Visualization of endogenous p53-mediated transcription in vivo using sodium iodide symporter.

PURPOSE: To develop a gamma camera imaging method for the determination of endogenous gene expression, we evaluated the expression of endogenous p53 gene using human sodium iodide symporter (hNIS) gene as reporter. EXPERIMENTAL DESIGN: We constructed cis-p53RE-hNIS reporter vector placed under control of an artificial enhancer (p53RE). Moreover, we transfected it into human hepatoma cell line SK-Hep1 by liposome. Geneticin was used for the selection of stable transfectant (SK-Hep1p53NIS). To evaluate the function of hNIS, the inhibition study was examined with 1 mmol/L potassium perchlorate. After treatment of Adriamycin with serial dose for 24 hours, we measured the uptake of 125I and did Western blot analysis to evaluate expression of p53 protein. Tumor xenografts were produced in nude mice by s.c. injection of SK-Hep1p53NIS cells. After 7 days, scintigraphic images of nude mice before and after Adriamycin treatment were obtained using [99mTc]-pertechnetate. RESULTS: In the SK-Hep1p53NIS cells, Adriamycin-treated cells accumulated up to three times higher than did nontreated cells. Potassium perchlorate inhibited completely the uptake of 125I. As Adriamycin dose increased, radioiodide uptake was significantly correlated with activated p53 as well as total p53 protein level. When Adriamycin (2 mg/kg) was treated in the same mice, a significantly higher uptake of [99mTc]-pertechnetate was observed in SK-Hep1p53NIS xenografts compared with nontreated xenografts (P < 0.05, unpaired t test). CONCLUSIONS: These results suggest that p53 expression level can be monitored by NIS gene expression using cis-p53RE-hNIS system in vitro and in vivo.     

Human sodium iodide symporter gene adjunctive radiotherapy to enhance the preventive effect of hMUC1 DNA vaccine

We demonstrate the use of combination therapy to overcome the limitations of cancer DNA vaccines by adding radioiodine gene therapy in an animal cancer model. We established a stable cell line (CT26/hMUC1-hNIS-Fluc: CMNF) expressing the hMUC1, hNIS and Fluc genes using a retro- and lentivirus system. The survival rates (%) of CMNF cells were determined using clonogenic assays after (131)I treatment. After i.m. immunization to 4 groups of Balb/c mice (pcDNA3.1, pcDNA3.1+(131)I, pcDNA3-hMUC1+PBS and pcDNA3-hMUC1+(131)I groups) with pcDNA3-hMUC1 or pcDNA3.1 once a week for 2 weeks, 1 x 10(5) CMNF cells were injected s.c. into the right thighs of mice in each group. Twenty-one days after tumor transplantation, (131)I was administered i.p. to the pcDNA3.1+(131)I and pcDNA3-hMUC1+ (131)I groups. Tumor progression was monitored in the 4 groups by bioluminescent and scintigraphic imaging and by taking caliper measurements. Tumor masses were extracted and weighted at 39 days post-tumor challenge. We confirmed that CMNF cells highly express hMUC1, hNIS and Fluc by FACS, (125)I uptake, and luciferase assay. The survival rates of CMNF were markedly reduced to (14.6 +/- 1.5)% after (131)I treatment compared with the survival rates of parental cells (p < 0.001). Tumor growth inhibition was significant only in the pcDNA3-hMUC1+ (131)I group at 39 days post challenge. Tumor masses in pcDNA3-hMUC1+ (131)I group were smaller than those of the other groups. This study shows that the weak preventive effects of cancer DNA vaccine can be overcome by radioiodine gene therapy utilizing sodium iodide symporter.     

Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.     

hTPO和hNIS共转染胶质瘤细胞碘摄取和^131I治疗的实验研究

目的构建含有人甲状腺过氧化物酶基因（hTPO）的重组腺病毒,并与人钠-碘转运体基因（hNIS）共转染至神经胶质瘤细胞中,研究其摄碘能力及对肿瘤细胞的杀伤能力,探讨131I治疗胶质瘤的可能性。方法应用AdEasy系统构建重组腺病毒AdTPO,利用重组质粒将hNIS基因转染入神经胶质瘤细胞系U251中获得hNIS—U251细胞系作为阴性对照组,再利用重组腺病毒AdTPO将TPO基因转染入hNIS—U251细胞系中获得AdTPO—hNIS—U251作为实验组,未转入hTPO和hNIS的细胞U251作为空白对照组。研究三组细胞的摄碘实验、过氯酸盐抑制实验、有机化测定实验检测其摄碘功能,细胞克隆形成实验评价131I对转染肿瘤的杀伤作用。结果成功构建出重组腺病毒AdTPO,并在稳定表达hNIS的U251细胞中实现了hTPO的共转染。hNIS—U251组（每分钟放射性计数55769．96±4353．26）比空白对照组（每分钟放射性计数507．67±57．69）摄碘能力增高约110倍,有效半衰期7分钟,有机化程度约为0．1％,细胞克隆形成率（9．08±2．86）％,较对照组减低约10倍。AdTPO—hNIS—U251组（每分钟放射性计数74647．53±3605．88）比空白对照组摄碘能力增高约147倍,有效半衰期延长至13分钟,有机化程度增高至10％,细胞克隆形成率（6．80±2．09）％,较对照组减低约13倍。结论将hTPO和hNIS共转染至神经胶质瘤细胞后,可有效提高细胞的摄碘能力,hTPO延长了放射性碘在细胞中的停留时间,131I对瘤细胞有较强的杀伤作用。     

MIDGE/hNIS vaccination generates antigen-associated CD8+IFN-gamma+ T cells and enhances protective antitumor immunity

Human sodium iodide symporter (hNIS) is a transmembrane protein that actively transports iodide ions into thyroid cells. hNIS is over-expressed in some cases of the thyroid cancers compared with the surrounding normal tissues and has been considered to be an attractive target for immunotherapy. The aim of this study is to determine the feasibility of utilizing the hNIS antigenic protein in enhanced-antigen-associated immunotherapy using image analysis with a gamma counter. To accomplish this, minimalistic immunogenically defined gene expression (MIDGE), either plain or coupled to a nuclear localization signal (NLS) peptide, was used as a vector system. Vaccination with MIDGE/hNIS, MIDGE/hNIS-NLS and pcDNA3.1/hNIS produced a significant increase in the number of hNIS-associated IFN-gamma-secreting CD8(+) T cells, with MIDGE/hNIS having the strongest effect. In addition, immunization with the hNIS encoding vectors induced antigen-mediated antitumor activity against NIS-expressing CT26 tumors in vivo, with the highest tumor free rate (100%) and lowest tumor growth being observed up to 40 days after the CT26/NIS tumor challenge with MIDGE/hNIS than those resulting from other immunization groups. Tumor progression could be followed noninvasively and repetitively by monitoring levels of hNIS gene expression in the tumors using scintigraphic image analysis. Overall, hNIS has a potential use as an antigen for immunization approaches, and vaccination with MIDGE/hNIS vectors is an effective means of generating hNIS-associated immune responses in mice.     

Combined E7-dendritic cell-based immunotherapy and human sodium/iodide symporter radioiodine gene therapy with monitoring of antitumor effects by bioluminescent imaging in a mouse model of uterine cervical cancer

Using a uterine cervical cancer cell line expressing human papillomavirus (HPV) 16 E7 antigen and bioluminescent imaging (BLI), we evaluated the therapeutic potential of combined immunotherapy using transfected dendritic cells (DC-E7) and human sodium/iodide symporter (hNIS) radioiodine gene therapy in a xenograft animal cancer model. Dendritic cells expressing either E7 antigen (DC-E7) or no-insert (DC-no insert) were made for immunization materials, and murine uterine cervical cancer cell line coexpressing E7, firefly luciferase, hNIS, and EGFP genes (TC-1/FNG) were prepared for the animal tumor model. C57BL/6 mice were divided into five therapy groups (phosphate-buffered saline [PBS], DC-no insert, DC-E7, I-131, and DC-E7+I-131 groups). Single therapy with either DC-E7 or I-131 induced greater retardation in tumor growth compared with PBS or DC-no insert groups, and it resulted in some tumor-free mice (DC-E7 and I-131 groups, 40% and 20%, respectively). Combination therapy with DC-E7 and I-131 dramatically inhibited tumor growth, thus causing complete disappearance of tumors in all mice, and these effects were further confirmed by BLI in vivo. In conclusion, complete disappearance of the tumor was achieved with combined DC-E7 vaccination and hNIS radioiodine gene therapy in a mouse model with E7-expressing uterine cervical cancer, and serial BLIs successfully demonstrated antitumor effects in vivo.     

