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Supplementary Figure 1 from The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16INK4a/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1−/− mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16INK4a expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was observed in tissues of mice mutant for the Ink4a/Arf locus. p16INK4a and p19ARF inhibited DNA synthesis in MCF7 cells. p16INK4a repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16INK4a occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19ARF repressed cyclin D1 through a novel distal cis-element at −1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

Supplementary Figure 2 from The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements

First-line therapy for men with metastatic or recurrent prostate cancer following definitive local therapy is medical or surgical castration. Though effective initially in most patients, the majority of tumors develop castration resistance, necessitating the addition of further therapy. The historic treatment paradigm of second-line androgen manipulation, followed by cytotoxic salvage chemotherapy, has changed in recent years with better understanding of mechanisms that lead to castration resistance. This review will outline the data supporting the use of targeted and chemotherapeutic agents for prostate cancer, review data leading to US Food and Drug Administration (FDA) approval of the newest agents abiraterone, enzalutamide, and cabazitaxel, as well as review ongoing studies of novel agents.

The PC12 pheochromocytoma cell line responds to nerve growth factor (NGF) by exiting from the cell cycle and differentiating to induce extending neurites. Cyclin D1 is an important regulator of G1/S phase cell cycle progression, and it is known to play a role in myocyte differentiation in cultured cells. Herein, NGF induced cyclin D1 promoter, mRNA, and protein expression via the p21RASpathway. Antisense- or small interfering RNA to cyclin D1 abolished NGF-mediated neurite outgrowth, demonstrating the essential role of cyclin D1 in NGF-mediated differentiation. Expression vectors encoding mutants of the Ras/mitogen-activated protein kinase pathway, and chemical inhibitors, demonstrated NGF induction of cyclin D1 involved cooperative interactions of extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase pathways downstream of p21RAS. NGF induced the cyclin D1 promoter via Sp1, nuclear factor-κB, and cAMP-response element/activated transcription factor sites. NGF induction via Sp1 involved the formation of a Sp1/p50/p107 complex. Cyclin D1 induction by NGF governs differentiation and neurite outgrowth in PC12 cells.

The NAD-dependent histone deacetylase Sir2 plays a key role in connecting cellular metabolism with gene silencing and aging. The androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens, including dihydrotestosterone (DHT). Here, SIRT1 antagonists induce endogenous AR expression and enhance DHT-mediated AR expression. SIRT1 binds and deacetylates the AR at a conserved lysine motif. Human SIRT1 (hSIRT1) repression of DHT-induced AR signaling requires the NAD-dependent catalytic function of hSIRT1 and the AR lysine residues deacetylated by SIRT1. SIRT1 inhibited coactivator-induced interactions between the AR amino and carboxyl termini. DHT-induced prostate cancer cellular contact-independent growth is also blocked by SIRT1, providing a direct functional link between the AR, which is a critical determinant of progression of human prostate cancer, and the sirtuins.

The position effect variegation in Drosophila and Schizosaccharomyces pombe, and higher-order chromatin structure regulation in yeast, is orchestrated by modifier genes of the Su(var) group, (e.g., histone deacetylases ([HDACs]), protein phosphatases) and enhancer E(Var) group (e.g., ATP [adenosine 5'-triphosphate]-dependent nucleosome remodeling proteins). Higher-order chromatin structure is regulated in part by covalent modification of the N-terminal histone tails of chromatin, and histone tails in turn serve as platforms for recruitment of signaling modules that include nonhistone proteins such as heterochromatin protein (HP1) and NuRD. Because the enzymes governing chromatin structure through covalent modifications of histones (acetylation, methylation, phosphorylation, ubiquitination) can also target nonhistone substrates, a mechanism is in place by which epigenetic regulatory processes can affect the function of these alternate substrates. The posttranslational modification of histones, through phosphorylation and acetylation at specific residues, alters chromatin structure in an orchestrated manner in response to specific signals and is considered the basis of a "histone code." In an analogous manner, specific residues within transcription factors form a signaling module within the transcription factor to determine genetic target specificity and cellular fate. The architecture of these signaling cascades in transcription factors (SCITs) are poorly understood. The regulation of estrogen receptor (ERalpha) by enzymes that convey epigenetic signals is carefully orchestrated and is reviewed here.

Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. Herein, cyclin D1 deficiency increased mitochondrial size and activity that was rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1), which induces nuclear-encoded mitochondrial genes, was repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis and mitochondrial function.

Sirtuins (Sir2-related enzymes) are a recently discovered class of NAD(+)-dependent protein deacetylases that regulate gene expression in a variety of organisms by deacetylation of modified lysine residues on histones, transcription factors and other proteins. Conservation of sirtuin regulation of the insulin-insulin-like growth factor I signaling pathway has been observed for Caenorhabditis elegans and mammals, indicating an ancient role for sirtuins in the modulation of organism adaptations to nutritional intake. The human sirtuin SIRT1 regulates a number of transcription factors that modulate endocrine signaling, including peroxisome proliferator-activated receptor gamma, peroxisome proliferator-activated receptor gamma coactivator 1alpha, forkhead-box transcription factors and p53.

