Human Mammary Carcinoma Cells Research Articles

The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells.

The αvβ3 integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the αvβ3 integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to αvβ3 requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances αvβ3 recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt αvβ3-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated αvβ3 integrins.

Evidence that mitogen-activated protein kinase phosphatase-1 induction by proteasome inhibitors plays an antiapoptotic role.

Inhibitors of the proteasome, a multicatalytic proteinase complex responsible for intracellular proteolysis, activate programmed cell death in part through the c-Jun-N-terminal kinase (JNK). Proteasome inhibitors also induce mitogen-activated protein kinase phosphatase-1 (MKP-1), however, which can inactivate JNK, and we therefore considered the hypothesis that MKP-1 induction may be antiapoptotic. Over-expression of MKP-1 in A1N4-myc human mammary epithelial and BT-474 breast carcinoma cells decreased proteasome inhibitor-mediated apoptosis. On the other hand, BT-474 cells stably expressing an MKP-1 small interfering RNA (siMKP-1) and MKP-1 knockout mouse embryo fibroblasts underwent enhanced apoptosis compared with their respective controls. MKP-1-mediated inhibition of apoptosis was associated with decreased phospho-JNK levels, whereas MKP-1 suppression or inactivation enhanced phospho-JNK. Anthracyclines repress MKP-1 transcription, suggesting that they could enhance proteasome inhibitor-mediated apoptosis. Such combinations induced increased cell death in association with enhanced phospho-JNK and decreased MKP-1 levels. Inhibition of JNK signaling decreased the proapoptotic activity of the anthracycline/proteasome inhibitor regimen. Xenograft studies showed the combination was more effective at inducing tumor growth delay, associated with suppression of MKP-1 and enhancement of apoptosis and phospho-JNK. Infection of anthracycline/proteasome inhibitor-treated A1N4-myc cells with Adenoviral-MKP-1 suppressed apoptosis and phospho-JNK. Finally, the anthracycline/proteasome inhibitor regimen activated apoptosis and phospho-JNK to a greater extent in BT-474/siMKP-1 cells than controls. These findings for the first time demonstrate that proteasome inhibitor-mediated induction of MKP-1 is antiapoptotic through inhibition of JNK. Furthermore, they suggest that a proteasome inhibitor/anthracycline regimen holds potential for enhanced antitumor activity in part through repression of MKP-1, supporting clinical evaluation of such combinations.

Phenotypic conversion of human mammary carcinoma cells by autocrine human growth hormone.

We report here that autocrine production of human growth hormone (hGH) results in a phenotypic conversion of mammary carcinoma cells such that they exhibit the morphological and molecular characteristics of a mesenchymal cell, including expression of fibronectin and vimentin. Autocrine production of hGH resulted in reduced plakoglobin expression and relocalization of E-cadherin to the cytoplasm, leading to dissolution of cell-cell contacts and decreased cell height. These phenotypic changes were accompanied by an increase in cell motility, elevated activity of specific matrix metalloproteinases, and an acquired ability to invade a reconstituted basement membrane. Forced expression of plakoglobin significantly decreased mammary carcinoma cell migration and invasion stimulated by autocrine hGH. In vivo, autocrine hGH stimulated local invasion of mammary carcinoma cells concomitant with a prominent stromal reaction in comparison with well delineated and capsulated growth of mammary carcinoma cells lacking autocrine production of hGH. Thus, autocrine production of hGH by mammary carcinoma cells is sufficient for generation of an invasive phenotype. Therapeutic targeting of autocrine hGH may provide a mechanistic approach to prevent metastatic extension of human mammary carcinoma.

