Detection Of Telomerase Activity In Tumor Cells With MR Imaging Mediated By HTERT Promoter In Vitro

ObjectiveMalignant tumor is a kind of systemic and fatal disease that severely harms the publichealth and life. Its early diagnosis is one of the most important factors that affect theprognosis of patients. Medical imaging equipment (such as CT, MRI, B ultrasound, PET/CT)provides reliable and noninvasive method for tumor diagnosis. Besides, the usage ofenhancement scan has more value in the differential diagnosis. However, there are somedifficulties in differentiating the benign and malignant of the early lesions, the small lesions(such as small pulmonary nodules), even the large lesions (such as soft tissue tumor), and thebenign lesions abundant in blood supply. At present, the confirmed diagnosis of malignanttumor relies principally on pathological examinations that diagnose diseases in cellular,molecular or genetic levels. Among which the detection of tumor markers has been widelyused in the diagnosis, directing therapy and estimating prognosis of malignant tumors.Telomerase activity has been found in the majority of malignant tumors (>85%), but notin normal tissues or benign lesions. Therefore, telomerase activity can be taken as molecularevidence for the diagnosis of malignant tumors. Telomerase activation is essential for theimmortality of tumor cells; hence, it is closely associated with the progression and prognosisof malignant tumors. Moreover, telomerase activity is independent of tumor origin. Therefore,compared to other makers (such as AFP, CEA, and PSA), the detection of telomerase activitycan be used to the diagnosis of broad-spectrum malignant tumors. At present, the telomeraseactivity of lesions has predominantly been determined until examination of the resectedspecimen, but sometimes, the anatomy of some lesions restricts the obtainment of specimen.Therefore, a new approach that can detect telomerase activity noninvasively is needed, whichcan also provide a new method for the diagnosis of malignant tumors in vivo.Many studies of home and abroad had confirmed that cells overexpressing ferritin canabsorb more iron ions and store them in the ferritins, which can increase the transverserelaxation rate significantly, and so change the MR signal intensity of themselves. These results suggest that the ferritin can be used as a MR reporter gene. Moreover, the expressionof ferritin can be regulated by a promoter, thus we can diagnose diseases in the molecularlevel. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomeraseand whether the hTERT is transcribed is the key factor in the regulation of the telomeraseactivity, indicating that the hTERT promoter possesses telomerase-targeting and can beregarded as a specific promoter for the broad-spectrum malignant tumors. Therefore, weassume that the ferritin gene can be expressed only in the telomerase-positive cells under thecontrol of hTERT promoter, and thus change the MR singal intensity of malignant tumors. Itis a promising way to detect telomerase activity of tumor cells noninvasively and achieve thegoal of diagnosis of malignant tumor in vivo with the use of MR ferritin reporter imagingtechnology mediated by hTERT promoter.To sum up, the aim of this study is to investigate the feasibility of detecting telomeraseactivity by MR imaging technology with the use of a lentiviral expression vector carrying theferritin heavy chain (Fth) reporter driven by the hTERT promoter, and thus to providetheoretic and experimental bases for the use of this technology for malignant tumor diagnosisin vivo.MethodsConstruction of Lenti-hTERT-Fth1-3flag-Puro and Lenti-CMV-Fth1-3flag-Puro vectorsThe Fth1gene tagged with3flag was synthesized and subcloned into the multiplecloning site of pCDH-CMV-MCS-EF1-Puro plasmid to form pCDH-CMV-Fth1-3flag-Puroplasmid, and then the CMV promoter was replaced by the hTERT promoter to form pCDH-hTERT-Fth1-3flag-Puro plasmid. The lentiviral vectors were packaged by co-transfecting the293T packaging cells with the reconstructed plasmids, pCD/NL-BH*DDD