Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector

Brightwell, Gale; Poirier, Véronique; Cole, Emily; Ivins, Sarah; Brown, Keith

doi:10.1016/s0378-1119(97)00178-9

Cited by 44 publications

(32 citation statements)

References 15 publications

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“…The 143B cell line was transfected under the control of the CMV promoter; whereas MNNG/HOS was transfected under control of EF1α, since the CMV promoter did not result in detectable luciferase activity in MNNG/HOS. The activity of the CMV promoter depends on the cell cycle stage and medium composition [41]. It was shown that changes in the CMV promoter activity, induced by DNA methylation and histone deacetylation, can silence the gene expression and decrease the signal intensity during in vivo imaging [42].…”

Section: Discussionmentioning

confidence: 99%

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi

Welling

Que

Henriquez

et al. 2007

Eur J Nucl Med Mol Imaging

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Purpose The purpose of this work was the development of an orthotopic model of osteosarcoma based on luciferaseexpressing tumour cells for the in vivo imaging of multidrug resistance (MDR) with 99m Tc-sestamibi. Methods Doxorubicin-sensitive (143B-luc + ) and resistant (MNNG/HOS-luc + ) osteosarcoma cell lines expressing different levels of P-glycoprotein and carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by bioluminescence imaging and X-ray. After tumour growth, a 99m Tcsestamibi dynamic study was performed. A subset of animals was pre-treated with an MDR inhibitor (PSC833). Images were analysed for calculation of 99m Tc-sestamibi washout half-life (t 1/2 ), percentage washout rate (%WR) and tumour/non-tumour (T/NT) ratio.Results A progressively increasing bioluminescent signal was detected in the proximal tibia after 2 weeks. The t 1/2 of 99m Tc-sestamibi was significantly shorter (p<0.05) in drugresistant MNNG/HOS-luc + tumours (t 1/2 =87.3±15.7 min) than in drug-sensitive 143B-luc + tumours (t 1/2 =161.0±47.4 min) and decreased significantly with PSC833 (t 1/2 =173.0± 24.5 min, p<0.05). No significant effects of PSC833 were observed in 143B-luc + tumours. The T/NT ratio was significantly lower (p<0.05) in MNNG/HOS-luc + tumours than in 143B-luc + tumours at early (1.55±0.22 vs 2.14± 0.36) and delayed times (1.12±0.11 vs 1.62±0.33). PSC833 had no significant effects on the T/NT ratios of either tumour. Conclusion The orthotopic injection of tumour cells provides an animal model suitable for functional imaging of MDR. In vivo bioluminescence imaging allows the non-invasive monitoring of tumour growth. The kinetic analysis of 99m Tc-sestamibi washout provides information on the functional activity of MDR related to P-glycoprotein expression and its pharmacological inhibition in osteosarcoma.

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Section: Discussionmentioning

confidence: 99%

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi

Welling

Que

Henriquez

et al. 2007

Eur J Nucl Med Mol Imaging

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“…This reduction in GFP expression could be the result of altered or blocked transcription from the CMV immediate-early promoter that drives the GFP cDNA. 13 Stimulation of infected quiescent cells with growth factors may induce transcription factors like NF-kB, which normally reside in the cytoplasm during quiescence to translocate to the nucleus to activate transcription of target genes. 14 dl01/07 was attenuated in both quiescent and proliferating normal cells, confirming previous findings.…”

