In order to better understand colon cancer, a model system reflecting the heterogenous nature of this disease was developed and used in the development of new cytotoxic and non-cytotoxic therapeutic approaches. A large bank of colon carcinoma cell lines was established from primary human colon carcinomas and grouped based on their tumorigenicity in athymic mice, their growth rates in soft agarose and in tissue culture, and their secreted levels of carcinoembryonic antigen. These cell lines were later characterized based on cell surface proteins and antigens detected with antisera raised against a differentiated colon carcinoma cell line. Although these biochemical markers correlated with the biological classification of these cell lines, there was still extensive heterogeneity within each group in all properties examined. This colon carcinoma cell system was used to study natural vs. selected resistance to the anticancer drug mitomycin C (MMC). The differing IC50 values in vitro were reflected in the inhibition by MMC of xenograft growth in athymic mice. A new, more readily bioactivatable analogue of MMC was tried and shown to be more active in vitro and in vivo, suggesting that rapid efflux of the drug before activation may be important in examining causes of resistance to MMC. Another approach to the treatment of colon cancer is the use of non-cytotoxic agents such as growth factors and differentiation agents to restore normal growth to the malignant cells. We have isolated and characterized two types of polypeptides from colon carcinoma cells and conditioned medium from these cells. The first, transforming growth factors (TGF's) confer a transformed phenotype on non-transformed fibroblasts while the second, tumor inhibitory factors (TIF's), inhibits the anchorage independent growth of transformed cells. The fact that extracts of colon carcinoma cells contain both activities suggests that the heterogeneity of the cell lines could be due to different levels of TGF's and TIF's produced. The effectiveness of differentiation agents to restore normal growth control using a transformed mouse embryo cell line was examined. Treatment of these cells with differentiation agents restored normal growth control to these cells. An increased synthesis of TGF's resulted from these treatments. Therefore, differentiation agents may be useful in non-cytotoxic treatment. The use of this model system for human colon carcinoma will hopefully lead to more effective drugs for the treatment of colon cancer in man.

An siRNA directed against the extracellular calcium‐sensing receptor (CaSR) was used to down‐regulate this protein in CBS colon carcinoma cells. In additional studies, we utilized a variant of the parental CBS line that demonstrates CaSR expression but does not upregulate this protein in response to extracellular Ca2+. In neither the siRNA‐transfected cells nor the Ca2+‐nonresponsive variant cells did inclusion of Ca2+ in the culture medium inhibit proliferation or induce morphological alterations. Extracellular Ca2+ also failed to induce E‐cadherin production or a shift in β‐catenin from the cytoplasm to the cell membrane. In mock‐transfected cells and in a Ca2+‐responsive variant line derived from the same parental CBS cells, Ca2+ treatment resulted in growth‐reduction. This was accompanied by increased E‐cadherin production and a shift in β‐catenin distribution from the cytoplasm to the cell membrane. Additionally, down‐regulation of c‐myc and cyclin D1 expression was observed in mock‐transfected cells and in the Ca2+‐responsive variant line (along with reduced T cell factor transcriptional activation). Neither c‐myc nor cyclin D1 was significantly down‐regulated in the siRNA‐transfected cells or in the Ca2+‐nonresponsive variant cells upon Ca2+ stimulation. In histological sections of human colon carcinoma CaSR was significantly reduced as compared to the level in normal colonic crypt epithelial cells. Where CaSR expression was high, strong surface staining for E‐cadherin and β‐catenin was observed. Where CaSR expression was reduced, β‐catenin surface expression was likewise reduced. © 2007 Wiley‐Liss, Inc.

