Antizyme (AZ) is known to be a regulator of
polyamine metabolism that inhibits
ornithine decarboxylase activity and
polyamine transport, thus restricting
polyamine levels. Transgenic mice with AZ expression targeted to the basal cell layer of the forestomach epithelium by the
keratin 5 promoter were used to investigate whether AZ overexpression inhibited uncontrolled cell proliferation in
zinc-deficient (ZD) mice and reduced their susceptibility to forestomach
carcinogenesis by
N-nitrosomethylbenzylamine (NMBA). Four-week-old
keratin 5/AZ and wild-type (Wt) littermates were placed on ZD or
zinc-sufficient (ZS) diets to form four groups: ZD:AZ, ZD:Wt, ZS:AZ, and ZS:Wt. After 5 weeks, 27-45 mice in each group were treated twice with NMBA and sacrificed 14 weeks later. Independent of
zinc intake, AZ mice had significantly lower forestomach
tumor incidence and
tumor multiplicity than respective Wt littermates (P < 0.001): 21% of ZD:AZ versus 76% of ZD:Wt mice and 3% of ZS:AZ versus 33% of ZS:Wt mice developed
tumors.
Spermidine content was reduced in NMBA-treated ZD:AZ forestomachs.
Zinc deficiency increased the forestomach cell proliferation in Wt mice, but this effect was blocked by AZ. Conversely, apoptosis was substantially higher in control and NMBA-treated ZD:AZ than respective ZD:Wt forestomachs. The restored ZD:AZ forestomach epithelium displayed strong expression of Bax, a proapoptotic
protein, and weak staining of
cyclin D1 and its catalytic partner Cdk4, key regulatory
proteins controlling G(1) to S progression. In contrast, proliferative ZD:Wt forestomach showed strong expression of Bcl-2, an antiapoptotic
protein, and overexpression of
cyclin D1/Cdk4. Treatment of ZD:Wt mice with
alpha-difluoromethylornithine, an inhibitor of
ornithine decarboxylase, had similar results to AZ in reducing
tumor incidence,
spermidine content, decreasing cell proliferation, and increasing apoptosis. These results demonstrate that AZ may act as a tumor suppressor gene stimulating apoptosis and restraining cell proliferation, thereby inhibiting forestomach
tumor development. Although effects of AZ on functions other than
polyamine metabolism are possible, alterations in
polyamines are the most likely explanation for the reduction in
tumors, supporting the use of strategies to modulate
polyamine levels for
cancer chemoprevention in individuals at high risk of developing
malignancies of the gastrointestinal tract.