Low Molecular Weight Isoforms Research Articles

A Differential Effect of FGF‐2 on Resistance to Doxorubicin‐Induced Necrotic Versus Apoptotic‐Like Damage in Neonatal Rat Cardiomyocyte Cultures

Therapeutic agents like doxorubicin (DOX), an anti‐cancer anthracycline, can increase the risk of cardiac damage. Strategies are needed to protect the heart that still allows the benefits of drug treatment. “Basic” fibroblast growth factor‐2 (FGF‐2), and specifically the low (18 kDa) molecular weight isoform is cardioprotective, but this has not been reported for DOX‐induced injury. The clinical effects of DOX have been modeled in rat cardiomyocytes, and 0.1–1.0 μM DOX is sufficient to induce cell damage. Here, we assessed the effect of 10 nM FGF‐2 on 0.5 μM DOX‐induced necrotic versus apoptotic‐related damage over 24 hours, as detected by the presence of lactate dehydrogenase in the culture medium (LDH) versus DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). DOX induced a decrease in apparent (attached) cell number and a significant increase in LDH within 6 hours. The remaining cells continued to display muscle striations based on alpha‐actinin staining, but an increase in TUNEL intensity from 6 to 24 hours was also seen. Pre‐treatment (30 min) with FGF‐2 resulted in a significant decrease in LDH but not apparent TUNEL intensity. Additional assays to characterize these effects are being pursued. These data suggest that FGF‐2 can increase resistance to DOX‐induced plasma membrane damage but not DNA fragmentation under the conditions tested.

The Structural Basis of Higher Activity of Truncated Cyclin E1-CDK2 Complexes in Tumor Cells

Hyperactivation of the enzyme, Cyclin Dependent Kinase Type II (CDK2) on binding with low molecular weight isoforms of its activator protein, Cyclin E has been reported in vivo. Such isoforms of Cyclin E are primarily detected in the nucleus of cancer cells. Here, we study the conformational variations of CDK2 complexed with various isoforms of Cyclin E, via Molecular Dynamics simulations. Results show significant energetic and structural changes in various key regions of the complexes including the substrate binding pocket of CDK2-Cyclin interface, Our results correlate very well with the data from experiments. The study could help designing specific inhibitors to target abnormal kinases.

P5-03-03: Identification of New Substrates for Breast Tumor Specific Low-Molecular-Weight Cyclin E Cyclin-Dependent-Kinase 2.

Abstract Background: Cyclin E overexpression occurs in 25% of breast cancer tumors and is linked to poor prognosis. In tumor cells full length cyclin E (FL-E) is processed by an elastase-like protease into low-molecular weight isoforms (LMW-E) that are biochemically hyperactive. When overexpressed in breast cancer cells, LMW-E induced genomic instability and resistance to p21, p27, and antiestrogens. LMW-E are tumor specific and are associated with the aggressive triple negative breast cancer (TNBC). Transgenic mice overexpressing LMW-E had increased incidence of mammary tumors and distant metastasis when compared to FL-E. Furthermore, when the role of CDK2 in LMW-E mediated mammary tumorigenesis was interrogated by crossing LMW-E transgenic mice with CDK2 knock out mice, we found that the mice are protected against mammary tumor formation. Additionally, treatment of LMW-E transgenic mice with roscovitine (a CDK2 inhibitor) delays mammary tumor formation. These results indicate that kinase inhibition may have therapeutic activity in LMW-E overexpressing tumors as shown in this preclinical model. These results lead us to hypothesize that the biological and biochemical differences between FL-E and LMW-E may be due to the phosphorylation of a distinct set of substrates when complexed with CDK2. Our goal is to identify distinct LMW-E/CDK2 substrates on a proteome-wide scale that could serve as novel therapeutic targets for the treatment of the aggressive LMW-E expressing TNBC. Material and Methods: We used two different approaches to identify LMW-E/CDK2 substrates. 1) We generated an analog sensitive CDK2 kinase, (F80A or F80G)-CDK2 to specifically radiolabel its substrates in cell extracts followed by their identification by mass spectroscopy. 2) We incubated ProtoArrayMicroarrays spotted with 3000 GST-tagged human protein on high density glass slides either with recombinant active FL-E/CDK2 or LMW-E/CDK2. Results: In the first approach, we expressed and purified wild-type CDK2 in complex with FL-E or LMW-E and CDK2 (F80A) and CDK2 (F80G) from insect cells. Although all 3 CDK2 kinases can use ATP to phosphorylate GST-Rb protein, only the F80G mutant can use PE-ATP-γ-S. In the second approach, we incubated protein arrays either with recombinant active FL-E/CDK2 or LMW-E/CDK2 at a concentration of 50 nM. Our first screen identified a total of 122 potential substrates to both FL-E/CDK2 or LMW-E/CDK2 kinase complexes. We only identified 1 protein that is phosphorylated by FL-E/CDK2 significantly more than by LMW-E/CDK2 as compared to the 32 potential substrates specific to LMW-E/CDK2 suggesting that by losing the N-terminal portion, the LMW-E/CDK2 kinase complex is able to specifically interact and phosphorylate novel proteins. Discussion: The identification of new physiological LMW-E/CDK2 substrates will lead to the development of novel targets for therapeutics and the identification of the biological function for the treatment of the aggressive LMW-E expressing TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-03-03.