Cyclin D1 is overexpressed in human tumors, correlating with cellular metastasis, and is induced by activating Rho GTPases. Herein, cyclin D1-deficient mouse embryo fibroblasts (MEFs) exhibited increased adhesion and decreased motility compared with wild-type MEFs. Retroviral transduction of cyclin D1 reversed these phenotypes. Mutational analysis of cyclin D1 demonstrated that its effects on cellular adhesion and migration were independent of the pRb and p160 coactivator binding domains. Genomewide expression arrays identified a subset of genes regulated by cyclin D1, including Rho-activated kinase II (ROCKII) and thrombospondin 1 (TSP-1). cyclin D1(-/-) cells showed increased Rho GTP and ROCKII activity and signaling, with increased phosphorylation of LIM kinase, cofilin (Ser3), and myosin light chain 2 (Thr18/Ser19). Cyclin D1 repressed ROCKII and TSP-1 expression, and the migratory defect of cyclin D1(-/-) cells was reversed by ROCK inhibition or TSP-1 immunoneutralizing antibodies. cyclin E knockin to the cyclin D1(-/-) MEFs rescued the DNA synthesis defect of cyclin D1(-/-) MEFs but did not rescue either the migration defect or the abundance of ROCKII. Cyclin D1 promotes cellular motility through inhibiting ROCK signaling and repressing the metastasis suppressor TSP-1.

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein and promotes progression through the G1-S phase of the cell cycle. Amplification or overexpression of cyclin D1 plays pivotal roles in the development of a subset of human cancers including parathyroid adenoma, breast cancer, colon cancer, lymphoma, melanoma, and prostate cancer. Of the three D-type cyclins, each of which binds cyclin-dependent kinase (CDK), it is cyclin D1 overexpression that is predominantly associated with human tumorigenesis and cellular metastases. In recent years accumulating evidence suggests that in addition to its original description as a CDK-dependent regulator of the cell cycle, cyclin D1 also conveys cell cycle or CDK-independent functions. Cyclin D1 associates with, and regulates activity of, transcription factors, coactivators and corepressors that govern histone acetylation and chromatin remodeling proteins. The recent findings that cyclin D1 regulates cellular metabolism, fat cell differentiation and cellular migration have refocused attention on novel functions of cyclin D1 and their possible role in tumorigenesis. In this review, both the classic and novel functions of cyclin D1 are discussed with emphasis on the CDK-independent functions of cyclin D1.

Post-translational modification of chromatin histones governs a key mechanism of transcriptional regulation. Histone acetylation, together with methylation, phosphorylation, ubiquitylation, sumoylation, glycosylation, and ADP ribosylation, modulate the activity of many genes by modifying both core histones and non-histone transcription factors. Epigenetic protein modification plays an important role in multiple cellular processes including DNA repair, protein stability, nuclear translocation, protein-protein interactions, and in regulation of cellular proliferation, differentiation and apoptosis. Histone acetyltransferases modify histones, coactivators, nuclear transport proteins, structural proteins, cell cycle components and transcription factors including p53 and nuclear receptors. The estrogen, PPARgamma and androgen receptor are members of the nuclear receptor (NR) superfamily. The androgen receptor (AR) and estrogen receptor alpha (ERalpha) are directly acetylated by histone acetyltransferases at a motif that is conserved between species and other NR. Point mutations at the lysine residue within the acetylation motif of the AR and ERalpha have been identified in prostate cancer as well as in breast cancer tissue. Acetylation of the NR governs ligand sensitivity and hormone antagonist responses. The AR is acetylated by p300, P/CAF and TIP60 and acetylation of the AR regulates co-regulator recruitment and growth properties of the receptors in cultured cells and in vivo. AR acetylation mimic mutants convey reduced apoptosis and enhanced growth properties correlating with altered promoter specificity for cell-cycle target genes. Cell-cycle control proteins, including cyclins, in turn alter the access of transcription factors and nuclear receptors to the promoters of target genes.

The androgen receptor (AR) regulates ligand-dependent gene transcription upon binding specific DNA sequences. The AR conveys both trans-activation and trans-repression functions, which together contribute to prostate cellular growth, differentiation, and apoptosis. Like histone H3, the AR is post-translationally modified by both acetylation and phosphorylation. The histone acetyltransferase p300 transactivates the AR and directly acetylates the AR in vitro at a conserved motif. Point mutations of the AR acetylation motif that abrogate acetylation reduce trans-activation by p300 without affecting the trans-repression function of the AR. The current studies assessed the functional relationship between acetylation and phosphorylation of the AR. Herein trans-activation of the AR acetylation site mutants were enhanced by the p42/p44 MAPK pathway but were defective in regulation by protein kinase A (PKA) signaling. PKA inhibition augmented ARwt activity but not AR acetylation mutant gene reporter activity and association at an androgen response element in chromatin immunoprecipitation assays. Mutations of the lysine residues at the AR acetylation site reduced trichostatin A (TSA) responsiveness and ligand-induced phosphorylation of the AR. The AR acetylation site mutant formed ligand-induced phosphorylation-dependent isoforms with distinguishable characteristics from wild type AR as determined with two-dimensional electrophoresis. Conversely, point mutation of a subset of AR phosphorylation sites reduced trichostatin A responsiveness and trans-activation by histone acetyltransferases. Together these studies suggest that acetylation and phosphorylation of the AR are linked events and that the conserved AR lysine motif contributes to a select subset of pathways governing AR activity.

