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Expression Is Reduced in Anaplastic Thyroid Cancer and Its Reexpression Results in Apoptosis
Laboratory of Endocrine Cell Biology, National Research Laboratory Program, Departments of Internal Medicine (H.K.C., J.M.S., H.K., K.C.P., D.W.K., E.S.H., J.H.S., B.-J.K., H.J.H., H.K.R., M.S.) and Pathology (J.M.K.), Chungnam National University College of Medicine, Daejeon 301-721; Neurogenex Inc. (Y.-W.Y., N.-C.J.), Seoul 151-744; and Department of Biological Science (D.K.), Korea Advanced Institute of Science and Technology, Daejeon 301-721, Korea
Address all correspondence and requests for reprints to: Minho Shong, M.D., Laboratory of Endocrine Cell Biology, Department of Internal Medicine, Chungnam National University School of Medicine, 640 Daesadong Chungku, Taejon 301-040, Korea. E-mail: minhos{at}cnu.ac.kr.
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Abstract |
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RNA was present at significantly lower levels in anaplastic cancer cells, compared with normal primary cultured thyrocytes. In addition, the adenovirus-mediated reexpression of Gadd45 significantly inhibited the proliferation of anaplastic thyroid carcinoma cells, ARO, FRO, and NPA cells, which was attributed to apoptosis. Furthermore, the adenovirus-mediated delivery of Gadd45 gene in anaplastic thyroid cancer resulted in the inhibition of tumor growth in vivo. This in vitro and in vivo activity of the adenovirus-mediated transduction of CR6/Gadd45, on anaplastic thyroid cancer cell growth suppression, was reminiscent of the effects of p53. This study demonstrates that the Gadd45 gene has potential use as a candidate gene for gene therapy in anaplastic thyroid cancer. |
Introduction |
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Martelli et al. (7) generated cell hybrids by fusing the cells with the anaplastic phenotypes (ARO and FRO) and differentiated phenotype. Although these cell hybrids contained alleles from the highly malignant parental cell lines, ARO and FRO showed a lower growth rate, compared with parental cell lines, and were unable to grow in soft agar and induce tumors after injection into athymic mice. These findings suggest that anaplastic carcinoma is achieved by the impairment of gene functions that negatively regulate cell growth, rather than by the activation of dominant oncogenes. Therefore, the identification of relevant genes involved in suppression of growth potentials of anaplastic thyroid cancers are important for the understanding and development of new therapeutics for anaplastic thyroid cancer.
The Gadd45 family (growth arrest and DNA damage-inducible gene family) consists of Gadd45
(8), Gadd45ß(MyD118) (9), and Gadd45(CR6/OIG37) (10, 11, 12). All Gadd45 family members are small acidic proteins that are expressed to different degrees in all tissues and its expression is mediated by both p53-dependent and -independent mechanisms (13 13A, 14, 15). Gadd45, which is identified as an activator of mitogen-activated protein/ERK kinases 4/MTK1 (11), an IL-2-induced immediate-early gene (CR6) (10), an oncostatin M-inducible gene (OIG37) (12), shows high amino acid sequence similarity to Gadd45 and Gadd45ß(Myd118) (68% and 70%, respectively). Gadd45 family proteins has been implicated in a variety of growth-regulatory mechanisms, including DNA replication and repair (16, 17), G2/M checkpoint control (18, 19), and apoptosis (11).
Because Gadd45 plays critical roles in G2/M arrest and induction of apoptosis, we analyzed he expression of Gadd45 in anaplastic thyroid cancer cell lines to test the potential utilities of Gadd45 gene for candidate for gene therapy in anaplastic thyroid cancer. We found that Gadd45 expression was significantly reduced in anaplastic cancer cell lines, compared with cultured normal thyrocytes. Reexpression of Gadd45 in anaplastic cancer cells inhibited proliferation. The inhibition of proliferation was results from apoptosis. Furthermore, adenovirus-mediated delivery of Gadd45 gene in anaplastic thyroid cancer results in inhibition of tumor growth in vivo. The in vitro and in vivo activity of growth suppression of anaplastic thyroid cancer cells by adenovirus-mediated delivery of CR6/Gadd45 were similar to p53. Here, we suggest that the Gadd45 gene has potential utilities as a candidate gene for gene therapy against anaplastic thyroid cancer.