Abstract P210: Human Atrial Nonmyocytes Secrete Prohypertrophic, High-Molecular-Weight FGF-2, Which Is Upregulated by Angiotensin II via AT-1 and AT-2 Receptors, as Well as ERK and MMP Activation

Background: Very little is known about the expression and role of fibroblast growth factor-2 (FGF-2) isoforms in the human heart. Using the rat model we have documented that high molecular weight Hi-FGF-2 rather than the commonly studied 18 kDa low molecular weight isoform Lo-FGF-2 is a potent inducer of cardiac hypertrophy in vitro and in vivo ; and that Hi-FGF-2 is expressed and secreted predominantly by cardiac non-myocytes (fibroblasts). We have now examined (i) the expression of Hi-FGF-2 in adult human heart (atria) and heart-derived non-myocytes (HDNM), and; (ii) signals regulating Hi-FGF-2 expression in HDNM. Methods/Results: Atrial tissue, obtained from patients undergoing cardiac surgery, (blinded study), was used to obtain extracts, and to isolate migratory cells (fibroblastic, HDNM). All tissue extracts (n=30) contained Hi- as well as Lo-FGF-2, assessed by Western blotting. Amounts of total FGF-2 varied from 1.5 - 25.5 pg per µg of extracted protein. Immunohistochemistry of paraffin-embedded atrial tissue sections and immunofluorescence of HDNM illustrated that human Hi-FGF-2 is localized mainly in the nucleus but is also present in cytoplasm. As was the case with rat- (ventricle and/or atria) derived fibroblasts, HDNM expressed predominantly Hi-FGF-2 (90% of total). The expression/secretion of Hi-FGF-2 by HDNM, as well as by human embryonic heart-derived fibroblasts, was significantly up-regulated by angiotensin II (Ang II). Simultaneous inhibition of both AT-1 as well as AT-2 receptors (by losartan and PD123319, respectively) was required to fully prevent Ang II-induced Hi-FGF-2 up-regulation. In addition, both inhibition of ERK activation (by U0126), or MMP activity (by MMP-2 Inhibitor I) fully prevented Ang II-induced up-regulation of human Hi-FGF-2. Conclusions: We have shown for the first time that human heart-derived fibroblastic cells express and secrete pro-hypertrophic Hi-FGF-2 in culture; and thus are likely to do so in vivo . Our data also suggest that the beneficial effects of drugs targeting Ang II signal transduction may be due, in part to their effects on Hi-FGF-2 accumulation.

Inhibition of testicular and Vipera russelli snake venom hyaluronidase activity by Butea monosperma (Lam) Kuntze stem bark

This study was carried out to investigate the anti-fertility and anti-venom activities of the extract of the stem bark of Butea monosperma by inhibiting hyaluronidase, which is a spreading factor and plays a role in fertilisation. Among ethanol, methanol and water extracts, the ethanol extract dose-dependently inhibited the ovine, mouse testicular and Vipera russelli snake venom hyaluronidase enzyme activities, with IC50 values 12.00 ± 0.45, 49.40 ± 1.58 µg and 125.42 ± 2.82 µg mL–1, respectively. In a zymogram assay, the extract showed differential inhibition towards hyaluronidase isoform preferentially with low-molecular weight isoforms. The V. russelli snake venom-induced hemorrhage was significantly reduced at 1:05 ratio of venom-to-extract in mouse. The high antioxidant activity and total phenolic content in the ethanolic extract strongly correlated with the hyaluronidase inhibition. The above results justify the traditional use of the stem bark of B. monosperma as a contraceptive and a strong antidote to snake venom.

Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread.

Cyclin E Low-Molecular-Weight Isoform as a Predictor of Breast Cancer in Japanese Women

Overexpression of low-molecular-weight isoforms (LMWI) of cyclin E in breast cancer cells is associated with poor prognosis and could serve a novel role in breast cancer progression. LMWI originate from proteolytic processing of cyclin E, which is deregulated and hyperactive. In this study, levels of full-form/LMWI cyclin E were determined with the use of Western blot analysis in 69 Japanese breast cancer patients. LMWI cyclin E levels were significantly correlated with known parameters such as tumor grade and estrogen/progesterone receptor expression. In multivariate analysis, patient survival was significantly correlated with tumor grade but not with either form of cyclin E. LMWI was not as strong a predictor as tumor grade in this study, whereas some cases of early relapse with LMWI overexpression and lower tumor grade were reported. Thus, LMWI might be a good complementary factor to other predictors for early relapse of breast cancer.

Histochemical localization of caldesmon isoforms in colon adenocarcinoma and lymph node metastases

Alternative splicing of the caldesmon gene results in high (h-caldesmon) and low (l-caldesmon) molecular weight isoforms of the cytoskeleton-associated protein caldesmon. h-Caldesmon is predominantly expressed not only in smooth-muscle cells but also in pericryptal fibroblasts in colon. l-Caldesmon is widely expressed and localized in podosomes/invadopodia. Studies with transformed and cancer cell lines suggest that a reduction in l-caldesmon facilitates podosome/invadopodia formation and metastasis. We investigated caldesmon isoforms in colon adenocarcinoma and their lymph node metastases using immunohistochemistry. Caldesmon immunoreactivity of colon adenocarcinoma primary tumors and lymph node metastases was very similar. l-Caldesmon immunoreactivity of cancer epithelial cells in primary tumors and lymph node metastases varied ranging from reduced to stronger as compared to immunoreactivity of normal areas. The variation did not show a consistent relation to the tumor center or invasive margin. While h-caldesmon immunoreactivity of pericryptal fibroblasts and blood vessels in the stroma of primary tumors and lymph node metastases was markedly reduced, cancer-associated fibroblasts and blood vessels in the stroma were strongly immunoreactive for l-caldesmon. Our results show a differential behavior of h- and l-caldesmon isoforms in epithelium and stroma of colon adenocarcinoma and lymph node metastases.

Abstract 4541: Binding and transcriptional activity of the androgen receptor isoforms in 22Rv1 prostate cancer cells

Abstract Prostate carcinoma (CaP) is a heterogeneous multifocal disease where gene expression and regulation is altered as the disease progresses but the androgen receptor (AR) continues to be a pivotal regulatory molecule. The 22Rv1 castrate resistant cell line, derived from the androgen dependent CWR22 xenograft model, continues to express the AR and remains androgen responsive. However, in additional to the full length AR, these cells also express several low molecular weight isoforms. These isoforms are the predominant (95%) forms present in the nucleus in castrate androgen levels. siRNA knockdown of all AR isoforms in castrate levels of androgen decreases cell growth indicating that AR is required for proliferation. Analysis of ChIP-promoter array data identified 128 binding sites (FDR<=0.05) in Rv1 cells proliferating in androgen-depleted media. A closer examination of these 128 binding sites revealed that 20% of the sites showed exactly the same start and end position in the absence or presence of DHT. The remainder (80%) were within ∼35-1000bp upstream of the start position or downstream of the end position. Only 6% of the binding sites contained the palendromic consensus AR-response element and 48% contained the AR half-site motif. The 128 binding sites were associated with 118 genes. To define the role of AR binding in regulating transcription, the expression of 20 of the 118 genes was analyzed following knockdown of AR in castrate levels of androgen. One third of these genes are transactivated, while 1/3 are repressed, arguing that AR activating and repressing complexes co-exist in these cells. Notably the expression of CDKN1B (p27) RNA and protein is highly activated following AR knockdown, suggesting that AR-dependent repression of this cell cycle inhibitor may be important for proliferation of 22Rv1 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2011-4541