Probasin promoter (ARR(2)PB)-driven, prostate-specific expression of the human sodium iodide symporter (h-NIS) for targeted radioiodine therapy of prostate cancer

Prostate cancer is one of the most promising candidates for sodium iodide symporter (NIS)-mediated gene therapy. Adenovirus-mediated expression of NIS that is driven by prostate-specific promoters induces generous radioiodine accumulation in prostate cancer cells that may be used for therapy with (131)I. We have recently developed a replication-deficient adenovirus carrying the human NIS cDNA linked to a composite probasin promoter, ARR(2)PB, aiming toward specific expression of the human NIS gene (h-NIS) in prostate tissue for targeted radioactive iodide therapy of prostate cancer (Ad-ARR(2)PB/hNIS). The ability of Ad-ARR(2)PB/hNIS to cause NIS expression in tumor cells was characterized by iodide uptake assay and compared with Ad-CMV/hNIS in which the h-NIS expression is driven by the cytomegalovirus (CMV) promoter. Androgen-dependent prostate cancer cell lines (LNCaP) and non-prostate origin tumor cell lines (SNU449, MCF-7, HCT116, OVCAR-3, and Panc-1) were infected with the viral constructs, and perchlorate-sensitive (125)I uptake and NIS protein expression were measured. Ad-ARR(2)PB/hNIS-infected LNCaP cells showed androgen-dependent and perchlorate-sensitive iodide uptake. Iodide accumulation in LNCaP cells infected with Ad-ARR(2)PB/hNIS, followed by incubation with synthetic androgen, was 5.3-fold increased compared with those coincubated with perchlorate (15,184 +/- 1,173 cpm versus 2,837 +/- 187 cpm). Ad-ARR(2)PB/hNIS-infected LNCaP cells revealed a 3.2-fold increase of iodide accumulation compared with those infected with Ad-CMV/hNIS (multiplicity of infection = 30). Iodide uptake in a panel of non-prostate tumor cell lines infected with Ad-ARR(2)PB/hNIS was no more than 2,500 cpm, demonstrating the tissue specificity of this construct. These results indicate that Ad-ARR(2)PB/hNIS can be used to achieve high-magnitude and tissue-specific expression of h-NIS in prostate tissue and is a promising candidate for cancer gene therapy of prostate cancer.     

脂质体介导入钠/碘同向转运体基因转染肺癌A549细胞及其蛋白表达

目的:研究人钠/碘同向转运体(NIS)基因转染肺癌细胞及其蛋白表达。方法:将体外培养的肺癌A549细胞分为实验组和对照两组:以脂质体Lipofectamihe2000为载体,分别介导转染重组质粒pcDNA3-hNIS和空质粒pcDNA3。NIS基因重组质粒pcDNA3-hNIS进行扩增、纯化,并经酶切鉴定和DNA测序。采用Western Blot法和免疫组化法分别检测转染肺癌细胞中NIS蛋白表达。结果:酶切鉴定和DNA测序结果表明重组质粒pcDNA3-hNIS中插入的hNIS基因片段大小、方向正确。WesternBlot法和免疫组化染色结果显示实验组有NIS蛋白表达,其阳性率达70.6%且主要分布于细胞膜而对照组无表达。两组比较有显著性差异(P=0.000)。结论:脂质体Lipofectamine 2000介导转染人钠/碘同向转运体基因pcDAN3-hNIS肺癌细胞能够成功地表达NIS蛋白。     

Transfection of the Human Sodium/Iodide Symporter （NIS） Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.     