Vitamin D (VD) protects against colon carcinogenesis by mechanisms not fully understood. We had earlier reported on the similarity in the biologic action of VD and that of the calcium-sensing receptor (CaSR) in human colon carcinoma cells. At the molecular level, the CaSR gene contains 2 VD response elements and VD stimulates the expression of CaSR. In this study, we investigated on the relationship between VD action and CaSR function. We determined and compared the action of VD in human colon carcinoma cells (CBS, Moser, Caco-2 and HCT116) and their CaSR knocked-down counterparts. VD inhibited cellular proliferation, cellular invasion, and anchorage-independent growth and stimulated the expression of p21/Waf1 but not in CaSR knocked-down cells. These results demonstrate, for the first time, that the known tumor-suppressive function of VD requires functional CaSR and knocking down CaSR expression abrogated this function of VD. We recently reported that activation of CaSR in human colon carcinoma cells downregulated the expression of thymidylate synthase (TS) and survivin and promoted a significant increase in sensitivity to cytotoxic drugs. We now demonstrate, for the first time, that VD suppressed the expression of TS and survivin, TS and survivin gene transcriptional activities and promoted a cytotoxic response to 5-FU in a CaSR-dependent manner. Ectopic expression of wild-type CaSR in colon carcinoma cells also inhibited the expression of TS and survivin and enhanced cellular sensitivity to 5-FU. VD, however, could no longer enhance cellular sensitivity to 5-FU in cells overexpressing CaSR.

Parabenzoquinone (1,4-benzoquinone) (PBQ) is a bioactve quinone present in cigarette smoke and diesel smoke, which causes severe genotoxic effects both in vitro and in vivo. In the previous study, we showed that the microtubules are one of the major targets of cigarette smoke-induced damage of lung epithelium cells. In the present study, we have investigated the effect of PBQ on cellular microtubules using human type II lung epithelial cells (A549) and also on purified tubulin. Cell viability experiments using A549 cells indicated a very low IC(50) value (approximately 7.5 microM) for PBQ. PBQ inhibited cell cycle progression and induced apoptosis of A549 cells. PBQ also induced the contraction and shrinkage of the A549 cells in a time- and concentration-dependent manner, which is proved to be a direct effect of the damage of the microtubule cytoskeleton network, and that was demonstrated by a immunofluorescence study. PBQ also inhibited the assembly of tubulin in lung cells and a in cell free system (IC(50) approximately 5 microM). Treatment with PBQ resulted in the degradation of tubulin in lung cells without affecting the actin network, and this was confirmed by a Western blot experiment. Upregulation of pro-apoptotic proteins such as p53 and Bax and downregulation of antiapoptotic protein Bcl-2 were observed in PBQ-treated A549 cells. Simultaneously, loss of mitochondrial membrane potential and activation of caspase-3 were also observed in the PBQ treated lung epithelium cells. Fluorescence and circular dichroism studies demonstrated that the denaturation of tubulin in a cell free system was caused by PBQ. However, in the presence of N-acetyl cysteine (NAC), damage of the microtubule network in A549 cells by PBQ was prevented, which led to a significant increase in the viability of A549 cells. These results suggest that microtubule damage is one of the key mechanisms of PBQ induced cytotoxity in lung cells.

Ca 2+ and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/ receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca 2+ (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-regulated the expression of 5-FU target, thymidylate synthase (TS) and the anti-apoptotic protein survivin. Cells that were resistant to drugs expressed little or no CaSR but abundant amount of survivin. Disruption of CaSR expression by shRNA targeting the CaSR abrogated these modulating effects of CaSR activation on the expression of NQO1, TS, survivin and cytotoxic response to drugs. It is concluded that activation of CaSR can enhance colon cancer cell sensitivity to MMC and 5-FU and can modulate the expression of molecules involved in the cellular responses to these cytotoxic drugs.