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16INK4a/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1−/− mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16INK4a expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was observed in tissues of mice mutant for the Ink4a/Arf locus. p16INK4a and p19ARF inhibited DNA synthesis in MCF7 cells. p16INK4a repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16INK4a occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19ARF repressed cyclin D1 through a novel distal cis-element at −1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

The Wnt/beta-catenin/Tcf and IkappaB/NF-kappaB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/beta-catenin signaling occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear beta-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IkappaB kinase complex (IKK) that phosphorylates IkappaB contains IKKalpha, IKKbeta, and IKKgamma. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/beta-catenin and IkappaB pathways in mitogenic signaling. Mitogenic induction of G(1)-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1(-/-) cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IkappaB/IKKalpha or beta-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKalpha. In IKKalpha(-/-) cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKalpha restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKalpha(-/-) cells, beta-catenin phosphorylation was decreased and purified IKKalpha was sufficient for phosphorylation of beta-catenin through its N-terminus in vitro. Because IKKalpha but not IKKbeta induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKalpha and IKKbeta may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.

The last decade has seen a substantial change in thinking about the role of acetylation in regulating diverse cellular processes. The correlation between histone acetylation and gene transcription has been known for many years. The cloning and biochemical characterization of the enzymes that regulate this post-translational modification has led to an understanding of the diverse role histone acetyltransferases (HATs) play in cellular function. Histone acetylases modify histones, transcription factors, co-activators, nuclear transport proteins, structural proteins and components of the cell cycle. This review focuses on the role of histone acetylases in coordinating hormone signaling and the cell cycle. Transition through the cell cycle is regulated by a family of protein kinase holoenzymes, the cyclin-dependent kinases (Cdks) and their heterodimeric cyclin partners. Recent studies have identified important cross-talk between the cell cycle regulatory apparatus and proteins regulating histone acetylation. The evidence for a dynamic interplay between components regulating the cell cycle and acetylation of target substrates provides an important new level of complexity in the mechanisms governing hormone signaling.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERalpha) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERalpha-regulated gene expression involves interactions with cointegrators (e.g. p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERalpha is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ERalpha at lysine residues within the ERalpha hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERalpha hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERalpha acetylation normally suppresses ligand sensitivity. These ERalpha lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERalpha acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

In addition to its documented role in the proteolytic processing of Notch-1 and the beta-amyloid precursor protein, presenilin 1 (PS1) associates with beta-catenin. In this study, we show that this interaction plays a critical role in regulating beta-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle. Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner. The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Deltacat restores Notch-1 proteolytic cleavage and Abeta generation in PS1-deficient cells, indicating that PS1 function in modulating beta-catenin levels can be separated from its roles in facilitating gamma-secretase cleavage of beta-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of beta-catenin in the absence of PS1, a phenotype that may account for the increased stability in PS1-deficient cells. Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

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.

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1) phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-kappaB (NF-kappaB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IkappaB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-kappaB and an ATF-2 binding site. Inhibiting NF-kappaB by overexpression of an NF-kappaB trans-dominant inhibitor (nonphosphorylatable IkappaBalpha) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-kappaB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of Rac(Leu-61) had comparable effects on NF-kappaB transcriptional activity and activation of the cyclin D1 promoter. The NF-kappaB factors Rel A (p65) and NF-kappaB(1) (p50) induced the cyclin D1 promoter, requiring both the NF-kappaB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-kappaB(1) to the cyclin D1 promoter NF-kappaB site. Activation of Rac1 in NIH 3T3 cells induces both NF-kappaB binding and activity and enhances expression of cyclin D1 through an NF-kappaB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-kappaB in cell cycle regulation through cyclin D1 and Rac1.

Cocoa-derived flavanols and procyanidins have been previously reported to exhibit anti-oxidant and anti-tumor properties. In this study, we have investigated the cellular growth inhibitory effect of chemically-synthesized procyanidin [3-O-galloyl]-(-)-epicatechin-(4beta,8)-(+)-catechin-3-O-gallate (GECGC) on a variety of human cancer cell lines. Among 16 human cancer cell lines tested, GECGC selectively inhibited proliferation of a subset of human cancer cell lines, especially those of short doubling time. In contrast, all 6 normal cell lines tested including human mammary epithelial cells and skin fibroblast were resistant to GECGC's cytotoxicity. Cell cycle analysis and apoptosis assay showed that GECGC increased sub-G(1) population and increased the population of propidium iodide and Annexin V staining cells in GECGC-sensitive cell lines, suggesting that cell growth inhibition by GECGC may be mediated through both apoptotic and non-apoptotic mechanisms. Further characterization of GECGC cytotoxicity on 30 genetically modified cell lines with overexpression or depletion of key proteins involved in cell cycle regulation and signal transduction pathways suggested that GECGC-mediated cell death involves IKKalpha and IKKgamma. Collectively, our observations indicate that synthesized GECGC has selective anti-proliferative effect on human cancer cells and warrant further evaluation as a preventive and chemotherapeutic reagent to human malignancies.

