Anchorage-dependent Cell Growth Research Articles

A HIC-5- and KLF4-dependent Mechanism Transactivates p21Cip1 in Response to Anchorage Loss

Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21(Cip1) transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.

Abstract 20: PHLDA1/2 contribute to tumor suppression in breast and lung cancer as downstream targets of oncogenic HER2 signaling

Abstract PHLDA1 (pleckstrin homology-like domain, family A, member 1) has been reported to act as a tumor suppressor gene in multiple cancers such as melanoma and breast cancer but appears to contribute to intestinal tumorigenesis. PHLDA2 is located in a cluster of imprinted genes on chromosome 11p15.5, an important tumor suppressor gene region. Alterations in this region may be associated with multiple cancers. Loss of imprinting of PHLDA2 was found to mediate placental growth retardation. The pleckstrin homology-like domain (PH domain) mediates protein recruitment to cellular membranes, which is of paramount importance for signal transduction. AKT and ERK are important protein kinases that play key roles in multiple cellular processes such as cell proliferation, apoptosis, transcription and cell migration. Activation and phosphorylation of AKT require binding to PIP through its PH domain. Another family member, PHLDA3 recently has been reported to be a major negative regulator of AKT signaling analogous to PTEN and it was shown that it functions as a negative AKT regulator by the quenching of membrane-bound phosphatidyl-inositol molecules. In our microarray studies of oncogenic EGFR and ErbB2 signaling using erlotinib-treated HCC827, CL-387,785-treated H1975 as well as lapatinib-treated Calu-3 and SKBR-3 cells, PHLDA1 and PHLDA2 were consistently identified as immediately downregulated genes upon oncogenic HER signaling inhibition and this was confirmed using quantitative RT-PCR as well as Western blotting studies. The regulation of PHLDA1/2 by HER signaling inhibition has not been investigated in detail previously. Our results show that the PI3K inhibitor, LY294002 significantly induces PHLDA1 expression but inhibits PHLDA2 expression in a time-dependent manner. Transient transfection mediated PHLDA1 over-expression in SKBR3 inhibited AKT activation and siRNA-mediated suppression of PHLDA2 in Calu3 cells dramatically induced ERK activation. Stable transfection mediated PHLDA1 over-expression in breast cancer cells inhibited both anchorage-dependent and independent cell growth in vitro. MTS cell proliferation studies showed that siRNA mediated suppression of PHLDA2 stimulated Calu3 cell growth. Studies are ongoing to test the potential role of PHLDA1/2 on inhibiting the direct activation of AKT by detailed PIP3 quantification studies. Our results provide strong evidence that PHLDA1/2 are downstream targets of HER2 signaling and suggest complex regulation of PHLDA1 and 2 by ERK and PI3K pathways. Our studies also suggest significant growth inhibitory effects for PHLDA1/2 and suggest that they might potentially act competitively with AKT and ERK to perform tumor suppression function. Our studies identify PHLDA1/2 as novel targets for therapeutic modulation in EGFR and ErbB2-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 20. doi:1538-7445.AM2012-20

HDGF-related protein-3 is required for anchorage-independent survival and chemoresistance in hepatocellular carcinomas

ObjectiveHepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of six members and are characterised by a conserved HATH domain. Among the family members, HDGF was the first to be...

Caveolin-1, antiapoptosis signaling, and anchorage-independent cell growth. Focus on “Caveolin-1 regulates Mcl-1 stability and anoikis in lung carcinoma cells”

caveolins (cav-1, -2, and -3) are integral membrane proteins that are essential components of caveolae, ∼50–100 nm invaginations found in cholesterol and sphingolipid-rich regions of the plasma membrane. In addition to functioning in transport pathways and cholesterol homeostasis, caveolae

Dextran-containing nanocarriers significantly promote greater anchorage dependent cell growth and density compared to microcarriers

