Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer.

BACKGROUND: The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC). METHODS: Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses. RESULTS: Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis. CONCLUSION: Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

The mammary gland iodide transporter is expressed during lactation and in breast cancer.

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.

BRCA1 inhibition of estrogen receptor signaling in transfected cells.

Mutations of the breast cancer susceptibility gene BRCA1 confer increased risk for breast, ovarian, and prostatic cancers, but it is not clear why the mutations are associated with these particular tumor types. In transient transfection assays, BRCA1 was found to inhibit signaling by the ligand-activated estrogen receptor (ER-alpha) through the estrogen-responsive enhancer element and to block the transcriptional activation function AF-2 of ER-alpha. These results raise the possibility that wild-type BRCA1 suppresses estrogen-dependent transcriptional pathways related to mammary epithelial cell proliferation and that loss of this ability contributes to tumorigenesis.

Activation of p300 histone acetyltransferase activity and acetylation of the androgen receptor by bombesin in prostate cancer cells.

Androgen receptor signaling in prostate cancer cells is augmented by the androgen receptor (AR) coactivator p300, which transactivates and acetylates the AR in the presence of dihydrotestosterone (DHT). As prostate cancer (PC) cells progress to androgen independence, AR signaling remains intact, indicating that other factors stimulate AR activities in the absence of androgen. We previously reported that neuropeptide growth factors could transactivate the AR in the presence of very low concentrations of DHT. Here, we examine the involvement of p300 in neuropeptide activation of AR signaling. Transfection of increasing concentrations of p300 in the presence of bombesin into PC-3 cells resulted in a linear increase in AR transactivation, suggesting that p300 acts as a coactivator in neuropeptide-mediated AR transactivation. P300 is endowed with histone acetyltransferase (HAT) activity. Therefore, we examine the effect of bombesin on p300 HAT activity. At 4 h after the addition of bombesin, p300 HAT activity increased 2.0-fold (P<0.01). Incubation with neutral endopeptidase, which degrades bombesin, or bombesin receptor antagonists blocked bombesin-induced p300 HAT activity. To explore the potential signaling pathways involved in bombesin-induced p300 HAT activity, we examined Src and PKCdelta pathways that mediate bombesin signaling. Inhibitors of Src kinase activity or Src kinase siRNA blocked bombesin-induced p300 HAT activity, whereas PKCdelta inhibitors or PKCdelta siRNA significantly increased bombesin-induced p300 HAT activity suggesting that Src kinase and PKCdelta kinase are involved in the regulation of p300 HAT activity. As AR is acetylated in the presence of 100 nM DHT, we next examined whether bombesin-induced p300 HAT activity would result in enhanced AR acetylation. Bombesin-induced AR acetylation at the same motif KLKK observed in DHT-induced acetylation. Elimination of p300 using p300 siRNA reduced AR acetylation, demonstrating that AR acetylation was mediated by p300. AR acetylation results in AR transactivation and the expression of the AR-regulated gene prostate-specific antigen (PSA). Therefore, we examined bombesin-induced AR transactivation and PSA expression in the presence and absence of p300 siRNA and found inhibition of p300 expression reduced bombesin-induced AR transactivation and PSA expression. Together these results demonstrate that bombesin, via Src and PKCdelta signaling pathways, activates p300 HAT activity which leads to enhanced acetylation of AR resulting in increased expression of AR-regulated genes.

NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1.

Accumulating evidence implicates the transcription factor NF-kappaB as a positive mediator of cell growth, but the molecular mechanism(s) involved in this process remains largely unknown. Here we use both a skeletal muscle differentiation model and normal diploid fibroblasts to gain insight into how NF-kappaB regulates cell growth and differentiation. Results obtained with the C2C12 myoblast cell line demonstrate that NF-kappaB functions as an inhibitor of myogenic differentiation. Myoblasts generated to lack NF-kappaB activity displayed defects in cellular proliferation and cell cycle exit upon differentiation. An analysis of cell cycle markers revealed that NF-kappaB activates cyclin D1 expression, and the results showed that this regulatory pathway is one mechanism by which NF-kappaB inhibits myogenesis. NF-kappaB regulation of cyclin D1 occurs at the transcriptional level and is mediated by direct binding of NF-kappaB to multiple sites in the cyclin D1 promoter. Using diploid fibroblasts, we demonstrate that NF-kappaB is required to induce cyclin D1 expression and pRb hyperphosphorylation and promote G(1)-to-S progression. Consistent with results obtained with the C2C12 differentiation model, we show that NF-kappaB also promotes cell growth in embryonic fibroblasts, correlating with its regulation of cyclin D1. These data therefore identify cyclin D1 as an important transcriptional target of NF-kappaB and reveal a mechanism to explain how NF-kappaB is involved in the early phases of the cell cycle to regulate cell growth and differentiation.

Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1.

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.

Ral GTPases contribute to regulation of cyclin D1 through activation of NF-kappaB.

Ral GTPases have been implicated as mediators of Ras-induced signal transduction from observations that Ral-specific guanine nucleotide exchange factors associate with Ras and are activated by Ras. The cellular role of Ral family proteins is unclear, as is the contribution that Ral may make to Ras-dependent signaling. Here we show that expression of activated Ral in quiescent rodent fibroblasts is sufficient to induce activation of NF-kappaB-dependent gene expression and cyclin D1 transcription, two key convergence points for mitogenic and survival signaling. The regulation of cyclin D1 transcription by Ral is dependent on NF-kappaB activation and is mediated through an NF-kappaB binding site in the cyclin D1 promoter. Ral activation of these responses is likely through an as yet uncharacterized effector pathway, as we find activation of NF-kappaB and the cyclin D1 promoter by Ral is independent of association of Ral with active phospholipase D1 or Ral-binding protein 1, two proteins proposed to mediate Ral function in cells.

Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.

Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.

Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.

Fos family members induce cell cycle entry by activating cyclin D1.

Expression of the fos family of transcription factors is stimulated by growth factors that induce quiescent cells to reenter the cell cycle, but the cellular targets of the Fos family that regulate cell cycle reentry have not been identified. To address this issue, mice that lack two members of the fos family, c-fos and fosB, were derived. The fosB-/- c-fos-/- mice are similar in phenotype to c-fos-/- mice but are 30% smaller. This decrease in size is consistent with an abnormality in cell proliferation. Fibroblasts derived from fosB-/- c-fos-/- mice were found to have a defect in proliferation that results at least in part from a failure to induce cyclin D1 following serum-stimulated cell cycle reentry. Although definitive evidence that c-Fos and FosB directly induce cyclin D1 transcription will require further analysis, these findings raise the possibility that c-Fos and FosB are either direct or indirect transcriptional regulators of the cyclin D1 gene and may function as a critical link between serum stimulation and cell cycle progression.

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1.

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.

Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway.

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.

NF-kappaB and cell-cycle regulation: the cyclin connection.

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.

TGFbeta and PTHrP control chondrocyte proliferation by activating cyclin D1 expression.

Exact coordination of growth plate chondrocyte proliferation is necessary for normal endochondral bone development and growth. Here we show that PTHrP and TGFbeta control chondrocyte cell cycle progression and proliferation by stimulating signaling pathways that activate transcription from the cyclin D1 promoter. The TGFbeta pathway activates the transcription factor ATF-2, whereas PTHrP uses the related transcription factor CREB, to stimulate cyclin D1 promoter activity via the CRE promoter element. Inhibition of cyclin D1 expression with antisense oligonucleotides causes a delay in progression of chondrocytes through the G1 phase of the cell cycle, reduced E2F activity, and decreased proliferation. Growth plates from cyclin D1-deficient mice display a smaller zone of proliferating chondrocytes, confirming the requirement for cyclin D1 in chondrocyte proliferation in vivo. These data identify the cyclin D1 gene as an essential component of chondrocyte proliferation as well as a fundamental target gene of TGFbeta and PTHrP during skeletal growth.

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Gut-enriched Krüppel-like factor represses cyclin D1 promoter activity through Sp1 motif.

