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Objective To examine the impact of hormones used for controlled ovarian hyperstimulation (COH) on normal and malignant breast cell growth and proliferation. Design In vitro study of cultured normal and malignant breast cell lines. Setting Academic medical center. Patient(s) None. Intervention(s) Normal and malignant breast cell lines cultured in two- and three-dimensional (2D and 3D) systems and treated with follicle-stimulating hormone (FSH), luteinizing hormone (LH), or FSH with LH or human chorionic gonadotropin (hCG). Main Outcome Measure(s) Effects of treatment on cell proliferation in 2D culture using the MTS assay and on colony growth in 3D culture. Result(s) Compared with untreated cells, normal MCF-10A cells showed a decrease in proliferation and colony size when exposed to a combination of FSH and hCG. The HCC 1937 cells treated with FSH and LH also showed a decrease in colony growth but no change in proliferation. None of the treatments had an effect on the proliferation or colony size of the MCF-7 cells. Conclusion(s) Follicle-stimulating hormone, LH, and hCG do not appear to cause an increase in cell proliferation or colony growth in either normal or malignant mammary epithelial cell lines. The potential risk for mammary cell transformation associated with these agents may be related to indirect endocrine effects on breast cell physiology.

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 50 of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNAbinding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels. US National Institutes of Health NIH: R01-AI044254 NIH: R01-GM070662 NIH Training Grant in Microbial Pathogenesis T32-AI49795

Previous studies using small numbers of serum samples from human patients and experimentally infected animals identified the frequent presence of antibodies recognizing RevA, a borrelial fibronectin-binding outer surface protein. We now demonstrate that most examined Lyme disease spirochetes from North America and Europe contain genes encoding RevA proteins, some with extensive regions of conservation and others with moderate diversity. Line blot analyses using recombinant RevA from two diverse Lyme disease spirochetes of RevA and serum samples from culture-confirmed human Lyme disease patients from the United States ( n = 46, mainly with early Lyme disease) and Germany (>500, with early and late manifestations of Lyme disease) were performed. The results indicated that a sizable proportion of patients produced antibodies that recognized recombinant RevA. Overall, RevA-based serological studies were less sensitive and less specific than other assay types, such as the VlsE-based C6 peptide assay. However, sera from patients in the initial stages of Lyme disease contained antibodies against RevA, demonstrating that this protein is expressed early in human infection. Thus, RevA may be a useful target for preventative or curative therapies.

The Lyme disease spirochaete,Borrelia burgdorferi, can invade and persistently infect its hosts' connective tissues. We now demonstrate thatB. burgdorferiadheres to the extracellular matrix component laminin. The surface-exposed outer-membrane protein ErpX was identified as having affinity for laminin, and is the first laminin-binding protein to be identified in a Lyme disease spirochaete. The adhesive domain of ErpX was shown to be contained within a small, unstructured hydrophilic segment at the protein's centre. The sequence of that domain is distinct from any previously identified bacterial laminin adhesin, suggesting a unique mode of laminin binding.

Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi . The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium's dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, ε-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.

Lyme borreliae naturally maintain numerous distinct DNA elements of the cp32 family, each of which carries a mono- or bicistronic erp locus. The encoded Erp proteins are surface-exposed outer membrane lipoproteins that are produced at high levels during mammalian infection but largely repressed during colonization of vector ticks. Recent studies have revealed that some Erp proteins can serve as bacterial adhesins, binding host proteins such as the complement regulator factor H and the extracellular matrix component laminin. These results suggest that Erp proteins play roles in multiple aspects of mammalian infection.

The Lyme disease spirochete,Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five differentB. burgdorferiouter surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded bycspA; BbCRASP-2, encoded bycspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded byerpP,erpC, anderpA, respectively. We now present analyses of the recently identified BbCRASP-2 andcspZexpression patterns throughout theB. burgdorferiinfectious cycle, plus novel analyses of BbCRASP-1 anderp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription ofcspZand synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, andcspZexpression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed byB. burgdorferito control expression of this borrelial infection-associated protein.