The endocrine signaling governing nuclear receptor (NR) function has been known for several decades to play a crucial role in the onset and progression of several tumor types. Notably among these are the estrogen receptor (ER) in breast cancer and androgen receptor (AR) in prostate cancer. Other nuclear receptors may be involved in cancer progression including the peroxisome-proliferator activating receptor gamma (PPARγ), which has been implicated in breast, thyroid, and colon cancers. These NR are phylogenetically conserved modular transcriptional regulators, which like histones, undergo post-translational modification by acetylation, phosphorylation and ubiquitination. Importantly, the transcriptional activity of the receptors is governed by the coactivator p300, the activity of which is thought to be rate-limiting in the activity of these receptors. Histone acetyltransferases (HATs) and histone deacetylases (HDACs), modify histones by adding or removing an acetyl group from the ɛ amino group of lysines within an evolutionarily conserved lysine motif. Histone acetylation results in changes in chromatin structure in response to specific signals. These enzymes can also directly catalyze the NRs themselves, thus modifying signals at the receptor level. The post-translational modification of NR which is regulated by hormones, alters the NR function toward a growth promoting receptor. The deacetylation of NR is mediated by TSA-sensitive and NAD-dependent deacetylases. The regulation of NR by NAD-dependent enzymes provides a direct link between intracellular metabolism and hormone signaling.

Histone modifier proteins have come to the forefront in the study of gene regulation. It is now known that histone methyltransferases, acetytransferases, kinases, ubiquitinases, deacetylases and demethylases orchestrate expression of target genes by modifying both histone and non-histone proteins. The nuclear receptor (NR) superfamily govern such diverse biological processes as development, physiology and disease, including human cancer. The involvement of NR in complexes with coactivators and corepressors is necessary for regulation of target genes. This review focuses on the newly recognized interactions between the NR and histone modifying enzymes. In addition to regulating histones, the histone modifying proteins directly modify and thereby regulate NR activity. In the same manner that signaling platforms exist within the histone tails that are post-translationally processed by histone modifying proteins, cascades of post-translational modification have been identified within the NR that coordinate their activity. This review focuses on the regulation of the NR estrogen receptor (ERalpha), androgen receptor (AR) and peroxisome proliferator activated receptor-gamma (PPARgamma), given their role in tumor onset and progression.

The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor α (ERα)–dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERα binding. Cyclin D1 and BRCA1 were both capable of binding ERα in a common region of the ERα hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERα and for rescue of BRCA1-mediated ERα transcriptional repression. In chromatin immunoprecipitation assays, 17β-estradiol (E2) enhanced ERα and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERα recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERα recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERα recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERα repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.

The cyclin D1 gene encodes the labile serum-inducible regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. Overexpression of cyclin D1 promotes cellular proliferation and normal physiological levels of cyclin D1 function to inhibit adipocyte differentiation in vivo. We have previously shown that cyclin D1 inhibits peroxisome proliferator-activated receptor (PPAR)gamma-dependent activity through a cyclin-dependent kinase- and retinoblastoma protein-binding-independent mechanism. In this study, we determined the molecular mechanism by which cyclin D1 regulated PPARgamma function. Herein, murine embryonic fibroblast (MEF) differentiation by PPARgamma ligand was associated with a reduction in histone deacetylase (HDAC1) activity. Cyclin D1-/- MEFs showed an increased propensity to undergo differentiation into adipocytes. Genetic deletion of cyclin D1 reduced HDAC1 activity. Reconstitution of cyclin D1 into the cyclin D1-/- MEFs increased HDAC1 activity and blocked PPARgamma-mediated adipogenesis. PPARgamma activity was enhanced in cyclin D1-/- cells. Reintroduction of cyclin D1 inhibited basal and ligand-induced PPARgamma activity and enhanced HDAC repression of PPARgamma activity. Cyclin D1 bound HDAC in vivo and preferentially physically associated with HDAC1, HDAC2, HDAC3, and HDAC5. Chromatin immunoprecipitation assay demonstrated that cyclin D1 enhanced recruitment of HDAC1 and HDAC3 and histone methyltransferase SUV39H1 to the PPAR response element of the lipoprotein lipase promoter and decreased acetylation of total histone H3 and histone H3 lysine 9. Collectively, these studies suggest an important role of cyclin D1 in regulation of PPARgamma-mediated adipocyte differentiation through recruitment of HDACs to regulate PPAR response element local chromatin structure and PPARgamma function.

The SIR2 family of nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases modulates diverse biological functions in different species, including longevity, apoptosis, cell cycle exit, and cellular differentiation. SIRT1, the closest mammalian ortholog of the yeast SIR2 (silent information regulator 2) gene, represses several transcription factors, including p53, NFkappaB and forkhead proteins. The p300 protein serves as a rate-limiting transcriptional cointegrator of diverse transcription factors either to activate or to repress transcription through modular subdomains. Herein, SIRT1 physically interacted with and repressed p300 transactivation, requiring the NAD-dependent deacetylase activity of SIRT1. SIRT1 repression involved the CRD1 transcriptional repression domain of p300. Two residues within the CRD1 domain (Lys-1020 and Lys-1024) were required for SIRT1 repression and served as substrates for SIRT1 deacetylation. These residues also serve as acceptor lysines for modification by the ubiquitin-like SUMO protein. The SUMO-specific protease SSP3 relieved SIRT1 repression of p300. SSP3 antagonism of SIRT1 required the SUMO-deconjugating function of SSP3. Thus, p300 serves as a deacetylase substrate for SIRT1 through a conserved SUMO consensus motif. Because p300 is a limiting transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve an important integration point during metabolism and cellular differentiation.

