A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines

Schäfer, Hubert; Schäfer, A; Kiderlen, AF; Kn, Masihi; Bürger, Reinhard

doi:10.1016/s0022-1759(97)00040-9

Cited by 45 publications

(45 citation statements)

References 18 publications

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“…Expression of enzymes not naturally occurring in mammalian cells, such as firefly luciferase, allows highly sensitive detection and a large dynamic range. 17 Current luciferase detection assays such as the Promega Bright-Glo Luciferase Assay System can detect concentrations of luciferase as low as 10 À20 mol. 31 Furthermore, as luciferase has only an approximate 30 min half-life in growth medium, 17 its assay should provide a measure that is temporally precise.…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study, we measured firefly luciferase released from human cancer cell spheroids to quantify tumor cell killing by a replicative Ad with a luciferase transgene expression cassette. 15 As Ads kill infected cells by effecting membrane disruption, detection of intracellular luciferase protein 17 indicates cellular membrane disruption and consequent cell death. This method, however, can only test CRAds that incorporate the luciferase gene, but clinical therapeutic agents rarely accommodate such reporter genes.…”

Section: Introductionmentioning

confidence: 99%

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A three-dimensional assay for measurement of viral-induced oncolysis

et al. 2007

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Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/ 3-D24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-D24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A three-dimensional assay for measurement of viral-induced oncolysis

et al. 2007

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“…2A). Moreover, caspase-1 autoactivation also results in the induction of cell death, which can be assayed by the release of cotransfected ␤-galactosidase into the medium (31,32). Additional coexpression of YopE WT , but not of the catalytically inactive mutant YopE M , inhibited the autocatalytic processing of procaspase-1 as well as the proteolytic maturation of proIL-1␤.…”

Section: Specific Yop Effector Proteins and Rho Gtpasesmentioning

confidence: 99%

Targeting Rac1 by the Yersinia Effector Protein YopE Inhibits Caspase-1-mediated Maturation and Release of Interleukin-1β

Schotte

Denecker

Broeke

et al. 2004

Journal of Biological Chemistry

127

124

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“…Since luciferase is an intracellular protein, it is expected to be present in the supernatant only if its producer cells undergo lysis 37,57 as they do during Ad replication. In addition, when luciferase is expressed from a nonreplicating vector, maximum production is observed between 24 and 72 hours.…”

Section: Discussionmentioning

confidence: 99%

“…Supernatant luciferase has been measured to quantify oncolysis. 37 We employed this assay using Bright-Glo Luciferase Assay Reagent until all luminometer readings reached baseline. Mock infection measurements were subtracted from the readings of the viral-infected groups.…”

Section: Immunostaining Of Tumor Primary Spheroidsmentioning

confidence: 99%

Replication of an integrin targeted conditionally replicating adenovirus on primary ovarian cancer spheroids

et al. 2003

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Replication competent viruses hold promise for treatment of advanced cancers resistant to available therapeutic modalities. Although preliminary clinical results have substantiated their efficacy, preclinical development of these novel approaches is limited by assay substrates. The evaluation of candidate agents could be confounded by differences between primary tumor cells and tumor cell lines, as discordance in the levels of surface receptors relevant for viral entry has been reported. Since primary tumor cells are difficult to analyze ex vivo for longitudinal observation of virus replication, we developed three-dimensional aggregates or spheroids of unpassaged and purified ovarian cancer cells as a means for prolonging primary tumor cell viability and as a threedimensional in vitro model for replicative viral infection. Ovarian cancer cells purified from ascites samples were sustained for 30 days while retaining the infection profile with tropism modified and unmodified adenoviruses (Ads). Cell line and primary cell spheroids were used to quantitate the replication and oncolytic potency of replicative Ads in preclinical testing for human ovarian cancer trials. Therefore, spheroids provide a method to sustain purified unpassaged primary ovarian cancer cells for extended periods and to allow evaluation of replicative viruses in a three-dimensional model.

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A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines

Cited by 45 publications

References 18 publications

A three-dimensional assay for measurement of viral-induced oncolysis

A three-dimensional assay for measurement of viral-induced oncolysis

Targeting Rac1 by the Yersinia Effector Protein YopE Inhibits Caspase-1-mediated Maturation and Release of Interleukin-1β

Replication of an integrin targeted conditionally replicating adenovirus on primary ovarian cancer spheroids

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