Abstract
Radioiodide is an effective therapy for thyroid cancer. This treatment modality exploits the thyroid -specific expression of the sodium iodide symporter ( NIS ) gene, which allows rapid internalization of iodide into thyroid cells. To test whether a similar treatment strategy could be exploited in nonthyroid malignancies, we transfected non -small cell lung cancer ( NSCLC ) cell lines with the NIS gene. Although the expression of NIS allowed significant radioiodide uptake in the transfected NSCLC cell lines, rapid radioiodide efflux limited tumor cell killing. Because thyroperoxidase ( TPO ) catalyzes iodination of proteins and subsequently causes iodide retention within thyroid cells, we hypothesized that coexpression of both NIS and TPO genes would overcome this deficiency. Our results show that transfection of NSCLC cells with both human NIS and TPO genes resulted in an increase in radioiodide uptake and retention and enhanced tumor cell apoptosis. These findings suggest that single gene therapy with only the NIS gene may have limited efficacy because of rapid efflux of radioiodide. In contrast, the combination of NIS and TPO gene transfer, with resulting TPO -mediated organification and intracellular retention of radioiodide, may lead to more effective tumor cell death. Thus, TPO could be used as a therapeutic strategy to enhance the NIS -based radioiodide concentrator gene therapy for locally advanced lung cancer. Cancer Gene Therapy ( 2001 ) 8, 612 -618 

L ung cancer is the leading cause of cancer death in both men and women in the United States and has an overall 5 -year survival of less than 15%. 1, 2 Because lung cancer mortality has changed minimally in the last 20 years, novel therapeutic approaches are needed. We hypothesized that the introduction of the sodium iodide symporter (NIS) gene would enable radioiodide concentration within non -small cell lung cancer (NSCLC ) cells and promote tumor cell killing. Optimization of this approach could lead to clinically applicable strategies for the treatment of solid tumors including regionally advanced NSCLC. Iodide uptake by the thyroid is mediated by the NIS and is inhibited by potassium perchlorate (KClO 4 ). 3, 4 The expression of NIS in thyroid cancer cells also explains the therapeutic efficacy of radioiodide in this malignancy. 5, 6 131 I rapidly accumulates in thyroid cancer cells resulting in DNA damage and tumor cell death. Thyroperoxidase (TPO ) present in thyroid cells catalyzes iodination of tyrosine residues of thyroglobulin and thus promotes iodide retention within thyroid cells. 7 Any iodide not organified by TPO undergoes rapid efflux by an active process. 3 -6 Thus, a balance between NIS -mediated iodide influx and TPO -inhibited efflux determines the intracellular concentration of iodide. Expression of both NIS and TPO provides the therapeutic efficacy of radioiodide in thyroid diseases. 8 Endogenous NIS expression has not been found in lung cancer and it is not present in normal lung tissue. 9, 13 Transfection of a variety of tumors including melanoma, colon carcinoma, ovarian adenocarcinoma, lung cancer, 10, 11 and prostate cancer 12 with an NIS -expressing vector results in increased radioiodide uptake. However, rapid radioiodide efflux from these transfected cells may limit the antitumor efficacy of this therapeutic strategy.

TPO in NSCLC cells overcomes this deficiency and enhances tumor cell killing.
METHODS

Human NSCLC lines and culture conditions
Human lung adenocarcinoma cell lines SKLU -1, 125, and A549 and lung squamous cell carcinoma lines H520 and SK -MES -1 were obtained from American Type Culture Collection ( Rockville, MD ). The human squamous cell carcinoma line RH2 was established in our laboratory. 19 The cells were grown at 378C under an atmosphere of 5% CO 2 in air as monolayers in 25 -cm 2 tissue culture flasks containing 5.0 mL of RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U /mL penicillin / streptomycin solution and 2 mM glutamine ( JRH Biosciences, Lenexa, KS ) .
Determination of NIS mRNA expression by reverse transcriptase -polymerase chain reaction ( RT -PCR )
The PCR primer pairs for human NIS were synthesized and purified in the UCLA DNA -primer synthesis core facility. The sequence of the human NIS sense primer is 5 0 -GCT GAG GAC TTC TTC ACC GGG GGC CG -3 0 and the sequence of the antisense primer is 5 0 -GTC AGG GTT AAA GTC CAT GAG GTT G -3 0 . These two primer pairs correspond to the coding region 349-374 and 930 -906, respectively, of the human NIS cDNA with an amplified DNA fragment of 558 bp. Total RNA isolation and RT-PCR were performed as described previously.