Human sodium iodide symporter added to multidrug resistance 1 small hairpin RNA in a single gene construct enhances the therapeutic effects of radioiodine in a nude mouse model of multidrug resistant colon cancer

The objective of this study was to investigate the therapeutic potential of 131I added to doxorubicin therapy in multidrug resistance (MDR) mouse colon cancer coexpressing the MDR1 small hairpin RNA (shRNA) and human sodium iodide symporter (hNIS) gene in a single gene construct and to visualize the antitumor effects using molecular nuclear imaging. HCT-15 coexpressing shRNA for MDR1 gene (MDR1 shRNA) and hNIS gene with a single construct was established (referred to as MN61 cell). Inhibition of P-gp function by MDR1 shRNA and functional activity of hNIS gene was assessed using a ??(m)Tc sestamibi uptake and 12?I uptake, respectively. Cytotoxic effects by a combination of doxorubicin and 131I were determined in parental (HCT-15) or MN61 cells using an in vitro clonogenic assay. Therapeutic effect of either combination therapy (doxorubicin and 131I) or single therapy (doxorubicin or 131I alone) was evaluated by tumor volume measurement. ??(m)Tc-sestamibi, 123I, and ??(m)Tc-pertechnetate images of mice were acquired to evaluate functional assessment in vivo. Cellular uptake of ??(m)Tc-sestamibi and 12?I was approximately 2-fold and 100-fold higher in MN61 cells than in parental cells, respectively. Combination of 131I and doxorubicin resulted in higher cytotoxcity in MN61 cells as compared with parental cells. Scintigraphic imaging showed higher uptake of ??(m)Tc-sestamibi and 123I in MN61 tumor as compared with parental tumor. In mice treated with doxorubicin, there was a slight delay in tumor growth in the MN61 tumor but not in the parental tumor. Cancer treatment with 131I or doxorubicin induced a rapid reduction of tumor volume in the MN61 tumor but not in the parental tumor. Combination therapy further generated a rapid reduction of tumor volume as compared with 131I therapy alone (p?相似文献   

Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter

To test the feasibility of using the survivin promoter to induce specific expression of sodium/iodide symporter (NIS) in cancer cell lines and tumors for targeted use of radionuclide therapy, a recombinant adenovirus, Ad-SUR-NIS, that expressed the NIS gene under control of the survivin promoter was constructed. Ad-SUR-NIS mediating iodide uptake and cytotoxicity was performed in vitro. Scintigraphic, biodistribution and radioiodine therapy studies were performed in vivo. PC-3 (prostate); HepG2 (hepatoma) and A375 (melanoma) cancer cells all exhibited perchlorate-sensitive iodide uptake after infection with Ad-SUR-NIS, approximately 50 times higher than that of negative control Ad-CMV-GFP-infected cells. No significant iodide uptake was observed in normal human dental pulp fibroblast (DPF) cells after infection with Ad-SUR-NIS. Clonogenic assays demonstrated that Ad-SUR-NIS-infected cancer cells were selectively killed by exposure to (131)I. Ad-SUR-NIS-infected tumors show significant radioiodine accumulation (13.3 ± 2.85% ID per g at 2 h post-injection), and the effective half-life was 3.1 h. Moreover, infection with Ad-SUR-NIS in combination with (131)I suppressed tumor growth. These results indicate that expression of NIS under control of the survivin promoter can likely be used to achieve cancer-specific expression of NIS in many types of cancers. In combination with radioiodine therapy, this strategy is a possible method of cancer gene therapy.     

Prostate-specific antigen (PSA) promoter-driven androgen-inducible expression of sodium iodide symporter in prostate cancer cell lines

Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study represents an initial step toward therapy of prostate cancer with radioiodine.     

肿瘤血管靶向分子探针多肽RRL（g2）的作用研究

[目的]设计合成分子结构更小的多肽RRL（g2）,并用放射性核素99mTc标记,得到新型优化肿瘤新生血管分子探针。[方法]应用化学合成法合成多肽RRL（g2）及对照肽片段GGG（g2）,应用高效液相色谱（HPLC）及电喷雾离子质谱（EMI-MS）对合成化合物的分子量及纯度进行鉴定,进而对放射性核素99mTc标记的RRL（g2）多肽探针及对照肽进行体内外生物学性质评价。[结果]应用化学合成法合成的RRL（g2）及GGG（g2）纯度达99%以上,放射性核素99mTc的标记率约73%,稳定性较好。静脉注射分子探针后30min,99mTc-RRL（g2）相对于对照肽99mTc-GGG（g2）,在肿瘤组织中有较高的摄取率,而且在肿瘤组织中能滞留较长时间。[结论]新型肿瘤新生血管多肽探针99mTc-RRL（g2）相比于131I-tRRL,在肿瘤新生血管分子显像应用中拥有更大的优势,如肿瘤摄取率的增加,是更有前景的新型肿瘤新生分子显像多肽探针。     