Heat shock protein 70 can interact with and also prevent the high molecular weight FGF2‐induced apoptotic chromatin compaction

Fibroblast growth factor 2 (FGF2) is translated as high molecular weight (hi) and low molecular weight (lo) isoforms. We reported that increased nuclear expression of hi but not lo FGF2 elicited chromatin compaction and apoptotic cell death. We have begun investigating the role of hi FGF2 isoform‐specific interacting partners in modulating its pro‐apoptotic effect. Isoform‐specific affinity chromatography was used to capture interacting proteins from cell cytosolic and nuclear fractions. Nuclear proteins that bound only to the hi FGF2‐affinity column were identified by MS/MS peptide sequencing, and included the heat shock protein 70 (hsp70). Hsp70 was confirmed as a hi (but not lo) FGF2 interacting partner by co‐immunoprecipitation experiments. Overexpession of hsp70 prevented the hi FGF2‐induced chromatin compaction in cardiomyocytes. While hsp70 was mainly cytosolic in the absence of over‐expressed hi FGF2, it translocated to the nucleus in the presence of hi FGF2 over‐expression. The nuclear pattern of hsp70 localization partially overlapped with that of hi FGF2, suggesting interaction in situ. In conclusion, hsp70 interacts with hi but not lo FGF2, and is capable of preventing nuclear compaction by hi FGF2. It is possible that an in situ interaction between hsp70 and hi FGF2 is preventing the latter from exerting its apoptotic chromatin compaction effects.

Cellular Uptake of Soluble Neutrophil Elastase Increases Cyclin E (CCNE) Isoform Expression and Significantly Augments Susceptibility of Breast Cancer Cells to Cytolysis by CCNE-Specific Cytotoxic T Lymphocytes

AbstractAbstract 2090We have shown that cytotoxic T lymphocytes (CTL) with specificity for the cyclin E (CCNE) derived HLA-A2-restricted peptide CCNE144-152 (ILLDWLMEV) specifically lyse myeloid and lymphoid leukemia in proportion to CCNE overexpression. Full length (FL) CCNE is also overexpressed in most solid tumors, including breast cancer where it is a poor prognostic factor. In addition, leukemia and many breast cancers express tumor-specific low molecular weight (LMW) isoforms of CCNE that result from post-translational processing of the FL protein. Neutrophil elastase (NE), derived from the primary granules of neutrophils, cleaves FL CCNE into LMW forms and NE has been identified in breast cancer tissue. Therefore, we hypothesized that CCNE may also be a breast cancer tumor antigen because the CCNE144-152 peptide is contained within the overexpressed FL and LMW forms, and that effective T cell immunity could be amplified by increased availability of LMW forms within tumor cells exposed to NE. The link between innate immunity, inflammation, and tumor immunity is poorly understood, and this mechanism could explain a role for tumor-infiltrating inflammatory cells in breast cancer. To test this, we elicited CCNE-CTL from peripheral blood lymphocytes of HLA-A2+ healthy donors by weekly stimulation with CCNE-pulsed T2 cells and low-dose IL-2. After 21 days, cytotoxicity of target cells by the lymphocytes was tested with a standard 4-hour calcein AM-based assay. The CCNE-CTL specifically lysed T2 cells pulsed with CCNE (30%) but not non-pulsed T2 cells (0%) at an effector:target (E:T) ratio of 20:1 (p < 0.01). Next, we tested whether CCNE-CTL killed HLA-A2+ MDA-MB-231 (231) breast cancer cells. First, we confirmed that FL CCNE and LMW forms were expressed in 231 cells, while low expression of FL CCNE and no expression of the LMW forms was observed in benign epithelial cells by Western blot. Next, we showed that CCNE-CTL mediated 49% lysis of 231 breast cancer cells at an E:T ratio of 20:1. To look for in vivo evidence of CCNE recognition, we studied peripheral blood lymphocytes from breast cancer patients by flow cytometry with CCNE/HLA-A2 tetramers and anti-CD8 antibodies. In 3 of 4 breast cancer patients we identified CCNE-CTL, with no detectable CCNE-CTL in healthy controls. Together, these results confirm that CCNE is also a tumor antigen in breast cancer. NE, which cleaves CCNE and is expressed in breast cancer tissue, may be produced endogenously by breast cancer cells, or exogenously by inflammatory cells in the tumor microenvironment. Therefore, we studied 231 cells and three other breast cancer cell lines (MDA-MB-453, MCF-7, and HER18) for NE expression. RT-PCR performed with NE-specific primers showed that none of the cells expressed NE mRNA and Western blot showed no NE protein expression, suggesting that NE in tumor tissue derives from neutrophils or other inflammatory cells. To determine whether NE is taken up by breast cancer cells, we used flow cytometry to show that 231 cells pulsed with soluble NE took up an increasing amount of NE and was maximal by 24 hours when intracellular NE expression in 231 cells was comparable to that of HL60 leukemia cells that express high levels of NE. In addition, LMW isoforms of CCNE were increased in NE-pulsed 231 cells, by Western blot. Importantly, CCNE-CTL specific lysis of NE-pulsed 231 cells was 2-fold higher compared to nonpulsed 231 (46% versus 22% at E:T 10:1, p = 0.01). Taken together, these data show that overexpressed CCNE144-152 is a novel breast cancer peptide antigen. Furthermore, exogenous NE is taken up by breast cancer cells, increasing LMW forms of CCNE and enhancing the susceptibility of breast cancer cells to CCNE-CTL-mediated cytolysis. This study links a specific enzyme secreted by neutrophils in the innate immune response to tumors to a specific adaptive immune response against breast cancer, and it suggests that immunotherapy targeting CCNE is warranted. Disclosures:No relevant conflicts of interest to declare.

