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Therapeutic Usefulness of Wild-Type p53 Gene Introduction in a p53-Null Anaplastic Thyroid Carcinoma Cell Line

The Departments of Pharmacology 1 (M.Na., Y.N., M.Ni.), Anatomy 1 (K.A.), and Surgery 1 (M.Na., H.A.), and the Department of Nature Medicine, Atomic Bomb Disease Institute (T.-t.Y., A.O., H.N., S.Y.), Nagasaki University School of Medicine, Nagasaki 852-8523; and the Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Showa University (M.Y., T.Y.), Tokyo 142, Japan

Address all correspondence and requests for reprints to: Yuji Nagayama, M.D., Department of Pharmacology 1, Nagasaki University School of Medicine, 1–12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: nagayama{at}net.nagasaki-u.ac.jp

	Abstract

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Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.

	Introduction

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THE TUMOR suppressor p53 is a transcription factor identified approximately a decade ago (1). Recent studies suggest that one of the most critical physiological roles of p53 is to mediate two different cellular responses after exposure to DNA-damaging stimuli such as ionizing radiation and chemotherapeutic agents: growth arrest at G1 phase of the cell cycle and apoptosis (1). After DNA damage, p53-mediated G1 arrest delays entry into S phase and facilitates DNA repair by allowing time for such functions to occur before DNA replication, whereas apoptosis induced by p53 prevents propagation of a cell that sustains stable genomic alterations. Loss of p53 function is known to render the cells susceptible to tumorigenesis. Indeed, p53 is one of the most frequently mutated genes in a wide range of human cancers (2). In thyroid carcinomas, the prevalence of p53 gene mutations is 70–85% in anaplastic carcinomas and 0–9% in differentiated carcinomas (3, 4, 5). p53 mutations are therefore considered to be the late genetic event associated with loss of the differentiated phenotype in thyroid carcinomas in the context of the multistage process of tumorigenesis and confer these tumors with aggressive properties.

Restoration of wild-type (wt) p53 expression by introducing the wt-p53 gene has recently been used in several experimental cancer models and human clinical trials as a cancer gene therapy (6, 7). The antitumorigenic and anti-proliferative potential of wt-p53 gene introduction has been described in tumor cells harboring mutant p53 (8, 9, 10, 11, 12, 13, 14, 15). We and others have previously reported that restoration of wt-p53 expression by stably transfecting the temperature-sensitive (ts) p53 gene, which functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively, results in attenuated cell growth, but not apoptotic cell death, in p53-null anaplastic thyroid carcinoma cell lines at 32 C (16, 17). These data suggest that introduction of the wt-p53 gene alone may be insufficient as a gene therapy for thyroid carcinomas harboring p53 mutations.

Recent studies have reported that p53 status also affects the chemosensitivity and radiosensitivity of tumor cells (6, 18). Thus, wt-p53 reexpression may alter sensitivity to chemotherapeutic agents and/or ionizing radiation. These effects may be very critical from a clinical point of view, because resistance of tumor cells to ionizing radiation and chemotherapy remains a significant obstacle in the treatment of cancers. Furthermore, wt-p53 has been reported to inhibit angiogenesis by suppressing vascular endothelial growth factor (VEGF), one of the potent angiogenic factors (19), and inducing thrombospondin-1 (TSP-1), one of the potent antiangiogenic factors (20).

In the present studies, therefore, we extended our previous study to further investigate the therapeutic benefits of recombinant wt-p53 reexpression in p53-null thyroid carcinoma cells.

	Materials and Methods

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Cells and culture condition

FRO cells, an anaplastic thyroid carcinoma cell line with no p53 expression (21), have been stably transfected with the ts-p53 expression plasmid, pCMV-p53Val138 (16). Parental FRO and tsFRO cells were cultured in RPMI 1640 with 10% FBS and appropriate antibiotics. Bovine aortic endothelial (BAE) cells were isolated as previously described (22) and were used at passages 5–11. BAE cells were maintained in Ham’s F-12 medium with 5% FBS.

