ras provides evidence for cross-talk between the p21 ras and cell cycle regulatory pathways.

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.The neu (c-erbB-2, HER-2) proto-oncogene encodes a receptor tyrosine kinase that is a member of a growth factor receptor family, which includes the epidermal growth factor (EGF) receptor (ErbB-1), ErbB-3, and ErbB-4. neu is overexpressed in 20 to 30% of human breast tumors (64). Both Neu and the EGF receptor stimulate proliferation of breast cancer cells, and overexpression of these two proteins correlates with progression of human breast cancer and poor patient prognosis (28,31,47). A substitution point mutation at residue 664 (Val3Glu) in the transmembrane domain of rat Neu (referred to as NeuT) encodes an activated transforming tyrosine kinase (7). Overexpression of either wild-type Neu or NeuT in transgenic mice under the control of the murine mammary tumor virus (MMTV) long terminal repeat induces mammary adenocarcinoma with high frequency (25, 41). Several independent transgenic strains bearing the identical MMTV-neuT transgene developed synchronous, multifocal mammary tumors involving all mammary glands (24), providing strong evidence that activated neu requires few if any additional genetic events to transform the epithelial cell.In mammary tumors of mice transgenic for the wild-type Neu receptor (MMTV-neu mice), the receptor's intrinsic tyrosine kinase activity was increased in association with inframe somatic mutations of the transgene (61). Introduction of these extracellular domain deletion (ECD) mutations into the wild-type Neu cDNA enhanced neu transforming potential (61). Transgenic mice expressing these Neu deletion mutants in the mammary gland (MMTV-NDL mice) developed multifocal mammary adenocarcinomas with high frequency and shorter latency compared with mice transgenic for the wildtype neu. In primary human breast tumors, a splice variant of ErbB-2 encoding a similar ECD deletion which can...

Cell-cycle progression is mediated by a coordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F͞DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G 1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G 1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at ؊954 with additional contributions from a CRE site at ؊57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97-103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stressactivated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen.

Coordinated interactions between cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at ؊127 bound Sp1, Sp3, and Sp4, and the distal region at ؊143 bound E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at ؊954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1 ؊/؊ ) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1 ؉/؉ fibroblasts but increased apoptosis in cyclin D1؊/؊ cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G 1 analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

Angiotensin II (AII) binds to specific G protein-coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. Since the cyclin D1 gene encodes the regulatory subunit of the cyclin D1-dependent kinase (CD 1 K) required for phosphorylation of the retinoblastoma protein (pRB), an essential and rate-limiting step in G 1 phase progression of the cell cycle, we examined the effect of AII on cyclin D1 expression and CD 1 K activity in the human adrenal cell line H295R. AII (10 ؊6 M) stimulated G 1 phase progression within 12 h, with a maximal effect after 72 h. This action was antedated by the induction of cyclin D1 mRNA (3-fold), cyclin D1 nuclear protein abundance (4-fold), and CD 1 K activity (4-fold). AII induced cyclin D1 promoter activity 4-fold, via the AT 1 receptor through an enhancer sequence at ؊954 base pairs. c-Fos and c-Jun bound the cyclin D1 ؊954 enhancer sequence, and the abundance of c-Fos within this complex was increased by AII treatment. AII induced extracellular signal-regulated kinase (ERK) activity 7-fold, and dominant-negative mutants of either p21 ras or ERK reduced AII-stimulated cyclin D1 promoter activity. These findings suggest that AII may stimulate mitogenesis by increasing CD 1 K activity through a p21 ras /ERK/activator protein 1 pathway.

Recent reports have suggested that polymorphisms in the human paraoxonase (HUMPONA) gene may be a genetic risk factor for coronary artery disease (CAD) in white populations. However, this association has not yet been confirmed in other ethnic populations. We studied 75 Japanese patients with CAD, whose coronary lesions were confirmed by angiography, and 115 Japanese control subjects with no history of CAD and a normal resting electrocardiogram. The assays for genotyping the two polymorphisms in the HUMPONA gene (192Arg/Gln and 55Leu/Met) were based on changes in restriction enzyme digestion patterns. For codon 192, the frequencies of the Arg-coding allele (B allele) in both patients and control subjects were much higher than those from published results of whites (.26 to .31), and the difference between patients (.74) and control subjects (.59) was statistically significant (P = .002). The patient group had a higher proportion of Arg/Arg (B/B) homozygotes (52.0% vs 32.2%, P = .006). For codon 55, the frequencies of the Leu-coding allele in control subjects and patients were much higher (.91 and .93, respectively) than those published results for whites, but there was no difference between Japanese control subjects and Japanese patients. When subjects with the 55Leu/Leu genotype only were analyzed, 192Arg/Arg homozygotes were still significantly more frequent in the patients than in the control subjects (55.4% vs 37.2%, P = .024), and the frequency of the 192Arg allele was also higher in patients than control subjects (P = .013). Logistic regression analysis including conventional coronary risk factors revealed that 192Arg is an independent risk factor for CAD. Thus, in the Japanese, the association of CAD with the 192Arg variant of HUMPONA (B-type enzyme) is similar to that reported for whites, although the allele frequencies for 192Arg and 55Leu are much higher in the former than the latter population.

The cyclin D1 gene is overexpressed in breast tumors and encodes a regulatory subunit of cyclin-dependent kinases that phosphorylate the retinoblastoma protein.