Cyclin D1 encodes a regulatory subunit, which with its cyclin-dependent kinase (Cdk)-binding partner forms a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. In addition to its Cdk binding-dependent functions, cyclin D1 regulates cellular differentiation in part by modifying several transcription factors and nuclear receptors. The molecular mechanism through which cyclin D1 regulates the function of transcription factors involved in cellular differentiation remains to be clarified. The histone acetyltransferase protein p300 is a co-integrator required for regulation of multiple transcription factors. Here we show that cyclin D1 physically interacts with p300 and represses p300 transactivation. We demonstrated further that the interaction of the two proteins occurs at the peroxisome proliferator-activated receptor γ-responsive element of the lipoprotein lipase promoter in the context of the local chromatin structure. We have mapped the domains in p300 and cyclin D1 involved in this interaction. The bromo domain and cysteine- and histidine-rich domains of p300 were required for repression by cyclin D1. Cyclin D1 repression of p300 was independent of the Cdk- and retinoblastoma protein-binding domains of cyclin D1. Cyclin D1 inhibits histone acetyltransferase activity of p300 in vitro. Microarray analysis identified a signature of genes repressed by cyclin D1 and induced by p300 that promotes cellular differentiation and induces cell cycle arrest. Together, our results suggest that cyclin D1 plays an important role in cellular proliferation and differentiation through regulation of p300. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported in part by Grants R01CA70896, R01CA75503, R01CA86072, R01CA93596, R01CA107382 from the National Institutes of Health (to R. G. P.) and NIDDK 1R21DK065220-02 from the National Institutes of Health (to M. F.).

Signal transduction pathways mediate cell-cell interactions and integrate signals from the extracellular environment through specific receptors at the cell membrane. They play a pivotal role in regulating cellular growth and differentiation and in mediating many physiological and pathological processes, such as apoptosis, inflammation, and tumor development. The mitogen- activated protein kinases (MAPKs) constitute a cascade of phosphorylation events that transmit extracellular growth signals through membrane-bound Ras to the nucleus of the cell. In this chapter, detailed protocols for analyzing the kinase activities of the key components of the MAPKs pathway MEK1, ERK1, JNK, and p38 MAPK are described. A brief introduction to the chemical inhibitors to the MAPKs pathway is provided in the method section of each kinase assay. Inhibitors of other signaling pathways are summarized in Table 1. The reporter assay of cyclin D1, a key downstream target gene of MAPKs pathway, is also described in detail.

Signal transduction pathways mediate cell-cell interactions and integrate signals from the extracellular environment through specific receptors at the cell membrane. They play a pivotal role in regulating cellular growth and differentiation and in mediating many physiological and pathological processes, such as apoptosis, inflammation, and tumor development. The mitogen- activated protein kinases (MAPKs) constitute a cascade of phosphorylation events that transmit extracellular growth signals through membrane-bound Ras to the nucleus of the cell. In this chapter, detailed protocols for analyzing the kinase activities of the key components of the MAPKs pathway MEK1, ERK1, JNK, and p38 MAPK are described. A brief introduction to the chemical inhibitors to the MAPKs pathway is provided in the method section of each kinase assay. Inhibitors of other signaling pathways are summarized in Table 1. The reporter assay of cyclin D1, a key downstream target gene of MAPKs pathway, is also described in detail.

Cyclin D1 integrates extracellular signals by coupling signals from cell surface receptors to transcription factors, thereby regulating diverse gene expression networks. Cyclin D1 has well defined roles in cell cycle progression that are aberrantly activated in many cancers, thus, it is an appropriate pathway to target for therapeutic intervention. This review will outline the basic regulatory machinery responsible for cell cycle control and describe the latest advances made in the field. Strategies for targeting cyclin D1 and cyclin D1-mediated signaling pathways as means of developing novel and perhaps more effective anticancer treatments will be discussed. Examples of novel cell cycle-targeting molecules that are currently being tested in clinical trials will be discussed as well.

Modification by acetylation occurs at -amino lysine residues of histones and transcription factors. Unlike phosphorylation, a direct link between transcription factor acetylation and cellular growth or apoptosis has not been established. We show that the nuclear androgen receptor (AR), a DNA-binding transcriptional regulator, is acetylated in vivo. The acetylation of the AR is induced by ligand dihydrotestosterone and by histone deacetylase (HDAC) inhibitors in living cells. Direct AR acetylation augmented p300 binding in vitro. Constructs mimicking neutral polar substitution acetylation (ARK630Q ,A R K630T) enhanced p300 binding and reduced N-CoR/HDAC/Smad3 corepressor binding, whereas charged residue substitution (ARK630R) reduced p300 binding and enhanced corepressor binding. The AR acetylation mimics promoted cell survival and growth of prostate cancer cells in soft agar and in nude mice and augmented transcription of a subset of growth control target gene promoters. Thus, transcription factor acetylation regulates coactivator/corepressor complex binding, altering expression of specific growth control genes to promote aberrant cellular growth in vivo. Prostate cancer is the second leading cause of cancer death in American males. Although potentially curable by radical prostatectomy or radiation therapy, metastatic disease is common at presentation and may occur subsequently in patients treated with curative intent. The androgen receptor (AR) is a classical nuclear receptor (NR) that binds testosterone and is required for the induction of male secondary sexual characteristics. The AR conveys several dissociable functions, including transactivation, transrepression, growth regulation, basal activity, and context-dependent cell survival or apoptosis functions. Aberrant AR function plays an important role in prostate cancer (1). The wild-type AR can induce cellular differentiation or cellular apoptosis in prostate cancer cells (2, 16, 47). Both AR-dependent and AR-independent mechanisms contribute to prostate cancer cellular growth. Somatic missense AR gene mutations have been detected in prostate cancer cell lines, xenografts, and primary and metastatic forms of prostate cancer (39–41).

