Study Of MR Molecular Imaging Of Malignant Tumors Based On An Optimal HTERT Promoter Driving The Experession Of Tyrosinase

1. Purpose and significanceMalignant tumor is one of the primary causes for human death. The early diagnosisand treatment of tumor is the major way for patients to survive. At present the clinicaldiagnosis of tumor mainly relies on imaging, but it is often detected at late stage, becauseCT, MRI and X-ray imaging are based on the abnormality of anatomical structures. Due to"the same diseases with different images, the different diseases with the same imaging", itis difficult to diagnose some atypical tumors (such as isolated nodules in lung) rare tumorsor tumors in early stages. PET/CT is regarded as the best method to qualitatively diagnosetumors in the early period. According to the uptake rate of glucose, check tracer18F-FDGused in PET can detect both benign and malignant tumors indirectly.18F-FDG is not atumor-specific tracer, so false positives and false negatives are not avoidable.Biopsy is a gold standard for qualitative diagnosis of tumors. But for some speciallesions (such as in heart, vessels), it is a high risk to take tissue samples for biopsy.Moreover, tumors which are able to undertake biopsy are usually in the middle or late stageand patients have missed the optimal time for treatment. Therefore, there is a need todevelop a non-invasive and effective method for early qualitative diagnosis. As thedevelopment of molecular biology and application of molecular probe, imaging hasdeveloped from the studies of anatomy and physiology to molecular imaging. Especially,the fast-growing molecular MR imaging has provided new ideas for early diagnosis oftumor.With the development of molecular biology and the application of molecular probe inimaging, images from the traditional medicine has been to anatomical and physiologicalfunction of deep into the molecular imaging, especially in recent years, the rapiddevelopment of MR molecular imaging of tumor early qualitative diagnosis provides new ideas.MR molecular imaging is currently getting a significant amount of attention in reportergene's expressions as imaging modes. MR reporter gene, as gene-expressed product, caninduce signal change by specially binding to molecular probe. Tyrosinase as a MR reportergene has been concerned by many researchers. Expression of tyr gene can producttyrosinase---a rate limiting enzyme which can produce melanin in various cells throughcatalytic reaction. One of significant characteristics of melanin is that it contains lots ofanions with high affinity to various metal cations (35%of the melanin's weight), whichabsorbs extracellular and intracellular metal cations, especially Fe3+. It is estimated thatmelanins per gram on average have1020absorption sites for Fe3+. Like Gd3+, Fe3+is aparamagnetic material, whose concentration closely correlates with change of MR signal. Inthe appropriate concentration range, it can significantly shorten T1relaxation time ofproton of surrounding tissues. The targeted tissue can be specifically displayed with highintensity on T1WI. A linear correlation can be found between signal increase and Fe3+concentration. Signals of T1WI depend on melanin production, and signal densitysignificantly correlates with tyrosine activity and melanin. Accordingly, the signal densityof T1WI can reveal the expression of tyr gene in transfected cells, and indirectly reflectactivity of marker gene, with a significant linear correlation. Melanin can act as a MRmolecular probe.The way that tyr gene can be expressed in tumor cells rather than in normal tissues orcells, is the key to achieving specific diagnosis of tumor. At present, tumor specific antigenpromoters are discovered with high specificity. For example, AFP promoter can beexpressed only in hepatoma cells, CEA only in some gastrointestinal tumors or pulmonarycarcinoma, and PSA promoter only in prostatic cancer. Expression of downstream tyr genedriven by these promoters only applies to malignant tumors with tyr antigen. The diagnosisscope is narrow. Therefore, tumor specific antigen promoter with broad-spectrum is needed.Under the action of this promoter, the downstream tyr reporter gene can be expressed intumor cells rather than in normal cells.As the requirement of infinite proliferation of tumor, telomerase can be expressed inmost of malignant tumors, but in few stem cells and somatic cells with strong hyperplasia.HTERT, as the rate-limiting enzymes of telomerase, plays an important role in its activity regulation. Our previous studies have suggested that hTERT can be expressed in malignanttumors and some precancerous tissues rather than normal cells. hTERT-basedimmunotherapy can kill, hTERT-positive tumor cells, without lethal activity for normalcells, especially for leukomonocyte with low hTERT