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Keywords:

  • thymidylate synthase;
  • cervix neoplasms;
  • 5-fluorouracil;
  • gene transfer;
  • SKG-II;
  • SKG-I

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The prognosis of cancers of various organs overexpressing thymidylate synthase (TS) has been reported to be poor. It has been suggested that the poor prognosis is partly due to a low sensitivity of TS-overexpressing tumors to TS-targeting 5-fluorouracil (5-FU). To investigate the relationship between TS expression and sensitivity to 5-FU, we used the TS-overexpressing cervical cancer cell line SKG-II/TS and SKG-I/TS that had been established by TS gene transfer. The 50% growth inhibitory concentration (IC50) of 5-FU for SKG-II/TS was 24 ± 6.0 μM, which was 6 times as high as that for the control (4.0 ± 1.1 μM), showing significantly decreased sensitivity to 5-FU (p < 0.01). The IC50 of 5-FU for SKG-I/TS was 90 ± 15 μM, which was over 2 times as high as that for the control (40 ± 0.6 μM), showing significantly decreased sensitivity to 5-FU (p < 0.05). Thus, TS-overexpressing tumors have decreased sensitivity to 5-FU, which may be one of the factors that determine the prognosis of these tumors. © 2003 Wiley-Liss, Inc.

Thymidylate synthase (TS; EC 2.1.1,45) is the key regulatory enzyme that catalyzes the methylation of deoxyuridine monophosphate in the de novo synthesis pathway of pyrimidine. Clinically, the relationship between TS expression and the degree of malignancy of tumors of various organs including colorectal cancer has attracted attention. TS-overexpressing tumors have been reported to be generally poor.1, 2, 3, 4, 5, 6, 7, 8 Although the mechanism leading to the poor prognosis is unknown, exaggerated cell proliferation in TS-overexpressing tumors,9, 10 rapid invasive growth and metastatic activity of tumors7 and decreased sensitivity chiefly of colorectal cancers to 5-fluorouracil (5-FU)1, 5 have been implicated. However, there is no direct evidence that TS overexpression decreases the sensitivity to 5-FU. In our study, we investigated whether TS overexpression decreases the sensitivity to 5-FU in a TS-overexpressing cervical cancer cell line established by TS gene transfer.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

TS-overexpressing human uterine cervical carcinoma transfectants

TS-overexpressing human uterine cervical carcinoma transfectant SKG-II/TS and control transfectant SKG-II/Luciferase (LUC) were created as described previously.11

SKG-I, the human uterine cervical carcinoma cell line, was a kind gift from Professor Nozawa, and was maintained as described.12 A TS-expressing vector described previously11 was transfected into SKG-I cells by the standard calcium phosphate precipitation method.13 The cells were selected in the presence of 10 mg/mL blasticidin S hydrochloride. Resistant cells were obtained after 4 weeks, and SKG-I/TS was obtained. The level of TS in SKG-I/TS was measured by the fluorodeoxyuridine monophosphate (FdUMP)-TS binding assay according to the method of Spears et al.,14 using [6-3H]FdUMP as substrate. Protein content was determined using the Bio-Rad (Veenendaal, The Netherlands) protein assay kit. The TS activity measured as the number of FdUMP-binding sites was calculated per mg protein.

Colorimetric assay

The sensitivity of the cells to 5-FU was investigated by colorimetric assay using a Cell Proliferation Kit II (XTT)(Boehringer Mannheim GmbH Biochemica, Mannheim, Germany). The cells were exposed to 5-FU (Kyowa Hakko, Tokyo, Japan) at concentrations of 0.2–200 μM for 72 hr. The viable cell count measured by colorimetric assay was presented as a percent ratio to the count of the control untreated with 5-FU. A dose-response curve was prepared and the 50% growth inhibitory concentrations (IC50) was obtained.

Statistical analysis

Significance of difference was analyzed by unpaired student's t-test. A p value < 0.05 was regarded as significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

First, we investigated the TS activity in SKG-I and SKG-I/TS by FdUMP-TS binding assay. The TS activity of SKG-I/TS was 0.6±0.03 pmol/mg protein, which was significantly higher than those of SKG-I (0.3±0.04 pmol/mg protein)(p < 0.01).