Cancer cells differ from normal cells in many characteristics including loss of differentiation and uninhibited cell proliferation. Recent studies have focused on the identification of factors contributing to cell growth and differentiation. Gut-enriched Krüppel-like factor (GKLF or KLF4) is a newly identified eukaryotic transcription factor and has been shown to play a role in regulating growth arrest. We have previously shown that GKLF mRNA levels were significantly decreased in colon cancer tissues, and that over-expression of GKLF in colonic adenocarcinoma cells (HT-29) resulted in reduction of cyclin D1 (CD1) mRNA and protein levels. The current study was undertaken to determine the mechanisms by which GKLF inhibited CD1 expression. In a transient transfection system, GKLF suppressed CD1 promoter activity by 55%. Sequential deletion and site-directed mutation analysis of the CD1 promoter have identified the sequence between -141 and -66, a region containing an Sp1 response element, to be essential for GKLF function. By electrophoretic mobility gel shift assay, recombinant GKLF and nuclear extracts from HT-29 cells were found to bind to the Sp1 motif on the CD1 promoter. The inhibitory effect of GKLF on the CD1 promoter activity was completely abolished by excessive amount of Sp1 DNA and GKLF significantly reduced the stimulatory function of Sp1 suggesting that GKLF and Sp1 may compete for the same binding site on the CD1 promoter. These results indicate that GKLF is a transcriptional repressor of the CD1 gene and that the inhibitory effect of GKLF is, in part, mediated by interaction with the Sp1 binding domain on its promoter.

Transforming growth factor-beta1 recruits histone deacetylase 1 to a p130 repressor complex in transgenic mice in vivo.

Transforming growth factor (TGF)-beta1 functions as a tumor suppressor in vivo. Using transgenic mice, we show that hepatic TGF-beta1 overexpression inhibits abundance of the cyclin-dependent kinase activating tyrosine phosphatase cdc25A protein. The reduction in cdc25A protein levels was associated with increased binding of histone deacetylase 1 to p130 in the hepatic extracts. In cultured cells, HDAC1/p130 overexpression inhibited activity of the cdc25A promoter through an E2F site. TGF-beta1 treatment enhanced p130 binding to the cdc25A promoter E2F site assessed in chromatin immunoprecipitation assays. Hepatic proliferation induced by partial hepatectomy was associated with a decrease in the amount of HDAC1 bound to p130, without a significant decrease in p130 abundance, suggesting that HDAC1 binding to p130 may be regulated by proliferative stimuli. The induction of cdc25A abundance induced by partial hepatectomy correlated with the induction of DNA synthesis. These studies suggest that TGF-beta1 may enhance HDAC1 binding to p130 in vivo, thereby inhibiting cdc25A gene expression. TGF-beta1 regulation of HDAC1/pocket protein associations may provide a link between chromatin remodeling proteins and cdk inhibition through induction of cdc25A in vivo.

A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells.

Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), a novel 3-substituted indolinone compound, binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb, an increase in caspase-3 activation (5-84%), and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents.

Disruption of BRCA1 LXCXE motif alters BRCA1 functional activity and regulation of RB family but not RB protein binding.

The tumor suppressor activity of the BRCA1 gene product is due, in part, to functional interactions with other tumor suppressors, including p53 and the retinoblastoma (RB) protein. RB binding sites on BRCA1 were identified in the C-terminal BRCT domain (Yarden and Brody, 1999) and in the N-terminus (aa 304-394) (Aprelikova et al., 1999). The N-terminal site contains a consensus RB binding motif, LXCXE (aa 358-362), but the role of this motif in RB binding and BRCA1 functional activity is unclear. In both in vitro and in vivo assays, we found that the BRCA1:RB interaction does not require the BRCA1 LXCXE motif, nor does it require an intact A/B binding pocket of RB. In addition, nuclear co-localization of the endogenous BRCA1 and RB proteins was observed. Over-expression of wild-type BRCA1 (wtBRCA1) did not cause cell cycle arrest but did cause down-regulation of expression of RB, p107, p130, and other proteins (e.g., p300), associated with increased sensitivity to DNA-damaging agents. In contrast, expression of a full-length BRCA1 with an LXCXE inactivating mutation (LXCXE-->RXRXH) failed to down-regulate RB, blocked the down-regulation of RB by wtBRCA1, induced chemoresistance, and abrogated the ability of BRCA1 to mediate tumor growth suppression of DU-145 prostate cancer cells. wtBRCA1-induced chemosensitivity was partially reversed by expression of either Rb or p300 and fully reversed by co-expression of Rb plus p300. Our findings suggest that: (1) disruption of the LXCXE motif within the N-terminal RB binding region alters the biologic function of BRCA1; and (2) over-expression of BRCA1 inhibits the expression of RB and RB family (p107 and p130) proteins.