319 Factors that govern the cell death response induced by inhibition of the molecular chaperone heat shock protein 90

The ginger rhizome is rich in bioactive compounds, including [6]-gingerol, [8]-gingerol, and [10]-gingerol; however, to date, most research on the anti-cancer activities of gingerols have focused on [6]-gingerol. In this study, we compared [10]-gingerol with [8]-gingerol and [6]-gingerol in terms of their ability to inhibit the growth of human and mouse mammary carcinoma cells. A colorimetric assay based on the enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide revealed that [10]-gingerol was more potent than [6]-gingerol and at least as potent as [8]-gingerol for the inhibition of triple-negative human (MDA-MB-231, MDA-MB-468) and mouse (4T1, E0771) mammary carcinoma cell growth. Further investigation of [10]-gingerol showed that it suppressed the growth of estrogen receptor-bearing (MCF-7, T47D) and HER2-overexpressing (SKBR3) breast cancer cells. The inhibitory effect of [10]-gingerol on the growth of MDA-MB-231 cells was associated with a reduction in the number of rounds of cell division and evidence of S phase-cell cycle arrest, as well as induction of apoptosis due to mitochondrial outer membrane permeabilization and the release of proapoptotic mitochondrial cytochrome c and SMAC/DIABLO into the cytoplasm. Surprisingly, killing of MDA-MB-231 cells by [10]-gingerol was not affected by a pan-caspase inhibitor (zVAD-fmk) or an anti-oxidant (N-acetylcysteine), suggesting that the cytotoxic effect of [10]-gingerol did not require caspase activation or the accumulation of reactive oxygen species. These findings suggest that further investigation of [10]-gingerol is warranted for its possible use in the treatment of breast cancer.

Cytotoxic and proapoptotic activity of ent-16β-17α-dihydroxykaurane on human mammary carcinoma cell line MCF-7

Here we describe the cytotoxic and proapoptotic effect of an ent-kaurane (ent-16β-17α-dihydroxykaurane), compound isolated from Croton malambo barks, on malignant cell growth. When MCF-7 mammary carcinoma cells were treated with increasing concentrations of the ent-kauranoid, its cytotoxic activity showed an IC 50 of 12.5 μg/ml, dose that is 2.66-fold lower than the corresponding value for non-malignant cells. At this growth inhibitory dose, both mRNA and protein levels for Bcl-2 as well as mRNA for hTERT were significantly reduced. The observed preapoptotic activity seemed to be triggered by a mechanism that is not directly affecting NF-κB binding ability. The potential use of this plant-derived compound as a cancer chemotherapy agent is discussed.

Emmprin promotes anchorage-independent growth in human mammary carcinoma cells by stimulating hyaluronan production.

Emmprin (CD147; basigin) is a plasma membrane glycoprotein, enriched on the surface of many cancer cells, which induces matrix metalloproteinase synthesis via cell-cell interactions. Elevated emmprin production causes increased growth in vivo of human mammary carcinoma cells. In this study, we show that elevation of emmprin expression in less aggressive human carcinoma cells, which normally express low emmprin levels, induces the ability to grow under anchorage-independent conditions. We also found that elevated emmprin expression stimulates hyaluronan production and that the effect of emmprin on anchorage-independent growth is dependent on hyaluronan. Furthermore, emmprin stimulates cell survival pathway signaling in a hyaluronan-dependent manner. From these and other studies we conclude that emmprin enhances several malignant properties of cancer cells, including anchorage-independent growth, invasiveness, and chemoresistance.

Controlling Distribution by Cleavage

Arachidonic acid (AA) stimulates the adhesion of cells, such as the metastatic breast carcinoma cell line MDA-MB-435, to extracellular matrix proteins, such as collagen. Kennett et al. show that exposure of cells to AA stimulates activation of protein kinase Cμ (PKCμ) and the p38 mitogen-activated protein kinase and promotes adhesion of MDA-MB-435 to collagen. Not only was PKCμ activated by phosphorylation in response to AA, it was translocated to the membrane and cleaved by calpain, which resulted in the accumulation of a truncated, activated cytosolic form of PKCμ. Pharmacological inhibition of PKCμ or calpain decreased cell adhesion in response to AA. Stimulation of phosphorylation of PKCμ or p38 was independent of each other, on the basis of pharmacological inhibition experiments. However, inhibition of p38 activity did decrease the calpain-mediated proteolysis of PKCμ. Thus, p38 may provide feedback on the PKCμ arm of the pathway not at the level of activation, but at the level of proteolysis by calpain and subsequent subcellular redistribution of the truncated active form of PKCμ. S. B. Kennett, J. D. Roberts, K. Olden, Requirement of protein kinase Cμ activation and calpain-mediated proteolysis for arachidonic acid-stimulated adhesion of MDA-MB-435 human mammary carcinoma cells to collagen type IV. J. Biol. Chem. 279 , 3300-3307 (2004). [Abstract] [Full Text]

HES-1, a novel target gene for the aryl hydrocarbon receptor.