Heritable and reversible changes in gene expression can occur without alterations in DNA sequence largely dependent upon the position of a gene within an accessible (euchromatic) chromatin environment. This position effect variegation in Drosophila and S. pombe, and higher order chromatin structure regulation in yeast, is orchestrated by modifier genes of the Su(var) group (e.g. histone deacetylases (HDACs), protein phosphatases) and enhancer E(var) group (e.g. ATP-dependent nucleosome remodeling proteins). Higher order chromatin structure is regulated in part by covalent modification of the N-terminal histone tails of chromatin and histone tails in turn serve as platforms for recruitment of signaling modules that include non-histone proteins such as HP1 and NuRD. As the enzymes governing chromatin structure through covalent modifications of histones (acetylation, methylation, phosphorylation, ubiquitination) can also target non-histone substrates, a mechanism is in place by which epigenetic regulatory processes can affect the function of these alternate substrates. The nuclear receptor (NR) superfamily consists of conserved modular transcriptional regulators. Herein, we review the functional properties of nuclear receptors regulated by their direct acetylation including ligand-dependent activation, cellular growth and apoptosis.

Most human tumors display inactivation of the p53 and the p16(INK4)/pRb pathway. The Ink4a/alternative reading frame (ARF) locus encodes the p16(INK4a) and p14(ARF) (murine p19(ARF)) proteins. p16(INK4a) is deleted in 40-60% of breast cancer cell lines, and p16(INK4a) inactivation by DNA methylation occurs inor =30% of human breast cancers. In mice genetically heterozygous for p16(INK4a) or Ink4a/Arf, predisposition to specific tumor types is enhanced. Ink4a/Arf(+/-) mice have increased E micro -Myc-induced lymphomagenesis and epidermal growth factor receptor-induced gliomagenesis. ErbB2 (epidermal growth factor receptor-related protein B2) is frequently overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in vivo. We determined the role of heterozygosity at the Ink4a/Arf locus in ErbB2-induced mammary tumorigenesis. Compared with mouse mammary tumor virus-ErbB2 Ink4a/Arf(+/-) mice, mouse mammary tumor virus-ErbB2 Ink4a/Arf(wt) mammary tumors showed increased p16(INK4a), reduced Ki-67 expression, and reduced cyclin D1 protein but increased mammary tumor apoptosis with no significant change in the risk of developing mammary tumors. These studies demonstrate the contribution of Ink4a/Arf heterozygosity to tumor progression is tissue specific in vivo. In view of the important role of Ink4a/Arf in response to chemotherapy, these transgenic mice may provide a useful model for testing breast tumor therapies.

This work was supported by grants from the NIH (R01CA86072 to R.G.P. and R01CA72038-01 to S.A.W.F.) and The Susan Komen Breast Cancer Foundation (to R.G.P.). R.T.H. and E.J. were supported by the Medical Research Council. Y.-G.Y. is supported by grant CA26504 to E. R. Stanley. Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institutes of Health grant 5-P30-CA13330-26. The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both traits repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-kappaS, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation. Publisher PDF

The cyclins are a family of proteins that are centrally involved in cell cycle regulation and which are structurally identified by conserved "cyclin box" regions. They are regulatory subunits of holoenzyme cyclin-dependent kinase (CDK) complexes controlling progression through cell cycle checkpoints by phosphorylating and inactivating target substrates. CDK activity is controlled by cyclin abundance and subcellular location and by the activity of two families of inhibitors, the cyclin-dependent kinase inhibitors (CKI). Many hormones and growth factors influence cell growth through signal transduction pathways that modify the activity of the cyclins. Dysregulated cyclin activity in transformed cells contributes to accelerated cell cycle progression and may arise because of dysregulated activity in pathways that control the abundance of a cyclin or because of loss-of-function mutations in inhibitory proteins.Analysis of transformed cells and cells undergoing mitogen-stimulated growth implicate proteins of the NF-kappaB family in cell cycle regulation, through actions on the CDK/CKI system. The mammalian members of this family are Rel-A (p65), NF-kappaB(1) (p50; p105), NF-kappaB(2) (p52; p100), c-Rel and Rel-B. These proteins are structurally identified by an amino-terminal region of about 300 amino acids, known as the Rel-homology domain. They exist in cytoplasmic complexes with inhibitory proteins of the IkappaB family, and translocate to the nucleus to act as transcription factors when activated. NF-kappaB pathway activation occurs during transformation induced by a number of classical oncogenes, including Bcr/Abl, Ras and Rac, and is necessary for full transforming potential. The avian viral oncogene, v-Rel is an NF-kappaB protein. The best explored link between NF-kappaB activation and cell cycle progression involves cyclin D(1), a cyclin which is expressed relatively early in the cell cycle and which is crucial to commitment to DNA synthesis. This review examines the interactions between NF-kappaB signaling and the CDK/CKI system in cell cycle progression in normal and transformed cells. The growth-promoting actions of NF-kappaB factors are accompanied, in some instances, by inhibition of cellular differentiation and by inhibition of programmed cell death, which involve related response pathways and which contribute to the overall increase in mass of undifferentiated tissue.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.

