Melanoma incidence is growing at a faster rate than any other human malignancy. Wild-type (wt) p53 is important in both G(1) and G(2) cell cycle arrest, and cyclin D1 (CD1) is necessary for G(1)-->S progression in melanoma cells. We reported that an adenoviral vector containing wt p53 significantly reduced [(3)H]thymidine uptake in melanoma cells containing mutant but not wt p53. Subsequently we showed that CD1 decreased melanoma proliferation and increased apoptosis. We now extend these findings by evaluating the effect on preformed melanomas of (1) intratumoral therapy with wt p53 alone, (2) wt p53 in combination with antisense (AS) CD1, both short (< or =14 days) and longer term, and (3) doubling the dose or repeat doses of wt p53 or AS CD1. Two melanoma cells lines that metastasize in SCID mice (451 and 1205) were used, one containing a p53 mutation (451) and the other a normal p53 gene sequence (1205). Compared to injection with a control adenoviral vector containing beta-galactosidase (LacZ), intratumoral injection of wt p53 slowed the growth of tumors formed from 451 cells. Using 5 x 10(8) plaque forming units as our standard intratumoral dose, neither doubling the dose of LacZ, p53 or AS CD1, nor repeat doses of the vectors, was as effective as combined therapy with wt p53+AS CD1, which resulted in the shrinkage of all tumors treated and 4/7 (57%) tumors vanished. No tumors treated with wt p53 or AS CD1 alone vanished. Wt p53+AS CD1 treatment resulted in significantly more cells undergoing apoptosis compared to either therapy alone. In summary, combining the separately effective treatment vectors p53 and AS CD1 led to an enhanced growth-suppressive and apoptotic effect, supporting a role for combination gene therapy to treat human malignant melanoma.