Abstract Microcarriers containing cellulose-derived materials have been successfully applied to enhance the growth of anchorage-dependent cells maintained especially in bioreactors. By replacing microcarriers with nanocarriers containing dextran, we hypothesized that the density of the anchorage-dependent cells would rise dramatically because the decreased particle size and associated enhancement in surface to volume ratios of nanoparticles contained within the nanoemulsion-based nanocarriers would increase the number of dextran molecules for the anchorage-dependent cells to attach to. Our studies utilized self-assembly nanoemulsions (SANE) formed by a modified phase inversion temperature (PIT) process to produce dextran oil and surfactant-containing nanocarriers having mean particle sizes of 26 nm compared to microcarriers which were greater than 6000 nm. Our results demonstrated that dextran-containing nanocarriers allowed up to 10 fold greater cell density,12% more media lactate concentration, 83% higher cell lysate protein and 59% greater glucose concentration, used as a measure of polymer levels in the nanocarriers compared to microcarriers. In conclusion, nanocarriers with increased numbers of dextran molecules, developed in these studies may be useful to further increase the production of anchorage-dependent animal cell-derived products or production of mass cell growth for other applications.

Abstract B106: Role of TGF-beta superfamily ligand and estrogen signaling in mediating osteoblastic tumorigenesis of prostate cancer cells.

Abstract Prostate cancer metastasizes to bone in 80% of patients suffering from advanced disease. The nature of bone lesions is predominantly osteoblastic resulting in severe bone pain, hypocalcemia and nerve compression syndromes. Transforming growth factor-beta (TGFβ), which is a multifunctional cytokine, is a critical regulator of osteogenesis. Our group has recently shown that TGFβ signaling helps in promoting osteoblastic metastasis by a novel human prostate cancer cell line, PacMetUT1. TGFβ has been suggested to be one of the physiological upregulatory factors of bone aromatase in human osteoblast-like cells. Recent data further implicates an aberrant aromatase expression and estrogen signaling in the development of prostate malignancy. However, the effect of TGFβ superfamily ligands on aromatase expression and estrogen production and the mechanism by which osteoblastic lesions are formed have not been intensively investigated in prostate cancer cells. We hypothesize that TGFβ and other growth factors released from tumor cells can induce aromatase gene expression and activity in bone cells leading to an enhanced estradiol synthesis which would in turn lead to an increase in bone formation as estrogen is a known osteogenic factor. We examined the effect of TGFβ and BMP-4/7 on aromatase expression and osteogenic differentiation of pre-osteoblastic cells in vitro. Treatment with TGFβ 1 and BMP-4/7 resulted in an increase in aromatase expression in both mouse pre-osteoblasts and human bone marrow-derived mesenchymal stem cells. Treatment with letrozole (aromatase inhibitor) as well as an anti-estrogen (ICI 182, 780) resulted in a decrease in osteoblastic differentiation of mesenchymal stem cells as measured by alkaline phosphatase activity assay and expression of osteogenic markers by quantitative real-time PCR analysis. Furthermore, 17-β estradiol treatment of PacMetUT1 cells resulted in an enhanced anchorage-dependent and independent cell growth in vitro. This effect was very significantly inhibited by tamoxifen treatment, which is a selective estrogen receptor modulator. 17-β estradiol also induced a change in cell morphology of PacMetUT1 cells with a reduction in E-cadherin expression. Our results demonstrate an aromatase-mediated induction in osteogenesis by TGFβ superfamily ligands in the pre-osteoblastic cells and a stimulatory effect of estrogen signaling on PacMetUT1 cell tumorigencity in vitro. Future studies will delineate whether the induction of aromatase mediates osteoblastic bone metastasis of prostate cancer cells in vivo and if combination treatment of TGFβ and aromatase inhibitors can be used as a novel therapeutic strategy, to alleviate bone metastasis in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B106.