expression. Obviously, activity oftelomerase is regulated by transcription of hTERT which is regulated by hTERT promoter.Because the telomerase can be expressed in more than85%malignant tumors or tissues, thehTERT promoter has great tumor-targeting, which can be considered as a tumor specificpromoter and be used into targeted diagnosis and treatment for tumor.Based on the above analysis, we suppose that hTERT promoter can drive theexpression of tyrosinase gene (MR reporter gene) as a tumor-targeting promoter. Thissubject will study the values of hTERT promoter driving Try gene expression in diagnosisof malignant tumor on MR, and provide a theoretical basis for application of MR inqualitative diagnosis of occupying lesions.Supported by the National Natural Science Foundation (No.30871150), our study hassuccessfully designed and constructed optimized hTERT promoters-pLenti-hTERTp/CD-IRES2-EGFP and pLenti-CMVp/CD-IRES2-EGFP. The in vitro and in vivostudies have revealed that cytosine deaminase suicide gene driven by the optimized hTERTpromoter could kill telomere positive tumor cells rather than telomere negative tumor cells.It is suggested that the designed and constructed hTERT promoter has significanttelomerase-targeting.In conclusion, based on previous studies, this study considers hTERT as the promoterof tyrosinase gene, takes advantage of telomerase specificity of hTERT promoter, andmakes the tyrosinase gene product melanin only in telomere positive malignant tumor cellswith absorption of extracellular and intracellular Fe3+, as to achieve early diagnosis oftumor with high definition MRI techniques. Hopefully, it can be proved by these studiesthat expression of tyrosinase reporter gene driven by hTERT promoter can providetheoretical basis and technical method for qualitative diagnosis of occupying lesion.Methods:1. Construction of pLenti-hTERT/Tyr-IRES2-EGFP and pLenti-CMV/Tyr-IRES2-EGFPFirst, Hum-ORF-M13containing the full-length gene encoding Tyrosinase as MR report gene is purchased from a company and transformed into DH5-É‘. Then Tyrosinasegene cDNA length is amplified by PCR and connected with T carrier to get pMD19-Tyrplasmid. The enzyme of the plasmid can result in Tyr segment. PlasmidhTERTp/CD-IRES2-EGFP and CMVp/CD-IRES2-EGFP successfully constructed before,are cut by double digestion of EcoR I and Sal I, with the acquisition of linear fragments ofhTERTp-EGFP and CMVp-EGFP. Full length cDNA of tyrosinase is amplified by usingPCR technique. Tyr fragment is respectively connected with linear fragment ofhTERTp-EGFP and CMVp-EGFP by by T4DNA, to get plasmid hTERT/Tyr-IRES2-EGFPand CMVp/Tyr-IRES2-EGFP our subject requires. These plasmids are confirmed byrestriction enzyme and DNA sequencing. Virus is packaged and concentrated byfour-plasmid system to determine virus titer and the best multiplicity of infection (MOI).2. In vitro study on detecting telomerase targeting of lentiviral expression vectorand protein function of MR reporter genepLenti-hTERT/Tyr-IRES2-EGFP and pLenti-CMV/Tyr-IRES2-EGFP infect thetelomere positive tumor cells (HepG2, SGC7901and SW480) in vitro, as well as thetelomere negative tumor cell (U2OS). These infected cells (8) and the non-infected cells (4)are served as the targeted cells. Among the12groups, the cells infected by the CMVpromoter are taken as the positive controls, the non-infected cells and the U2OS infected bythe hTERT promoter as the negative controls, the telomere positive tumor cells infected bythe hTERT promoter as the experimental group. Confocal laser is used to observe theexpression of EGFP. Tyr mRNA expression in the targeted cells is detected by RT-PCR.Absorbance at490nm within the targeted cells is measured by colorimetric method,determining tyr activities of these cells. Melanin production is detected by Fontana staining.After the co-incubation with the targeted cells at different times, effect on signal change isexamined by MR scanning, and the absorption change of ferri ion over time is detected byPrussian blue staining.3. in vivo study on specific MR diagnosis of tumor by lentiviral expression vectorSCID mice underwent bilaterally subcutaneous inoculations (left: non-transfectedSGC7901and U2OS cells, right: the transfected SGC7901and U2OS cells), turning intotumor-bearing mice models. On a3.0T MR scanner, we observed signal changes ofCMV/hTERT promoter driven tumor specific expressions and telomere positive/negative tumors, as well as of the transfected and non-transfected cells; and studied the feasibilitythat tyr gene driven by hTERT promoter could provide a qualitative diagnosis for thetelomerase positive tumor. On a model fluorimager, we observed green fluorescent proteinexpressions of all the tumors in vivo and in vitro; and performed Fontana stainging andPrussian blue staining on the tumor specimens, observing intracellular