Carnosic acid inhibits the proliferation and migration capacity of human colorectal cancer cells.

Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. The objective of this study was to examine whether carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., would inhibit the cell viability of three CRC cell lines: Caco-2, HT29 and LoVo in a dose-dependent manner, with IC50 values in the range of 24-96 µM. CA induced cell death by apoptosis in Caco-2 line after 24 h of treatment and inhibited cell adhesion and migration, possibly by reducing the activity of secreted proteases such as urokinase plasminogen activator (uPA) and metalloproteinases (MMPs). These effects may be associated through a mechanism involving the inhibition of the COX-2 pathway, because we have determined that CA downregulates the expression of COX-2 in Caco-2 cells at both the mRNA and protein levels. Therefore, CA modulates different targets involved in the development of CRC. These findings indicate that carnosic acid may have anticancer activity and may be useful as a novel chemotherapeutic agent.

TNF-alpha enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B.

Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-alpha on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-alpha exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-kappaB. Importantly, activation of NF-kappaB was required for estrogen-induced proliferation and cyclin D1 expression. TNF-alpha enhanced the estrogen response by increasing the levels and availability of NF-kappaB. Chromatin immunoprecipitation analysis suggested that the action of estrogens is mediated by a protein complex that contains the activated estrogen receptor, the nuclear receptor coactivator RAC3 and a member of the NF-kappaB family. Finally, our results demonstrate that activation of this transcription factor could be one of the key signals for estrogen-mediated response.

Differential expression of CPD1 during postnatal development in the mouse cerebellum.

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.

NF-kappaB activation is involved in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) by interleukin-1beta.

Interleukin-1 beta (IL-1beta) regulates the levels of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein in the T84 human carcinoma cell line. Here, we studied the role of the transcription factor NF-kappaB in this regulation. Initially, T84 cells were pretreated with the NF-kappaB inhibitor pyrrolidine dithiocarbamate. Cells were then stimulated with IL-1beta, and CFTR mRNA levels were determined after 4 h by Northern blot analysis. As a result of PDTC treatment, IL-1beta stimulation of CFTR mRNA was blocked. On the other hand, daunorubicin, an NF-kappaB activator, increased the steady-state levels of CFTR mRNA. Furthermore, after treatment with IL-1beta for 1 h, cytoplasmic IkappaBalpha degradation occurred simultaneously with translocation of p65 into the nucleus. The T84 cells were also transduced with an adenoviral vector expressing a dominant negative form of IkappaBalpha, which prevents IkappaBalpha phosphorylation and the subsequent nuclear translocation of NF-kappaB. After viral transduction, the cells were stimulated with IL-1beta for 4 h, and CFTR mRNA levels were measured by Northern blot analysis. The stimulation of CFTR, induced by IL-1beta, was also blocked in the presence of the dominant negative mutant. These results indicate that NF-kappaB is involved in the pathway by which IL-1beta regulates CFTR.

Interleukin-1beta regulates CFTR expression in human intestinal T84 cells.

Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.

Development of monoclonal oligobodies and chemically synthesized oligobodies.

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.

Development of monoclonal oligobodies and chemically synthesized oligobodies

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.

Development of monoclonal oligobodies and chemically synthesized oligobodies.

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.

Oligobodies: bench made synthetic antibodies.

Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.

Oligobodies: bench made synthetic antibodies.

Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as [quot ]synthetic antibodies[quot ]. The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named [quot ]oligobodies[quot ], has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.

TGF-beta1 up-regulates the mRNA for the Na+/Ca2+ exchanger in neonatal rat cardiac myocytes.

Northern analyses of neonatal cardiac myocytes demonstrated that TGF-beta1 (5 ng/ml) stimulates and IL-1beta (5 ng/ml) decreases the steady-state levels of the mRNA coding for the Na+/Ca2+ exchanger. This is in agreement with the effects of TGF-beta1 and IL-1beta on beating rate and calcium uptake, suggesting that such effects might be mediated, at least partially, through up-regulation of the Na+/Ca2+ exchanger. Basal and TGF-beta1 stimulated mRNA levels were inhibited by the PKC inhibitors H7 (10 microM) and GF109203X (250 nM). In addition, apigenin (12.5 microM), a MAP kinase inhibitor, was able to inhibit basal mRNA levels for the exchanger. Cycloheximide (35.5 microM) had no effect on basal mRNA levels for the exchanger but steady-state levels were diminished in cells treated with TGF-beta1. Finally, actinomycin D (10 microM) inhibited both basal and TGF-beta1 stimulated mRNA levels, though with a more pronounced effect in the presence of TGF-beta1. These results suggest that a complex mechanism of regulation exists for the exchanger and that PKC and possibly MAP kinases might be involved. The up-regulation of this important protein for calcium extrusion, induced by TGF-beta1, might prepare cells to better overcome the calcium overload which occurs under cellular stress and might explain some of the cytoprotective effects of TGF-beta1.

Identification by differential display of a mRNA specifically induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in T84 human colon carcinoma cells.

12-o-Tetradecanoylphorbol-13-Acetate (TPA) down-regulates the expression of the gene responsible for cystic fibrosis (CFTR). To understand the mechanism by which TPA down-regulates CFTR, we decided to study genes specifically induced by this phorbol ester in T84 human colon carcinoma cells, which highly express CFTR, using differential display. Several strategies that allowed us to overcome false-positive reactions in differential displays are described. We have detected different cDNAs obtained from mRNAs specifically induced by TPA. A cDNA fragment corresponding to a mRNA of approximately 2.2 kb was sequenced. Part of this sequence has been reported by others in GenBank and corresponds to a cDNA from a human lung library. The function is unknown and does not have any significant homology with other sequences. It is expressed after two hrs in T84 cells treated with TPA, reaching a maximum response by four hrs. The dose-response curve shows increased mRNA levels starting at 10 ng/ml of TPA and reaching a maximum by 50 ng/ml TPA (10-fold stimulation over control). This mRNA shows a rapid and large response to TPA and it might be involved in the differentiation of T84 cells induced by TPA.

Increased secretory activity and estradiol receptor expression are among other relevant aspects of MCF-7 human breast tumor cell growth which are expressed only in the absence of serum.

We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.

Role of thrombin in the proliferative response of T-47D mammary tumor cells. Mitogenic action and pleiotropic modifications induced together with epidermal growth factor and insulin.

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell line from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 32P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells.

A novel mechanism of resistance to alpha-difluoromethylornithine induced by cycloheximide. Growth with abnormally low levels of putrescine and spermidine.

Treatment of the chemically transformed fibroblasts BP-A31 and other cell lines with low concentrations of cycloheximide (CHM) for 72 h followed by the removal of the protein synthesis inhibitor leads to the proliferation of alpha-difluoromethylornithine (DFMO)-resistant phenotypes. These drug-resistant cells contain almost no ornithine decarboxylase (ODC) activity and concomitantly very low levels of putrescine and spermidine. Southern blot analysis and measurements of ODC activity and intracellular polyamine levels showed that the described mechanism of inducing resistance to DFMO triggered by CHM does not involve ODC gene amplification, altered transport of the drug or reduced affinity of the enzyme for DFMO.