A number of distinct surveillance systems are found in mammalian cells that have the capacity to interrupt normal cell-cycle progression. These are referred to as cell cycle check points. Surveillance systems activated by DNA damage act at three stages, one at the G1/S phase boundary, one that monitors progression through S phase and one at the G2/M boundary. The initiation of DNA synthesis and irrevocable progression through G1 phase represents an additional checkpoint when the cell commits to DNA synthesis. Transition through the cell cycle is regulated by a family of protein kinase holoenzymes, the cyclin-dependent kinases (Cdks), and their heterodimeric cyclin partner. Orderly progression through the cell-cycle checkpoints involves coordinated activation of the Cdks that, in the presence of an associated Cdk-activating kinase (CAK), phosphorylate target substrates including members of the "pocket protein" family. One of these, the product of the retinoblastoma susceptibility gene (the pRB protein), is phosphorylated sequentially by both cyclin D/Cdk4 complexes and cyclin E/Cdk2 kinases. Recent studies have identified important cross talk between the cell-cycle regulatory apparatus and proteins regulating histone acetylation. pRB binds both E2F proteins and histone deacetylase (HDAC) complexes. HDAC plays an important role in pRB tumor suppression function and transcriptional repression. Histones are required for accurate assembly of chromatin and the induction of histone gene expression is tightly coordinated. Recent studies have identified an important alternate substrate of cyclin E/Cdk2, NPAT (nuclear protein mapped to the ATM locus) which plays a critical role in promoting cell-cycle progression in the absence of pRB, and contributes to cell-cycle regulated histone gene expression. The acetylation of histones by a number of histone acetyl transferases (HATs) also plays an important role in coordinating gene expression and cell-cycle progression. Components of the cell-cycle regulatory apparatus are both regulated by HATs and bind directly to HATs. Finally transcription factors have been identified as substrate for HATs. Mutations of these transcription factors at their sites of acetylation has been associated with constitutive activity and enhanced cellular proliferation, suggesting an important role for acetylation in transcriptional repression as well as activation. Together these studies provide a working model in which the cell-cycle regulatory kinases phosphorylate and inactivate HDACs, coordinate histone gene expression and bind to histone acetylases themselves. The recent evidence for cross-talk between the cyclin-dependent kinases and histone gene expression on the one hand and cyclin-dependent regulation of histone acetylases on the other, suggests chemotherapeutics targeting histone acetylation may have complex and possibly complementary effects with agents targeting Cdks.

Publisher Summary This chapter focuses on the Ras regulation of Cyclin D 1 promoter. Cyclin D l, the regulatory subunit of cyclin-dependent kinases (CDK) 4 and 6, is required for, and capable of, shortening, the G j phase of the cell cycle through the formation of holoenzyme complexes (cyclin-CDK) that phosphorylate retinoblastoma protein (pRB). Ras transformation is inhibited by antisense to cyclin D1 mRNA. The Ras-related proteins, Rac and Rho, also induce the cyclin D1 promoter. Racl regulates several distinct pathways; no single Rac effect is necessary or sufficient for transformation. Platelet-derived growth factor (PDGF) activates the extracellular signal-regulated kinase (ERK) pathway, which has been shown to induce DNA synthesis, increase cyclin D1 protein levels, and stimulate the transcription of the cyclin D1 promoter. Therefore, Rac 1 is neither necessary nor sufficient to activate the ERK pathway, suggesting that Rac regulation of cyclin D1 is ERK independent. The cyclin DI promoter is used as a molecular probe of the signal transduction pathways involved in Ras signaling. The valid assessment of promoter activation by Ras requires the use of a reporter system that is not itself responsive to components of the Ras signaling pathway.

