Examination of a panel of ER positive breast cancer cell lines showed that they were dierentially growth inhibited by vitamin D 3 and its analogue EB1089. EB1089 treatment of the breast cancer cell lines MCF-7 E, BT20, T47D, and ZR75 demonstrated a correlation between a reduction in Cdk2 kinase activity towards phosphorylation of histone H1 and a decrease in DNA synthesis, while no modulation of Cdk2 activity was observed in the vitamin D 3 and EB1089 resistant cell line MCF-7 L. This was accompanied by a time dependent decrease in the percentage of S phase cells in the responsive lines. Characterization of the expression levels of Cdk2 and its related cell cycle proteins in MCF-7 E cells showed that after EB1089 treatment, there was a concentration and time dependent up-regulation of p21 as well as a decrease in cyclin A proteins. Paradoxically, cyclin E levels were increased as a function of treatment. Analysis of cyclin-Cdk2-Cdki complex formation showed that in EB1089 treated MCF-7 E cells, Cdk2, cyclin A and cyclin E immunoprecipitates contained an increased abundance of p21. In contrast to MCF-7 E cells, increases in both p21 and p27 as well as their complex formation with Cdk2 were observed in BT20 and ZR75 cells. These ®ndings indicate that up-regulation of p21 as well as p27 in some cell types may account for the inactivation of Cdk2 activity and a G 1 block of the cell cycle following EB1089 treatment.

In view of the tumor suppressor role of the transforming growth factor-␤ (TGF␤) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGF␤ growth inhibition activity, was significantly inhibited by vitamin D 3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGF␤, was not affected by these two compounds. TGF␤-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGF␤ activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3-4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells. Transforming growth factor-␤ (TGF␤)1 comprises a family of hormone-like polypeptides that affects cell growth, adhesion, and differentiation (1). They act as growth inhibitors for most epithelial cells and some cancer cells. Two pathways are primarily involved in mediating effects of TGF␤ on cell growth and differentiation. One pathway involves blockade of cell cycle transit, while the other involves alteration of the extracellular matrix environment.TGF␤s elicit their effects by binding to cell surface receptors. Three major types of receptors have been shown to be present in most TGF␤-responsive cell lines. They are designated as type I (RI), type II (RII), and type III (RIII), respectively. RIII is a 280 -330-kDa glycoprotein that has no functional signaling domain but rather serves as a ligand storage protein and presents TGF␤ to the signaling receptors (2). RI and RII, which are glycoproteins of ϳ55 and 85 kDa, respectively, form a heteromeric receptor complex. Both are serine/threonine kinases, and each appears to be indispensable for TGF␤ signaling (3-5). The direct involvement of both RI and RII in conferring TGF␤ effects indicates that loss of either of the functional receptors would contribute to loss of autocrine TGF␤ activity. Loss of negative autocrine TGF␤ activity results in a growth advantage caused by an imbalance in positive and negative regulators, possibly leading to tumor formation and progression (6, 7). Recent evidence has shown a loss of RII is often associated with the failure to respond to autocrine and exogenous TGF␤. We have previously demonstrated that re-expression of this receptor in an RII-deficient breast cancer cell line (late passage MCF-7) leads to restoration of TGF␤ sensitivity and reduced malignancy in athymic nude mice (6). In addition, it has been shown that mutational inactivation of...

Transforming growth factor beta (TGF-beta) receptor expression and signal transduction in human breast cancer are reviewed as a function of estrogen receptor (ER) expression. ER+ breast cancer cells are generally resistant to the inhibitory effects of TGF-beta. The only known exception appears to be MCF-7 early passage cells which are initially sensitive to TGF-beta, but gain resistance after long-term passage in tissue culture. A number of studies have shown that loss of sensitivity is due to inadequate TGF-beta type II (TGFRII) receptor expression. Stable transfection of TGFRII into ER+ breast cancer cell lines results in the acquisition of TGF-beta sensitivity and reversion of malignancy. Although there are exceptions, ER- breast cancer cells usually express TGFRII, but nevertheless show a low level of sensitivity to TGF-beta. Thus resistance in these cells implies a postreceptor mechanism. Given the frequency with which loss of TGF-beta sensitivity has been associated with loss of TGFRII, the ER- breast cancer cell lines may represent valuable models for identifying postreceptor mechanisms of resistance.

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