In vitro cell growth

FRO and tsFRO cells, cultured at 37 or 32 C for 3 days, were seeded at 10³-10⁴/well in 48-well culture plates (day 0). The viable cell numbers were calculated on days 2, 4, and 6 by trypan blue exclusion test.

In vitro chemosensitivity

The cells, maintained at 37 or 32 C for 3 days, were seeded at a density of 3–10 x 10³/well in 96-well microplates. On the next day, the cells were exposed to anticancer drugs [cisplatin (Nihon Kayaku Co., Tokyo, Japan), 5-fluorouracil and doxorubicin (Kyowa Hakko Co., Tokyo, Japan)]. Cell viability was determined 3 days later with a cell counting kit (Wako, Osaka, Japan).

In vitro radiosensitivity

The cells maintained at 37 or 32 C for 3 days were seeded at low density in 10-cm dishes. One day later, the cells were irradiated with graded doses using an EX-300 -irradiator (200 kV; 15 mA; filter, 0.5 mm aluminum; 1.41 Gy/min; Toshiba, Tokyo, Japan). Colonies larger than 5 mm were counted after 2 weeks (the cells cultured at 37 C), 3 weeks (FRO at 32 C), or 6 weeks (tsFRO at 32 C), because of the different cell growth rates (see Fig. 1).

View larger version (21K): [in this window] [in a new window]   Figure 1. Growth rates of FRO and tsFRO cells cultured at nonpermissive and permissive temperatures. The cells, cultured at 37 or 32 C for 3 days, were seeded at 103-104/well in 48-well culture plates. The viable cell numbers were calculated 2, 4, and 6 days later by trypan blue exclusion test. Data are representative of three separate experiments; each point represents the mean ± SD (n = 3) and is expressed as a percentage relative to the cell number seeded on day 0.

Total RNA was extracted as previously described (23). Twenty-five micrograms of RNA were subjected to Northern blot as previously described (23), with human VEGF complementary DNA (cDNA) (24), TSP-1 cDNA (a gift from Prof. W. A. Frazier, Washington University, St. Louis, MO), and cyclophilin cDNA as probes.

Immunoblot

Conditioned media were obtained by incubating the cells for 2 days in Ham’s F-12 medium with 0.1% FBS at 32 C and were concentrated 10 times using an Ultrafree centrifugal filter (Millipore Corp., Bedford, MA). Twenty microliters of concentrated media were subjected to immunoblot as previously described (23) with monoclonal anti-human TSP-1 (Genzyme Corp., Cambridge, MA).

In vitro capillary endothelial migration assay

The BAE cell migration assay was performed as previously described (25) with minor modifications. A polycarbonate membrane culture insert (12-µm pore size; Iwaki Glass, Chiba, Japan) was coated with 50 µg Cellmatrix collagen type I-A (Nitta Gelatin, Inc., Osaka, Japan) in 20 µL Ham’s F-12 medium containing 0.22% NaHCO₃, 20 mmol/L HEPES, and 0.005 N NaOH; dried at 37 C; and placed in a 24-well culture plate. The cells (1–3 x 10⁴), suspended in Ham’s F-12 with 2% FBS, were added to the upper chamber. Conditioned medium, obtained by incubating the cells for 2 days in Ham’s F-12 medium with 0.2% FBS at 32 C and concentrated up to 10 times, as mentioned above, in Ham’s F-12 medium-2% FBS containing 25 ng basic fibroblast growth factor (bFGF; Sigma Chemical Co., St., Louis, MO), or in Ham’s F-12 medium-2% FBS alone, was placed in the lower compartment of the chamber. After 24 h at 37 C, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab. The filters were fixed in ethanol and stained with trypan blue. The cells that migrated to the lower surface were then counted.