Determination of NIS mRNA expression by reverse transcriptase -polymerase chain reaction ( RT -PCR )
14 Briefly, 5Â10 6 NSCLC cells were lysed in guanidine thiocyanate (4 M) / 2-mercaptoethanol (1 mM ) solution and a mixture of acidified phenol and choloroform was added. Total RNA was precipitated by adding an equal volume of isopropanol and the RNA pellet was washed twice with 75% ethanol and dried briefly under vacuum. The dried RNA pellet was dissolved in 0.1% diethyl pyrocarbonate -treated, doubledistilled H 2 O (DEPC -ddH 2 O ) . RT-PCR was performed using 1 g of total RNA. The initial reverse transcription was at 428C for 1 hour in a solution containing 4 L MgCl 2 (25 mM ) , 2 L 10Â PCR buffer (500 mM KCl, 100 mM Tris -
Determination of NIS mRNA expression by reverse transcriptase -polymerase chain reaction ( RT -PCR )
PCR amplification was performed by using an RT-PCR kit (Perkin -Elmer Cetus, Norwalk, CT ). A PTC -100 -60 thermal cycler (MJ Research, Watertown, MA ) was programmed as follows: 2 minutes at 948C for 1 cycle; 1 minute at 948C, 1 minute at 588C, 1 minute at 728C for 35 cycles; and 7 minutes at 728C for 1 cycle. Twenty -five microliters of PCR products from each reaction were analyzed by 1.5% agarose/ ethidium bromide gel electrophoresis.
Southern blot analysis
The amplified PCR products were immobilized on a nylon membrane by a UV cross -linker (Stratagene, La Jolla, CA ) . The membrane was hybridized with a 32 P -labeled EcoRI fragment of NIS cDNA probe. After washing, the membrane was exposed to an X -ray film (Sigma, St. Louis, MO ) for 4 hours at À 708C.
Transfection of human NSCLC cell lines
The 7.8-kb pcDNA3 /NIS plasmid vector containing a 2.4-kb full -length functional human NIS insert driven by a CMV promoter was described previously. 15 The 6.0-kb pBShTPO plasmid vector containing a 3.0 -kb full -length functional human TPO insert driven by a CMV promoter was provided by Dr 15 The medium containing 125 I was removed and the cell monolayers were washed once with the same buffer. The cell -associated radioactivity of each sample was measured with a -counter.
Time course of
125 I uptake and 125 I efflux in NIS -transfected and NIS and TPO cotransfected RH2 cells
For the time course studies of NIS -mediated
125 I uptake in NSCLC, NIS -and control vector -transfected RH2 cells were incubated with 125 I (100 nmol /L ) . At various time points, the medium containing radioiodide was removed. The cell monolayers were rinsed with buffer and remaining cell -associated radioactivity was measured with acounter. For the 125 I efflux studies, NIS -transfected RH2 cells, NIS and TPO cotransfected RH2 cells, and RH2 cells receiving control vectors only were incubated with 125 I (100 nmol /L ) for 1 hour and the medium containing 125 I was removed. The cell monolayers were rinsed with buffer once and nonradioactive fresh medium was added. At various time points, the remaining cell -associated radioactivity of each sample was measured with a -counter. HUANG, BATRA, KOGAI, ET AL: NIS/TPO GENE THERAPY FOR LUNG CANCER was removed. TCA precipitation for determination of radioiodinated protein content was performed as described. 16 Briefly, cell lysates from each condition were prepared in buffer containing 1% sodium dodecyl sulfate (SDS ) and adjusted to a final volume of 3 mL containing 50 L of 2% sodium deoxycholate. Proteins in the cell lysates were precipitated by the addition of 1 mL of TCA to yield a final concentration of 10%. The assay test tubes were centrifuged at 3300Âg for 30 minutes and the supernatants containing free radioiodide were carefully removed by aspiration with a Pasteur pipette. The precipitated proteins were dissolved in 0.5 mL Hank's buffer and the associated radioactivity was measured with a -counter.
Inhibition of TPO by MMI
MMI is a TPO -specific inhibitor that acts by uncoupling TPO -catalyzed oxidative iodination. 17 Briefly, the transfected RH2 cells were incubated in culture containing 300 M MMI at 378C for 24 hours. The cells were exposed to 125 I (100 nmol /L ) solution at 378C for 1 hour and the medium containing radioiodide was removed. The cell monolayers were rinsed with buffer once. Cell lysates were prepared and intracellular proteins were precipitated by a TCA method as noted above. The associated radioactivity from the protein fraction of each sample was measured with a -counter. The results were compared with cells without MMI treatment.