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Materials and Methods |
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Cells were cultured in RPMI 1640 (Life Technologies, Inc., Burlington, Ontario, Canada) containing 10% fetal bovine serum (FBS) (Life Technologies, Inc.) with 1% penicillin/streptomycin (Life Technologies, Inc.). Tetrazolium [dimethylthiazoldiphenyltetra-zoliumbromide (MTT), M2128] was purchased from Sigma Chemical Co. (St. Louis, MO), and a stock solution was prepared by dissolving 5 mg MTT in 1 ml PBS and filtering the solution to remove particulates. The enhanced chemiluminescence detection system was purchased from Amersham, Inc. (Arlington Heights, IL). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) was performed using a commercial apoptosis detection kit (Promega Corp., Madison, WI). [-32P] was purchased from Amersham. A polyclonal mouse anti-p53 antibody was purchased from Transduction Laboratories Inc. (Mississauga, Ontario, Canada).
Antibody preparation
A peptide (DIVRVGDVQRL) (5 mg), corresponding to the midregion of human Gadd45, was coupled to the keyhole limpet hemocyanin (10 mg; Calbiochem, La Jolla, CA) by glutaraldehyde. After dialyzing against PBS, the resultant conjugate was mixed with an equal volume of Freund’s complete adjuvant and inoculated sc into two rabbits (1 mg each). The rabbits were given a booster dose every 2 wk and were bled 1 wk later. The IgG fraction was purified from the collected antisera using a Protein A-Sepharose (Pharmacia Biotech Inc., Uppsala, Sweden) column. The prepared antibodies were used for Western blot analysis. The specificity of the antibodies was confirmed by Western blot analysis using the recombinant GST-Gadd45 protein.
Cell culture
Thyroid tissues were obtained by total thyroidectomy from the patients with papillary thyroid cancer. Informed consent was obtained from all living patients, and the study was approved by the ethical committee of the Chungnam National University Hospital. The normal thyrocytes were isolated from the contralateral lobe as described previously (20). After the tissues were rinsed in PBS with penicillin (100 U/ml) and streptomycin (100 µg/ml), connective tissue was removed and thyroid tissue was finely minced. The obtained thyroid tissue fragments were stirred in flasks containing 100 µg/ml collagenase (Sigma) and 3.33 mg/ml dispase (BD Bioscience, Mississauga, Ontario, Canada) in Hank’s balanced salt solution on a magnetic stirrer, at 37 C for 30 min. The suspension was passed through a nylon filter and mixed with RPMI 1640 containing 10% heat-inactivated fetal calf serum. The filtrate was centrifuged at 1000 rpm for 5 min, and pellets were defined as thyrocytes.
Three human thyroid carcinoma cell lines (ARO, FRO, and NPA) were grown in RPMI 1640 medium supplemented with 10% FBS and ampicillin/streptomycin. The ARO and FRO cells were both derived from anaplastic carcinomas and harbor a defective and mutant (R273H) p53 gene (4). The NPA cells were derived from poorly differentiated papillary thyroid carcinomas also containing the mutant (G266E) p53 gene (4).
Immunohistochemistry
The anti-Gadd45 antibodies were obtained from New Zealand White rabbits that were immunized with the conjugated synthetic peptide corresponding to the midregion of human Gadd45, as described above. The antibodies were titrated against the GST-Gadd45 fusion protein by an enzyme-linked immunosorbent assay, and the serum exhibiting the highest titer (1:25,600) was used in the subsequent experiments. The tissue specimens were fixed in 10% formalin, embedded in paraffin, and sectioned. They were then stained with hematoxylin and eosin to evaluate the histological type. The samples used in this study were confirmed histologically as being either benign or malignant.