Known mainly for its role as a toxin sensor, the aryl hydrocarbon receptor (AhR) complex is also involved in homeostasis regulation and differentiation processes and activated by xenobiotic compounds like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hairy and Enhancer of Split homolog-1 (HES-1) is a key regulator not only in differentiation, but also in the cell cycle, and we show here that HES-1 is a new target gene for AhR regulation. HES-1 is up-regulated by TCDD both at protein and mRNA levels in T47D human mammary carcinoma cells. Actinomycin D experiments have shown that the AhR-mediated up-regulation of HES-1 mRNA is caused by transcriptional activation of the HES-1 gene, and we have identified a functional AhR response element (XRE) at -48/-42 in the upstream regulatory region of human HES-1. The HES-1 protein down-regulates expression of its own gene, and the HES element overlaps the XRE. Our data indicate that HES-1 and the AhR complex compete for binding to the composite HES/XRE element. Also, we have previously shown that HES-1 is down-regulated by the estrogen receptor ligand 17beta-estradiol (E2). Up-regulation of HES-1 expression is correlated with suppression of cell proliferation, and the E2-mediated down-regulation of HES-1 therefore increases cell proliferation. It is known that TCDD exerts antiestrogenic action in breast tissue both in vivo and in vitro. Our observation that both the estrogen receptor and AhR signaling pathways regulate HES-1, but with opposing effects, suggests the existence of a new pathway by which AhR represses E2-signaling.

Requirement of Protein Kinase Cμ Activation and Calpain-mediated Proteolysis for Arachidonic Acid-stimulated Adhesion of MDA-MB-435 Human Mammary Carcinoma Cells to Collagen Type IV

Arachidonic acid (AA) stimulation of adhesion of human metastatic breast carcinoma cells to collagen type IV depends on the protein kinase C (PKC) pathway(s) and is associated with the translocation of PKC mu from the cytoplasm to the membrane. In the present study, we have further explored the role of PKC mu in AA-stimulated adhesion. PKC mu activation site serines 738/742 and autophosphorylation site serine 910 are rapidly phosphorylated, and in vitro PKC mu kinase activity is enhanced in response to AA treatment. Inhibition of PKC mu activation blocks AA-stimulated adhesion. A phosphorylated, truncated species of PKC mu was detected in AA-treated cells. This 77-kDa protein contains the kinase domain but lacks a significant portion of the regulatory domains. Inhibition of calpain protease activity blocks generation of the truncated protein, promotes accumulation of the activated, full-length protein in the membrane, and blocks the AA-mediated increase in adhesion. p38 MAPK activity is also required for AA-stimulated adhesion. Activation of PKC mu and p38 are independent events. However, inhibition of p38 activity reduces calpain-mediated proteolysis of PKC mu and in vivo calpain activity, suggesting a role for p38 in regulation of calpain activity and a point for cross-talk between the PKC and MAPK pathways. These results support the hypothesis that AA stimulates activation of PKC mu, which is cleaved by calpain at the cell membrane. The resulting truncated kinase, as well as the full-length kinase, may be required for increased cell adhesion to collagen type IV. Additionally, these studies present the first evidence for calpain cleavage of a non-structural protein leading to the promotion of tumor cell adhesion.

