P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endothelium and platelets. The major ligand for P-selectin on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin can also bind to carcinoma cells, but the nature of the ligand(s) on these cells is unknown. Here we investigated the P-selectin binding to a breast and a small cell lung carcinoma cell line that are negative for PSGL-1. We report that CD24, a mucin-type glycosylphosphatidylinositol-linked cell surface molecule on human neutrophils, pre B lymphocytes, and many tumors can promote binding to P-selectin. Latex beads coated with purified CD24 from the two carcinoma cell lines but also neutrophils could bind specifically to P-selectin-IgG. The binding was dependent on divalent cations and was abolished by treatment with O-sialoglycoprotein endopeptidase but not endoglycosidase F or sialidase. The beads were stained with a monoclonal antibody (MoAb) to CD57 (HNK-1 carbohydrate epitope) but did not react with MoAbs against the sialylLex/a epitope. The carcinoma cells and CD24-beads derived from these cells could bind to activated platelets or P-selectin transfected Chinese hamster ovary cells (P-CHO) in a P-selectin–dependent manner and this binding was blocked by soluble CD24. Transfection of human adenocarcinoma cells with CD24 enhanced the P-selectin–dependent binding to activated platelets. Treatment of the carcinoma cells or the CD24 transfectant with phosphatidylinositol-specific phospholipase C reduced CD24 expression and P-selectin–IgG binding concomitantly. These results establish a role of CD24 as a novel ligand for P-selectin on tumor cells. The CD24/P-selectin binding pathway could be important in the dissimination of tumor cells by facilitating the interaction with platelets or endothelial cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor ( ⌬ Raf-1:ER), which can be activated with estradiol. ⌬ Raf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27 kip1 , and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by ⌬ Raf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors. ( J. Clin. Invest. 1998. 101:153-159.) Key words: SCLC • activated raf • MAP kinase • cell cycle • p27 kip1 • MEK inhibitor PD098059

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16 ink4a , PSM-RB is also a potent inhibitor of cell cycle progression in RB-de®cient tumor cells. Surprisingly, we identi®ed a tumor cell line that is resistant to the cell cycle inhibitory e ects of PSM-RB. This ®nding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this`resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the ®rst to show that RB inhibits cellular proliferation through at least two distinct mechanisms ± inhibition of cell cycle progression and induction of cell death.Keywords: apoptosis; cyclin; phosphorylation; cell death; RB The retinoblastoma tumor suppressor protein, RB, is inactivated in over 60% of studied human tumors (Bartek et al., 1997;Sherr, 1996). Inactivation of RB can be achieved through multiple distinct mechanisms, including direct loss of functional protein due to mutation, binding of oncoproteins of DNA tumor viruses, or by overt phosphorylation of RB, which inactivates its growth-suppressing function (Bartek et al., 1997;Sherr, 1996;Wang et al., 1994). It has been hypothesized that RB must be phosphorylated and inactivated by cyclin dependent kinase (CDK)/cyclin complexes in cells to allow for cell cycle progression, and in tumor cells this process is often deregulated (Bartek et al., 1997;Sherr, 1996). For example, ampli®cation of the proto-oncogenes CDK4 or Cyclin D1 results in excessive phosphorylation and inactivation of RB. Likewise, loss of the tumor suppressor protein p16 ink4a which normally serves to attenuate the activity of CDK4 and cyclin D1, results in deregulated phosphorylation and inactivation of RB. It is thought that unphosphorylated (i.e. active) RB prevents cellular proliferation by sequestering a host of factors and assembling complexes which inhibit cell cycle progression (De Pinho, 1998;Sidle et al., 1996;Wang et al., 1994). Phosphorylation of RB by CDK/cyclin complexes causes release of these proteins, and thus enables cell cycle advancement.Studies aimed at directly de®ning the role of RB in cell cycle processes have been hindered by the fact that ectopically expressed wild-type RB is a very poor inhibitor of cellular proliferation, since it is phosphorylated and inactivated by endogenous CDK/Cyclin activity (Wang et al., 1994;Zacksenhaus et al., 1993). Because of ...