Nuclear matrix protein ( NMP ) assay
Apoptosis was quantified by measuring the amount of released NMP from apoptotic cells. 18 In this assay, the amount of released NMP is a function of the number of apoptotic cells. Transfected RH2 cells were incubated in 125 I ( 100 nmol /L ) solution at 378C for 10 minutes and the medium containing radioiodide was removed. The cell monolayers were rinsed with buffer and incubated in medium without mark27@example.org. After 24-hour incubation, the culture medium from each sample was collected and radioiodide -induced apoptosis was quantified using an NMP ELISA kit ( Oncogene Research Products, Cambridge, MA ).
Statistical analysis
Results are presented as means ± SD from a single experiment that is representative of at least three replicate experiments. When error bars are not visible, they are obscured by the symbol for the mean. Significance between experimental versus control values was calculated using the Student's t test.
RESULTS

NIS -mediated
125 I uptake in NSCLC Six different NSCLC cell lines (RH2, H520, 125, SK -Mes -1, SKLU -1, A549 ) were transiently transfected with pcDNA3/ NIS vectors or control empty vectors. Utilizing RT-PCR, the NIS mRNA expression was only detectable in the NIS -transfected NSCLC cell lines but not in the parental lines ( Fig 1A ) . Radioiodide uptake was measured to assess NIS function in the transfectants. After 48 hours, cells were incubated with 125 I ( 100 nmol /L ) with or without KClO 4 for 1 hour. The medium containing 125 I was removed and the cell monolayers were washed with the same buffer. The cellassociated radioactivity of each sample was measured with a -counter. A 2 -to 10 -fold increase in 125 I uptake over the baseline was observed in six NIS -transfected NSCLC cell lines ( Fig 1B ) . The 125 I uptake in NIS -transfected lung cancer cells could be inhibited by KCIO 4 , consistent with the function of NIS in thyroid cells. Thus, these results suggest that the increase in radioiodide uptake in transfected lung cancer cells is specifically NIS -mediated. Time course of NIS -mediated 125 
I uptake and efflux in RH2 cells
To determine the kinetic parameters of radioiodide transport in the NIS -transfected NSCLC, we performed 125 I time course and efflux studies in a representative human lung squamous cell carcinoma cell line, RH2. First, the NIStransfected RH2 cells and control RH2 cells (transfected with empty vectors) were incubated with 125 I ( 100 nmol / L ) for various durations and cell -associated radioactivity was measured. Maximal 125 I uptake in the NIS -transfected RH2 cells plateaued within 10 minutes and cells maintained this level when radioiodide was present in the medium (P < .01 at all time points ) (Fig 2A ) . Because cells did not further concentrate iodide, the results suggest that a balance of radioiodide uptake and efflux in the NIS -transfected RH2 cells was established. When cells were then only exposed to radioiodide for 1 hour followed by removal of 125 I -containing medium, a rapid efflux of radioiodide from the cells was evident (Fig 2B) .
NIS and TPO cotransfected RH2 cells show significant increase in radioiodide uptake and retention
Because the efficacy of radioiodide concentrator gene therapy may be related to both radioiodide uptake and retention, we sought to increase intracellular radioiodide retention using TPO -mediated organification. Accordingly, we transfected RH2 cells with the NIS and TPO genes. After 48 hour transfection, cells were exposed to 125 I for 1 hour. Medium containing 125 I was then removed and replaced with fresh medium. At various time points, the remaining radioactivity from each sample was measured with a -counter. NIS and TPO cotransfected lung cancer cells exhibited both an increased radioiodide uptake ( P <.001 at 0 minutes, Fig 3 ) and radioiodide retention ( P <.01 at 5 -30 minutes, Fig 3 ) compared to the NIStransfected cells.
The increase in radio -iodinated intracellular proteins in NIS and TPO cotransfected RH2 cells is TPO -dependent
To confirm that increased radioiodide uptake and retention were secondary to an increased TPO -mediated iodination I ( 100 nmol / L ) . At various time points ( 5 -120 minutes ) , the medium containing radioiodide was removed. The cell monolayers were rinsed with buffer and remaining radioactivity was measured with acounter. Maximal 125 I uptake in the NIStransfected RH2 cells plateaued within 10 minutes and cells maintained this level while radioiodide was present in the medium ( P < .01 at all time points ) . B: Rapid 125 I efflux following uptake in the NIS -transfected RH2 cells. When cells were transiently exposed to radioiodide for 1 hour followed by removal of 125 I, a rapid efflux of radioiodide from the cells was evident ( P < .01 at time points 5 -30 minutes compared to 0 minutes ) . HUANG, BATRA, KOGAI, ET AL: NIS/TPO GENE THERAPY FOR LUNG CANCER of intracellular proteins, we exposed cells to the TPO inhibitor MMI and quantified the iodinated protein fraction in transfected cells. As predicted, cells transfected with NIS alone had a small but significant portion of radioiodinated intracellular proteins ( P <.05 compared to controls ) that was not sensitive to MMI inhibition ( Fig  4 ) . In contrast, the NIS /TPO cotransfected cells showed a marked increase in radio -iodinated intracellular proteins ( P <.01 compared to both NIS alone and controls ) that was blocked by MMI ( Fig 4) . These results indicated that TPO, which organifies thyroglobulin in thyrocytes, was capable of organifying alternative protein substrates within lung cancer cells.