and pLTR-Gpackaging plasmids. After collected and concentrated, a quantitative PCR method was used todetermine the titer of viruses. The constructed lentiviral vectors were named asLenti-hTERT-Fth1-3flag-Puro and Lenti-CMV-Fth1-3flag-Puro respectively.Detection of telomerase activity in tumor cells by MR imagingTelomerase-positive cells (A549, SKOV3and293T) and telomerase-negative cells(U2OS and HPDLF) were infected with Lenti-hTERT-Fth1-3flag-Puro or Lenti-CMV-Fth1-3flag-Puro vector. The telomerase-positive cells infected with Lenti-hTERT-Fth1-3flag-Purovector were served as the experimental groups, the cells infected with Lenti-CMV-Fth1- 3flag-Puro vector as the positive control groups, the telomerase-negative cells infected withLenti-hTERT-Fth1-3flag-Puro vector and the uninfected cells as the negative control groups.The expression of flag in all cells was detected by immunofluorescence staining, and theexpression level of Fth determined by ELISA assay. Cells were grown in mediumsupplemented with FAC (1mM) for48hours. A CCK-8kit was used to test the effect of Fthoverexpression or FAC on cell viability. Prussian blue iron staining and a total iron reagent kitwere used to determine iron accumulation in cells. MR scanning was performed to detect thesignal changes of cells in vitro.ResultsBy restriction enzyme and DNA sequence analysis, we confirmed that the recombinantplasmids containing the Fth1gene under the control of hTERT promoter or CMV promoterwere constructed successfully. After packaging, the lentivirus titer was about5x108TU/ml.Immunofluorescence staining showed that the expression of flag was observed only intelomerase-positive cells infected with Lenti-hTERT-Fth1-3flag-Puro and in all cells infectedwith Lenti-CMV-Fth1-3flag-Puro, while absent in telomerase-negative cells infected withLenti-hTERT-Fth1-3flag-Puro or uninfected ones. As supported by ELISA assay, the level ofFth1in telomerase-positive cells infected with Lenti-hTERT-Fth1-3flag-Puro and in all cellsinfected with Lenti-CMV-Fth1-3flag-Puro was increased significantly compared to theuninfected ones （p<0.05）, while there was no significant difference between thetelomerase-negative cells infected with Lenti-hTERT-Fth1-3flag-Puro and the uninfected ones（p<0.05）.By using CCK-8assay reagent, we found that there was no significant difference inviability between the cells in WT, CMV, and hTERT groups, or in the cells maintained inmedium with and without FAC (1mM) for48hours（p>0.05）. Prussia blue iron staining andtotal iron detection assay showed that the telomerase-positive cells infected withLenti-hTERT-Fth1-3flag-Puro and the cells infected with Lenti-CMV-Fth1-3flag-Puroaccumulated more iron than the uninfected ones, while the telomerase-negative cells infectedwith Lenti-hTERT-Fth1-3flag-Puro did not.MR scanning revealed that the R2*values of telomerase-positive cells infected withLenti-hTERT-Fth1-3flag-Puro and cells infected with Lenti-CMV-Fth1-3flag-Puro weresignificantly increased than the uninfected ones （p<0.05）, while there was no significant difference between the telomerase-negative cells infected with Lenti-hTERT-Fth1-3flag-Puroand the uninfected ones（p>0.05）. Correspondingly, telomerase-positive cells infected withLenti-hTERT-Fth1-3flag-Puro and cells infected with Lenti-CMV-Fth1-3flag-Puro showedlower signal in T2*WI compared with telomerase-negative cells infected withLenti-hTERT-Fth1-3flag-Puro and the uninfected cells.ConclusionIn this experiment the lentiviral vector containing the MR Fth1reporter gene controlledby the hTERT promoter (Lenti-hTERT-Fth1-3flag-Puro) had strict targeting for telomerase.When infected with this vector, only the telomerase-positive cells could overexpress Fth1andaccumulate more iron and thus change their MR signals finally. In summary, we can detect thetelomerase activity of cells with the help of MR ferritin reporter imaging technology mediatedby hTERT promoter noninvasively. This study provides a theoretic and experimental basis forfurther using MR imaging technology in diagnosing malignant tumors in vivo.