Differentiation-associated staining with anti-pimonidazole antibodies in head and neck tumors

Background and purpose Hypoxia is a strong negative prognostic factor for all three major treatment modalities for cancer. The bioreductive drug pimonidazole is currently under clinical investigation as a hypoxia marker. In human head and neck tumors, in addition to staining patterns typical of chronic hypoxia, staining was seen specifically around areas of keratinization, raising the question of whether this is hypoxia-related. This could influence quantitative hypoxia estimates using this marker. We investigated here whether the differentiation-related staining was caused by locally high reductive enzyme levels. Patients and methods The nitrotetrazolium compound NBT was used, which is reduced by nitroreductases to yield a blue color. The assay was validated on three genetically related MDA231 human mammary carcinoma cell lines: wildtype, overexpressing DT-diaphorase (DT1), and overexpressing cytochrome p450 reductase (R4). Increased NBT staining under normoxia was indeed seen for both R4 and DT1 lines. Pimonidazole staining under normoxia was only seen in the R4 line. Results Frozen tumor sections from 20 patients with head and neck cancer injected with pimonidazole were incubated with NBT. Parallel sections were stained for pimonidazole. Staining patterns were then compared on matched images, and areas of keratinization scored for the presence or absence of pimonidazole and NBT. Pimonidazole staining was seen in 56% of keratinized areas, and of these, 78% showed increased NBT staining, indicating that high reductase levels are not a necessary requirement for differentiation-associated pimonidazole staining. In a second series, frozen sections of tumors from 15 patients not receiving pimonidazole were incubated with NBT and compared with staining after incubation with pimonidazole under both oxic and hypoxic conditions. Pimonidazole staining of some keratinizing areas under oxic conditions was seen. Of these areas, only a proportion (70%) showed increased NBT staining, confirming the lack of correspondence between keratin-associated pimonidazole staining and reductase levels. Conclusion Hypoxia-independent pimonidazole staining can occur in more differentiated head and neck tumors, necessitating caution in hypoxia quantification. These data argue against a causative role for locally high reductase levels in differentiation-associated staining. DT-diaphorase appears to play no role in pimonidazole reduction.

Expression profile of genes associated with antimetastatic gene: nm23-mediated metastasis inhibition in breast carcinoma cells.

Metastases of various malignancies have been shown to be inversely related to the abundance of nm23 protein expression. However, the downstream pathways involved in nm23-mediated suppression of metastasis have not been elucidated. In the present investigation, we used cDNA microarrays to identify novel genes and functional pathways in nm23-mediated spontaneous breast metastasis. Microarray experiments were performed in a pair of cell lines, namely, C-100 (only vector transfected; highly metastatic) and H1-177 (nm23 transfected; low metastatic), derived from human mammary carcinoma cell line MDA-MB-435. The cDNA microarray analysis using GeneSpring software revealed significant as well as consistent alterations in the expression (up- and downregulation) of 2158 genes in a total of 18889 genes between high and low metastatic cells. Some of these genes were grouped into 6 functional categories, namely, invasion and metastasis, apoptosis and senescence, signal transduction molecules and transcription factors, cell cycle and repair, adhesion, and angiogenesis to extrapolate an association between these genes and different functional pathways involved in nm23-regulated metastasis. The results suggest that nm23 gene plays a major role in metastasis and its mechanism of action of metastasis suppression may involve downregulation of genes associated with cell adhesion, motility (integrins alpha2, -8, -9, -L and -V, collagen type VIII alpha1, fibronectin 1, catenin, TGF-beta2, FGF7, MMP14 and 16, ErbB2) and possibly certain tumor/metastasis suppressors (2 members of SWI/SNF-related matrix-associated proteins 2 and 5 and PTEN).

Identification of Genes Associated with the Invasive Status of Human Mammary Carcinoma Cell Lines by Transcriptional Profiling

We derived the transcriptional profiles of four invasive and four noninvasive mammary carcinoma cell lines by Affymetrix GeneChip^® Technology with the profile of human mammary epithelial cells as a reference. We focused on the identification of genes which are upregulated in the invasive cell lines based on the following threshold levels: -fold change of 2 or higher in at least three or more cell lines. According to the scoring criteria as described above, we identified 18 transmembrane receptors, 18 secreted proteins and 5 kinases. Several of the genes described have already been put into context with respect to invasion of mammary carcinoma. We therefore focused on deregulated genes for which such an association has not been described before: transmembrane receptor tyrosine kinase DDR2, transmembrane receptors PMP22 and EMP3 and cell adhesion molecule N-cadherin. Making use of real-time PCR, consistently increased steady-state levels of mRNAs for these genes were found in an extended panel of invasive and noninvasive mammary cancer cell lines.

