Immunotherapy based on the administration of the mycobacterium bacillus Calmette-Guerin has been successfully used in the treatment of in situ transitional cell bladder cancer, and may be applicable to the treatment of cutaneous malignant melanoma. Antigen 85A (Ag85A) and heat shock protein 65 kDa (hsp65) are major secreted proteins of Mycobacterium species and potent stimulators of cell-mediated immunity. This study evaluated the ability of Ag85A and hsp65 gene transfection to limit tumor growth by B16-F10 mouse melanoma cells. Immunoblotting confirmed protein expression and secretion by B16-F10 cells that were transiently transfected with plasmid DNA containing the Ag85A or hsp65 gene. Groups of syngeneic C57BL/6 mice were injected subcutaneously with 1x105 untransfected B16-F10 cells or B16-F10 cells transiently transfected with either empty vector or vector containing the Ag85A or hsp65 gene. Ag85A-expressing B16-F10 cells exhibited a dramatic 76% reduction (p<0.05, Mann-Whitney U test) in tumor weight in comparison to empty vector controls at 14 days post-inoculation. In contrast, hsp65-transfected B16-F10 cells did not show any change in tumorigenicity. Decreased tumorigenicity by Ag85A-transfected B16-F10 cells was not due to a reduced ability of Ag85A-transfected B16-F10 cells to proliferate since both mock- and Ag85A-transfected B16-F10 cells showed increased in vitro proliferation in comparison to untransfected cells. Hematoxylin and eosin staining revealed that Ag85A-transfected B16-F10 tumors contained an inflammatory leukocyte infiltrate that was not present in hsp65-transfected tumors. Reduced tumor progression by Ag85A-transfected B16-F10 melanoma cells suggests that immunotherapy based on the transient induction of Ag85A expression may be an effective approach for the treatment of cutaneous malignant melanoma.

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