Gadd45 expression was analyzed by immunohistochemistry in thyroid cancer tissues. The samples were microwaved for 10 min, and slides were incubated with the anti-Gadd452 antiserum (1:800) at room temperature for 1 h. The slides were washed in PBS and incubated with a linking solution containing biotinylated goat antirabbit IgG (LSAB kit; DAKO Laboratories) at room temperature for 30 min. The slides were then sequentially incubated with streptavidin peroxidase at room temperature for 30 min and the 3,3'-diaminobenzidine chromogen substrate solution (DAKO Laboratories), counterstained with Meyer’s hematoxylin, and mounted. The preimmune sera did not stain the tissues at a 1:800 dilution.
Northern blot analysis
Complementary DNA probes for Gadd 45, -ß, and - were radiolabeled with Klenow DNA polymerase-directed incorporation of 32P deoxycytidine 5'-triphosphate, primed by random deoxynucleotide monophosphate hexamers. The probe was purified by removal of unincorporated 32P-labeled deoxycytidine 5'-triphosphate by Sephadex-G-50 spin column. Prehybridization and hybridization were carried out in QuickHyb hybridization solution according to the manufacturer’s protocol (Stratagene, La Jolla, CA). The stringency of the washing conditions used in the Northern blot experiments is as follows: low-stringency wash was done at room temperature x 30 min with three changes of wash solution containing sodium chloride 300 mmol/liter, sodium citrate 300 mmol/liter [2 x saline sodium citrate (SSC)], and 0.1% sodium dodecyl sulfate (SDS). High-stringency wash was done at 50 C x 40 min with two changes of wash solution containing 0.2x SSC, 0.1% SDS.
Generation and transduction of recombinant adenovirus
Recombinant adenoviral vectors were generated by standard homologous recombination method (21). Briefly, each cDNA encoding p53 or Gadd45 were amplified by PCR and cloned into the shuttle vector, pxcx2dCMV (cytomegalovirus). Adp53 and AdGadd45 adenovirus was generated through homologous recombination between cotransfected pBHG10 plasmid and shuttle plasmids expressing p53 or Gadd45 in 293 cells as described previously (21). The AdGFP or AdCMVGAL virus used as a control was an E1 deletion recombinant adenovirus containing a ß-galactosidase and green fluorescent gene driven by CMV promoter. Recombinant adenoviruses were amplified after plaque purification and titrated using plaque assay as described (22). All expression cassettes were confirmed by DNA sequencing and Western blot.
Western blot analysis for Gadd45 and p53 expression
The subconfluent cells in a 10-cm dish were infected with either Adp53 or AdGadd45 at the multiplicity of infection (MOI) indicated. Two days later, total cell lysates were prepared and 20 µg protein were subjected to Western blot analysis in 12% SDS-PAGE under denaturing and reducing conditions. Antihuman p53 monoclonal antibody (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and polyclonal anti-Gadd45 antiserum were used to detect expressed p53 and Gadd45 proteins.
MTT assay
Cell proliferation levels were evaluated using MTT as a marker for cellularity after defined periods of culture. Cells were harvested using a 0.25% trypsin/EDTA solution and resuspended in serum-reduced medium (RPMI + 1% FBS). Cell suspensions of 2.5 x 104 cells/100 µl were plated into 96-well plates. Ninety-six-well plates were then incubated for 48 h, without any additional supplementation, at 37 C in a 5% CO2 incubator. Cells were infected with AdGadd45 and Adp53 at MOI 50 for indicated times. At the end of the incubation, each well received 25 µl of 0.5% MTT solution. The plates were then returned to the incubator for a period of 2 h. At the completion of this second incubation, 100-µl extraction buffer [20 µg SDS in 80 ml N,N-dimethyl formamide and water, to a volume of 100 ml (pH 4.7)] was added and mixed thoroughly. The plates were then incubated overnight at 37 C in a 5% CO2 incubator. The plates were read on a spectrophotometer at an absorbance of 540 nm.
