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Evaluation of thymidylate synthase RNA expression by polymerase chain reaction
R M Elledge1, N Okuyama, S D Fitzgerald
1University of Texas Health Science Center, Division of Medical Oncology, San Antonio 78284-7884.
Abstract:
5-Fluorouracil (5FU), an antimetabolite often used for the treatment of breast cancer, binds to and inactivates the enzyme thymidylate synthase (TS). Measurement of TS levels may be useful in examining resistance to 5FU, but current methods involving ligand binding assays present considerable problems due to the low basal level of this enzyme in many tissues. Therefore, a semiquantitative polymerase chain reaction (PCR) technique to measure TS RNA concentration in breast tumour cell lines has been developed with potential for measuring TS RNA levels in individual breast tumours. Using less than 1 microgram of total RNA, we determined that the T47D breast cancer cell line expressed TS levels 1.3 to 4.4 times lower than those of four other breast cancer cell lines, MCF-7, MDA-MB-468, MDA-MB-231, and MDA-A1R. Using the MCF-7 cell line as a representative of relatively high TS expression, the relationship between the S-phase fraction (SPF), and TS levels in MCF-7 and T47D cells was next examined because cell cycle kinetics can also influence drug response. TS expression was directly related to SPF. MCF-7 cells had a higher SPF, corresponding to their higher TS level relative to T47D cells. Lastly, 5FU sensitivity was assayed for and it was found that MCF-7 cells were more sensitive to 5FU than T47D, despite having higher TS levels. A semiquantitative technique for assessing TS RNA expression by PCR in breast cancer cell lines has been demonstrated. In the cell lines tested, there was no direct relationship between TS level and 5FU sensitivity, though there was a direct relationship between cell proliferation and sensitivity to 5FU.
Insights
A new semiquantitative polymerase chain reaction (PCR) method accurately measures thymidylate synthase (TS) RNA in breast cancer cells. This technique reveals TS levels are linked to proliferation, not directly to 5-fluorouracil (5FU) sensitivity.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- 5-Fluorouracil (5FU) is a key breast cancer drug targeting thymidylate synthase (TS).
- Measuring TS levels is crucial for predicting 5FU resistance, but current methods are problematic due to low enzyme levels.
- A novel, sensitive method is needed to quantify TS expression in breast tumors.
Purpose of the Study:
- To develop and validate a semiquantitative polymerase chain reaction (PCR) technique for measuring TS RNA levels in breast cancer cell lines.
- To investigate the relationship between TS RNA levels, S-phase fraction (SPF), and 5FU sensitivity in different breast cancer cell lines.
Main Methods:
- Developed a semiquantitative PCR assay to measure TS RNA concentration using minimal total RNA (<1 microgram).
- Compared TS RNA expression across multiple breast cancer cell lines (T47D, MCF-7, MDA-MB-468, MDA-MB-231, MDA-A1R).
- Assessed the correlation between TS levels, S-phase fraction (SPF), and 5FU sensitivity in MCF-7 and T47D cell lines.
Main Results:
- The T47D cell line exhibited significantly lower TS RNA levels (1.3-4.4 times lower) compared to other tested cell lines.
- TS RNA expression was found to be directly proportional to the S-phase fraction (SPF), indicating a link to cell proliferation.
- Contrary to expectations, MCF-7 cells, with higher TS levels, showed greater sensitivity to 5FU than T47D cells.
Conclusions:
- A reliable semiquantitative PCR method for assessing TS RNA expression in breast cancer cell lines has been established.
- TS RNA levels correlate with cell proliferation (SPF) but do not directly predict 5FU sensitivity.
- Cell proliferation rate, rather than TS expression alone, appears to be a key determinant of 5FU sensitivity in these breast cancer models.