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Growth and intestinal differentiation are independently regulated in HT29 colon cancer cells
P C Schroy1, A K Rustgi, E Ikonomu
1Department of Medicine, Boston University Medical Center, Massachusetts 02118.
Abstract:
The polar-planar compound hexamethylene bisacetamide (HMBA) can inhibit HT29 colon carcinoma cell growth and induce a more benign phenotype, as defined by decreased anchorage-independent clonogenicity, loss of a cell surface malignancy marker, and decreased in vivo tumorigenicity. The principle aim of this study was to determine whether HMBA's effects on HT29 cell growth and biologic behavior correlate with effects on intestinal differentiation. Parallel studies were performed with sodium butyrate (NaBT), a potent inducer of intestinal differentiation. HT29 cell growth, proliferation, and markers of intestinal differentiation were assayed after short- and long-term treatment with HMBA, NaBT, or the combination. Both 5 mM HMBA and 5 mM NaBT were potent inhibitors of monolayer growth; in combination their effects were nearly additive. Inhibition of DNA synthesis was detectable within 6 h of treatment and was preceded by down-regulation of c-myc expression. Soft agar clonogenicity was also decreased by 90%, > 99%, and > 99% by HMBA, NaBT, and the combination, respectively. Despite these parallel effects on growth and in vitro markers of a benign phenotype, effects on intestinal differentiation were discordant. NaBT induced significant increases in membrane-associated alkaline phosphatase activity, cytosolic mucin content, PAS+/diastase-resistant cells, and ultrastructural evidence of intestinal cell differentiation. HMBA not only failed to induce markers of intestinal differentiation, but attenuated NaBT's effects when used in combination. These data suggest that growth and intestinal differentiation may be independently regulated in HT29 cells. They also suggest that expression of intestinal markers of differentiation is not a prerequisite for the acquisition of a more benign phenotype.
Insights
Hexamethylene bisacetamide (HMBA) reduces colon cancer cell growth and malignancy but does not induce intestinal differentiation. Growth inhibition and differentiation appear to be independently regulated processes in HT29 cells.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Biology
Background:
- Hexamethylene bisacetamide (HMBA) is a polar-planar compound known to inhibit HT29 colon carcinoma cell growth.
- HMBA induces a less malignant phenotype in these cells, characterized by reduced clonogenicity and tumorigenicity.
- The study investigates the correlation between HMBA's effects on cell growth and its impact on intestinal differentiation markers.
Purpose of the Study:
- To determine if HMBA's inhibition of HT29 colon carcinoma cell growth correlates with effects on intestinal differentiation.
- To compare the effects of HMBA with sodium butyrate (NaBT), a known inducer of intestinal differentiation.
- To investigate the independent or combined effects of HMBA and NaBT on HT29 cell growth and differentiation.
Main Methods:
- HT29 cells were treated with HMBA, NaBT, or a combination for short and long durations.
- Cell growth, proliferation, and DNA synthesis were measured.
- Markers of intestinal differentiation, including alkaline phosphatase activity and mucin content, were assessed.
- Soft agar clonogenicity and c-myc expression were analyzed.
Main Results:
- Both HMBA and NaBT significantly inhibited HT29 cell monolayer growth and DNA synthesis, with nearly additive effects when combined.
- Treatment with HMBA and NaBT markedly reduced soft agar clonogenicity.
- While NaBT induced significant markers of intestinal differentiation, HMBA failed to do so and even attenuated NaBT's differentiation-inducing effects.
- Down-regulation of c-myc expression preceded the inhibition of DNA synthesis.
Conclusions:
- HT29 colon carcinoma cell growth and intestinal differentiation appear to be independently regulated.
- HMBA's ability to induce a benign phenotype does not necessitate the induction of intestinal differentiation markers.
- These findings suggest distinct molecular pathways govern proliferation and differentiation in colon cancer cells.