重组腺病毒携带钠碘转运体基因介导放射性碘治疗小鼠胶质瘤

目的 验证转染人钠碘转运体基因(hNIS)介导放射性碘治疗肿瘤的有效性.方法 利用重组腺病毒将hNIS基因及人甲状腺过氧化物酶(hTPO)基因转染入人胶质瘤细胞系U251中,使肿瘤细胞获得hNIS和 hTPO基因,然后进行体外摄125I实验、过氯酸盐抑制实验、体外125I反流及内流实验.应用转染hNIS和hTPO基因及未转染hNIS和hTPO基因的细胞株,分别建立荷瘤裸鼠模型,并检测131I对肿瘤的抑制作用.结果 利用腺病毒可以将hNIS和hTPO基因成功转染到U251细胞系中,转染上述基因的肿瘤细胞系可以摄取碘,而且这种摄碘的功能是由hNIS基因所介导的,转染后的hNIS-U251细胞系摄碘能力是U251细胞的121.2倍,hNIS-hTPO-U251细胞系摄碘能力是U251细胞的172.0倍.131I对裸鼠移植瘤的治疗结果表明,在131I作用下,对照组肿瘤均继续迅速生长,而hNIS转染组及hNIS和hTPO共转染组移植瘤体积均有所减小.结论 在肿瘤细胞中转染hNIS和hTPO基因后,可以提高其摄取12I的活性.131I 可以有效杀伤荷瘤裸鼠体内的中瘤细胞.

Abstract：

Objective To explore the possibility of tranfecting hNIS and hTPO genes into gliomas cells by recombinant adenovirus for radioactive iodide treatment. Methods To tranfect hNIS gene into human glioma cell line U251 by recombinant adenovirus.The biological functions of the cells stably expressing hNIS and hTPO genes were assessed by 1251 uptake assay,125I influx-course and 12I-effluxcourse.A glioma model was established with inoculation of the U251 cells in nude mice,and the inhibiting effect of 131 I on the tumor growth was tested in the mouse models. Results The hNIS and hTPO genes were successfully transfected into human gliomas cell line U251 cells by recombinant adenovirus.The radioactive iodide could be intaken by the tumor cells mediated by hNIS gene.The uptake of 125I was higher in cell lines hNIS-U251 and hNIS-hTPO-U251 cells than in cell line U251 cells.The tumor volume of the mice after 131I treatment was significantly decreased in comparison with that before treatment.Conclusion Radioactive 131I treatment after HNIS-based gene transfer can be enhanced and effectively inhibite the tumor growth in nude mice.     

Sodium/iodide symporter gene transfection increases radionuclide uptake in human cisplatin-resistant lung cancer cells

The sodium/iodide symporter (NIS) is involved in iodide uptake and has been used for the diagnosis and treatment of thyroid cancer. Transfection of the NIS gene in A549 human lung cancer cells can induce radioactive iodine (¹³¹I) and radioactive technetium (^99mTc) uptake. The aim of the present study was to assess the role of NIS in ^99mTc and ¹³¹I uptake by the A549/DDP human cisplatin-resistant lung cancer cell line. To do so, recombinant adenovirus, adenovirus-enhanced green fluorescent protein-human NIS (Ad-eGFP-hNIS) and Ad-eGFP-rat NIS (Ad-eGFP-rNIS) vectors were established. These vectors were transfected into A549/DDP cells and xenograft tumors in nude mice. Assessment of ^99mTc and ¹³¹I uptake was performed. Results showed that the transfection efficiency of Ad-eGFP-hNIS and Ad-eGFP-rNIS in A549/DDP cells was at least 90 % in all experiments, and that the uptake ability of ^99mTc and ¹³¹I was highly enhanced (14–18 folds for ^99mTc, and 12–16 folds for ¹³¹I). However, the radionuclide concentration in transfected NIS genes’ A549/DDP cells reached a plateau within 30–60 min, indicating that NIS transport led rapidly to ^99mTc and ¹³¹I saturation in cells. In xenograft tumor models, uptake of ^99mTcO₄ ^? was obviously higher in the hNIS and rNIS groups compared with controls. In conclusion, these results support the hypothesis that A549/DDP cells can effectively uptake ^99mTc and ¹³¹I when transfected with the hNIS and rNIS gene. The rNIS or hNIS gene could be used as an effective method for the effective delivery of radioactive products to specific tissues for imagery and/or treatment.     

Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter

Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.     

Pharmacokinetics of 99mTc(Sn)- and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice

The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of 99mTc(Sn) and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant 125I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake [48.6% injected dose (ID)/g +/- 8.1% (SD)] and low tumor uptake (1.5% ID/g +/- 0.6%) for 99mTc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for 131I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the 131I-labeled fragments and poor tumor visualization with predominant kidney uptake for 99mTc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.     

Uptake of the cation hexakis(2-methoxyisobutylisonitrile)-technetium-99m by human carcinoma cell lines in vitro

Uptake of the cationic compound hexakis(2-methoxyisobutylisonitrile)-technetium-99m ([99mTc]MIBI) was examined in nine human tumor cell lines. The concentration of [99mTc]MIBI after a 1-h incubation with the compound varies from 5 to 28% of the activity in the external medium. In contrast, normal V79 cells (Chinese hamster lung fibroblasts) and human peripheral blood mononuclear cells exhibit a minimal uptake of less than 2% of the activity in the medium. Kinetic experiments with SW-13 cells indicate a rapid uptake over time (t1/2 of 10 min) until a steady state is approached whose concentration appears directly correlated with the extracellular concentration of [99mTc]MIBI with no evidence of saturation over the range tested (10(-12)-10(-9) M). [99mTc]MIBI is taken up by a temperature dependent process that is restricted to living cells. Microautoradiography demonstrates that [99mTc]MIBI is clustered in the cytoplasm around the nucleus. Given that depolarizing the plasma membrane potential in high K+ buffer results in lowering the uptake of [99mTc]-MIBI and that alteration of the mitochondrial membrane potential with valinomycin or nigericin induces, respectively, a significant decrease or increase of [99mTc]MIBI uptake, we propose that the plasma and mitochondrial membrane potentials play a major role in the uptake. These data suggest that the gamma emitter [99mTc]MIBI exhibits interesting tumor cell interaction characteristics with promise for in vivo tumor imaging.     

新型肿瘤新生血管靶向分子探针99mTc-RRL的合成及初步研究

[目的]探讨新型肿瘤新生血管显像剂99mTc-RRL的合成与标记及其在不同肿瘤动物模型中的初步显像效果,评价99mTc-RRL用于肝癌裸鼠模型体内的生物分布特点。[方法]固相合成法合成RRL,与99mTc进行标记。制备不同类型肿瘤动物模型,并进行单光子计算机断层（SPECT）显像。荷瘤HepG2肝癌裸鼠尾静脉注射99mTc-RRL后,于不同时刻处死动物,取感兴趣器官和组织称重并测量其放射性计数,计算各器官或组织的%ID/g值。同时,高锝酸钠阴性对照组、非标记RRL竞争阻断组采用同样方法,计算其6h时刻各脏器或组织的%ID/g值。HE染色和CD34免疫组化方法用于评价瘤体新生血管状态及分子探针检测阈值的相关性分析。[结果]99mTc-RRL标记率达80%,放射化学纯度高,体外稳定性好。SPECT显像示肿瘤部位见显影,轮廓清晰,放射性明显高于其他器官,随时间延长,肿瘤部位有持续放射性聚集。生物分布数据显示99mTc-RRL血液清除快,肿瘤部位显示放射性持续滞留,提供良好靶/非靶组织比值。HE染色和免疫组化结果显示了瘤体旺盛的血管生成;瘤体大小与探针摄取量呈明显正相关关系。[结论]99mTc-RRL能够特异性地聚集于不同类型肿瘤组织。体内生物分布好,可提供良好的靶/非靶组织比值,是具有应用价值的肿瘤新生血管靶向示踪剂。