bcl-2 expression is reciprocal to p53 and c-myc expression in metastatic human colorectal cancer

Abstract

Apoptosis (programmed cell death) inhibition may be an important mechanism by which gastrointestinal mucosal cells containing damaged DNA evade normal clearance mechanisms and grow to become invasive tumours. Since bcl-2 is an apoptosis inhibitor, bcl-2 mRNA expression was measured in 21 metastases of colorectal cancer using reverse transcription–polymerase chain reaction analysis. The mean bcl-2 mRNA expression (0.45 U, P<0.0001) was lower than that of normal mucosal controls (=1 U). p53 expression was inversely correlated with bcl-2 expression (P=0.021) in 19 evaluable samples, and in tumours where p53 expression was over twice that of normal colonic mucosal values, bcl-2 mRNA was significantly decreased (mean 0.30, P=0.0052). c-myc was also inversely correlated with bcl-2 expression (P=0.025). Decreased bcl-2 expression in metastatic colorectal cancer may be partly due to allelic loss, given the proximity of bcl-2 to the frequently deleted DCC gene on chromosome 18q. However, the inverse correlation to p53/c-myc suggests an active downregulation of bcl-2, possibly following delegation of its apoptosis inhibiting role to other genes.

Introduction

In colorectal cancer, the accumulation of multiple genetic alterations in oncogenes and tumour suppressor genes results in impaired cellular growth control mechanisms causing tumour development and metastasis[1]. The most common genetic changes associated with advanced colorectal cancer are ‘loss of function’ mutations or deletions at p53, ras, DCC, APC/FAP loci and c-myc overexpression. Mechanisms that influence clearance of cells containing DNA damage may also be essential in preventing the establishment of pathological clones leading to tumour development, especially in tissues with high mitotic activity, such as normal gastrointestinal mucosa.

Apoptosis (programmed cell death) has been shown to be an important process by which somatically mutated cells are eliminated. Several genes are known to regulate apoptosis. The bcl-2 proto-oncogene is an apoptosis inhibitor originally described in association with the t(14;18)(q32;q21) translocation in follicular B cell lymphoma, which places the bcl-2 gene under the stimulatory control of the IgH promoter-enhancer at 14q32, resulting in increased bcl-2 mRNA and protein[2]and inhibition of apoptosis. bcl-2 expression and immunoreactivity has also been demonstrated in non-haemopoietic long lived cells and complex epithelia[3], suggesting a general role for bcl-2 in regulating apoptosis. However, its part in the development or progression of epithelial malignancies is not yet understood. bcl-2 oncoprotein has been described in normal colonic mucosa4, 5, 6, 7, 8, where it is restricted to the epithelial regenerative compartment, the intestinal crypt bases. Dysplastic colonic epithelium and normal epithelium immediately adjacent to adenocarcinomas have shown increased bcl-2 staining both in intensity and the number of bcl-2 positive cells5, 7, 8, 9. Adenomas have been found to express more bcl-2 than mucosal controls4, 5, 7, 8, as have carcinomas in some reports4, 5, but in other studies, adenocarcinomas displayed decreased bcl-2 immunoreactivity7, 8, 9. The importance of bcl-2 in colonic tumorigenesis and the interactions of bcl-2 with other genes involved in colorectal tumour growth and metastasis are, however, not yet understood. This is partly due to the fact that all available data on the relationship between bcl-2 and p53 in colorectal cancer is immunohistochemical. However, p53 immunohistopositivity gives no information on the mutational state of the p53 gene[10]and recent evidence[11]suggests that p53 mRNA and protein accumulation can occur in cells carrying the wild-type p53 gene as well as in p53 mutants. Determination of p53 mutational status, mRNA expression and protein accumulation is important in order to allow comment on p53 function, which is crucial in cell cycle checkpoint control and the maintenance of genome integrity by the initiation of apoptosis[12]. The cellular proto-oncogene c-myc is similarly involved in the regulation of cell proliferation, transformation and apoptosis and has been shown to interact with bcl-2 in apoptosis[13].

In this study, we determined bcl-2 mRNA expression in normal colonic mucosa and 21 colorectal tumour metastases, which we correlated to our findings of p53 expression and mutation status, expression and amplification of c-myc, as well as mutations in the ki-ras oncogene, in the same collection of colorectal cancer specimens14, 15, 16.