NIS and TPO cotransfected RH2 cells show enhanced radioiodide -induced apoptosis
Radioiodide is well known as an established, efficient, and safe therapy for hyperthyroidism and thyroid cancer. Radiation -induced tumor cell apoptosis has been well documented. 10 -12,18,20 To test whether the combination of NIS / TPO -mediated increased uptake and retention of radioiodide results in tumor cell killing, we quantified radioiodide -induced apoptosis in the transfected NSCLC RH2 cells. NMP ELISA detects solubilized NMRs released by apoptotic cells. This assay is based on the observation that there is a linear relationship between the portion of apoptotic cell population and the amount of NMRs released to the culture medium during cell apoptosis. In our preliminary studies, we determined that one NMP unit equals approximately 365 apoptotic RH2 cells (data not shown) . To induce apoptosis, the transfected cells were incubated with 125 I (100 nmol / L) for 10 minutes. The cell monolayers were rinsed with buffer and incubated in medium without radioiodide for 24 hours. Both NIS -transfected and the combination of NIS and TPO cotransfected lung cancer cells showed significant radioiodide-induced apoptosis compared to the controls (P < .01 ) ( Fig 5 ) . Furthermore, NIS and TPO cotransfected RH2 cells showed a greater degree of radioiodide-induced apoptosis compared to the NIS -transfected RH2 cells (P < .05 ) ( Fig 5) . These results indicate that an effective elevation of intracellular radioiodide levels resulted in an increase of apoptosis in NSCLC.
DISCUSSION
The mortality associated with lung cancer has changed minimally in the last 20 years, in part because there is no curative or consistently effective therapy for patients with advanced lung cancer. We have chosen to further develop a recently introduced gene -based strategy for potentially I medium was removed. The cells were washed in buffer and radio -iodinated protein content from each sample was determined by TCA precipitation. Control = control vector -transfected RH2 cells. NIS = NIS -transfected RH2 cells. NIS / TPO = NIS and TPO cotransfected RH2 cells. NIS versus control = P < .05; NIS / TPO versus control = P < .01; NIS / TPO versus NIS = P < .01. Figure 5 . The NIS and TPO cotransfected RH2 cells show enhanced radioiodide -induced apoptosis. NIS -transfected, NIS and TPO cotransfected, and control vector -transfected RH2 cells were exposed to 125 I ( 100 nmol / L ) for 10 minutes and 125 I medium was removed. The cell monolayers were rinsed with buffer and incubated in medium without radioiodide at 378C for 24 hours. The culture medium from each sample was collected for NMP ELISA. One NMP unit equals approximately 365 apoptotic RH2 cells. Both NIStransfected and NIS and TPO cotransfected lung cancer cells showed significant radioiodide -induced apoptosis compared to the controls ( P < .01 ) . Additionally, NIS and TPO cotransfected RH2 cells showed a greater degree of radioiodide -induced apoptosis compared to the NIS -transfected RH2 cells ( P < .05 ) .
DISCUSSION
Cancer Gene Therapy, Vol 8, No 8, 2001 treating regional lung cancer. This strategy introduces the NIS gene into tumor cells to promote radioiodide uptake, and consequently, to induce cytotoxicity. Importantly, our preliminary results using gene transfection suggested that the feasibility of this strategy would be limited by the lack of intracellular retention of the internalized radioiodide. Our current findings introduce a novel intervention that represents a conceptual advance to NIS-based gene therapy. We have documented enhanced retention of radioiodide by promoting its organification, and as a result, enhanced the cytotoxic efficacy of this approach.