Apoptosis analysis
To demonstrate that the cells were apoptotic, we used a DNA laddering and TUNEL method (23). The 106 cells were used to prepare samples for DNA laddering. Cells were lysed and digested for 1 h at 50 C in 20 µl of a solution containing 5 mM EDTA, 25 mM Tris-HCl (pH 8.0), 5 mg/ml lauryl sarcosine, and 0.5 mg/ml proteinase K. RNase A (10 µl) from a stock solution of 0.5 mg/ml was added to the samples, which were incubated for an additional hour at 50 C. The samples were heated to 70 C, and 10 µl of melted loading buffer containing 10 M EDTA, 1% (wt/vol) low-melting-point agarose, 40% (wt/vol) sucrose, and 0.25% bromphenol blue were added to each sample. The samples were immediately dry loaded into a gel of 2% agarose type II (Sigma) and 0.7% Infinity Agarose Enhancer (Oncor, Gaithersburg, MD). To make up the gel, the agarose and enhancer were slurried in 7 ml ethanol and then made up to 70 ml with Tris acetate buffer [40 mM Tris, 5 mM sodium acetate, and 1 mM EDTA (pH 8.1)] and supplemented with 0.5 µg/ml ethidium bromide. The gel was run at 40 V for 5 h in Tris-acetate buffer.
TUNEL was performed using a commercially available apoptosis detection kit (Promega) with the following modifications: Samples were fixed with 4% paraformaldehyde (methanol-free) for 10 min at room temperature, washed twice with PBS for 5 min, and then incubated with 0.2% Triton X-100 for 15 min at room temperature. After two washes of 5 min each with PBS, the samples were incubated with equilibration buffer (from kit) for 10 min at room temperature. The equilibration buffer was drained, and reaction buffer containing equilibration buffer, nucleotide mix, and TdT enzyme was added and incubated in a humid atmosphere at 37 C for 1 h in the dark. The reaction was terminated by immersing the samples in 2x SSC for 15 min. Samples were washed three times for 5 min to remove unincorporated fluorescein-deoxy uridine 5-triphosphate. Fluorescent bleaching was minimized by treating slides with an enhancing reagent (Prolong solution). Immunofluorescence microscopy was performed using fluorescence microscope (Leica). ARO, FRO, and NPA cells were identified by red fluorescence, and DNA fragmentation was detected by localized green and yellow fluorescence within the nucleus of apoptotic cells. The proportion of apoptotic populations was calculated by counting 10,000 nuclei in each slide under a microscope, and three sample slides for each treatment were counted to obtain an average.
Treatment of tumors with AdGadd45
Six- to 7-wk-old male nude mice (Charles River Laboratories, Wilmington, MA) were injected sc, on both sides of the flanks, with 5 x 106 ARO cells in 100 µl PBS. Tumor sizes were measured regularly and the volume was calculated using the formula for an ellipsoid, i.e. tumor volume (mm3) = 
/6 x W x L2, where L = length of the tumor and W = width of the tumor. When the tumors had reached approximately 100 mm3, mice (n = 7) were randomly divided into four treatment groups: 1) Not infected, 2) AdCMVGAL, 3) Adp53, and 4) AdGadd45. Viruses (109 pfu) in 150 µl serum-free medium were administered intratumorally one time every day and the tumor sizes were monitored. All mice were maintained in Animal Facility of Chungnam National University School of Medicine. All animal studies were approved by the Animal Care Committee and were conducted in accordance with the principles and the procedures outlined in the Guide for the Care and Use of Laboratory Animals in Chungnam National University School of Medicine.
Statistical analysis was performed using an unpaired t test with StatView 4.02 software (Abacus Concepts Inc., Berkeley, CA).
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Results |
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RNA in normal thyroid cells and ARO, FRO, and NPA cells
The expression of Gadd45 family genes is evaluated in normal human thyroid cells and anaplastic thyroid cancer cell lines ARO, FRO, and NPA. Gadd45, Myd118/Gadd45ß, and CR6/Gadd45 RNA were detected in primary cultured human thyroid cells (Fig. 1
). ARO, FRO, and NPA cells showed a similar level of Gadd45 and Gadd45ß expression, compared with normal primary cultured thyrocytes. Although primary cultured thyrocytes showed significant level of Gadd45 RNA expression, the cell lines, ARO, FRO, and NPA, did not show a detectable level of Gadd45 RNA expression. These observations suggest that regulation of Gadd45 expression was altered in anaplastic thyroid cancer cell lines ARO FRO, and NPA.