Regenerating islet‐derived family member, 4 modulates multiple receptor tyrosine kinases and mediators of drug resistance in cancer

Regenerating islet-derived family member, 4 (Reg IV) is a secreted protein and member of the C-type lectin superfamily. Expression analyses have characterized Reg IV as a prognostic marker for certain cancers; however, the functional role of Reg IV in cancer, including downstream signaling, has only begun to be elucidated. To investigate the biological role of Reg IV in cancer, phosphorylation events were studied in cancer cell lines in the context of either Reg IV stimulation (HCT116 cells) or knockdown of endogenous Reg IV (PC3 and KM12 cells). In addition to the previously observed impact on epidermal growth factor receptor and Akt phosphorylation, we observed modulation in the phosphorylation of multiple additional receptor tyrosine kinases (RTKs), including insulin receptor, insulin-like growth factor receptor as well as their downstream effectors, mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways. Furthermore, knockdown of Reg IV impacted the ability of insulin and EGF to stimulate downstream tyrosine phosphorylation. Knockdown of Reg IV in cancer cell lines inhibited anchorage-dependent and anchorage-independent (both soft-agar and spheroid assays) cell growth and induced cell cycle arrest. This was accompanied by upregulation of p21 and p27. Transiently silencing Reg IV in cancer cells induced apoptosis and downregulated Bcl-2. Conversely, stimulation of HCT116 cells with recombinant Reg IV induced Bcl-2. Hsp27, a molecule implicated in drug resistance, was similarly modulated by Reg IV. Consistent with our observations with Reg IV siRNA-mediated knockdown, monoclonal antibodies directed against Reg IV inhibited PC3 and KM12 cell growth. Collectively, Reg IV plays an important role in cancer by modulation of key signaling molecules including Hsp27, Bcl-2 and multiple RTKs.

Combination of Farnesyltransferase and Akt Inhibitors Is Synergistic in Breast Cancer Cells and Causes Significant Breast Tumor Regression in ErbB2 Transgenic Mice

The Akt activation inhibitor triciribine and the farnesyltransferase inhibitor tipifarnib have modest to little activity in clinical trials when used as single agents. In this article, preclinical data show that the combination is more effective than single agents both in cultured cells and in vivo. Combination index data analysis shows that this combination is highly synergistic at inhibiting anchorage-dependent growth of breast cancer cells. This synergistic interaction is also observed with structurally unrelated inhibitors of Akt (MK-2206) and farnesyltransferase (FTI-2153). The triciribine/tipifarnib synergistic effects are seen with several cancer cell lines including those from breast, leukemia, multiple myeloma and lung tumors with different genetic alterations such as K-Ras, B-Raf, PI3K (phosphoinositide 3-kinase), p53 and pRb mutations, PTEN, pRB and Ink4a deletions, and ErbB receptor overexpression. Furthermore, the combination is synergistic at inhibiting anchorage-independent growth and at inducing apoptosis in breast cancer cells. The combination is also more effective at inhibiting the Akt/mTOR/S6 kinase pathway. In an ErbB2-driven breast tumor transgenic mouse model, the combination, but not single agent, treatment with triciribine and tipifarnib induces significant breast tumor regression. Our findings warrant further investigation of the combination of farnesyltransferase and Akt inhibitors.

Overexpression of the dynein light chain km23‐1 in human ovarian carcinoma cells inhibits tumor formation in vivo and causes mitotic delay at prometaphase/metaphase

km23-1 is a dynein light chain that was identified as a TGFβ receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells. More importantly, induction of km23-1 expression substantially reduced the tumorigenicity of SKOV-3 cells in a xenograft model in vivo. Fluorescence-activated cell sorting analysis of SKOV-3 and IGROV-1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho-MEK, phospho-ERK and cyclin B1 were elevated, as was the mitotic index, suggesting that km23-1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, although the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/- cells, it was inappropriately retained at the metaphase kinetochore in Dox- cells. Thus, the mechanism by which high levels of km23-1 suppress ovarian carcinoma growth in vitro and inhibit ovary tumor formation in vivo appears to involve a BubR1-related mitotic delay.

Small Molecule Inhibitors as Probes for Estrogen and Androgen Receptor Action

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.

Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma

Although expression of non-protein-coding RNA (ncRNA) can be altered in human cancers, their functional relevance is unknown. Ultraconserved regions are noncoding genomic segments that are 100% conserved across humans, mice, and rats. Conservation of gene sequences across species may indicate an essential functional role, and therefore we evaluated the expression of ultraconserved RNAs (ucRNA) in hepatocellular cancer (HCC). The global expression of ucRNAs was analyzed with a custom microarray. Expression was verified in cell lines by real-time PCR or in tissues by in situ hybridization using tissue microarrays. Cellular ucRNA expression was modulated with siRNAs, and the effects on global gene expression and growth of human and murine HCC cells were evaluated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells compared with nonmalignant hepatocytes. Among these ucRNAs, the greatest change was noted for ultraconserved element 338 (uc.338), which was dramatically increased in human HCC compared with noncancerous adjacent tissues. Although uc.338 is partially located within the poly(rC) binding protein 2 (PCBP2) gene, the transcribed ncRNA encoding uc.338 is expressed independently of PCBP2 and was cloned as a 590-bp RNA gene, termed TUC338. Functional gene annotation analysis indicated predominant effects on genes involved in cell growth. These effects were experimentally demonstrated in both human and murine cells. siRNA to TUC338 decreased both anchorage-dependent and anchorage-independent growth of HCC cells. These studies identify a critical role for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.

A Noncompetitive Small Molecule Inhibitor of Estrogen-regulated Gene Expression and Breast Cancer Cell Growth That Enhances Proteasome-dependent Degradation of Estrogen Receptor α

The mechanisms responsible for 17β-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic (75)Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 +/- 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G(2)-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle.

Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

The contribution of lipid metabolic pathways to malignancy is poorly understood. Expression of the fatty acyl-CoA synthetase ACSVL3 was found to be markedly elevated in clinical malignant glioma specimens but nearly undetectable in normal glia. ACSVL3 levels correlated with the malignant behavior of human glioma cell lines and glioma cells propagated as xenografts. ACSVL3 expression was induced by the activation of oncogenic receptor tyrosine kinases (RTK) c-Met and epidermal growth factor receptor. Inhibiting c-Met activation with neutralizing anti-hepatocyte growth factor monoclonal antibodies reduced ACSVL3 expression concurrent with tumor growth inhibition in vivo. ACSVL3 expression knockdown using RNA interference, which decreased long-chain fatty acid activation, inhibited anchorage-dependent and anchorage-independent glioma cell growth by approximately 70% and approximately 90%, respectively. ACSVL3-depleted cells were less tumorigenic than control cells, and subcutaneous xenografts grew approximately 60% slower than control tumors. Orthotopic xenografts produced by ACSVL3-depleted cells were 82% to 86% smaller than control xenografts. ACSVL3 knockdown disrupted Akt function as evidenced by RTK-induced transient decreases in total and phosphorylated Akt, as well as glycogen synthase kinase 3beta, via a caspase-dependent mechanism. Expressing constitutively active myr-Akt rescued cells from the anchorage-dependent and anchorage-independent growth inhibitory effects of ACSVL3 depletion. These studies show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function.

EphA2 overexpression promotes ovarian cancer growth

Background: Silencing EphA2 has been shown to result in anti-tumor efficacy. However, it is not known whether increasing EphA2 expression specifically results in increased tumor growth and progression. We examined the effects of stable EphA2 transfection into poorly invasive ovarian cancer cells with regard to in vitro invasive and in vivo metastatic potential. Methods: EphA2 gene was introduced into A2780 cells by retroviral infection. The effects of increased EphA2 expression were examined on cellular morphology, and anchorage-dependent and independent cell growth. Furthermore, the effect of EphA2 overexpression on metastatic ability was determined using an orthotopic nude mouse model of ovarian carcinoma. Results: In low cell density, EphA2-overexpressing A2780 cells (A2780-EphA2) displayed less cell-cell contact, increased cell-extracellular matrix (ECM) attachment and anchorage-independent cell growth compared to empty vector controls. There was no significant effect on anchorage-dependent cell proliferation, migration, or invasion. Increased expression of EphA2 promoted tumor growth and enhanced the metastatic potential in A2780-EphA2 human ovarian cancer xenografts. The overexpression of EphA2 resulted in enhanced microvessel density (MVD), but had no effect on tumor cell proliferation. Conclusions: EphA2 promotes tumor growth by enhancing cell-ECM adhesion, increasing anchorage-independent growth, and promoting angiogenesis.

