Expression of D-type cyclins in colon cancer and in cell lines from colon carcinomas

Abstract

Cyclins D1, D2 and D3 play important roles in cell proliferation and differentiation. Although their abnormal expression has been linked to cancer development and progression in a number of tissues, the expression of cyclin D2 and D3 proteins in colon cancer has not yet been characterised. In this study, we examined cyclin D1, D2 and D3 protein expression by Western blot analysis in tumour and adjacent normal colon tissues of 57 patients. In addition, we examined D-type cyclins protein expression in HT29 and LoVo39 cell lines from colon carcinomas, as a function of induced proliferation and differentiation. In both cell lines, the expression of the three D-type cyclins increased as a result of induced proliferation, whereas the expression of cyclin D3 increased as a result of induced differentiation. In colon tumours, cyclin D1 was overexpressed in 44%, cyclin D2 was overexpressed in 53% and cyclin D3 was overexpressed in 35% of the cases. We also found that in 16% of the cases, cyclin D3 protein expression was reduced in the tumour, as compared to the adjacent normal tissue. Examination of D-type cyclin protein overexpression in relation to the TNM stage of the tumours revealed that overexpression of cyclins D1 and/or D2, but not cyclin D3, is linked to colon carcinogenesis and that overexpression of cyclin D2 may be related to a higher TNM stage of the tumour.

Figure 1

Expression of D-type cyclins during induction of proliferation. HT29 cells (left panels) and LoVo39 cells (right panels) were arrested in G1 phase of the cell cycle by serum starvation and then released. As a function of time following release, cell cycle phase distribution was analysed by flow cytometry (A), and D-type cyclin and β-actin protein expressions were analysed by Western blot analysis (B, C). In (B), representative Western blot analysis is presented. The locations of bands of the D-type cyclins and β-actin proteins, based on their molecular weight, are indicated by arrows. Numbers on top of each Western blot image represent the normalised expression of the various proteins (see Materials and Methods), relative to their expression at time of release from G1 arrest (0 h). Panel (C) displays the average relative expression of cyclin D1 (circles), D2 (squares) and D3 (triangles) as a function of time following release. The points and bars represent average and s.d. of three independent experiments.

Figure 2

D-type cyclins expression during induction of differentiation by NaB. HT29 cells (left panels) and LoVo39 cells (right panels) were treated with NaB for 24 h. (A) Alkaline phosphatase (ALP) activity measured as a function of increased NaB concentrations (indicated on the abscissa). Columns and bars represent averages and s.d. of 3–4 experiments. (B) Cyclins D3, D2 and D1 and β-actin protein expression as a function of 24 h treatment with 1 and 5 mM NaB. Numbers at the top of the panels indicate protein levels of the different D-type cyclins, relative to β-actin expression and normalised to the relative expression in the control cells (0 mM NaB). Data are representative of 3–4 similar experiments. (C) mRNA expression of CEA, MUC3, cyclin D3 and β-actin (indicated on the right) as a function of 24 h treatment with 1 and 5 mM NaB. Numbers on top of the CEA, MUC3 and cyclin D3 panels represent mRNA expression relative to β-actin and normalised to relative expression of the corresponding gene in control cells (0 mM NaB). Data are representative of three experiments.

Figure 3

Expression of D-type cyclins in colorectal tumours. Western blot analysis of cyclin D1 (A), cyclin D2 (B) and cyclin D3 (C) protein expression in tumour (T) and adjacent normal (N) colon tissues. Representative results of several patients are shown (indicated by case number, on top). Protein extract from HT29 cells was used as a positive control for cyclin D1 and D3 protein expression and as a negative control for cyclin D2 protein expression. Protein extract from LoVo39 cells was used as a positive control for cyclin D2 expression. The locations of bands of the D-type cyclins and β-actin proteins, based on their molecular weight, are indicated by arrows. Numbers on the bottom of each Western blot image represent the normalised expression of D-type cyclin proteins in the tumour tissue (T), relative to their expression in the adjacent normal tissue (N).