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expression in the primary thyroid tissues. The Gadd45 expression level in the papillary thyroid cancer samples were compared with the surrounding normal thyroid tissue. The Gadd45 expression level in the papillary thyroid cancer tissue was similar to the normal tissue (Fig. 1B
). The Gadd45 and -ß expression level in the tumor tissue showed a similar RNA expression level, compared with the normal tissue (data not shown). These observations suggest that Gadd45 expression may not be altered in differentiated thyroid cancer, such as papillary thyroid cancer.
Two patients (C1 and C2) were selected after a review of the histology of the surgical samples to observe Gadd45 expression in the poorly differentiated thyroid cancers. Gadd45 expression was examined in poorly differentiated thyroid cancers, which originated from a papillary cancer as determined by immunohistochemical analysis (Fig. 1C). The expression of Gadd45 was ubiquitous (data not shown) and normal thyroid epithelial cells exhibited strong immunoreactivity to the anti-Gadd45 antibodies. Gadd45 immunoreactivity was confined to within nucleus and the nuclear Gadd45 immunoreactivity was strongly positive in the normal thyroid epithelial cells. However, the nucleus of the cancer cells showed a markedly lower Gadd45 immunoreactivity (Fig. 1C). The infiltrated lymphocytes within the tumors showed a high level of Gadd45 immunoreactivity in their nuclei, suggesting the uniformity of staining in the slides. These findings indicate that Gadd45 expression may be lost in certain steps of tumor progression.
Expression of Gadd45 and p53 in ARO, FRO, and NPA cells by infections of AdGadd45 and Adp53 adenovirus
We tried to observe the effects of reexpression of Gadd45 in anaplastic thyroid cancer cell lines. For the expression of Gadd45 in those cancer cell lines, we transduced AdGadd45 into ARO, FRO, and NPA cells. At the same time, we also wanted to observe the cellular effects of p53 expression in same cell lines to compare the effects of Gadd45. The efficiency of adenovirus gene transfer into anaplastic thyroid cancer cells was determined by fluorescent microscopy 48 h after infection with AdGFP. AdGFP efficiently transferred the GFP gene into the cells; more than 95% of staining or fluorescence was obtained at an MOI of 50 in ARO, FRO, and NPA cells (data not shown). The expression of Gadd45 and p53 protein by AdGadd45 and Adp53 infection was confirmed by Western blot analysis (Fig. 2). Uninfected ARO, FRO, and NPA cells showed an extremely low level of Gadd45 protein. ARO cells expressed a low level of mutant p53 protein, but FRO and NPA cells did not express a detectable level of p53 protein. These p53 expression patterns are consistent with their p53 gene status as described in previous studies (24). Infection of AdGadd45 resulted in a high level of Gadd45 expression in ARO, FRO, and NPA cells (Fig. 2A). The significant expression of Gadd45 was achieved by the dose of AdGadd45 at 30 MOI in all three above cell lines (Fig. 2A). Infection of Adp53 also showed increases of p53 protein level in ARO and NPA cells. However, FRO cells showed inefficient expression of p53 by transduction of Adp53 (Fig. 2B). An infection with AdGFP and AdCMVGAL did not induce any endogenous Gadd45 expression in the ARO, FRO, and NPA cells (data not shown). From these observations, we found AdGadd45 are effective expression measures for Gadd45 in ARO, FRO, and NPA cells.
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transduction on proliferation in anaplastic thyroid cancer cells
To evaluate the effects of Gadd45 transduction on in vitro proliferation and viability of anaplastic thyroid cancer cells, ARO, FRO, and NPA cells infected with AdGadd45 at MOI of 100. The effects of Gadd45 on cell proliferation were compared with AdGFP as a negative control and Adp53 as a positive control. The ARO, FRO, and NPA cells were infected with AdGFP, Adp53, and AdGadd45, the cytostatic effects are analyzed using an MTT assay (Fig. 3). As shown in Fig. 3, infection of AdGFP at an MOI of 100 had little effects on cell proliferation of ARO, FRO, and NPA, but infection of AdGadd45 led to a significant decrease of cell number in all the cells, ARO, FRO, and NPA. The significant decrease of viable cell number after infection of AdGadd45 was noted from 48 h after infection. In ARO, FRO, and NPA cells infected with Adp53 at 50 MOIs, the viable cell number was clearly decreased (Fig. 3). Adp53-mediated cytostatic effects were less in FRO cells, compared with ARO and NPA cells. Simultaneous expression of p53 and Gadd45 resulted in additive effects on the inhibition of proliferation of ARO, FRO, and NPA cells (data not shown). From these observations, we found that reexpression of Gadd45 resulted in significant decrease of cell proliferation potentials of ARO, FRO, and NPA cells. And the cytostatic effects of Gadd45 on anaplastic cancer cells were comparable with p53 with the same MOI in ARO and NPA cells. Interestingly, the cytostatic effects of Gadd45 on FRO cells was more pronounced, compared with p53.
