Alkylphospholipids (ALKs) are a novel class of antineoplastic compounds that display potent antiproliferative activity against several in vitro and in vivo human tumor models. However, the mechanism by which these agents exert this desired effect is still unclear. In this study, we investigated the effect of perifosine, a p.o.-bioavailable ALK, on the cell cycle kinetics of immortalized keratinocytes (HaCaT) as well as head and neck squamous carcinoma cells. All cells were sensitive to the antiproliferative properties of perifosine with an IC(50) of similar0.6-8.9 microM. Cell cycle arrest at the G(1)-S and G(2)-M boundaries was observed in HN12, HN30, and HaCaT cells independent of p53 function, and this effect was preceded by loss in cdc2 and cyclin-dependent kinase (cdk) 2 activity. Analysis of cdk complexes in vitro demonstrated that perifosine, up to 20 microM, did not directly interfere with these enzymes. However, aphidicolin-synchronized HN12 cells released in the presence of perifosine (10 microM) demonstrated increased expression of total p21(WAF1) and increased association of p21(WAF1) with cyclin-cdk complexes resulting in reduced cdc2 activity. HCT116 isogenic cell lines were used to assess the role of p21(WAF1) induction by perifosine. This compound (20 microM) induced both G(1)-S and G(2)-M cell cycle arrest, together with p21(WAF1) expression in both p53 wild-type and p53(-/-) clones. By contrast, p21(-/-) variants demonstrated no p21(WAF1) induction or cell cycle arrest. Similar results were obtained with other ALK congeners (miltefosine and edelfosine). These data, therefore, indicate that perifosine blocks cell cycle progression of head and neck squamous carcinoma cells at G(1)-S and G(2)-M by inducing p21(WAF1), irrespective of p53 function, and may be exploited clinically because the majority of human malignancies harbor p53 mutations.