The causative agent of Lyme disease,Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development ofB. burgdorferiinfection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways inB. burgdorferisurvival during mammalian infection. While it was predicted thatB. burgdorferishould be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis ofB. burgdorferiacquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection byB. burgdorferiand indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.

background: Breast cancer development involves a series of mutations in a heterogeneous group of proto-oncogenes/tumor suppressor genes that alter mammary cells to create a microenvironment permissive to tumorigenesis. Exposure to hormones during infertility treatment may have a mutagenic effect on normal mammary epithelial cells, high-risk breast lesions and early-stage breast cancers. Our goal was to understand the association between infertility treatment and normal and cancerous breast cell proliferation. methods: MCF-10A normal mammary cells and the breast cancer cell lines MCF-7 [estrogen receptor (ER)-positive, well differentiated] and HCC 1937 (ER-negative, aggressive, BRCA1 mutation) were treated with the weak ER activator clomiphene citrate and hormones that are increased during infertility treatment. Direct effects of treatment on cell proliferation and colony growth were determined. results: While clomiphene citrate had no effect on MCF-10A cells or MCF-7 breast cancer cells, it decreased proliferation of HCC 1937 versus untreated cells (P ¼ 0.003). Estrogen had no effect on either MCF-10A or HCC 1937 cells but, as expected, increased cell proliferation (20– 100 nM; P ≤ 0.002) and colony growth (10–30 nM; P , 0.0001) of MCF-7 cells versus control. Conversely, progesterone decreased both proliferation (P ¼ 0.001) and colony growth (P ¼ 0.01) of MCF-10A cells, inhibited colony size of MCF-7 cells (P ¼ 0.01) and decreased proliferation of HCC 1937 cells (P ¼ 0.008) versus control. hCG (100 mIU/ml) decreased both proliferation (P ≤ 0.01) and colony growth (P ≤ 0.002) of all three cell lines. conclusions: Although these data are preclinical, they support possible indirect estrogenic effects of infertility regimens on ER-positive breast cancer cells and validate the potential protective effect of pregnancy-related exposure to hCG.

Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3, or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1), or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.

Smad3, a component of the transforming growth factor β signaling cascade, contributes to G1 arrest in breast cancer cells. Cyclin D1/cyclin-dependent kinase 4 (CDK4) promotes G1-S–phase transition, and CDK phosphorylation of Smad3 has been associated with inhibition of Smad3 activity. We hypothesized that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through CDK4-mediated phosphorylation and inhibition of Smad3 and release of G1 arrest. Real-time quantitative reverse transcription-PCR and immunoblotting were used to evaluate expression of study proteins in cyclin D1–overexpressing breast cancer cells. Smad3 transcriptional activity and cell cycle control were examined in cells transfected with wild-type (WT) Smad3 or Smad3 with single or multiple CDK phosphorylation site mutations (M) in the presence or absence of the CDK4 inhibitor or cotransfection with cdk4 small interfering RNA (siRNA). Transfection of the Smad3 5M construct resulted in decreased c-myc and higher p15INK4B expression. Compared with WT Smad3, overexpression of the Smad3 T8, T178, 4M, or 5M mutant constructs resulted in higher Smad3 transcriptional activity. Compared with cells transfected with WT Smad3, Smad3 transcriptional activity was higher in cells overexpressing Smad3 mutant constructs and treated with the CDK4 inhibitor or transfected with cdk4 siRNA. Cells transfected with Smad3 T8 or T178 and treated with the CDK4 inhibitor showed an increase in the G1 cell population. Inhibition of CDK-mediated Smad3 phosphorylation released cyclin D1–regulated blockade of Smad3 transcriptional activity and recovered cell cycle arrest in breast cancer cells. Targeted inhibition of CDK4 activity may have a role in the treatment of cyclin D–overexpressing breast cancers. Mol Cancer Res; 8(10); 1375–87. ©2010 AACR.

Background Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind. Results Both the H. influenzae and the E. coli YbaB proteins bound to tested DNAs. DNA-binding was not well competed with poly-dI-dC, indicating some sequence preferences for those two proteins. Analyses of binding characteristics determined that both YbaB orthologs bind as homodimers. Different DNA sequence preferences were observed between H. influenzae YbaB, E. coli YbaB and B. burgdorferi EbfC, consistent with amino acid differences in the putative DNA-binding domains of these proteins. Conclusion Three distinct members of the YbaB/EbfC bacterial protein family have now been demonstrated to bind DNA. Members of this protein family are encoded by a broad range of bacteria, including many pathogenic species, and results of our studies suggest that all such proteins have DNA-binding activities. The functions of YbaB/EbfC family members in each bacterial species are as-yet unknown, but given the ubiquity of these DNA-binding proteins among Eubacteria, further investigations are warranted.

Borrelia burgdorferi , the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32.

