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Updated: Oct 4, 2026
An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Published on: September 18, 2013
Modulation of endogenous beta-tubulin isotype expression as a result of human beta(III)cDNA transfection into
S Ranganathan1, R A McCauley, D W Dexter
1Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Abstract:
Increases of individual beta tubulin isotypes in antimicrotubule drug resistant cell lines have been reported by several laboratories. We have previously described elevations in beta(III)and beta(IVa)isotypes in estramustine and paclitaxel resistant human prostate carcinoma cells. To investigate further the function of beta tubulin isotypes in antimicrotubule drug response, human prostate carcinoma cells that normally have very low to undetectable levels of beta(III)were stably transfected with beta(III)cDNA in pZeoSV system. An 18 bp haemagglutinin (HA) epitope tag was added at the 3' end prior to cloning into the vector. Cells were transfected with pZeoSV or pZeoSV-beta(III)plasmids and selected in the presence of Zeocin. Immunofluorescent staining of the transfectant cells have shown significant expression and incorporation of HA-tagged beta(III)tubulin into cellular microtubules. Quantitation of Western blots revealed the HA-tagged beta(III)levels to be approximately 7-fold higher than the vector control cells. RT-PCR analysis confirmed the increase at the transcript level and also revealed a collateral increase of beta(II)and beta(IVb)transcripts. Cell viability assays indicated that sensitivity of beta(III)transfected cells to various antimicrotubule agents was similar to vector transfected cells: IC50 values for estramustine, paclitaxel, colchicine and vinblastine were 4 microM, 4 nM, 22 nM and 2 nM, respectively for both cell lines. Thus, overexpression of beta(III)isotype in human prostate carcinoma cells by stable transfection failed to confer antimicrotubule drug resistance to these cells. Counterregulatory increases of endogenous beta(II)and beta(IVb)tubulin isotypes in these beta(III)transfected cells may be a compensatory mechanism used by the cells to overcome the effects of elevated beta(III)levels on the cellular microtubules. These results highlight the difficulty in isolating the contribution of single tubulin isotypes in drug response studies.
Insights
Overexpressing beta(III) tubulin in prostate cancer cells did not increase resistance to antimicrotubule drugs. Compensatory increases in other tubulin isotypes may explain this lack of drug resistance.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Antimicrotubule drugs are crucial cancer treatments.
- Elevated beta tubulin isotypes are linked to drug resistance.
- The specific role of beta(III) tubulin in drug response requires further investigation.
Purpose of the Study:
- To determine if increased beta(III) tubulin expression confers resistance to antimicrotubule drugs in human prostate carcinoma cells.
- To investigate the functional impact of beta(III) tubulin overexpression on cellular microtubules and drug sensitivity.
Main Methods:
- Stable transfection of human prostate carcinoma cells with beta(III) tubulin cDNA.
- Expression analysis using immunofluorescence and Western blotting.
- Transcriptional analysis via RT-PCR.
- Cell viability assays to assess drug sensitivity (IC50 values).
Main Results:
- Successfully generated cell lines with significantly increased beta(III) tubulin expression (approx. 7-fold).
- Observed collateral increases in beta(II) and beta(IVb) tubulin transcripts.
- Beta(III) tubulin overexpression did not alter sensitivity to estramustine, paclitaxel, colchicine, or vinblastine.
Conclusions:
- Overexpression of beta(III) tubulin alone does not confer antimicrotubule drug resistance in this model.
- Counterregulatory increases in other tubulin isotypes may represent a compensatory mechanism.
- Isolating the contribution of single tubulin isotypes to drug response is complex.
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