melanin productionand absorption of ferri ions.Results:1. The construction and packaging of lentiviral expression vectors(pLenti-hTERT/Tyr-IRES2-EGFP and pLenti-CMV/Tyr-IRES2-EGFP)Restriction enzyme demonstrated that the constructed plasmids had a band with avalue of1600bp as same as the theoretic value of tyr gene. cDNA sequencing verified thatthe constructed plasmids had full length tyr genes with correct sequencing as comparedwith that in the GENE BANK. It was illustrated that plasmid hTERT/Tyr-IRES2-EGFP andCMV/Tyr-IRES2-EGFP were successfully constructed. After the packing of lentiviral4plasmids system, virus titer was detected with a value of108.2. In vitro study on the telomerase targeting detection and on MR image oflentiviral expression vectorUnder laser scanning cofocal microscope (LSCM), EGFP expressions were observedin the telomerase positive tumor cells((SGC-7901gastric cancer cell,HepG2lung cancercell,SW480colon cancer cell) after infected by the pLenti-hTERT/Tyr-IRES2-EGFP andpLenti-CMV/Tyr-IRES2-EGFP. They were also found in the telomerase positive U2OSosteosarcoma after infected by the pLenti-CMV/Tyr-IRES2-EGFP rather than by thepLenti-hTERT/Tyr-IRES2-EGFP. As suggested by RT-PCR and western blot (WB), tyrmRNA and tyr protein were expressed in the telomerase positive tumor cells after infectedby the pLenti-hTERT/Tyr-IRES2-EGFP and pLenti-CMV/Tyr-IRES2-EGFP, while theywere only observed in the telomerase negative tumor cells infected by thepLenti-CMV/Tyr-IRES2-EGFP rather than by the pLenti-hTERT/Tyr-IRES2-EGFP. Asrevealed by A490, greater tyr activity represented in the telomerase positive tumor cellsinfected by the pLenti-hTERT/Tyr-IRES2-EGFP and pLenti-CMV/Tyr-IRES2-EGFP andthe telomerase negative tumor cells infected by the pLenti-CMV/Tyr-IRES2-EGFP. It wassignificantly better than that in the non-transfected cells and the telomerase negative tumor cells infected by the pLenti-hTERT/Tyr-IRES2-EGFP (p<0.001). In coverglass, Fontanastaining showed brow silver granules in cytoplasm with tyr activity, and absence of these inthe cells without tyr activity. Prussian blue staining displayed that, after the incubation withthe ferri ions at different times, deposition of blue-stain iron particles largely appeared inthe cells with tyr activity as the time increasing; the deposition number increased tomaximum after36h-48h. And it was slightly observed in the cells without tyr activity; asthe time increasing, the deposition number didn't increase. MR scanning revealed that,signal intensity of T1WI in the cell mass with tyr activity increased as the ferri ions'incubation prolonging, which was significantly higher than that in the cell mass without tyractivity(p<0.01).3. in vivo study on MR molecule image diagnostic of lentivirus expression vectorto different cancer cellsIn the inoculated SCID mice, T1WI signal densities of SGC7901and U2OS infectedby pLenti-CMV/Tyr-IRES2-EGFP, were significantly higher than those of thenon-transfected SGC7901and U2OS (p<0.05); T1WI signal densities of the U2OS infectedby pLenti-hTERT/Tyr-IRES2-EGFP is similar with those of the non-transfected U2OS(p>0.05); T1WI signal densities of the SGC7901infected bypLenti-hTERT/Tyr-IRES2-EGFP were significantly higher than those of the non-transfectedSGC7901(p<0.05). Model fluorescence imaging indicated that green fluorescence wasvisible in the tumors with increase of T1WI signals, and vice versa; change of the greenfluorescence was obviously more remarkable than that of the T1WI signal changes. Fontanastaining showed that deposition of black particles largely appeared in tumor specimens withthe increase of MR signals, suggesting production of melanin; but not in those without theincrease of MR signals. Prussian blue staining suggested that deposition of blue-stain ironparticles largely appeared in tumor specimens with increase of T1WI signals, and slightly intumor specimens without increase of MR signals.Conclusion:1. Based on the preliminary studies, we have successfully constructed lentiviralexpression vector containing MR reporter gene (tyr gene) for hTERT promoter driventumor specific expression of telomerase. Our in-vivo and in-vitro studies have confirmedthat the lentiviral expression virus has strict targeting for telomerase. 2. Our in-vivo and in-vitro studies have verified that the telomerase positive tumorcells infected by the lentiviral expression vector have great tyr activity, and product melaninwith absorption of ferri ions surrounding the cells, improving the specificity of T1WIsignal.3. Tyr gene can be served as MR reporter gene during in vivo and in vitro studies.According to the specificity of hTERT promoter driven telomerase, it can providetheoretical bases for qualitative diagnosis of tumor, and for the application of tyr gene asMR reporter gene in scientific research.