Macrophages and/or dendritic cells were found to play significant roles in both antibody-dependent and -independent control of B. hermsii infection, while bacterial binding of host factor H was found to be nonessential. Relapsing fever spirochetes, such as Borrelia hermsii, proliferate to high levels in their hosts’ bloodstream until production of IgM against borrelial surface proteins promotes bacterial clearance. The mechanisms by which B. hermsii survives in host blood, as well as the immune mediators that control this infection, remain largely unknown. It has been hypothesized that B. hermsii is naturally resistant to killing by the alternative pathway of complement activation as a result of its ability to bind factor H, a host complement regulator. However, we found that Cfh−/− mice were infected to levels identical to those seen in wild-type mice. Moreover, only a small minority of B. hermsii in the blood of wild-type mice had detectable levels of factor H adhered to their outer surfaces. In vitro, complement was found to play a statistically significant role in antibody-mediated inactivation of B. hermsii, although in vivo studies indicated that complement is not essential for host control of B. hermsii. Depletion of mφ and DC from mice had significant impacts on B. hermsii infection, and depleted mice were unable to control bloodstream infections, leading to death. Infection studies using muMT indicated a significant antibody-independent role for mφ and/or DC in host control of relapsing fever infection. Together, these findings indicate mφ and/or DC play a critical role in the production of B. hermsii-specific IgM and for antibody-independent control of spirochete levels.

Serological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferi sensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferi type strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection.

Host complement is widely distributed throughout mammalian body fluids and can be activated immediately as part of the first line of defense against invading pathogens. The agent of Lyme disease, Borrelia burgdorferi sensu lato (s.l.), is naturally resistant to that innate immune defense system of its hosts. One resistance mechanism appears to involve binding fluid-phase regulators of complement to distinct borrelial outer surface molecules known as CRASPs (complement regulator acquiring surface proteins). Using sensitive molecular biology techniques, expression patterns of all three classes of genes encoding the CRASPs of B. burgdorferi sensu stricto (BbCRASPs) have been analyzed throughout the natural tick-mammal infection cycle. Each class shows a different expression profile in vivo and the results are summarized herein. Studies on the expression of B. burgdorferi genes using animal models of infection have advanced our knowledge on the ability of the causative agent to circumvent innate immune defenses, the contributions of CRASPs to spirochete infectivity, and the pathogenesis of Lyme disease.

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease (Lyme borreliosis), is well-adapted to maintain a natural cycle of alternately infecting vertebrates and blood-sucking ticks. During this cycle, B. burgdorferi interacts with a broad spectrum of vertebrate and arthropod tissues, acquires nutrients in diverse environments and evades killing by vertebrate and tick immune systems. The bacterium also senses when situations occur that necessitate transmission between hosts, such as when an infected tick is taking a blood meal from a potential host. To accurately accomplish the requirements necessary for survival in nature, B. burgdorferi must be keenly aware of its surroundings and respond accordingly. In this review, we trace studies performed to elucidate regulatory mechanisms employed by B. burgdorferi to control gene expression, and the development of models or "paradigms" to explain experimental results. Through comparisons of five borrelial gene families, it is readily apparent that each is controlled through a distinct mechanism. Furthermore, those results indicate that current models of interpreting in vitro data cannot accurately predict all aspects of B. burgdorferi environmental sensing and gene regulation in vivo.