Section: Discussionmentioning

confidence: 99%

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

Vaillancourt¹,

Atencio²,

Quijano³

et al. 2005

Cancer Gene Ther

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Cultured primary human cells have been widely used to assess the selectivity of oncolytic viruses as potential anticancer agents. As culture conditions can potentially have a significant impact on virus replication and ultimately cell killing, we evaluated the effects of dl309, a wild-type adenovirus, and dl01/07, a conditionally replicating adenovirus mutant, on quiescent and proliferating primary mammary epithelial cells. When primary cells were induced into quiescence, both viruses exhibited similar attenuated cell killing. However, cell killing by dl309 was superior to dl01/07 in proliferating primary cells. Analysis of viral effects at the level of entry, E2F activation, DNA replication, and late gene expression indicated that attenuation of dl309 in quiescent cells correlated with decreased expression of viral late genes such as hexon. In contrast, attenuation of dl01/07 in quiescent cells correlated with inefficient induction of E2F activity and inability to undergo efficient DNA replication. In proliferating cells, dl309 replicated efficiently, whereas dl01/07 still showed attenuated replication. In summary, our results indicate the intrinsic preference of wildtype adenoviruses for killing proliferating cells, which is an attractive feature for using adenoviruses as oncolytic agents. These results also highlight the need for the use of appropriate growth conditions for primary cells in vitro to distinguish subtle differences in cell killing among various oncolytic viruses. Cancer Gene Therapy (2005) R eplication-competent adenoviruses are currently being evaluated as novel anticancer agents. A number of modifications in the genome of the adenovirus have been reported to result in viruses that are capable of selectively replicating in and killing cancer cells. 1,2 Based on their ability to selectively kill tumor cells in cell culture and antitumor activity in xenograft tumor models, a few of these engineered viruses are currently being evaluated as oncolytic agents in the clinic. 3 As human adenoviruses do not replicate well in nonhuman cells, it has not been possible to study toxicity related to replication in nontarget tissues in preclinical animal models. In the absence of suitable in vivo models, analysis of selective replication is widely performed by studying these viruses for their effect in vitro on tumor cells lines and primary nontransformed cells. 4 The effect of oncolytic viruses in vitro has been determined at the level of viral DNA replication, late gene expression, induction of cell death, and production of progeny virion. 5 In the in vitro assays, whether cells are rapidly growing or arrested can potentially have a significant effect on virus replication and ultimately cell killing.In the present study, we sought to compare the effect of the wild-type adenovirus and a mutant that was previously reported to replicate preferentially in tumor cells for their effects in proliferating and quiescent normal cells. Our results show that in vitro, replication competent adenoviruses including wild ...

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“…To determine the subcellular localization of the noncoding WT1-AS RNAs, the WT1/WT1-AS-expressing cell line 7.92 (Brightwell et al 1997) was fractionated into nuclear and cytoplasmic compartments and RNA expression assayed in each fraction by real-time RT-PCR. Successful separation of nucleus and cytoplasm was demonstrated by the presence of higher molecular weight, unprocessed ribosomal RNA in only the nuclear fraction, and by the localization of unspliced WT1 pre-mRNA predominantly in the nuclear fraction (Fig.…”

Section: Wt1-as Transcripts Are Transported Into the Cytoplasm And Fomentioning

confidence: 99%

Alternately spliced WT1 antisense transcripts interact with WT1 sense RNA and show epigenetic and splicing defects in cancer

Dallosso¹,

Hancock²,

Malik³

et al. 2007

RNA

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Many mammalian genes contain overlapping antisense RNAs, but the functions and mechanisms of action of these transcripts are mostly unknown. WT1 is a well-characterized developmental gene that is mutated in Wilms' tumor (WT) and acute myeloid leukaemia (AML) and has an antisense transcript (WT1-AS), which we have previously found to regulate WT1 protein levels. In this study, we show that WT1-AS is present in multiple spliceoforms that are usually expressed in parallel with WT1 RNA in human and mouse tissues. We demonstrate that the expression of WT1-AS correlates with methylation of the antisense regulatory region (ARR) in WT1 intron 1, displaying imprinted monoallelic expression in normal kidney and loss of imprinting in WT. However, we find no evidence for imprinting of mouse Wt1-as. WT1-AS transcripts are exported into the cytoplasm and form heteroduplexes with WT1 mRNA in the overlapping region in WT1 exon 1. In AML, there is often abnormal splicing of WT1-AS, which may play a role in the development of this malignancy. These results show that WT1 encodes conserved antisense RNAs that may have an important regulatory role in WT1 expression via RNA:RNA interactions, and which can become deregulated by a variety of mechanisms in cancer.

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Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector

Cited by 44 publications

References 15 publications

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

Alternately spliced WT1 antisense transcripts interact with WT1 sense RNA and show epigenetic and splicing defects in cancer

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