The pesticide malathion induces alterations in actin cytoskeleton and in cell adhesion of cultured breast carcinoma cells.

We have studied the effects of the organophosphorous pesticide malathion on cell viability, actin cytoskeleton, cell adhesion complex E-cadherin/beta-catenin, and Rho and Rac1 GTPases from the human mammary carcinoma cell line MCF-7. Malathion induced cell lethality, determined by the MTT assay, depending on the treatment conditions. Cells incubated with low concentrations of malathion, 16-32 microg/ml, showed high survival rates (>95%) at any evaluated time (1-5 days), whereas complete cell lethality was found using 512 microg/ml and 5 days of treatment. Deep morphological changes were induced with high doses of 64 and 128 microg/ml, and long incubation time (5 days); cells showed perinuclear vacuoles, rounding, shrinkage, and a gradual loss of adhesion. These changes were related to a decrease in the expression of the adhesion molecules, E-cadherin and beta-catenin, and to the distribution and reactivity of actin microfilaments to TRITC-phalloidin. Disruption of microfilaments, accompanied by the collapse of actin to perinuclear region, were characteristic of cells with loss of adhesion. At lower concentrations, some cells presented deformations on the plasma membrane as lamellipodia-like structures, which were particularly evident from 32 to 128 microg/ml. Conversely, we observed an increase in the expression of Rho and Rac1 GTPases, modulators of actin cytoskeleton and cell adhesion.

Alternative splicing of the human estrogen receptor α primary transcript: Mechanisms of exon skipping

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.

Negative regulation of BRCA1 gene expression by HMGA1 proteins accounts for the reduced BRCA1 protein levels in sporadic breast carcinoma.

A drastic reduction in BRCA1 gene expression is a characteristic feature of aggressive sporadic breast carcinoma. However, the mechanisms underlying BRCA1 downregulation in breast cancer are not well understood. Here we report that both in vitro and in vivo HMGA1b protein binds to and inhibits the activity of both human and mouse BRCA1 promoters. Consistently, murine embryonic stem (ES) cells with the Hmga1 gene deleted display higher Brca1 mRNA and protein levels than do wild-type ES cells. Stable transfection of MCF-7 cells with the HMGA1b cDNA results in a decrease of BRCA1 gene expression and in a lack of BRCA1 induction after estrogen treatment. Finally, we found an inverse correlation between HMGA1 and BRCA1 mRNA and protein expression in human mammary carcinoma cell lines and tissues. These data indicate that HMGA1 proteins are involved in transcriptional regulation of the BRCA1 gene, and their overexpression may have a role in BRCA1 downregulation observed in aggressive mammary carcinomas.

Human Growth Hormone-regulated HOXA1 Is a Human Mammary Epithelial Oncogene

Increased mammary epithelial expression of the human growth hormone (hGH) gene is associated with the acquisition of pathological proliferation. We report here that autocrine hGH production by human mammary carcinoma cells increased the expression and transcriptional activity of the homeobox domain containing protein HOXA1. Forced expression of HOXA1 in human mammary carcinoma cells resulted in increased total cell number primarily by the promotion of cell survival mediated by the transcriptional up-regulation of Bcl-2. HOXA1 also abrogated the apoptotic response of mammary carcinoma cells to doxorubicin. Forced expression of HOXA1 in mammary carcinoma cells, in a Bcl-2-dependent manner, resulted in dramatic enhancement of anchorage-independent proliferation and colony formation in soft agar. Finally, forced expression of HOXA1 was sufficient to result in the oncogenic transformation of immortalized human mammary epithelial cells with aggressive in vivo tumor formation. Herein, we have therefore provided a molecular mechanism by which autocrine hGH stimulation of human mammary epithelial cells may result in oncogenic transformation.