Downregulation of Aurora-A overrides estrogen-mediated growth and chemoresistance in breast cancer cells

Estrogen is known to play a causative role in the development of sporadic breast cancers and chemoresistance. However, studies on the mechanism and proteins involved in mediating the oncogenic effects of estrogen are very limited. Recently, Aurora-A, a centrosomal protein kinase, which induces centrosome amplification and genomic instability, has been shown to be upregulated during long-term treatment of rats with estrogen and was implicated in estrogen-induced oncogenesis. Herein, we present results of the studies carried out in short-term in vitro cultures to understand the regulation of Aurora-A by estrogen and the effect of downregulation of Aurora-A on estrogen-induced breast tumorigenesis and chemoresistance. Treatment of breast cancer cells with 10 nM 17beta-estradiol (E(2)) resulted in the upregulation of Aurora-A levels in an estrogen receptor-dependent manner. However, the upregulation by E(2) was not restricted to Aurora-A alone. Following release from the tamoxifen-induced arrest, the appearance of Aurora-A in the presence of estradiol in MCF7 cells coincided with the appearance of other mitotic markers suggesting that the spike in Aurora-A levels is an indirect consequence of estrogen-mediated cell proliferation. Thus, at least in short-term in vitro studies, Aurora-A is not a specific direct target of estrogen. However, downregulation of Aurora-A by RNA interference led to a significant decrease in estrogen-induced, anchorage-dependent, and independent growth of MCF7 cells. Moreover, knockdown of Aurora-A could overcome estrogen-induced decrease in docetaxel sensitivity of MCF7 cells. Cumulatively, we propose that the upregulation of Aurora-A by estrogen in short-term in vitro cultures is an indirect consequence of estrogen-induced cell proliferation. Nevertheless, Aurora-A inhibitors could be exploited to override the effects of estrogen on breast tumorigenesis and chemoresistance.

A Small Molecule Disruptor of Rb/Raf-1 Interaction Inhibits Cell Proliferation, Angiogenesis, and Growth of Human Tumor Xenografts in Nude Mice

Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251-mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents.

Growth Suppression of Lung Cancer Cells by Targeting Cyclic AMP Response Element-Binding Protein

Genes regulated by cyclic AMP-response element-binding protein (CREB) have been reported to suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis. However, it is not clear whether CREB is critically involved in lung carcinogenesis. We found that non-small cell lung cancer (NSCLC) cell lines exhibited elevated constitutive activity in CREB, in its immediate upstream kinases (ribosomal s6 kinase and extracellular signal kinase), and in the CREB-regulated cell survival proteins Bcl-2 and Bcl-xL. We hypothesized that constitutively active CREB is important to lung cancer cell growth and survival and therefore could be a potential therapeutic target for NSCLC. Ectopic expression of dominant repressor CREB and transfection with small interfering RNA against CREB suppressed the growth and survival of NSCLC cells and induced apoptotic cell death. Furthermore, treating H1734 NSCLC cells with an inhibitor of the CREB signaling pathway Ro-31-8220 inhibited CREB activation by blocking the activity of extracellular signal kinase and ribosomal s6 kinase, arrested the cell cycle at the G(2)-M phase, and subsequently induced apoptosis with the suppression of Bcl-2 and Bcl-xL expression. Ro-31-8220 suppressed both the anchorage-dependent and independent growth of NSCLC cells, but its cytotoxic effect was much less prominent in normal bronchial epithelial cells. Our results indicate that active CREB plays an important role in NSCLC cell growth and survival. Thus, agents that suppress CREB activation could have potential therapeutic value for NSCLC treatment.