Developmental expression of high molecular weight tropomyosin isoforms in Mesocestoides corti

Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia.

SiRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model

Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers.

Antagonistic effects of hi-FGF-2 on myocardial ischemia/reperfusion injury in rats

Objective To investigate the effect of CUG-initiated, high molecular weight fibroblast growth factor-2 (hi-FGF-2) on myocardial ischemia/reperfusion injury in rats. Methods Ex vivo rat hearts were subjected to 30 rain ischemia and 60 min reperfusion. Normal saline, hi-FGF-2 or low molecular weight FGF-2 (lo-FGF-2) (10 μg in 10 ml normal saline) was perfused with perfusate into the heart during the first 10 min of reperfusion. Distribution of exogenous FGF-2, changes of vinculin as well as cardiac mechanic functions were examined. Results Cardiac functions including systolic pressure, dp/dtmax and developed pressure were significantly improved in hi- and lo-FGF-2 groups compared to saline controls (P <0.05 or 0. 01 ). The exogenous hi- or lo-FGF-2 was broadly distributed into the myocardium during 1 h reperfusion. Intensive and intact vinculin staining was seen in both lo- and hi-FGF-2, but not in normal saline, administered hearts. Conclusion hi-FGF-2, like its low molecular weight isoform, is cardioprotective against myocardial ischemia/reperfasion injury. Key words: FGF-2; Myocardial ischemia; Reperfusion injury

Preferential accumulation and export of high molecular weight FGF-2 by rat cardiac non-myocytes

fibroblast growth factor-2 (FGF-2), implicated in paracrine induction of cardiac hypertrophy, is translated as high molecular weight (Hi-FGF-2) and low molecular weight (Lo-FGF-2) isoforms. Paracrine activities are assigned to Lo-FGF-2, whereas Hi-FGF-2 is presumed to have nuclear functions. In this work, we re-examined the latter presumption by asking whether: cardiac non-myocytes (CNMs) accumulate and export Hi-FGF-2 in response to pro-hypertrophic [angiotensin II (Ang II)] stimuli; an unconventional secretory pathway requiring activated caspase-1 affects Hi-FGF2 export; and secreted Hi-FGF-2 is pro-hypertrophic. using neonatal rat heart-derived cultures and immunoblotting, we show that CNMs accumulated over 90% Hi-FGF-2, at levels at least five-fold higher than cardiomyocytes (CMs). Pro-hypertrophic agents (Ang II, endothelin-1, and isoproterenol) up-regulated CNM-associated Hi-FGF-2. The Ang II effect was mediated by Ang II receptor-1 but not Ang II receptor-2 as it was blocked by losartan but not PD123319. CNM-derived Hi-FGF-2 was detected in two extracellular pools: in conditioned medium from Ang II-stimulated CNMs and in association with the cell surface/matrix, eluted with a gentle 2 M NaCl wash of the cell monolayer. Conditioned medium from Ang II-treated CNMs increased neonatal CM size, an effect prevented by anti-FGF-2-neutralizing antibodies. The caspase-1 inhibitor YVAD prevented the Ang II-induced release of Hi-FGF-2 to both extracellular pools. CNMs are major producers of Hi-FGF-2, up-regulated by hypertrophic stimuli and exported to the extracellular environment by a mechanism requiring caspase-1 activity, suggesting a link to the innate immune response. Hi-FGF-2 is likely to promote paracrine induction of myocyte hypertrophy in vivo.