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation.

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

Monosialyl-Gb5 organized with cSrc and FAK in GEM of human breast carcinoma MCF-7 cells defines their invasive properties

Two human mammary carcinoma cell variants, MCF-7/AZ and MCF-7/6, show the same composition in their glycosphingolipid-enriched microdomain (GEM) with regard to globo-series structures Gb3, Gb4, Gb5, monosialyl-Gb5, GM2, and cSrc and FAK. Both variants are non-invasive into collagen gel layer, and showed similar motility in wound migration assay. Whereas invasiveness and motility of MCF-7/AZ cells were enhanced greatly by treatment with mAb RM1 directed to monosialyl-Gb5, the same RM1 treatment had no effect on MCF-7/6. cSrc and FAK of MCF-7/AZ, but not MCF-7/6, were activated by RM1 treatment. Thus, malignancy of MCF-7 is highly dependent on monosialyl-Gb5, and its activation of cSrc and FAK in GEM.

Induction of fascin spikes in breast cancer cells by activation of the insulin-like growth factor-I receptor

Insulin-like growth factor-I receptor (IGF-IR) signaling contributes to the formation of mammary carcinomas and has chiefly been studied with regard to the proliferative and anti-apoptotic effects of IGF-IR signaling. However, IGF-IR activation also affects the actin cytoskeleton and alterations in cell migratory behavior are of known importance for the malignant conversion and metastasis of epithelial cells. The actin-binding protein fascin is found in cell projections and spikes that are involved in the locomotion of mesenchymal cells. Fascin expression is typically low in normal epithelial cells, but is markedly upregulated in several types of carcinomas. Here, we also demonstrate increased fascin expression in breast carcinoma cell lines and adopt MCF-7 human mammary carcinoma cells that over-express wild-type or kinase-inactivated forms of the IGF-IR as a model system to test the hypothesis that IGF-IR activation induces fascin projections. We show that the time-dependent dissociation of cell colonies that occurs upon receptor activation by IGF-I involves the formation of dynamic, fascin-containing lateral cell projections that co-localize with ruffling membranes in association with protrusive activity and cell migratory phenotype. The molecular mechanism of these effects is completely dependent on IGF-IR tyrosine kinase activity and is mediated by a phosphatidylinositol (PI) 3-kinase-dependent process. In demonstrating transduction of fascin spike assembly by activation of a peptide growth factor receptor, these novel data reveal a wide role for fascin spikes in cell motility and provide new insight into the complex effects of IGF-IR signaling on actin cytoskeletal organization.

Studies on the inhibition of tumor cell growth and microtubule assembly by 3-hydroxy-4-[( E)-(2-furyl)methylidene]methyl-3-cyclopentene-1,2-dione, an intensively coloured Maillard reaction product

Very recently, 3-hydroxy-4-[( E)-(2-furyl)methylidene]methyl-3-cyclopentene-1,2-dione (1) has been successfully identified as an intensively coloured Maillard product formed from glucose and l-proline upon thermal food processing. Using a biomimetic synthetic strategy, reference material of compound 1 was prepared and purified, and then used to study its effect on the growth of human tumor cells. Compound 1 was found to potently inhibit the growth of human tumor cells in vitro. Using a reporter gene assay we could show that in growth inhibitory concentrations compound 1 effectively inhibits the phosphorylation of the transcription factor Elk-1. In addition, 1 was found to affect the microtubule skeleton. The human mammary carcinoma cell line MCF-7 exhibits a decrease of the microtubule organisation when treated for 24 h with 1 (⩾20 μ m). At concentrations of 30 μ m and above a loss of microtubule integrity is observed after 1 h incubation. In vitro studies demonstrated that the polymerisation and, to a minor extent, also the depolymerisation of tubulin, isolated and purified from bovine brain, is inhibited in a dose-dependent manner at concentrations of 30 μ m and above. This is the first time that a non-enzymatically formed browning compound of known structure was reported to effectively inhibit tumor cell growth and microtubule assembly.