Effect of increased glucocorticoid responsiveness in transformed mouse lung cells
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Abstract:
Many transformed mouse lung cells, including LM2 cells, contain activating mutations in the Ki-ras gene and show reduced responsiveness to growth inhibition by glucocorticoids. LM2GR cells, which are LM2 cells stably transfected with a rat glucocorticoid receptor (GR) gene, were used to determine whether increasing glucocorticoid responsiveness can influence aspects of the transformed phenotype. LM2GR cells grew slower and had a lower final saturation density than the parental LM2 cells. Expression of growth-related genes was examined by northern blot analysis. The cells were serum-deprived and treated with fetal bovine serum (FBS), steroid-stripped FBS (ssFBS), dexamethasone, or 12-O-tetradecanoylphorbol-13-acetate. The level and pattern of Ki-ras mRNA expression was similar in both LM2 and LM2GR cells, but histone H4 mRNA was expressed in a more regulated fashion in LM2GR cells. The induction of c-jun and c-fos mRNA expression lasted longer in the LM2GR cells treated with ssFBS; however, the maximal induction was greater in the LM2 cells treated with FBS. LM2GR cells demonstrated similar activator protein-1 (AP-1) activity but higher GR activity than LM2 cells as determined by using AP-1-chloramphenicol acetyltransferase (CAT) and mouse mammary tumor virus-CAT transient transfection assays, consistent with the higher level of GR mRNA in LM2GR cells. Both cell lines exhibited the ability to grow in soft agar and to form tumors in nude mice. These results indicate that introduction of a functional GR transgene into LM2 cells can increase glucocorticoid responsiveness and alter the expression of genes involved in growth regulation but cannot overcome anchorage-independent cell growth or tumorigenicity, apparently because of the presence of an activated Ki-ras gene.
Insights
Introducing a glucocorticoid receptor (GR) gene into mouse lung cancer cells increased glucocorticoid responsiveness and altered gene expression. However, it did not overcome anchorage-independent growth or tumorigenicity due to an activated Ki-ras gene.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Transformed mouse lung cells (LM2) with activating Ki-ras mutations exhibit reduced glucocorticoid responsiveness.
- Glucocorticoids are known regulators of cell growth and differentiation.
Purpose of the Study:
- To investigate if enhanced glucocorticoid responsiveness in LM2 cells influences their transformed phenotype.
- To determine the impact of a functional glucocorticoid receptor (GR) transgene on gene expression and cellular behavior.
Main Methods:
- Stable transfection of LM2 cells with a rat GR gene to create LM2GR cells.
- Northern blot analysis to examine gene expression (Ki-ras, histone H4, c-jun, c-fos).
- Transient transfection assays (AP-1-CAT, MMTV-CAT) to assess GR and AP-1 activity.
Main Results:
- LM2GR cells showed slower growth and lower saturation density compared to LM2 cells.
- Histone H4 mRNA expression was more regulated in LM2GR cells.
- c-jun and c-fos induction kinetics differed between cell lines and treatments, with LM2GR cells showing prolonged induction with steroid-stripped FBS.
- LM2GR cells exhibited higher GR activity but similar AP-1 activity compared to LM2 cells.
Conclusions:
- Introduction of a functional GR transgene enhances glucocorticoid responsiveness and modulates growth-related gene expression in LM2 cells.
- Despite altered gene expression, enhanced GR responsiveness did not overcome anchorage-independent growth or tumorigenicity.
- The activated Ki-ras gene appears to be a key factor limiting the full reversal of the transformed phenotype.