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resulted in apoptosis in anaplastic cancer cells
To identify the molecular mechanisms of growth-inhibitory effects of Gadd45 in ARO, FRO, and NPA cells, we observed the apoptosis in the cells infected with AdGadd45. The induction of DNA internucleosomal cleavage, a hallmark of apoptosis, in Gadd45 expressed ARO, FRO, and NPA cells were investigated by agarose gel electrophoresis. Representative results of these experiments are shown in Fig. 4. There is a clear induction of the DNA ladder, the result of DNA cleavage, in AdGadd45- and Adp53-infected ARO, FRO, and NPA cells (Fig. 4).
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induced apoptosis, we assessed apoptosis by TUNEL assay. ARO, FRO, and NPA cells were infected with AdCMVGAL and AdGadd45, and TUNEL assay was performed after 24 h (Fig. 5). No apoptosis was observed in the uninfected cells. However, the cells that were infected with AdGadd45 showed apoptosis in the three cell lines, as indicated by the appearance of the TUNEL labeling (Fig. 5A). Figure 5B is a graphical quantitation of the TUNEL staining, which shows that AdGadd45 induced significant apoptosis as opposed to AdCMVGAL. The ARO and NPA cells showed more apoptotic cells, compared with the FRO cells in the same infected viral dose of AdGadd45 (Fig. 5B).
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gene in anaplastic thyroid cancer cells is enough to induce apoptosis and this apoptotic effects are one of major mechanisms in the inhibition of Gadd45-mediated suppression of proliferation in ARO, FRO, and NPA cells.
Effect of AdGadd45 infection on growth of thyroid tumors established in nude mice
In vivo efficacy of AdGadd45 infection was studied in thyroid carcinoma cell lines, ARO. AdCMVGAL, Adp53, and AdGadd45 were injected directly into the sc ARO tumors established in nude mice. We examined whether AdGadd45 is effective in inhibition of tumor growth; in addition, we tried to compare the efficacy of AdGadd45 with Adp53 infection. The animals having xenografted ARO tumors were subjected to four groups made of five mice in each group: not infected, AdCMVGAL, Adp53, and AdGadd45 groups after 2 wk of tumor establishment. The animals were treated with adenoviruses 109 pfu by single intratumoral injection everyday after 2 wk of tumor establishment. The mean tumor volume of control groups (not infected and AdCMVGAL group) reached as 198 mm3 and 186 mm3, respectively. Interestingly, the mean tumor volumes in Adp53 (134 mm3) and AdGadd45 (142 mm3) groups showed significantly less volume, compared with not infected and AdCMVGAL groups. The inhibitory effects on tumor growth by AdGadd45 infection were noted until 2 wk of repeated infection in survived mice. The growth-inhibitory effects between Adp53 and AdGadd45 are not significantly different at 1 and 2 wk of adenovirus treatment. These findings suggest that the delivery of Gadd45 in vivo also showed tumor inhibitory effects, and this inhibitory effect is comparable with p53.
