycetous yeast was found to accumulate intracellularly energy-storing carbohydrates (trehalose and glycogen) along with polyols (glycerol and erythritol) up to stationary growth phase. In trehalose-loaded cell, osmotic-stress resulted in the rapid generation of glycerol, and oxidative stress with menadione resulted in the rapid generation of erythritol. Under either of these conditions, the levels of the energy-storing carbohydrates were depleted, while little glucose uptake was observed. These results suggested that the intracellular pools of trehalose and glycogen were rapidly converted to glycerol in response to osmotic stress, and to erythritol in response to oxidative stress and altered redox balance. Expression of tps1 encoding trehalose synthetic enzymes paralleled trehalose accumulation in the cell during the culture in 2% glucose, in contrast, expression of tpp1 or tpp2 encoding trehalose-6-phosphate phosphatase was little increased under the same condition. Expression of tre (tre1/tre2) encoding trehalose hydrolase (trehalase) increased with time associated with depletion of trehalose during oxidative stress. From these results, we concluded that glycerol and erythritol, the compatible solutes in M.megachiliensis were metabolically interrelated to energy-storing carbohydrates such as trehalose or glycogen during conditions of osmotic or oxidative stress.

SIRT1 ameliorates age-related senescence of mesenchymal stem cells via modulating telomere shelterin.

Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD+-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.

Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line

Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined.

Telomere-Binding Protein TPP1 Modulates Telomere Homeostasis and Confers Radioresistance to Human Colorectal Cancer Cells

BackgroundRadiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear.Principal FindingsIn this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation.ConclusionsWe demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer.

Effect of trastuzumab on telomere-related proteins and the relationship to the sensitivity of HER2-amplified human gastric cancer cells to oxaliplatin and cisplatin.

53 Background: HER2 amplification occurs in about 20% of gastric cancers, and trastuzumab in combination with cisplatin based chemotherapy has been reported to improve oncological outcomes in gastric and gastro-oesophageal junction cancer with HER2 gene amplification. The aim of this study was to evaluate the potentially useful combined antitumor efficacy of trastuzumab and platinum agents in gastric cancer cells and to elucidate further the mechanisms possibly involved in the interaction between the trastuzumab and platinum agents. Methods: Gene expression was determined by using real-time quantitative PCR in gastric cancer cell lines. The chemosensitivity of gastric cancer cells to platinum agents and the apoptotic effect of drugs in vitro were evaluated using cellTiter 96 Aqueous One Solution Cell Proliferation Assay kit and double staining with both Annexin-V-FITC and PI, respectively. Results: Treatment with 1.0μg/ml trastuzumab for 48h could significantly increase sensitivity of oxaliplatin or cisplatin in HER2 amplified gastric cancer cells, and the IC50 of oxaliplatin and cisplatin were reduced to about 3.29 times and 6.91 times, respectively. Apoptosis analysis also indicated that trastuzumab significantly increased both oxaliplatin and cisplatin-induced apoptosis in NCI-N87 cells. Analysis of telomere-related genes revealed that trastuzumab singly and pretreatment with trastuzumab for 48h followed by oxaliplatin or cisplatin for another 48h could significantly downregulate the mRNA expression of TPP1, TRF1, TRF2, TRF2IP, POT1 and TIN2 genes. Conclusions: Our results describe the potential role of low dose trastuzumab to increase sensitivity of oxaliplatin and cisplatin in HER2 amplified gastric cancer cells, which may be partially through downregulating the expression levels of telomere-related genes.

