Clonal overexpression of metallothionein is induced by somatic mutation in morphologically normal colonic mucosa

Metallothionein (MT) overexpression occurs frequently in human tumours but the underlying mechanism is unknown. Morphologically normal-appearing mucosa from human colorectal carcinoma resection specimens and of the colons of ageing laboratory mice contains scattered single crypts whose cells show uniformly increased MT immunostaining, suggesting that MT overexpression arises directly from random crypt stem cell somatic mutation, followed by colonization of the clonal unit by the mutated progeny. This hypothesis has now been tested by quantifying the frequency of immunocytochemically detectable monocryptal colorectal MT overexpression, 5 and 30 days after injection of 8-week-old mice with a single dose of the mutagen dimethylhydrazine (DMH, 30 mg/kg subcutaneous). Otherwise normal-appearing MT-positive crypts were recorded as either wholly or partially involved by the overexpressing phenotype. Five days after DMH injection, the median frequency of partially involved MT-positive crypts was 11.7 x 10(-4), declining significantly to 1.8 x 10(-4) at 30 days (Mann-Whitney U, P < 0.01). In contrast, the median frequency of wholly involved crypts was 0.2 x 10(-4) at 5 days, increasing significantly (P < 0.005) to 12.9 x 10(-4) at 30 days. The frequency of MT-positive crypts and the time course of evolution of partially involved to wholly involved forms were similar to those described for mutation-induced crypt-restricted loss of glucose-6-phosphate dehydrogenase activity in mice treated with an identical DMH regimen. The findings indicate that cellular MT overexpression can occur as a direct consequence of somatic mutation, either cis-activating mutation(s) of the MT gene itself, or trans-activating mutation(s) of other genes involved in controlling MT expression. This is likely to be an important mechanism underlying MT overexpression in neoplasia. Such mutation-induced aberrant MT expression may be involved in the acquisition of selective cellular growth of survival advantage during tumour progression.