Immortalization of Human Adult Normal Prostatic Epithelial Cells by Liposomes Containing Large T-SV40 Gene

Cussenot, Olivier; Berthon, Ph.; Berger, Roland; Mowszowicz, Irène; Faille, A; Hojman, F; Teillac, P.; Duc, A. Le; Calvo, Fabien

doi:10.1016/s0022-5347(17)37953-3

Cited by 130 publications

(101 citation statements)

References 13 publications

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“…The SV40-immortalized human prostate epithelial cell line PNT1A was previously established and characterized. 40 (a) Cell growth of PNT1A cells with standard RPMI culture medium supplemented with 10% FBS and 1% L-glutamine (&), serum-free, low-Ca 2 þ KSFM culture medium supplemented with epidermal growth factor (EGF, 5 ng/ ml), bovine pituitary extract (BPE, 50 mg/ml) and 1% L-glutamine (n) or KSFM culture medium supplemented with either 1.8 mM Ca 2 þ (}) or 1 mM z-VAD-fmk (J). Cell proliferation was assessed using the crystal violet dye method.…”

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confidence: 99%

Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

et al. 2004

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Clusterin (CLU) is a secreted heterodimeric glycoprotein thatcan be produced almost ubiquitously in mammalians tissues.1Its gene expression is subjected to complex regulation and canchange enormously according to different stimuli.2 Cloned andidentified as the most potently induced gene in the regressingrat ventral prostate following androgen-ablation,3 CLU wasalmost simultaneously characterized and isolated by differentresearch groups working in widely divergent areas.2 CLU iscoded by a single copy gene, located on chromosome 8.4 Thegene codes for an initial precursor peptide glycosylated andcleaved into two a and b chains of 40 kDa each, held togetherby a unique five disulfide bond motif in the extracellular matureform.1 This secreted form of CLU has been suggested to act asa molecular chaperone following stress-induced injury,5clearing extracellular debris.6 However, it has been reportedthe existence of an inactive, cytoplasmic form of CLUproduced by alternative splicing that is converted by ionizingirradiation to a truncated mature nuclear isoform,7 which bindsthe Ku70/Ku80 complex in cell-free systems8 inhibiting cellgrowth and survival7 probably by a caspase-3-independentmechanism.9 Other alternative CLU isoforms, produced eitherby exon skipping10 or by post-translational modificationsactivated by apoptosis,11,12 were recently described. Thesedifferent isoforms of CLU have been suggested to beantiapoptotic6,13 or proapoptotic.7,10,12,14–16These controversial reports on the role of CLU might berelated to specific proteomic profiles that are produced bydifferent apoptotic stimuli (i.e. the general protein pattern ofCLU and the relative ratio between different CLU isoforms).This might explain why CLU has been involved in a plethora ofpathophysiological processes, including cell–cell and cell–matrix adhesion, cell differentiation, transformation, aging17,18and cancer,19 but its biological role still remains to be clearlyestablished. Reports suggesting that CLU may be a potentialtumor suppressor gene include the finding that CLU suppressesNF-kB activity and the metastatic phenotype ofneuroblastoma cells.20 We have previously reported that CLUoverexpression inhibits cell cycle progression of simian virus40(SV40)-immortalized human prostate PNT2 and PNT1Aepithelial cells.21To further assess the role of CLU in apoptotic processes wehave studied its expression pattern during the regulation ofcalcium homeostasis. Ca2þ is an important regulator ofapoptosis and cell survival.22,23,24 Both pathological increaseof Ca2þ concentration in the cytosol compartment byinophores25 and depletion of intracellular Ca2þ stores maytrigger apoptosis by disrupting intracellular architecture andallowing effectors to gain access to their substrates.24,26,27Activation of apoptotic endonucleases28 eliciting DNA cleavageand chromatin condensation has beenwell documented.29,30,31A tight buffering of intracellular Ca2þ is required for normalgrowth. In fact, apoptosis can be induced by Ca2þ mobilizationfrom intracellular pools,23,24,27 chelati...

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Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

et al. 2004

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“…To investigate the potential of targeting polymer-coated adenovirus vectors to a6b1 integrins on prostate cells using the -SIKVAV-peptide, a range of cell lines were tested representing various stages of cancer progression ranging from the SV40T antigen immortalized normal epithelia (PNT1a and PNT2), [27][28][29] through the P4E6 cell line derived from a well-differentiated tumor, 30,31 to the commonly used PC-3 and LNCaP lines derived from bone and supraclavicular lymph node metastases, respectively. Our analysis of the expression of the a6 and b1 integrin subunits in PC-3 and LNCaP cells was in agreement with previous studies, 13,14 with the reduced a6 integrin expression on LNCaP cells reflected in their semiadherent growth pattern on laminin-coated surfaces.…”

Section: Discussionmentioning

confidence: 99%

“…90112714), PNT1a and PNT2-C2 normal human prostate glandular epithelial cells immortalized with SV40, [27][28][29] and 293 adenovirus transformed human embryo kidney cells (ATCC No. CRL-1573), were each maintained in Dulbecco's minimal essential medium (DMEM), 10% foetal calf serum (FCS), 2 mM glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin (Invitrogen, Paisley, UK).…”

Section: Cellsmentioning

confidence: 99%

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via α6-integrins

Stevenson

Hale

et al. 2007

Cancer Gene Ther

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Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via a6b1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an a6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV-peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV-peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-a6 integrin and anti-b1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased a6b1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.

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“…pHPMA-ONp coating of CELO virus [55][56][57] SKOV-3 human metastatic ovarian adenocarcinoma cells (ATCC No. HTB-77) and primary chick embryo fibroblasts were each maintained in Dulbecco's minimal essential medium (DMEM) containing 10% fetal calf serum (fcs), 2 mM glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin (GibCoBRL).…”

Section: Retargeting Of Celo Virus M Stevenson Et Almentioning

confidence: 99%

Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells

et al. 2005

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Immortalization of Human Adult Normal Prostatic Epithelial Cells by Liposomes Containing Large T-SV40 Gene

Cited by 130 publications

References 13 publications

Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via α6-integrins

Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells

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