Next, we investigated the effect of TS expression on 5-FU-sensitivity in vitro. The IC50 of 5-FU for SKG-II/TS was 24 ± 6.0 μM, which was 6 times as high as that for SKG-II/LUC (4.0 ± 1.1 μM), showing significantly decreased sensitivity to 5-FU (p < 0.01, Fig. 1). The IC50 of 5-FU for SKG-I/TS was 90 ± 15 μM, which was over 2 times as high as that for SKG-I (40 ± 0.60 μM), showing significantly decreased sensitivity to 5-FU (p < 0.05, Fig. 2). Therefore, the expression of the TS resulted in reduced 5-FU-sensitivity.

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Figure 1. Dose-response curves of SKG-II transfectants exposed to 5-FU. The 50% growth inhibitory concentration (IC50) of 5-FU for SKG-II/TS was 24 ± 6.0 μM, which was 6 times as high as that for SKG-II/LUC (4.0 ± 1.1 μM), showing significantly decreased sensitivity to 5-FU (p < 0.01). Bars, SD.

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thumbnail image

Figure 2. Dose-response curves of SKG-I and SKG-I/TS exposed to 5-FU. The 50% growth inhibitory concentration (IC50) of 5-FU for SKG-I/TS was 90 ± 15 μM, which was over 2 times as high as that for SKG-I (40 ± 0.60 μM), showing significantly decreased sensitivity to 5-FU (p < 0.05). Bars, SD.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Studies have shown a relationship between TS expression in tumors and sensitivity to 5-FU. In an in vitro study using gastrointestinal cancer cell lines, Habara et al. found a significant correlation between TS activity and sensitivity to 5-FU.15 Wang et al. examined TS gene expression in five 5-FU-resistant colorectal cancer cell lines using complementary DNA (cDNA) microarrays, and reported that these cell lines showed a higher TS gene expression than the 5-FU-sensitive parent cell lines.16 These in vitro studies suggest that TS expression in tumors determines the sensitivity to 5-FU. In our study, we examined the effect of TS expression on 5-FU sensitivity in TS-overexpressing cell lines established by TS gene transfer, and showed that TS expression was associated with decreased sensitivity to 5-FU. These findings suggest that the poor prognosis of TS-overexpressing tumors is partly due to their decreased sensitivity to 5-FU. The mainstay of treatment for cervical cancer is surgery and radiotherapy, and therapy with 5-FU or other anticancer agents is rarely the treatment of choice. Therefore, it is difficult to speculate from the 5-FU sensitivity that TS expression determines the prognosis of cervical cancer. However, based on clinical analysis of patients with cervical cancer treated by radiotherapy, we reported that TS-overexpressing patients had a poorer prognosis than patients with low TS expression.17 In addition, we found in an in vitro study that TS-overexpressing cervical cancer cells were less sensitive to radiation.11 Recently, chemoradiation for advanced cervical cancer, which combines radiation and chemotherapy with 5-FU and cisplatin, has attracted attention.18, 19 Thus, analysis of TS expression in cervical cancers may predict the efficacy of chemoradiation.

The TS overexpression may decrease the sensitivity to 5-FU by the following mechanism. 5-FU is an anticancer agent targeting TS, which is converted in vivo to FdUMP by thymidine kinase, and this reportedly binds to TS to inhibit it, thereby exerting a cell-killing effect.20, 21 Thus, a larger dose of 5-FU is required to exert its effect on TS-overexpressing tumors, which means a decreased sensitivity to 5-FU.

In addition to decreased sensitivity to 5-FU and radiation, tumor invasion or distant metastasis has recently been implicated in the poor prognosis of TS-overexpressing tumors: immunostaining of rectal cancer specimens from clinical patients has shown that TS expression is an independent prognostic factor for local recurrence, distant metastasis, disease-free and overall survival.22 TS may be involved in tumor invasion and metastasis. Therefore, the investigation of invasive and metastatic activity and angiogenesis using our TS transfectants should be performed in the future.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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