We have previously characterized the diversity of cellular responses to transforming growth factor (TGF)-beta 1 from human colon carcinoma cells. We now show that morphological alteration and part of the growth-inhibitory responses elicited by growth factor (GF) are associated with the secondary effect of the induction of synthesis of extracellular matrix (ECM) glycoproteins. Specifically, morphological alteration is associated with the ECM glycoprotein laminin, and growth inhibition is associated with both laminin and fibronectin. Both TGF-beta 1 and TGF-beta 2 down-modulate the expression of nucleolar protein B23 (also known as numatrin or nucleophosmin, a positive regulator of cell proliferation). With one exception, the biological effects of both TGF-beta 1 and TGF-beta 2 on these human colon carcinoma cell lines are identical. Both GFs up-modulate the expression of carcinoembryonic antigen (CEA) and CEA-related gene products. However, some of these products are differentially regulated by TGF-beta 1 and TGF-beta 2. The differences in the profile of the induction of these CEA and CEA-related gene products, in the responsive cells, functionally distinguish TGF-beta 1 from TGF-beta 2. Finally, we identified and characterized some of the cellular proteins, the expression of which is up-modulated by GF. These proteins are epithelium-associated, differentiation-related cytokeratins. Both TGF-beta 1 and TGF-beta 2 up-modulate expression of the acidic and basic subtypes of human keratins in the responsive human colon carcinoma cells. Both the responsive and unresponsive cells, however, possess receptors for GFs.

The effects of elastomer type on the morphology, flammability, and mechanical properties of high‐impact polystyrene (HIPS)/polystyrene (PS)‐encapsulated magnesium hydroxide (MH) were investigated. The ternary composites were characterized by cone calorimetry, mechanical testing, and scanning electron microscopy. Morphology was controlled with poly[styrene‐b‐(ethylene‐co‐butylene)‐b‐styrene] (SEBS) triblock copolymer or the corresponding maleinated poly[styrene‐b‐(ethylene‐co‐butylene)‐b‐styrene] (SEBS‐g‐MA). The HIPS/SEBS/PS‐encapsulated MH composites exhibited separation of the filler and elastomer, whereas the HIPS/SEBS‐g‐MA/PS‐encapsulated MH composites exhibited encapsulation of the filler by SEBS‐g‐MA. The flame‐retardant and mechanical properties of the ternary composites were strongly dependent on microstructure. The composites with an encapsulation structure showed higher flame‐retardant properties than those with a separation structure at the optimum use level of SEBS‐g‐MA. Furthermore, the composites with a separation structure showed a higher modulus and impact strength than those with an encapsulation structure. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci 2008

Transforming growth factor (TGF)-beta 1 modulates the expression of extracellular matrix (ECM) glycoproteins, fibronectin and laminin and the adhesion of Moser colon cancer cells to these glycoproteins. Since adhesion can be altered through expression of cell-surface receptors, binding affinities of adhesion molecules for receptors, or both, we investigated the effect of TGF-beta 1 on the binding properties of fibronectin and laminin to their cell-surface receptors by saturation binding and Scatchard analyses using radiolabeled fibronectin and laminin. Fibronectin bound to its cell-surface receptor with high affinity (Kd = 1.25 x 10(-9) M), Moser cells had approximately 7.1 x 10(4) fibronectin-binding sites per cell. TGF-beta 1 treatment rapidly up-modulated the number of cell-surface fibronectin-binding sites by 1.9-fold. The binding affinity of fibronectin for the receptor, however, was not altered. Laminin was found to bind to a higher-affinity and a lower-affinity receptor. Moser cells expressed approximately 1.1 x 10(3) higher-affinity laminin-binding sites and approximately 3.1 x 10(4) lower-affinity-binding sites per cell. TGF-beta 1 rapidly increased the expression of the higher-affinity sites 3-fold and the lower-affinity sites 5-fold. The binding affinity of both the higher-affinity and lower-affinity laminin receptors increased 3-fold after 2 and 6 hr of TGF-beta 1 treatment respectively. Concurrent with receptor modulation, TGF-beta 1 induced the secretion of fibronectin and laminin from Moser cells. Northern hybridization analyses showed a concurrent stimulation of the expression of the mRNAs for ligands (fibronectin and laminin) and the mRNAs for the integrin species of the fibronectin and laminin receptors (alpha 5 and alpha 6 subunits). Thus the production of fibronectin and laminin and the expression of their receptors were tightly co-regulated by TGF-beta 1.