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Discussion |
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gene for treatment of anaplastic thyroid carcinoma. The Gadd45 gene was initially identified as a gene whose mRNA was rapidly induced by agents that cause DNA damage (8) such as UV radiation, methylmethane sulfonate, and x-ray irradiation. Gadd45 was widely studied as a marker for p53 activation because appreciable induction of Gadd45 by x-ray irradiation occurred only in cells having a wild-type p53 phenotype (13, 17). Gadd45-null mice exhibited several phenotypes characteristic of p53-deficient mice (25). Gadd45 is identified as a gene that is induced by IL-2 (10) and oncostatin M (12). Takekawa and Saito (11) discovered that Gadd45 are associated with MTK1, which in turn activated both p38 and Jun-terminal kinase (JNK) pathway leading to apoptosis in response to environmental stresses. Gadd45 also has been shown to be associated with proliferating cell nuclear antigen (26) and p21 (27) and involved in negative growth control. These observations suggest that Gadd45 is one of important regulatory proteins in the regulation of cell cycle and cell proliferation. However, there are few studies of the physiological and pathological roles of Gadd45 in endocrine cells and endocrine tumor, respectively.
Gadd45 are relatively highly expressed in endocrine organs, especially testis, adrenal gland, and thyroid gland (data not shown). Zhang et al. (28) have found that the mRNA expression of the Gadd45 gene is significantly different between normal human pituitary tissue and clinically nonfunctioning pituitary adenomas using cDNA-representational difference analysis. Although Gadd45 mRNA was found in normal human pituitary tissue, it was detectable in only one of 18 clinically nonfunctioning pituitary tumors by RT-PCR. Furthermore, this gene was not expressed in the majority of GH- or prolactin-secreting pituitary tumors. In colony formation assays, transfection of human Gadd45 cDNA into the human pituitary tumor-derived cell line results in a dramatic decrease in cell growth by 88%. Gadd45 also reduces colony formation in other pituitary tumor-derived cell lines, AtT20 and GH4, by approximately 60 and 50%, respectively, confirming its function in controlling cell proliferation in the pituitary tumor cells. These data indicate that Gadd45 is a powerful growth suppressor controlling pituitary cell proliferation, and Gadd45 represents the first identified gene whose expression is lost in the majority of human pituitary tumors.
We found that Gadd45 gene expression is significantly decreased in anaplastic thyroid cancer cell lines, ARO, FRO, and NPA, compared with primary cultured thyroid cells. Gadd45 and Gadd45ß did not show differences in expression between primary cultured thyroid cells and anaplastic thyroid cancer cell lines. These findings suggest that anaplastic cancer cells may specifically lose the certain regulatory mechanisms involved in the expression of Gadd45. Gadd45 and Gadd45ß has been indentified as p53 regulatory genes; however, recent studies suggest that Gadd45 family gene expression showed independence to p53 (29, 30, 31). Because the anaplastic cancer cells used in this experiment showed structural and functional inactivation of p53, Gadd45 and Gadd45ß expression may independent of p53. The dependency of p53 in the expression of Gadd45 is not fully studied. The findings that transduction of Adp53 did not induce Gadd45 expression in ARO, FRO, and NPA cells (data not shown) suggest that Gadd45 may not be a downstream gene of p53 in anaplastic thyroid cancer cells.
We proposed that the growth-inhibitory effects of Gadd45 in anaplastic thyroid cancer cells results from apoptosis. We showed transduction of AdGadd45 results in extensive apoptosis in ARO, FRO, and NPA cells. The mechanism of apoptosis may result from activation stress-responsive kinases, p38 and JNK, because Gadd45 has been shown to activate these kinase pathways (11, 32). Gadd45 that bound to an N-terminal domain of MTK1 and activated MTK1 kinase activity, both in vivo and in vitro (11, 32) was identified. Expression of the Gadd45 induces p38/JNK activation and apoptosis, which can be partially suppressed by coexpression of a dominant inhibitory MTK1 mutant protein. However, we could not observe the phosphorylation of p38 kinase after transduction of AdGadd45 in ARO, FRO, and NPA cells (data not shown). These observations suggest that Gadd45-induced apoptosis may not accompany the activation of p38 kinase in anaplastic cancer cells. The studies using embryo fibroblasts from Gadd45-null mice revealed no deficiency in JNK/p38 activation in Gadd45-/- fibroblasts and give support that the Gadd45-mediated apoptosis may have another apoptosis-triggering pathway (33).
