Constitutive c-myc expression suppresses cell cycle arrest, promotes entry into S phase, and results in the growth factor-independent expression of ornithine decarboxylase (ODC; EC 4.1.1.17). The ODC gene contains a conserved repeat of the Myc binding site, CACGTG, in intron 1. In this report, we demonstrate that c-Myc is a potent transactivator of ODC promoter-reporter gene constructs in fibrobLsts that requires the CACGTG repeat. These sites conferred Myc responsiveness on heterologous promoter constructs, suggesting that ODC is regulated by Myc at the level of tnscription initiation. Analysis of deletion and point mutants of c-myc revealed that domains required for bansactivation of the ODC promoter did not include the leucine zipper of the Myc protein. This suggests that Myc may interact with transcription factors other than Max to transactivate the ODC gene.

The expression of cyclin-dependent kinase inhibitor p27 kip1 in human tumors and normal tissues was investigated using a panel of novel anti-p27 kip1 mAbs. An inverse correlation between expression of p27 kip1 and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27 kip1 expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27 kip1 and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27 kip1 and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27 kip1 may be a useful prognostic marker in these cancers.

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 protein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apoptosis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the structure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammalian cells. We demonstrate that, in addition to the previously identified 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a second 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have distinct subcellular localizations, suggesting that they are functionally distinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5' sequences demonstrated that human and murine Bag-1 cDNAs contain longer open reading frames than originally suspected. We determined which open reading frames gave rise to the Bag-1 isoforms in human cells. Surprisingly, translation of neither protein initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag-1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently localized Bag-1 isoforms generated by alternative translation initiation, and Bag-1 proteins may play a dual role in regulating apoptosis and steroid hormone-dependent transcription.

The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G 0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.Members of the E2F family of transcription factors are thought to regulate cell cycle progression by activating the transcription of a set of genes necessary for the induction of S phase (30, 53). E2F DNA binding activities are dependent on growth factors (52), and their function as transcription factors is temporally regulated throughout the cell cycle by complex formation with the tumor suppressor protein pRb and the pRb-related proteins p107 and p130 (9,10,13,43,69). E2F is activated by adenovirus E1A binding to pRb and its related proteins, and release of E2F from pRb is critical for transformation induced by both E1A (18, 61) and pRb inactivation (29,31,57,58).The DNA binding activity originally termed free E2F (3) is now recognized to be a heterodimer containing the product of an E2F gene family member (E2F1 to E2F5) and a DP family member (5, 21-23, 27, 34, 38, 44, 72, 76). E2F can bind DNA in vitro, whereas DP proteins bind DNA only weakly (23). Dimerization of DP proteins with E2F proteins increases the transcriptional activity of E2F and is required for association of E2F with pRb or pRb-related proteins (4,5,21,28,41). Furthermore, enforced DP-1 expression augments E2F-mediated transformation of primary rat embryo fibroblast cells in cooperation with an activated ras oncogene (5,21,35).Microinjection of serum-starved fibroblasts with an E2F-1 expression plasmid (37) or glutathione S-transferase-E2F-1 fusion protein (17), or activation of E2F-1 expression in transfected cell lines (59,67), is sufficient to drive quiescent cells into S phase. Inappropriate entry of these cells into S phase, in the absence of survival factors, is associated with the activation