	Results

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We have previously found that tsFRO, an anaplastic thyroid carcinoma cell line FRO stably expressing ts-p53, displayed growth retardation with G1 arrest without apoptotic cell death, compared with the mock-transfected FRO cells, at a permissive temperature of 32 C (16). To confirm these findings, we calculated the cell doubling times of FRO and tsFRO cells. As shown in Fig. 1, the doubling times of FRO and tsFRO cells at a nonpermissive temperature of 37 C were essentially identical (21.5 ± 3.4 vs. 23.2 ± 3.6 h; mean ± SD of three independent experiments), whereas the cell doubling time of tsFRO cells was significantly longer than that of FRO cells at permissive temperature (36.0 ± 10.6 vs. 62.4 ± 3.4 h; P < 0.01). However, no significant increase in cell death determined by the dye exclusion test was observed in tsFRO cells compared to parental FRO cells at permissive temperature (data not shown).

We then evaluated whether wt-p53 induction might affect the chemosensitivity of FRO cells. Cisplatin, 5-fluorouracil, and doxorubicin were employed, which have been widely used in chemotherapy for thyroid carcinomas (26). FRO and tsFRO cells cultured at either nonpermissive or permissive temperature for 3 days were treated with various concentrations of the anticancer drugs, and viable cell number was determined 3 days later by colorimetrical assay. As shown in Fig. 2, the IC₅₀ values (drug concentrations required for 50% growth inhibition) for cisplatin, 5-fluorouracil, and doxorubicin were very similar between FRO and tsFRO cells at both nonpermissive and permissive temperatures, suggesting that p53 induction does not sensitize FRO cells to these anticancer drugs.

View larger version (20K): [in this window] [in a new window]   Figure 2. In vitro chemosensitivity of FRO and ts FRO cells at nonpermissive and permissive temperatures. The cells, cultured at 37 or 32 C for 3 days, were seeded at a density of 3–10 x 103/well in 96-well microplates. On the next day, the cells were exposed to various concentrations of anticancer drugs, including cisplatin, 5-fluorouracil, and doxorubicin. The cell viability was determined 3 days later with a cell counting kit. Data are representative of three separate experiments; each point represents the mean ± SD (n = 3) and is expressed as a percentage relative to the value in untreated cells.

View larger version (48K): [in this window] [in a new window]   Figure 3. In vitro radiosensitization of FRO and tsFRO cells at nonpermissive and permissive temperatures. The cells, cultured at 37 or 32 C for 3 days, were seeded at low density in 10-cm dishes. One day later, the cells were irradiated with graded doses. Colonies larger than 5 mm were counted 2 weeks (the cells cultures at 37 C), 3 weeks (parental FRO cultured at 32 C), or 6 weeks (tsFRO cultured at 32 C) later. Data are representative of two separate experiments; each bar represents the mean ± SD (n = 3) and is expressed as a percentage relative to the value in untreated cells. *, Statistically significant decrease between FRO and tsFRO cells cultured at permissive temperature (P < 0.05, by Student’s t test).

View larger version (29K): [in this window] [in a new window]   Figure 4. A, Northern blot analysis of VEGF and TSP-1 mRNA transcripts in FRO and tsFRO cells at nonpermissive and permissive temperatures. Twenty-five micrograms of total RNA extracted from the cells cultured at 37 or 32 C for 3 days were analyzed for expression of VEGF and TSP-1 mRNAs. The relative amount of mRNA was estimated by hybridization to cyclophilin cDNA. B, Immunoblot analysis of TSP-1 expression in FRO and tsFRO cells at 37 or 32 C. Ten-fold concentrated medium conditioned by FRO and tsFRO cells at 32 C for 2 days was used.

View larger version (19K): [in this window] [in a new window]   Figure 5. The in vitro capillary endothelial migration assay with the conditioned medium from FRO and tsFRO cells cultured at permissive temperature. Conditioned media were obtained from confluent cells cultured in a 10-cm dish at 32 C for 2 days, concentrated, and tested in the presence or absence of bFGF (25 ng/well) for the ability to induce endothelial cell migration or inhibit bFGF-induced endothelial cell migration. Each bar represents the mean ± SD (n = 4) of two separate experiments determined in duplicate. *, Statistically significant decrease compared to bFGF alone (P < 0.05, by Student’s t test).

