Recent evidence has established different functions for the tumor suppressor protein, p16(INK4A) aside from controlling the cell cycle. Here we report that cdk4/6 inhibition blocked both human umbilical vein endothelial cells (HUVEC) spreading on a vitronectin matrix and HUVEC migration on vitronectin. p16 can also act as an anti-angiogenic molecule in vitro since HUVEC and HMEC cells transfected with Ad-p16 or treated with Antennapedia p16 peptides are unable to differentiate on a Matrigel matrix. Both, p16, cyclin D1, cdk4 and cdk6 were immuno-colocalized with Ezrin, Rac, Vinculin, alphav-integrin, and FAK proteins in the ruffles and lamellipodia of migratory cells. Our results indicate that p16 is a key component of a new cytoplasmic pathway controlling angiogenesis of endothelial cells via the alphavbeta3-integrin-mediated migration.

The development and the effect of immunoliposomes directed against human breast cancer cells overexpressing p185/HER2 are described. These immunoliposomes carry an antisense oligonucleotide directed toward the translational start site of dihydrofalate reductase (DHFR) RNA, which causes high cytotoxicity. To prepare the immunoliposomes, we followed two methodologies based on the high affinity between streptavidin and biotin and the use of biotinylated antibodies. In the first approach, the streptavidin molecule is covalently attached to the phospholipid DOPE, which is mixed with the cationic liposome DOTAP complexed with the antisense oligonucleotide. The second approach, which is much easier to perform, involves the binding of streptavidin to antibody and oligonucleotide, both biotinylated, and the latter complexed with DOTAP. The formation of the intermediary complexes of this immunoliposome was studied sequentially by gel electrophoresis. The uptake of the oligonucleotide carried by the immunoliposome was monitored by flow cytometry and confocal microscopy. As a model, we used SKBR3 cells that overexpress p185. The full immunoliposomes were more toxic than the antisense oligonucleotide in the absence of the antibody, thus increasing the sensitivity of the treatment.

The effect of incubations with anti‐sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'‐untranslated region, the translational start, the splice sites and branch point of intron 1 and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation‐start site, at a range of concentrations between 1 and 4 μM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]‐ or fluorescein‐labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. Formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co‐transfected DHFR minigenes. Cell incubation with ATNL caused a time‐dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. Our results set the basis for a possible cancer therapy with anti‐sense oligonucleotides using DHFR as the target. Int. J. Cancer 81:785–792, 1999. © 1999 Wiley‐Liss, Inc.