DISCUSSION
131 I therapy is the standard treatment for differentiated thyroid cancers that have the capacity to accumulate iodide. 7 Because the treatment is very effective for thyroid cancer, Mandell et al 10 initially proposed NIS -based radioisotope concentrator gene therapy for extrathyroidal cancer. These investigators demonstrated that NIS -transduced cancer cells could accumulate significantly more radioiodide than parental cells, and consequently, be selectively killed in in vitro and in vivo models. Spitzweg et al 12 introduced the prostate -specific antigen ( PSA ) promoter to specifically regulate NIS transcription, and demonstrated tumor cell selectivity in a prostate cancer model. Most recently, Boland et al 11 generated an adenoviral vector encoding NIS, and presented data suggesting that a variety of tumor cell lines, including the NSCLC cell line A549, increased radioiodide uptake 35-to 225 -fold following adenovirus -mediated NIS gene transfer.
DISCUSSION
To evaluate this therapeutic approach for the treatment of lung cancer, we transfected six human NSCLC cell lines with the full -length functional NIS gene in a plasmidexpression vector (Fig 1 ) . We observed that there was indeed a rapid and significant increase in radioiodide uptake. The peak 125 I uptake occurred rapidly and cells maintained a high steady-state intracellular concentration provided that radioiodide was present in the medium. However, if radioiodide was removed from the medium, the cells displayed a rapid loss of iodide, suggesting that there was a pronounced egress of radioiodide in effect ( Fig  2 ) . In fact, this rapid efflux of radioiodide was also observed in a variety of tumor types. 10 -12 Taken together, these data indicated that the steady state intracellular concentration was both a function of internalized radioiodide following NIS gene transfer, and the rapid egress of 125 I that followed. This led us to conclude that the cytotoxic efficacy of NIS gene transfer may be limited by radioiodide efflux, and that enhancing the intracellular retention of radioiodide may confer a therapeutic advantage. Accordingly, before embarking on the construction of viral vectors for optimizing gene transfer, we set out to establish a proof -of -concept that we could augment intracellular retention of 125 I by promoting organification of the radionuclide. In addition, we wanted to evaluate whether increased retention corresponded to increased cytotoxicity.
DISCUSSION
We hypothesized that we could enhance the intracellular retention of radioiodide by promoting its organification using TPO, and that the increased retention would yield more cytotoxicity. To test this hypothesis, we constructed an expression vector that encoded TPO, and cotransfected this vector with the NIS gene. As indicated, the combination of NIS and TPO gene transfer resulted in increased uptake and retention of radioiodide compared to NIS transfection alone ( Fig 3 ) . In the thyroid, TPO iodinates thyroglobulin tyrosine residues resulting in iodide accumulation within thyrocytes. Similarly, as indicated by an increase in radio -iodinated intracellular proteins, the increased intracellular concentrations of 125 I were attributable to TPO -mediated organification, with a resultant lower radioiodide efflux from the cells ( Fig 4) . Because thyroglobulin is not present in NSCLC, TPO presumably utilizes other tyrosine -rich protein substrates to achieve efficient iodination of intracellular proteins in the NIS /TPO -transfected lung cancer cells. The confirmation and identification of these alternative protein substrates for TPO in NSCLC will require further investigation. Lastly, we have also established that increased intracellular retention confers greater cytotoxicity in lung cancer cells (Fig 5) .
DISCUSSION
In addition to thyroid cancer, breast cancer also expresses the NIS gene. 20 NIS gene expression is up -regulated by retinoic acid in breast cancer cells, and as a result, uptake of radioiodide by the breast cancer cells is further augmented by retinoic acid. 20 Because the efflux of radioiodide is relatively slow compared to lung cancer, the therapeutic efficacy of radioiodide incorporation is not affected in breast cancer cells. 20 Thus, although TPO may enhance the NIS-based radioiodide concentrator gene therapy in lung cancer, TPO may not be beneficial in different cancer types such as breast cancer in which radioiodide efflux has little effect on the efficacy of radiation -induced tumor cell death. We have tested whether expression of the NIS gene can be influenced or up -regulated by NSCLC in response to retinoic acid, but have found no induction of NIS response as measured by radioiodide uptake ( data not shown ).
DISCUSSION
In conclusion, the current studies suggest that the addition of TPO to NIS gene transfer contributes to radioiodide uptake and retention, and leads to greater tumor cell death. Following intratumoral administration, this combination gene therapy may play a role in controlling tumor growth in locally advanced NSCLC. Our studies support the development of efficient gene transfer vectors that encode both NIS /TPO for further preclinical evaluation of this gene therapy approach. Future work will evaluate the antitumor efficacy utilizing bicistronic adenoviral vectors expressing both NIS and TPO genes in an in vivo murine lung cancer model. We anticipate that these studies will provide information that will assist in both understanding and improving the efficacy of NIS /TPO radioiodide concentrator gene therapy.

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