Ral proteins are small GTPases that have been implicated in the control of cell proliferation and Ras-mediated oncogenic transformation (14, 48). The two known Ral isoforms, RalA and RalB, are 85% identical and comprise a distinct family within the Ras superfamily of GTPases (10). Ral proteins are more than 50% identical to Ras, have overall structural features similar to those of Ras, but do not share any known effector or regulatory proteins with Ras (6, 14). Like Ras GTPases, Ral proteins become biologically active upon exchange of bound GDP for GTP. This exchange is catalyzed in vivo by Ral-specific guanine nucleotide exchange factors (RalGEFs) (14). Several RalGEFs which contain carboxy-terminal Ras binding domains have been identified (14, 42). The observations that activated Ras can associate directly with RalGEFs (14) and activate the enzymatic activity of RalGEFs in vitro and in transfected cells (14, 55, 62) and that mitogen-dependent activation of Ral proteins requires Ras activation (63) have lead to the hypothesis that RalGEFs are Ras effector proteins. Consistent with this hypothesis is the observation that activation of Ral proteins appears to be required for Ras-induced oncogenic growth and morphological transformation (50, 55, 60) and induction of DNA synthesis (38). In addition, expression of RalGEFs or activated Ral proteins can cooperate with activation of other Ras effector cascades to transform cells (50, 55, 60). These observations suggest that Ral proteins may be important mediators of Ras-induced proliferative signals. However, the mechanism by which Ral may contribute to Ras signaling is unknown. In addition to effects on proliferation, Ral has been directly implicated in receptor-mediated endocytosis (40), Src kinase activation (20), phospholipase D1 (PLD1) activation (16, 23), and regulation of the actin cytoskeleton (42). Active PLD1 (23, 36), Ral-binding protein 1 (RalBP1) (9, 25, 44), and filamin (42) have been identified as Ral-interacting proteins and may function as Ral effectors. PLD1 is constitutively associated with Ral protein in cells (23). However, activation of Ral cooperates with ADP ribosylation factor GTPases to activate PLD1, perhaps by contributing to the formation of a PLD1 activation complex (28, 35). There is some evidence that active PLD1 can contribute to proliferation (13). For example, transfected PLD1 can contribute to oncogenic transformation of fibroblasts overexpressing epidermal growth factor (EGF) receptors (34). Unlike PLD1, RalBP1 associates with Ral in a GTP-dependent manner (9, 25, 44). The functional significance of a Ral-RalBP1 interaction is unknown; however, RalBP1 contains a GTPase-activating protein (GAP) domain that has activity toward Cdc42 and Rac GTPases (9, 25, 44). This observation has led to the hypothesis that Ral may negatively regulate the activity of these GTPases. In support of this, studies using PC12 cells suggest that RalGEFs can interfere with neurite differentiation in a Rac-dependent fashion (19). However, a direct effect of Ral on Rac or Cdc42 regulation has not yet been demonstrated. Finally, the Ral-filamin interaction may influence regulation of the actin cytoskeleton. Filamin binds Ral-GTP, and Ral-GTP will induce filopodia in human melanoma cells in a filamin-dependent fashion. In contrast to a potential role of Ral upstream of Rac/Cdc42 via RalBP1, Ral-mediated generation of filopodia is likely downstream of Cdc42 activation (42). To further elaborate the mechanism by which Ral GTPases may contribute to proliferation and transformation, we have examined the consequences of Ral activation on gene induction events that have been defined as critical convergence points for multiple mitogenic signaling cascades. We show here that activated Ral is sufficient to induce NF-κB transcription factor activity and accumulation of cyclin D1 protein. Ral activation of cyclin D1 expression is NF-κB dependent and is mediated by NF-κB binding sites in the cyclin D1 promoter. Activation of NF-κB and cyclin D1 expression by Ral is independent of Ral association with either PLD1 or RalBP1 and likely proceeds through a novel effector pathway. Ral-dependent regulation of NF-κB and cyclin D1 provides a mechanistic explanation for the positive role of Ral proteins in the regulation of proliferation and oncogenic transformation.

It recently was reported that Duchenne muscular dystrophy (DMD) patients and mdx mice have elevated levels of caveolin-3 expression in their skeletal muscle. However, it remains unknown whether increased caveolin-3 levels in DMD patients contribute to the pathogenesis of DMD. Here, using a genetic approach, we test this hypothesis directly by overexpressing wild-type caveolin-3 as a transgene in mice. Analysis of skeletal muscle tissue from caveolin-3- overexpressing transgenic mice reveals: ( i ) a dramatic increase in the number of sarcolemmal muscle cell caveolae; ( ii ) a preponderance of hypertrophic, necrotic, and immature/regenerating skeletal muscle fibers with characteristic central nuclei; and ( iii ) down-regulation of dystrophin and β-dystroglycan protein expression. In addition, these mice show elevated serum creatine kinase levels, consistent with the myo-necrosis observed morphologically. The Duchenne-like phenotype of caveolin-3 transgenic mice will provide an important mouse model for understanding the pathogenesis of DMD in humans.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.

The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease.

The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics. In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxyl-terminal to the zinc finger DNA-binding domain. [(14)C]acetate-labeling experiments demonstrated that AR acetylation by p300 in cultured cells requires the same residues identified in vitro. Point mutation of the AR acetylation site (K632A/K633A) abrogated dihydrotestosterone-dependent transactivation of the AR in cultured cells. Mutation of the p300 CH3 region or the p300/cAMP-response element-binding protein histone acetylase domain reduced ligand-dependent AR function. The identification of the AR as a direct target of histone acetyltransferase co-activators has important implications for targeting inhibitors of AR function.

Prostate cancer is the most common cause of non-cutaneous cancer in men and although frequently latent is the second commonest cause of death. Screening for the disease was historically based on symptoms of urethral obstruction, clinical examination of the prostate gland and serum measurements of prostate specific antigen. As prostate cancer growth in the early stages is enhanced by androgens, the mainstay of therapy has been androgen ablation by pharmaco-therapeutic or surgical means. The subsequent development of androgen therapy resistant prostate cancer in many patients, for whom therapeutic options remain limited, has led researchers to focus attention on understanding the molecular genetics of prostate cancer. The array of genetic abnormalities observed in prostate tumors, which include changes in components of the cell cycle, suggest the disease is quite heterogeneous and may require further sub-classification based on genetic markers. Such analyses may lead to identification of relevant new prognostic and therapeutic indicators. The advent of transgenic mouse models of prostate cancer may provide a critical tool for the implementation of rational genetic based therapeutics and alternate drug design.

The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras, neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between -81 to -73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1.