Introduction: Overexpression of cyclin D1 is associated with poor prognosis in breast cancer. Cyclin D1 regulates the noncanonical phosphorylation of Smad3, a member of the TGFΒ signaling cascade, through CDK4 and inhibits Smad3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through a CDK4 mediated inhibition of Smad3 cell cycle control.Methods: To examine the impact of cyclin D1 overexpression on Smad3 function, constructs containing empty vector (V), WT Smad3 (WT), and Smad3 with single or multiple CDK phosphorylation site mutations were cotransfected with a Smad3 reporter into cyclin D1 overexpressing MCF-7 and T47D cells. Smad3 containing mutant CDK sites is resistant to inhibitory CyclinD1/CDK4 phosphorylation. Study cells were also transfected with V, WT or mutant Smad3 and treated with a CDK4 inhibitor. Smad3 function was examined by luciferase reporter assays and cell cycle analysis. Levels of mRNA for cell cycle regulators cyclin dependent kinase inhibitor (cdki) p15 and c-myc were measured by qRT-PCR.Results: As expected, higher amounts of c-myc mRNA and lower amounts of cdki p15 mRNA were observed in parental, vector control and cyclin D1 overexpressing cells. While subsequent transfection with WT Smad3 resulted in a decrease in c-myc and an increase in p15 mRNA levels, the greatest decrease in c-myc and increase in p15 mRNA was found after transfection with CDK mutated Smad3, most prominently in the cyclin D1 overexpressing cells. Transfection of all individual Smad3 CDK phosphorylation site mutations resulted in an increase in Smad3 reporter activity as compared to transfection with WT Smad3. Transfection of Smad3 constructs with 4 and 5 CDK sites mutated resulted in highest overall Smad3 reporter activity, compared to WT Smad3, in the study cells. A dose dependent increase in Smad3 reporter activity was shown when MCF-7 and T47D cells were transfected with WT Smad3 and treated with increasing concentrations of a specific CDK4 inhibitor. Comparatively higher Smad3 reporter activity was found when the study cells were transfected with individual Smad3 mutant constructs and treated with the CDK4 inhibitor. Interestingly, transfection with the Smad3 construct with all CDK sites mutated and treatment with the CDK4 inhibitor resulted in inhibition of Smad3 reporter activity in the study cells. Lastly, cell cycle analysis revealed that MCF-7 and T47D cells treated with the CDK4 inhibitor showed an increase in the G1 cell cycle population.Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 appear to facilitate release of cyclin D1/CDK4 mediated inhibition of Smad3 in breast cancer cells. To this end, restoration of Smad3 activity resulted in increasing p15 and decreasing c-myc mRNA levels in the study cells. However, mutation of all Smad3 CDK phosphorylation sites and treatment with the CDK4 inhibitor resulted in a decrease in Smad3 reporter activity, indicating that some CDK activity is necessary for Smad3 function. By helping to restore cell cycle control, inhibition of CDK4 activity may have a role in the treatment of primary and metastatic breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2152.

Abstract #5033 Introduction: The transcription factor Smad 3, a member of the TGFβ signaling cascade, contributes to G1 cell cycle arrest in breast cancer cell lines. Overexpression of the cell cycle regulatory protein cyclin E, and its low molecular weight (LMW) counterparts, has been associated with poor prognosis in breast cancer. Cyclin E has been shown to mediate phosphorylation within or near the linker region of Smad 3 by cyclin dependent kinase 2 (CDK 2), and to inhibit Smad 3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin E may exert tumorigenic effects in breast cancer cell lines through the functional inhibition of Smad 3 mediated by CDK 2 phosphorylation. Methods: Endogenous levels of Smad 3 and phosphorylated Smad 3 in parental, vector only control, full length (FL), and two LMW forms of cyclin E overexpressing MCF-7 breast cancer cell lines were determined by western blotting. To elucidate the impact of cyclin E overexpression on Smad 3 function, constructs containing an empty vector, WT Smad 3, Smad 3 with a CDK phosphorylation site mutation within the linker region (Thr178), and outside the linker region (Thr8) were co-transfected with a Smad 3 reporter into the panel of MCF-7 cells. CDK2 or scrambled control siRNA was also transfected into the study panel of cells. Smad 3 function was then evaluated by luciferase reporter assays. Results: MCF-7 cells overexpressing FL or LMW cyclin E had a higher level of P- SMAD 3 as compared to the parental and vector only control cells. Total SMAD 3 levels were similar in all cells examined. Transfection of WT Smad 3 elicited an increase in reporter activity in parental and vector control cells, while both FL and LMW forms of cyclin E overexpressing cells were resistant to WT Smad 3 reporter induction. In parental and vector control MCF-7 cells, expression of the Smad 3 Thr178 mutation resulted in a three-fold induction in Smad 3 reporter activity as compared to the WT Smad 3 response. In FL and LMW cyclin E overexpressing cells, induction by the Thr178 mutation was more pronounced, as seen by a 5-fold and 8-fold induction of reporter activity in FL and LMW cyclin E cells, respectively. Transient transfection of the Thr8 mutation failed to evoke a reporter response in any of the panel of MCF-7 cells. MCF-7, FL, and LMW cyclin E cells demonstrated the greatest fold induction of Smad 3 reporter activity when transfected with CDK 2 siRNA. Conclusions: Overexpression of cyclin E may circumvent the growth inhibitory effects of Smad 3 signal transduction through the modulation of Smad 3 function. Specifically, this work indicates that cyclin E mediates Smad 3 inhibition in a CDK 2 dependent fashion in breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5033.