Adiponectin Multimers and ADIPOQ T45G in Coronary Artery Disease in Caribbean Type 2 Diabetic Subjects of African Descent

Ethnic differences may affect the association of adiponectin (Ad) multimers with coronary artery disease (CAD). We analyzed the associations of total Ad, Ad multimers, and T45G polymorphism of ADIPOQ gene with pre-existing CAD. We carried out a cross-sectional study of 216 Afro-Caribbean type 2 diabetic (T2D) subjects. Levels of total Ad, high molecular weight (HMW), middle molecular weight (MMW), and low molecular weight (LMW) isoforms were measured. Subjects were genotyped. Of the subjects studied, 57 had pre-existing CAD, 77% of whom have had myocardial infarction. Subjects with CAD had lower Ad levels (total and multimers) and a higher frequency carried the minor allele 45G, GG/TG, (18% vs. 8%, P = 0.03) than subjects without CAD. In logistic regression analysis, the models used evaluate Ad in the context of adjustment for metabolic syndrome characteristics. The adjusted odds ratio (OR) of CAD was increased significantly (by factors of 1.05-3.27) for males, older subjects, low high-density lipoprotein cholesterol (HDL-C), high triglycerides (TGs), and carriers of the 45 G allele. For Ad, in model 1 (including only total Ad) the adjusted OR was 2.30; P = 0.03 and, in model 2 (including the three multimers, but not total Ad), the adjusted ORs were 0.73; P = 0.52 (HMW), 2.90; P = 0.01 (MMW), and 2.08; P = 0.09 (LMW). The T45G polymorphism in the ADIPOQ gene and hypoadiponectinemia were associated with CAD in our T2D subjects of predominantly African background. This effect of Ad level was mainly related to the MMW Ad form.

Cyclin E Deregulation Impairs Mitotic Progression through Premature Activation of Cdc25C

The cyclin E-cyclin-dependent kinase 2 (CDK2) complex accelerates entry into the S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. The effect of low molecular weight isoforms of cyclin E (LMW-E) overexpression on mitotic progression and its link to genomic instability were the focus of this study. Here, we show that full-length cyclin E (EL) and LMW-E overexpression impairs the G(2)-M transition differently by targeting dual-specificity phosphatase Cdc25C activity. We identify Cdc25C as an interaction partner and substrate for cyclin E/CDK2 kinase. Specifically, the cyclin E/CDK2 complex phosphorylates Cdc25C on Ser(214), leading to its premature activation, which coincides with higher cyclin B/CDK1 and Polo-like kinase 1 (PLK1) activities in an S-phase-enriched population that result in faster mitotic entry. Whereas EL overexpression leads to hyperactivation of Cdc25C, cyclin B/CDK1, and PLK1 in a G(2)-M-enriched population, LMW-E overexpression causes premature inactivation of Cdc25C and PLK1, leading to faster mitotic exit. In addition, LMW-E-overexpressing cells showed a reduction in the mitotic index in the presence of a spindle poison and faster degradation of cyclin B, suggesting an increased rate of mitotic slippage and adaptation to the spindle checkpoint. Lastly, downregulation of Cdc25C inhibits LMW-E-mediated chromosome missegregation, anaphase bridges, and centrosome amplification. These results suggest that the high levels of LMW-E isoforms found in breast cancer may contribute to cellular transformation and genomic instability by impairing mitotic progression involving Cdc25C.

Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Overexpression of the low molecular weight isoforms (LMW-E) of cyclin E induces chromosome instability; however, the degree to which these tumor-specific forms cause genomic instability differs from that of full-length cyclin E (EL), and the underlying mechanism(s) has yet to be elucidated. Here, we show that EL and LMW-E overexpression impairs the G(2)-M transition differently and leads to different degrees of chromosome instability in a breast cancer model system. First, the most significant difference is that EL overexpression prolongs cell cycle arrest in prometaphase, whereas LMW-E overexpression reduces the length of mitosis and accelerates mitotic exit. Second, LMW-E-overexpressing cells are binucleated or multinucleated with amplified centrosomes, whereas EL-overexpressing cells have the normal complement of centrosomes. Third, LMW-E overexpression causes mitotic defects, chromosome missegregation during metaphase, and anaphase bridges during anaphase, most of which are not detected on EL induction. LMW-E induces additional mitotic defects in cooperation with p53 loss in both normal and tumor cells. Fourth, LMW-E-overexpressing cells fail to arrest in the presence of nocodazole. Collectively, the mitotic defects mediated by LMW-E induction led to failed cytokinesis and polyploidy, suggesting that LMW-E expression primes cells to accrue chromosomal instability by shortening the length of mitosis. Lastly, LMW-E expression in human breast cancer tissues correlates with centrosome amplification and higher nuclear grade. These results suggest that LMW-E overexpression leads to higher centrosome numbers in breast cancer, which is a prerequisite for genomic instability.

A novel interaction between HER2/neu and cyclin E in breast cancer

HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. Since both are involved in cell cycle regulation we investigated whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P < .0001) In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 siRNA or treatment with trastuzumab. Cyclin E expression levels were determined, and functional effects investigated using kinase assays, MTT assays to assess cell viability as a marker of proliferation, and FACS analysis to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional effects, including decreased cyclin E-associated kinase activity and decreased proliferation, due to increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies demonstrate that HER2-mediated signaling effects LMW cyclin E expression, which in turn effects cell cycle regulation. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.

Abstract 3880: The natural dietary phytochemical indole-3-carbinole (I3C) sensitizes ovarian cancer cells to the proteasome inhibitor bortezomib through inhibition of cyclin E activity

Abstract Introduction: Cyclin E deregulation appears to be an important event in the pathogenesis of a subset of epithelial ovarian cancers associated with poor outcome. One mechanism of cyclin E deregulation is through enhanced p27 degradation, which eliminates a powerful negative regulator of cyclin E. The proteasome inhibitor bortezomib has been shown to inhibit the growth of both ovarian and colorectal tumor cell lines through upregulation of p27 and induction of apoptosis, giving it a potential therapeutic role in the subset of ovarian cancers that overexpress cyclin E. Studies demonstrate that as many as five low molecular weight (LMW) isoforms of cyclin E exist, while only the 50-kDa cyclin E form is typically expressed in normal tissues. These LMW isoforms are tumor-specific and cause increased cell proliferation, elevated kinase activity and increased clonogenicity. LMW cyclin E isoforms are generated via proteolysis of the normal 50-kDa cyclin E form by elastase, which itself can be selectively inhibited by indole-3-carbinol (I3C), a natural component of Brassica vegetables. I3C exhibits potent anticarcinogenic properties and has recently been shown to shift the stable accumulation of cyclin E from the LMW to 50-kDa cyclin E form. By taking advantage of the specific inhibitory properties of I3C and bortezomib in the processing and potential expression of cyclin E, respectively, we hypothesize that ovarian cancers overexpressing cyclin E may demonstrate an enhanced response to targeted combination therapy with I3C and bortezomib. Methods: A panel of ovarian cancer cell lines was screened for cyclin E and p27 protein levels through western blotting. Representative cell lines with high and low cyclin E expression were treated with I3C and bortezomib and evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay and western blotting analysis. Results: OVCAR3 was found to express high levels of cyclin E and moderate levels of p27. In contrast, OVCAR5 was found to express low levels of cyclin E and high levels of p27. Treatment of OVCAR3 cells with I3C stabilized the expression of the normal 50-kDa cyclin E form in a dose-dependent manner. The combination of I3C and bortezomib treatment in OVCAR3 and OVCAR5 cells caused a significantly greater degree of cytotoxicity compared to either drug alone. While the effect was seen in both cell lines, inhibition of cell proliferation was more enhanced in the cyclin E overexpressing cell line OVCAR3. Conclusions: Indole-3-carbinol is a natural dietary substance that sensitizes ovarian cancer cells to the proteasome inhibitor bortezomib through stabilization and inhibition of cyclin E activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3880.