Altered expression of TPP1 in fibroblast-like synovial cells might be involved in the pathogenesis of rheumatoid arthritis

We undertook this study to determine whether the altered expression of telomeric proteins TPP1 and POT1 in fibroblast-like synovial cells (FLS) could provide insights into the pathogenesis of rheumatoid arthritis (RA). FLS were isolated from patients with RA, osteoarthritis (OA) and traumatic joint disease, and cultured in vitro. TPP1 and POT1 mRNA level of FLS were measured using real-time quantitative polymerase chain reaction (RT-qPCR) in 42 RA, 23 OA and 13 healthy cases. Immunofluorescence staining and Western blot were used to detect the expression of TPP1 and POT1 protein. Expression of TPP1 and POT1 mRNA was significantly reduced in RA cases (P < 0.001, respectively), and no significant difference was observed between OA and healthy cases (P > 0.05, respectively). Confocal microscopy images showed TPP1 and POT1 proteins mainly located in nucleus of FLS. Western blot demonstrated that TPP1 protein level was significantly reduced in RA cases (P < 0.001), and POT1 protein expression was not statistical significance among RA, OA patients and healthy cases (P > 0.05). Significant negative correlation was observed between level of TPP1 mRNA and titers of anti-CCP antibody (P < 0.001), RF (P < 0.01). Altered expression of TPP1 might contribute to persistent proliferation of FLS in RA, further study on functions of telomeric proteins in RA would be needed.

Changes in the expression of telomere maintenance genes might play a role in the pathogenesis of systemic lupus erythematosus

Previous studies demonstrated that telomerase activity increased while telomere length shortened in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE). This study aimed to examine the changes of telomere maintenance genes and their clinical significance in SLE. The mRNA level of telomeric proteins in PBMCs, including shelterin complex (TRF1, TRF2, POT1, TPP1, TIN2 and hRAP1), a set of multifunctional proteins involved in telomere maintenance (MRE11, KU80 and RPA1), and Ki67, was measured using real-time quantitative PCR in 56 SLE patients (36 treated and 20 untreated; 32 with renal involvement and 24 without renal involvement) and 46 healthy subjects (controls). The expression of TPP1, TIN2, POT1 and KU80 was significantly reduced while that of TRF2 and MRE11 increased in SLE patients (p < 0.05, respectively); significant difference was not found in the expression of TRF1, hRAP1, RPA1 and Ki67 (p > 0.05, respectively). The expression of TRF2, MRE11 and Ki67 was much higher in untreated SLE patients than in controls or treated SLE patients (p < 0.05, respectively); the expression of hRAP1 was much higher in SLE patients with renal involvement than in controls or SLE patients without renal involvement (p < 0.05, respectively). Significant positive correlation was found between level of KU80 and C3, TPP1 and TIN2, TPP1 and POT1, while significant negative correlation was found between KU80 and serum total globulins, TIN2 and RF, TPP1 and SLEDAI score (p < 0.05, respectively). In conclusion, altered expression of telomere maintenance genes might be involved in the pathogenesis of SLE. Further study in expression and functions of telomeric proteins would be needed.

Changes in the expression of telomere maintenance genes suggest global telomere dysfunction in B-chronic lymphocytic leukemia

In this study, we explored the telomeric changes that occur in B-chronic lymphocytic leukemia (B-CLL), in which telomere length has recently been demonstrated to be a powerful prognostic marker. We carried out a transcriptomic analysis of telomerase components (hTERT and DYSKERIN), shelterin proteins (TRF1, TRF2, hRAP1, TIN2, POT1, and TPP1), and a set of multifunctional proteins involved in telomere maintenance (hEST1A, MRE11, RAD50, Ku80, and RPA1) in peripheral B cells from 42 B-CLL patients and 20 healthy donors. We found that, in B-CLL cells, the expressions of hTERT, DYSKERIN, TRF1, hRAP1, POT1, hEST1A, MRE11, RAD50, and KU80 were more than 2-fold reduced (P < .001), contrasting with the higher expression of TPP1 and RPA1 (P < .001). This differential expression pattern suggests that both telomerase down-regulation and changes in telomeric proteins composition are involved in the pathogenesis of B-CLL.

TPP1 is a homologue of ciliate TEBP-β and interacts with POT1 to recruit telomerase

Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-alpha/beta heterodimer. Human telomeres contain duplex telomeric repeats with 3' single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-alpha homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-beta and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1-POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1-TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1-TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin-yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.