The dose and cell-type specificity of Gadd45-induced apoptosis is not fully understood. Although apoptosis was observed after the reexpression of Gadd45 in the anaplastic thyroid cancer cells that showed a low level of endogenous Gadd45 expression, it is not clear whether the preexisting endogenous Gadd45 affected the AdGadd45-mediated cellular effects. Because AdGadd45 is an effective means to introduce Gadd45 into the cells, FRTL-5 rat thyroid cells, which are untransformed and have a significant level of rat Gadd45 RNA expression in the cultured condition, were infected with them. AdGadd45 was also able to induce significant apoptosis in the FRTL-5 rat thyroid cells (data not shown). In addition, the AdGadd45 effects were observed in several other cancer cells, such as HeLa, Saos-2, and HCT116 (data not shown). All these cells also showed growth inhibition and apoptosis with AdGadd45 transduction. This suggests that the cellular effects such as apoptosis by AdGadd45-mediated expression is not dependent on the endogenous level of Gadd45 and its apoptotic effects are not specific to particular cell types.
We showed that AdGadd45 has inhibitory effectors on growth of xenotransplanted ARO tumors; however, growth suppression was not complete. Recent studies suggested that Adp53 gene therapy sensitizes the anaplastic thyroid cancer cells to chemotherapeutic agents (24, 34). In addition, ectopic expression of Gadd family proteins also was found to sensitize the cells to apoptosis induced by genotoxic agents such as UV, methylmethane sulfonate, -irradiation, and VP16 (35). These observations raise some possibilities that the concomitant use of chemotherapeutic agents with AdGadd45 may increase the therapeutic effects to anaplastic cancer cells.
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Footnotes |
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Abbreviations: CMV, Cytomegalovirus; FBS, fetal bovine serum; Gadd45, growth arrest- and DNA damage-induced gene; JNK, Jun-terminal kinase; MOI, multiplicity of infection; MTT, dimethylthiazoldiphenyltetra-zoliumbromide; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick end labeling.
Received December 26, 2002.
Accepted April 17, 2003.
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B downregulates pro-apoptotic JNK signalling. Nature 414:308–313[CrossRef][Medline]
) with proliferating cell nuclear antigen impedes negative growth control. J Biol Chem 276:2766–2774, a growth inhibitory gene, in human pituitary adenomas: implications for tumorigenesis. J Clin Endocrinol Metab 87:1262–1267/) sensitizes neoplastic cells to genotoxic stress-induced apoptosis. Int J Oncol 18:749–757[Medline]This article has been cited by other articles:
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  A. Scuto, M. Kirschbaum, C. Kowolik, L. Kretzner, A. Juhasz, P. Atadja, V. Pullarkat, R. Bhatia, S. Forman, Y. Yen, et al. The novel histone deacetylase inhibitor, LBH589, induces expression of DNA damage response genes and apoptosis in Ph- acute lymphoblastic leukemia cells Blood, May 15, 2008; 111(10): 5093 - 5100. [Abstract] [Full Text] [PDF]
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 J. Ying, G. Srivastava, W.-S. Hsieh, Z. Gao, P. Murray, S.-K. Liao, R. Ambinder, and Q. Tao The Stress-Responsive Gene GADD45G Is a Functional Tumor Suppressor, with Its Response to Environmental Stresses Frequently Disrupted Epigenetically in Multiple Tumors Clin. Cancer Res., September 15, 2005; 11(18): 6442 - 6449. [Abstract] [Full Text] [PDF]
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 E. S. Hwang, D. W. Kim, J. H. Hwang, H. S. Jung, J. M. Suh, Y. J. Park, H. K. Chung, J. H. Song, K. C. Park, S. H. Park, et al. Regulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT1-Dependent Genes by RET/PTC (Rearranged in Transformation/Papillary Thyroid Carcinoma) Oncogenic Tyrosine Kinases Mol. Endocrinol., November 1, 2004; 18(11): 2672 - 2684. [Abstract] [Full Text] [PDF]
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 S. Papa, F. Zazzeroni, C. G. Pham, C. Bubici, and G. Franzoso Linking JNK signaling to NF-{kappa}B: a key to survival J. Cell Sci., October 15, 2004; 117(22): 5197 - 5208. [Abstract] [Full Text] [PDF]
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