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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 24, 2005 as doi:10.1096/fj.05-4551fje. |
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,1
* The Molecular Imaging Program at Stanford (MIPS), The Department of Radiology and Bio-X Program, Stanford University;
The James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA;
The Department of Bioengineering, Stanford University; and
Department of Medicine, Division of Cardiology, Stanford University, Palo Alto, California, USA
1Correspondence: Stanford University School of Medicine, Edwards Bldg., R306 Stanford, CA 94305-5344, USA. E-mail: joewu{at}stanford.edu
SPECIFIC AIMS
To better understand stem cell biology and physiology in vivo, one must be able to track them noninvasively after transplant. Thus, the recent development of novel molecular imaging techniques for visualizing stem cell survival and proliferation in living subjects has attracted much attention. However, the effects of epigenetic modulation on reporter gene silencing have not been systemically addressed. In this study, we investigated the long-term stability of firefly luciferase expression in a stable embryonic rat cardiomyoblast cell line and tracked their survival in living subjects using bioluminescence imaging (BLI).
PRINCIPAL FINDINGS
1. Silencing of firefly luciferase gene expression is observed after repeated cell passages
After transfecting the rat H9c2 embryonic cardiomyoblast cell line with the pCMV-Fluc-SV40-neo plasmid, five single clones resistant to G418 were selected. To assess the stability of Fluc transgene expression, these clones were passaged serially for 3 months. The average firefly luciferase enzyme activity for all five clones at passage-25 (123±35) was only 28 ± 7% compared with passage-1 (647±155 relative light unit per microgram protein (RLU/µg)). [Henceforth, Fluc refers to firefly luciferase gene and FL to protein]. Clone number 3, denoted as H9c2-Fluc.3, had the strongest signal and was followed for an additional five months. The FL enzyme activity for this clone decreased significantly from passage-1 (843±28) to passage-20 (250±10) to passage-40 (44±3) to passage-60 (3±1) (P<0.05 vs. passage-1). At 8 months, the H9c2-Fluc.3 activity was only 0.01% compared with passage-1.
2. Rescue of firefly luciferase expression by DNA methylation inhibitor
We hypothesize that epigenetic modulation may be involved in silencing the Fluc reporter gene activity. Accordingly, we treated H9c2-Fluc.3 cells at passage-60 with 5-azacytidine (DNA methylation inhibitor), trichostatin A (histone deacetylation inhibitor), and retinoic acid (transcriptional activator) to rescue Fluc expression. Among these three, 5-azacytidine induced the highest level of FL activity. Treatment with trichostatin A also showed remarkable enhancement of FL activity with increasing drug dosages (P<0.05 for 200–800 nM vs. control). Retinoic acid had no significant induction of FL activity (P=N.S.). The relative ranges of 5-azacytidine that can induce Fluc gene expression without affecting H9c2 proliferation were 
50 µM of 5-azacytidine, 50 nM of trichostatin A, and 10 nM of retinoic acid. The Live/Dead cell viability assay showed similar patterns as the CyQuant cell proliferation assay (data not shown).
3. Dissecting the molecular mechanisms of reporter gene silencing
To assess whether the loss of FL activity was due to excessive DNA methylation of the cytomegalovirus (CMV) promoter, which can prevent binding of transcriptional factors, we treated H9c2-Fluc.3 cells at passage-60 with increasing concentrations of 5-azacytidine for 48 h. Then cell lysates were subjected to firefly luciferase enzyme assay, Western blot, and RT-PCR (Fig. 1
B, C). To assess the methylation status of the CMV promoter (position 437-463 and 562-588 on the sequence), DNA was treated with sodium bisulfite, which converts unmethylated cytosines to uracils (replicated as thymines after 2 rounds of PCR amplification), but methylated cytosines are not converted by bisulfite treatment and are replicated as cytosines during PCR. Therefore, a difference in methylation status is detected as a difference in DNA sequence. Increasing dosages of 5-azacytidine treatment led to a reduction in the degree of methylation at the 8 CpG sites examined within the CMV promoter (Fig. 1C
). These data suggest that 5-azacytidine acts by inhibiting DNA methyltransferase (DNMT) enzyme, which leads to more unmethylated CpG sites to allow better access of transcriptional factors to the CMV promoter, resulting in higher firefly luciferase mRNA, protein, and enzyme activity.