420. Tropism-Modified Adeno-Associated Virus Vector Mediates Targeting of Brain Vascular Endothelium In Vivo

Top of pageAbstract Adeno-associated virus (AAV) is an attractive vector for gene delivery because of its low toxicity and ability to express transgenes for many months. Collectively, the different serotypes of AAV show broad tissue and cell tropism. However, AAV does not preferentially transduce endothelia when administered in vivo. In our approaches to develop gene therapy for the CNS manifestations of the lysosomal storage diseases (mucopolysaccharidoses; [e.g. Beta-glucuronidase deficiency]) and the ceroid lipofuscinoses (e.g. CLN2 or tripeptidyl protease I [TPP-1] deficiency) endothelia remain an attractive target. Endothelial expression of TPP-1 or beta-glucuronidase results in both apical and basolateral secretion of enzyme; in the brain, this would allow exposure of recombinant enzyme to the underlying brain parenchyma. We used phage display to identify peptides that mediate selective and efficient binding to brain endothelial cells in vivo. Selected peptides were genetically incorporated into the AAV2 capsid, and peptide modified viruses (PM-AAVs) tested in vitro and in vivo. AAV2-ITR genomes were efficiently packaged into PM-AAVs and viral titers were comparable to those obtained with native AAV2 (5-10^11 vector genomes/ml). PM- and native-AAV2 vectors were subsequently injected intravenously via tail vein and vector genomes in various tissues quantified 28 days later. Approximately 10^4 more PM-AAV was present in brain relative to native AAV2. Moreover, while AAV2 genomes were predominantly found in liver, PM-AAV was highest in brain. Our primary data indicate that PM-AAV2 can be retargeted to brain by use of in vivo phage display-derived peptides. This strategy may prove valuable for correction of the CNS deficiencies of several fatal metabolic diseases.

92. Metabolically Biotinylated Adeno-Associated Virus (AAV) Types 1, 2, 3, 4 and 5 for Scalable Purification and Vector Retargeting

Adeno-associated virus (AAV) is an attractive vector for gene delivery because of its low toxicity and ability to express transgenes for many months. Collectively, the different serotypes of AAV show broad tissue and cell tropism. Despite this, AAV does not efficiently transduce endothelia. In our approaches to develop gene therapy for the CNS manifestations of the lysosomal storage diseases (mucopolysaccharidoses; [e.g., beta-glucuronidase deficiency]) and the ceroid lipofuscinoses (e.g., CLN2 or tripeptidyl protease I [TPP-1] deficiency) endothelia remain an attractive target. Endothelial expression of TPP-1 or beta-glucuronidase results in both apical and basolateral secretion of enzyme; in the brain, this would allow exposure of recombinant enzyme to the underlying brain parenchyma. As a first step in developing endothelial-targeted AAV, we panned, in vivo, for brain specific epitopes by phage display. Beta-Glucuronidase deficient and age matched wildtype littermates were used for panning. This was accomplished by canulating the internal carotid artery, injecting phage, and perfusing the mice 5 min later with DMEM. Phage were recovered by homogenizing the brain and incubating the homogenate with bacteria and culture media. After five rounds of panning we isolated a panel of targeting peptides that bound selectively and efficiently to brain endothelia. The motifs isolated from beta-glucuronidase deficient mice were distinct from those panned from heterozygous littermates. Selective peptides have been genetically incorporated into AAV2 VP1 capsid protein at amino acid position 587, a site previously shown to accommodate peptide insertion for directed gene transfer. Recombinant viruses will be assessed for specific delivery to brain relative to AAV2 with native VP1 capsid, in beta-glucuronidase and wildtype mice. Our studies provide possible strategies for correction of CNS deficiencies associated with metabolic diseases, by retargeting AAV2 to brain endothelia with motifs identified in vivo from phage display libraries.

Characterization of tpp1(+) as encoding a main trehalose-6P phosphatase in the fission yeast Schizosaccharomyces pombe.

We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase (tpp1(+)). Disruption of tpp1(+) caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40 degrees C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1(+) gene and containing a plasmid with tpp1(+) under the control of the thiamine-repressible promotor correlated with tpp1(+) repression. The level of tpp1(+) mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of tpp1(+) during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.