The biphasic induction of p21 and p27 in breast cancer cells by modulators of cAMP is posttranscriptionally regulated and independent of the PKA pathway.

Cyclic AMP (cAMP) elevation affects growth arrest and differentiation in a wide variety of breast cell lines; however, the mechanisms associated with this process are poorly understood. Previous studies linked cAMP-mediated growth arrest in breast tumor cells to increased levels of cyclin kinase inhibitor (CKI), p21. In the present study we examined the role of cAMP-dependent protein kinase (PKA) on p21 and p27 induction in the breast cancer cell line, MDA-MB-157. The induction of the CKIs by modulators of cAMP such as cholera toxin (CT) + 1-isobutyl-3-methylxanthine (IBMX) and lovastatin fluctuates with biphasic kinetics (although the kinetics of CKI induction with CT + IBMX treatment are different from that of lovastatin) and is depicted by the periodic accumulation of lower molecular weight forms of p21 and p27 which also correlate with fluctuations in CDK2 activity. Using three different approaches we show that the cAMP-mediated induction of CKIs is independent of PKA activity. In the first approach we treated MDA-MB-157 cells with a variety of cAMP modulators such as CT + IBMX, and forskolin in the presence or absence of H-89, a potent PKA inhibitor. This analysis revealed that the cAMP activators were capable of inducing p21 even though PKA activity was completely eliminated. In the second approach PKA dominant negative stable clones of MDA-MB-157 treated with CT + IBMX or forskolin also resulted in p21 induction, in the absence of any PKA activity. Last, treatment of MDA-MB-157 cells with lovastatin, another known cAMP modulator which also causes growth arrest, resulted in the induction of p21 and p27 without any increase in PKA activity. Collectively, the above results suggest that the induction of p21 by cAMP is through a novel pathway, independent of PKA activity.

t-complex-associated embryonic surface antigen homologous to mLAMP-1. I. Biochemical and molecular analyses.

Previously we described an embryonic cell surface glycoprotein, ESGp, associated with the t-embryonic lethal alleles of the mouse t complex. This antigen is expressed on the cell surface of both early mouse embryos and embryonal carcinoma (EC) cell lines. The antigen is localized to areas of cell-cell contact in EC lines and redistributes to the outer edges of the blastomeres during compaction, thereby indicating a potential role in embryonic cell-cell interaction. We now report that this t-complex-associated ESGp is homologous to the mouse lysosomal-associated membrane protein-1 (LAMP-1). Limited protein sequence analyses of the amino terminal and an internal peptide indicate considerable homology with the LAMP-1 protein. Biochemical parameters such as protein core size, sulfation and phosphorylation status, and resistance to proteolysis also demonstrate homology. While we detect only a single message with a mouse LAMP-1 cDNA probe via Northern blotting, Southern analyses indicate the existence of at least two homologous LAMP-1 genes. Additionally, we present evidence suggesting that ESGp/LAMP-1 serves as a substrate which may be differentially glycosylated by the activities of the gene products of the different t-lethal alleles.

t-complex-associated embryonic surface antigen homologous to mLAMP-1. II. Expression and distribution analyses.

We have previously demonstrated that a mouse t-complex-associated antigen (ESGp) is present on the cell surface, specifically at areas of cell-cell contact, of cleavage stage embryos and embryonal carcinoma cell lines. In the accompanying paper, we document isolation, purification, and partial sequencing of this molecule. Amino-terminal and internal peptide fragments are highly homologous to the mouse lysosomal-associated membrane protein-1 (mLAMP-1). We also demonstrate considerable similarity, though not necessarily identity, between these two antigens through both biochemical and molecular analyses. In this report we use immunological techniques to compare the expression and distribution of these antigens in various cell lines as well as mouse tissues and preimplantation stage embryos. The data presented indicate that there are: (1) different antigenic forms of the ESGp/ mLAMP-1 homologues and (2) changes in the molecular composition and expression of these forms during embryogenesis and differentiation. It is obvious from these studies that mLAMP-1 is not strictly a static molecule whose sole function is to protect the lumenal side of the lysosomal membrane but rather is a dynamic molecule of potential import in many other cellular and organismal functions.

Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion.

Cyclin-dependent kinase inhibitors (CKIs) p21, p27, p16, and p15 are an essential and integral part of cell cycle regulation. Studies on the expression of these inhibitors in normal versus tumor human breast cancer cells revealed that although p27 and p16 are expressed at higher levels in tumor cells, p21 and p15 expression were higher in normal cells. Analysis on the expression pattern of these proteins throughout the cell cycle in synchronized cells demonstrated a substantial increase in p21 during the S-phase in normal cells and barely detectable expression of p21 in any phase of the tumor cell cycle. Levels of p15, p16, and p27 remained relatively constant throughout the cell cycle of normal and tumor cells. Synchronization of tumor cells by lovastatin, which arrests cells in G1, resulted in increased levels of p21 and p27 with a concomitant decrease in cyclin-dependent kinase 2-associated kinase activity. Synchronization of cells by double-thymidine block did not result in the induction of p21 or p27. These observations suggest that lovastatin causes a profound cell cycle-independent alteration of CKI expression which is distinct from growth factor deprivation or thymidine block.

Cyclin E, a redundant cyclin in breast cancer.

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

Neu differentiation factor (Heregulin) activates a p53-dependent pathway in cancer cells.

Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by up-regulation of the p53 inducible gene, p21CIP1/WAF1, in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells. The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wildtype p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.