	Discussion

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With regard to p53-mediated cell death and cell growth inhibition, it remains unknown how p53 regulates these two distinct critical functions. This decision may be dependent on p53 expression level, cell type, and/or the cellular environment. Different tumor types may, therefore, exhibit distinct responses to reintroduction of wt-p53. Thus, wt-p53 induces apoptotic cell death in some tumor cells (8, 9, 10) and only attenuates cell growth in other tumor cell types (11, 12, 13, 14, 15). Our present and previous results demonstrating p53-mediated cell growth inhibition in tsFRO cells are consistent with the previous reports (17, 27) using other un- or poorly differentiated thyroid carcinoma cell lines with mutant p53. Taken together, reexpression of wt-p53 induces cell cycle arrest, not apoptotic cell death, in anaplastic thyroid carcinomas with mutant p53.

Radiosensitization by wt-p53 reexpression has also been described in some cancer cells (28, 29, 30). Close correlation is also present between p53 mutations and radioresistance in several tumor cells (31, 32). One possible mechanism for the p53-mediated radiosensitization may be at least in part due to reconstitution of the wt-p53-dependent apoptotic pathway. We have previously shown that FRO cells are more radioresistant than normal thyroid cells with wt-p53 (21). This radiosensitizing effect not only enhances the therapeutic effect of ionizing radiation, but also makes it possible to reduce the radiation dose required for effective treatment of anaplastic thyroid carcinomas. p53-defective anaplastic thyroid carcinomas may thus benefit from the multimodality approach of p53 gene therapy in combination with radio-therapy.

Lack of wt-p53-mediated chemosensitization in FRO cells suggests that anaplastic thyroid carcinoma cells may have another resistance mechanism(s) for anticancer agents that overwhelms the effect of reexpressed wt-p53. This idea is consistent with a recent study (33) in which the sensitivities of several thyroid anaplastic carcinoma cell lines to chemotherapeutics were evaluated; chemoresistance has been shown not to be necessarily correlated with p53 gene status, although KOA2 cells harboring the p53 mutation are resistant to all four chemotherapeutics examined. It may also be suggested that chemosensitivity in carcinoma cells may not be solely regulated by wt-p53 and that the mechanism(s) for p53-mediated radiosensitization may not be identical to those for p53-induced chemosensitization.

VEGF and TSP-1 are important factors for angiogenesis and antiangiogenesis, respectively, of tumor tissues. A highly tumorigenic potential is associated with elevated VEGF expression in human thyroid carcinoma cell lines, and anaplastic thyroid carcinoma tissues overexpress VEGF (34). TSP-1 overexpression is also reported to suppress tumor growth or metastasis in some tumor cells (35). Recent studies have revealed that wt-p53 may inhibit angiogenesis by suppressing VEGF expression and/or by inducing TSP-1 expression (19, 20), although the former was questioned by Agani et al. (36). The data obtained in this work, however, contradict these reports; wt-p53 introduction did not change VEGF mRNA levels and did suppress TSP-1 expression in FRO cells. In addition, contrary to the previous report (20) in which p53-null fibroblasts express no TSP-1, p53-null FRO cells express readily detectable levels of TSP-1. Nevertheless, we could not detect any difference in antiangiogenic activity between medium conditioned by FRO and tsFRO cells at permissive temperature in the in vitro capillary endothelial migration assay. As this assay examines only one aspect of angiogenesis (endothelial cell invasion), the possibility cannot be excluded that variations on TSP-1 expression levels may be reflected in other aspects of this process. Alternatively, wt-p53 reexpression may not significantly modulate the angiogenic activity of FRO cells as the net effect by altering the expression of other angiogenic/antiangiogenic factors. Further study will be necessary to clarify the pathological significance of TSP-1 in thyroid tumor growth and angiogenesis.

Received March 30, 1998.

Revised May 28, 1998.

Revised June 25, 1998.

Accepted July 1, 1998.

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