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4. Longitudinal BLI of H9c2-Fluc cells transplanted into skeletal muscles of living rats
To demonstrate that the reversal of reporter gene silencing can be maintained in vivo, one million H9c2-Fluc.3 cells at passage-60 were treated with 50 µM of 5-azacytidine for 48 h and then implanted into the left thigh of Spraque Dawley rats (n=10). The right thigh was injected with one million untreated H9c2-Fluc cells as control. Animals were imaged repetitively using D-luciferin as the reporter probe starting at 6 h after transplant (Fig. 2
A). On day 1, the BLI signal for treated cells was significantly higher compared with untreated cells (5.6x106±8.5x105 vs. 9.7x104±2.2x104 photons/s/cm2/sr; P<0.05). After 8 days, untreated cells implanted at the right thigh could not be readily distinguished from the background signal (
7000 photons/s/cm2/sr). By contrast, treated cells implanted at the left thigh showed a visible signal for up to 14 days (1.1x104±5.8x103 photons/s/cm2/sr). Because these animals were not immunosuppressed, there was gradual donor cell death within the first 2 weeks after cell transplantation in both legs, consistent with data from previous studies (Fig. 2B
). Postmortem histological analysis of both legs at 4 weeks did not identify any remaining cells (data not shown).
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CONCLUSIONS AND SIGNIFICANCE
Our major findings are as follows: 1) rat H9c2 embryonic cardiomyoblasts stably transfected with Fluc gradually lost their transgene expression over a span of 8 months; 2) the silenced gene expression could be reversed most impressively by a DNA methylation inhibitor (5-azacytidine), less by a histone deacetylase inhibitor (trichostatin A), and minimally by a transcriptional activator (retinoic acid); 3) the molecular mechanism of DNA methylation was validated by bisulfite sequencing as well as RT-PCR, Western, and enzyme assays; finally, 4) noninvasive BLI of living animals confirmed that 5-azacytidine treated H9c2-Fluc cells had significantly higher signal activity compared with untreated cells. Taken together, the data suggest that cellular control of exogenous transgene expression by epigenetic modulation can be reversed in vitro and extended to in vivo imaging.
There are 30,000 CpG islands in the mammalian genome of which 50–60% are found within the promoter region. DNA methylation plays critical roles in regulating gene expression during mammalian development, genomic imprinting, and tumorigenesis. It involves the addition of a methyl group to cytosine residues at CpG dinucleotides, which is catalyzed by DNMT enzymes. Afterward, proteins containing methylcytosine binding domain (MBD) targets to these methylated CpG sites, which then block the access of transcription factors. MBD proteins also recruit histone deacetylase enzymes (HDACs), which further cause a tighter packing of DNA. The promoter region becomes inaccessible to transcription factors, resulting in gene silencing. Because DNA methylation is potentially reversible, pharmacologic inhibitors provide a conceptually attractive approach for rescuing silenced gene expression (Fig. 3
).
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In conclusion, molecular imaging of reporter genes will continue to play an increasingly important role for monitoring stem cells noninvasively, repetitively, and quantitatively. Because the loss of reporter gene expression poses a difficult challenge for molecular imaging of stem cells, we believe our current in vitro and in vivo assay systems provide a useful experimental platform to study the mechanisms underlying gene silencing. Further studies are needed to determine whether similar processes are involved in other promoters/enhancers and reporter genes. Our ongoing efforts focus on using endogenous promoters such as ß-actin or ubiquitin to circumvent this issue. The answers to these questions will be particularly relevant as the fields of molecular imaging and gene/cell therapies move forward.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4551fje;
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-tubulin protein was used as the control. B) RT-PCR of control H9c2 show absent Fluc mRNA as expected. H9c2-Fluc.3 at passage-60 showed progressive increases in Fluc levels with higher dosages of 5-Aza. The 