Segregated Mathematical Model for Growth of Anchorage-Dependent MDCK Cells in Microcarrier Culture

To describe the growth behavior of anchorage-dependent mammalian cells in microcarrier systems, various approaches comprising deterministic and stochastic single cell models as well as automaton-based models have been presented in the past. The growth restriction of these often contact-inhibited cells by spatial effects is described at levels with different complexity but for the most part not taking into account their metabolic background. Compared to suspension cell lines these cells have a comparatively long lag phase required for attachment and start of proliferation on the microcarrier. After an initial phase of exponential growth only a moderate specific growth rate is achieved due to restrictions in space available for cell growth, limiting medium components, and accumulation of growth inhibitors. Here, a basic deterministic unstructured segregated cell model for growth of Madin Darby Canine Kidney (MDCK) cells used in influenza vaccine production is described. Four classes of cells are considered: cells on microcarriers, cells in suspension, dead cells, and lysed cells. Based on experimental data, cell attachment and detachment is taken explicitly into account. The model allows simulation of the overall growth behavior in microcarrier culture, including the lag phase. In addition, it describes the time course of uptake and release of key metabolites and the identification of parameters relevant for the design and optimization of vaccine manufacturing processes.

Activation of peroxisome proliferator‐activated receptor γ inhibits cell growth via apoptosis and arrest of the cell cycle in human colorectal cancer

To investigate the expression of peroxisome proliferator-activated receptors (PPAR)gamma and the effects of PPARgamma ligands on cells growth in colorectal cancer (CRC) cell line HT-29, and to explore whether the activation of PPARgamma by its selective ligand can induce apoptosis and the arrest of the cell cycle in these cells. A CRC cell line, HT-29, was used in this study. PPARgamma mRNA and the protein expressions were measured by reverse transcriptase-polymerase chain reaction and Western blot. The HT-29 cells were treated with two specific PPARgamma ligands: rosiglitazone and 15-d-PGJ2. The effects of PPARgamma activated by rosiglitazone and 15-d-PGJ2 on the anchorage-dependent and anchorage-independent growth of the cells were assessed by methylthiazolyl terazolium (MTT) and soft agar colony assay, respectively. Apoptosis was measured by TUNEL staining and flow cytometry (FCM) assay by CaspSCREEN Flowcytometric Apoptosis Detection Kit (BioVision, Palo Alto, USA). Furthermore, the caspase-3 expression was determined by a immunocytochemical staining method before and after treatment with rosiglitazone and 15-d-PGJ2 for 48 h. The cell cycles were measured by flow cytometric analysis using propidium iodide (PI). PPARgamma mRNA and protein expressions were observed in the HT-29 cells. The MTT assay showed that treatment of these cells with 0, 0.1, 1 or 10 micromol/L PPARgamma activators rosiglitazone or 15-d-PGJ2 for 0, 24, 48 or 72 h resulted in the inhibition of anchorage-dependent cell growth in a dosage- and time-dependent way. Rosiglitazone treatment during cell growth resulted in the reduction of colony formation and the effects were not immediately reversible in the cell culture. TUNEL staining showed DNA fragmentation in positive cells after treatment with rosiglitazone and 15-d-PGJ2 for 48 h. In addition, FCM showed that the apoptosis rates were 14.8+/-0.8% and 28.5+/-1.3% or 15+/-0.7% and 40+/-1.2% after the cells were incubated with 10 micromol/L rosiglitazone or 15-d-PGJ2 for 24 h and 48 h, while the apoptosis rates of cells without treatment were 3.8+/-0.4% and 8.8+/-0.4%, respectively. Consistent with these results, the positivity rates of caspase-3 expression in cells treated with rosiglitazone or 15-d-PGJ2 increased significantly when compared with the control group. To explore whether the regulation of the cell cycle was involved in the effect of PPARgamma ligands on cell growth, FCM using PI staining was assessed. The ratio of G0/G1 phase cells increased after incubated with 10 micromol/L rosiglitazone or 15-d-PGJ2 for 24 h and 48 h. Our results showed that PPARgamma was expressed in HT-29 cells and PPARgamma activation could inhibit cell growth through inducing apoptosis and suppressing the cell cycle. PPARgamma may be considered as a new therapeutic target for colon cancer in humans.