Research Article
Comparative study of cellular kinetics of reporter probe [131I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

https://doi.org/10.1016/j.nucmedbio.2008.10.016Get rights and content

Abstract

Aim

Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images.

Methods

Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyluracil) was labeled with 131I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [131I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry.

Results

FAU could be labeled with 131I, and the labeling efficiency and radiochemical purity rates were 53.82±2.05% and 94.85±1.76%, respectively. Time-dependent increase of the accumulation of [131I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55±0.37% and 2.09±0.34%, respectively. Greater uptakes of [131I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978–38.253, P<.01). The exogenous gene expression by polymerase chain reaction in adenovirus vector-infected cardiac myocytes was significantly higher than that in pDC316-tk/lipoplex-transducted ones (semiquantitative analysis, 3.11±0.14 versus 1.60±0.05, P<.01). Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rates were 81.70±0.40% in Ad5-tk and 22.06±0.32% in liposome (P<.01).

Conclusions

Both adenovirus and liposome could transfer reporter gene into cardiac myocytes successfully, and the expressed exogenous protein could form functional enzymes efficiently. However, the adenovirus vector acted more efficiently than did liposome, with a higher uptake rate of the reporter probe. Thus, adenovirus is competent for gene transfer in cardiac reporter gene imaging.

Introduction

Cardiac gene therapy holds great promise as a therapeutic strategy for several heart diseases, such as ischemic heart disease and cardiac failure [1], [2], [3]. Since 1998, more than 20 clinical trials of cardiac gene therapy had been under evaluation; however, most of them had been stopped for lack of good methods for monitoring the transgene expression and therapeutic effects [4], [5], [6]. Therefore, there is increasing interest in the development of imaging strategies that allow for noninvasive evaluation of the location, magnitude and duration of transgene expression in the heart over time [7], [8]. Imaging the expression of reporter genes by use of reporter genes has great potential in this regard [9], [10]. Suitable reporter genes can be coexpressed together with therapeutic genes, which enable external monitoring of the expression of the therapeutic gene. The principle of reporter gene nuclear imaging is based on reporter genes expressing a gene product that is normally absent in host tissue, which can be imaged with positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

HSV1-tk (herpes simplex virus type 1 thymidine kinase) is one of the most commonly used reporter genes and one of the most exciting developments in current molecular imaging studies that has been used in radiolabeled probe detection, SPECT and PET. Tjuvajev et al. [11], [12], [13] developed a system in which the HSV1-tk gene product reacts with a radiolabeled reporter substrate and converts it to a metabolite that is selectively trapped in the transduced cells. An enzyme, the product of HSV1-tk gene, can phosphorylate some compounds to its monophosphate forms. Once they are phosphorylated, the substrates are unable to be transported out of the cells, so they accumulate in the transduced cells. Therefore, cells in which successful gene transfer has occurred can be distinguished from nontransfected cells using the imaging equipments noninvasively. [Note: “HSV1-tk” refers to the gene, while “HSV1-TK” refers to the corresponding enzyme.] This system has been widely used in studies on tumor cells in culture and in vivo tumor models; however, only few studies focused on its usage in the myocardium [14], [15].

Improvement of the HSV1-tk reporting technique can be achieved by optimizing each component of the system. The radiolabeled probes are one of the most important parts. Some studies focused on the radiolabeled substrates, during which many radiolabeled reporter probes were developed. Among them, FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil) and [18F]FHBG [9-(4-[18F]fluoro-3-hydroxymethylbutyl guanine)] are the two most commonly used radiolabeled probes [16], [17].

Another important part is how to transfer the reporter gene into the cells efficiently, which may be related to gene expression degree and influence the quality of reporter gene images indirectly. As we have known, various gene transfer systems that are capable of penetrating cells have been developed to deliver DNA to the nucleus and result in the expression of the target protein. These systems include replicant-deficient viral vectors (e.g., adenovirus, adeno-associated virus), synthetic nonviral vectors (e.g., liposomes, polylysine, dendrimers), physical methods (e.g., gene gun), naked DNA or combinations of these various technologies [18], [19]. For the reporter gene transfer, most studies used adenoviral vectors because of their high transfecting efficacy. Few studies used other methods to transfer the reporter gene into myocardial cells for cardiac reporter gene imaging.

When different vectors are used for reporter gene transfer, we wonder if the degree of gene expression and the cellular uptake characteristic of radiolabeled probes are different in cardiac myocytes. In order to address this issue in greater detail, in this study, we directly compared the uptake characteristics of radiolabeled FIAU on the transferred HSV1-tk cardiac myocytes between two vectors, adenovirus and liposome. The aims of this study were to find the better vector and to provide theoretical foundation for good nuclide cardiac reporter gene images.

Section snippets

Gene transfer vectors

The plasmid vector pDC316-tk and the virus vector Ad5-tk (E1/E3-deleted replication-defective recombinant adenovirus type 5), carrying the HSV1-tk gene under the transcriptional control of the cytomegalovirus promoter, were constructed and purified in Vector Gene Technology (Beijing, China). The HSV1-tk gene in the recombinant vectors was identified by polymerase chain reaction (PCR). The viral titer of Ad5-tk was 1.6×1010 IU/ml as determined by the TCID50 method. A replication-defective

Neonatal primary cardiac myocyte culture

The growth condition of cardiac myocytes obtained from single collagenase digestion was observed under an inverted microscope. We found that the cells had strong viability and grew in contact with one another, resulting in cluster formation. Some cells began to beat in the cluster at 24 h, and several clusters reached the same beat rate at 48 h (shown in Fig. 1).

Radiolabeling and stability

The radiolabeling efficiency of [131I]FIAU was 53.82±2.05% (n=5). After purification on Sep-Pak C-18 column, the radiochemical purity

Discussion

One of the most important systems for obtaining clear reporter gene images is the transport system, which is capable of transferring reporter genes into cells with high efficiency. Until now, most in vitro transfection experiments worked on tumor and immortalization cells; few studies focused on primary cells [12]. Similar to gene therapy, to achieve reporter gene imaging, the genes of interest should be successfully delivered in to the target cells by delivery vectors to ensure their

Acknowledgments

This work was supported by the National Nature Science Foundation of China (Grant Nos. 30400176, 30571816, 30772208 and 30830041).

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