Culture of Tumorigenic Cells on Protein Fibers Reveals Metastatic Cell Behaviors.

Tumorigenic cell behaviors can be suppressed or enhanced by their physicochemical environment. As a first step toward developing materials that allow tumorigenic behaviors to be observed and manipulated, we cultured related MCF10 breast cell lines on fibers composed of the Drosophila protein Ultrabithorax (Ubx). These cell lines, originally derived from fibrocystic breast tissue, represent a continuum of tumorigenic behavior. Immortal but nontumorigenic MCF10A cells, as well as semitumorigenic MCF10AT cells, attached and spread on Ubx fibers. MCF10CA-1a cells, the most highly transformed line, secreted high concentrations of matrix metalloproteinases when cultured on Ubx materials, resulting in differences in cell attachment and cytoskeletal structure, and enabling invasive behavior. Because the mechanical and functional properties of Ubx fibers can be genetically manipulated, these materials provide a valuable tool for cancer research, allowing creation of diverse microenvironments that allow assessment of invasive, metastatic behavior.

Materials composed of the Drosophila Hox protein Ultrabithorax are biocompatible and nonimmunogenic.

Although the in vivo function of the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) is to regulate transcription, in vitro Ubx hierarchically self-assembles to form nanoscale to macroscale materials. The morphology, mechanical properties, and functionality (via protein chimeras) of Ubx materials are all easily engineered. Ubx materials are also compatible with cells in culture. These properties make Ubx attractive as a potential tissue engineering scaffold, but to be used as such they must be biocompatible and nonimmunogenic. In this study, we assess whether Ubx materials are suitable for in vivo applications. When implanted into mice, Ubx fibers attracted few immune cells to the implant area. Sera from mice implanted with Ubx contain little to no antibodies capable of recognizing Ubx. Furthermore, Ubx fibers cultured with macrophages in vitro did not lyse or activate the macrophages, as measured by TNF-α and NO secretion. Finally, Ubx fibers do not cause hemolysis when incubated with human red blood cells. The minimal effects observed are comparable with those induced by biomaterials used successfully in vivo. We conclude Ubx materials are biocompatible and nonimmunogenic.

Materials composed of the Drosophila melanogaster protein ultrabithorax are cytocompatible.

The Drosophila melanogaster Hox protein ultrabithorax (Ubx) has the interesting ability to hierarchically self-assemble in vitro into materials that have mechanical properties comparable to natural elastin. Ubx materials can be easily functionalized by gene fusion, generating potentially useful scaffolds for cell and tissue engineering. Here, we tested the cytocompatibility of fibers composed of Ubx or an mCherry-Ubx fusion protein. Fibers were cultured with three primary human cell lines derived from vasculature at low passage: umbilical vein endothelial cells, brain vascular pericytes, or aortic smooth muscle cells. No direct or indirect toxicity was observed for any cell line, in response to fibers composed of either plain Ubx or mCherry-Ubx. Cells readily adhered to Ubx fibers, and cells attached to fibers could be transferred between tissue cultures without loss of viability for at least 96 h. When attached to fibers, the morphology of the three cell lines differed somewhat, but all cells in contact with Ubx fibers exhibited a microtubular network aligned with the long axis of Ubx fibers. Thus, Ubx fibers are cytocompatible with cultured primary human vascular cells.

Vascular smooth muscle cells: isolation, culture, and characterization.

Vascular smooth muscle cells (VSMCs) are the cellular components of the normal blood vessel wall that provides structural integrity and regulates the diameter by contracting and relaxing dynamically in response to vasoactive stimuli. The differentiated state of the VSMC is characterized by specific contractile proteins, ion channels, and cell surface receptors that regulate the contractile process and are thus termed contractile cells. In addition to these normal functions, in response to injury or during development, VSMCs are responsible for the synthesis of extracellular matrix proteins, become migratory and proliferate. This phenotype has been termed synthetic cells. To better understand the mechanisms regulating these and other processes, scientists have depended on cultured cells that can be manipulated in vitro. In this chapter, we will discuss in detail the explant method for isolation of VSMC and will compare it to the enzymatic digestion method. We will also briefly describe methods for characterizing the resulting cells.

Modulation of cyclin dependent kinase inhibitor proteins and ERK1/2 activity in allylamine-injured vascular smooth muscle cells.

Chronic oxidative injury by allylamine (AAM) induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. The proliferative advantage of AAM vSMC compared to control cells is maintained with serial passage of the cells and the advantage is nullified when AAM cells are seeded on a collagen substrate. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p27 and p21, and mitogen activated protein (MAP) kinases, ERK1/2, in mediating the proliferative advantage of AAM stressed vSMC over control cells on plastic or collagen substrates. p27 levels in randomly cycling cells were comparable in both cell types irrespective of the substrate. In contrast, basal levels of p21 were 1.9 +/- 0.3 (P < 0.05)-fold higher in randomly cycling AAM cells seeded on plastic compared to controls, a difference that was lost on a collagen substrate. Following G0 synchronization, basal levels of both p27 and p21 were higher in AAM cells seeded on plastic compared to controls (1.7 +/- 0.2 and 2.0 +/- 0.3-fold, respectively, P < 0.05), but these differences were lost upon mitogenic stimulation. Pyrrolidine dithiocarbamate (PDTC) decreased p27 and p21 levels in cycling AAM cells relative to controls in a substrate-dependent manner. AAM cells seeded on plastic exhibited enhanced ERK1/2 activation upon mitogenic stimulation; seeding on collagen nullified this advantage. The duration of ERK1/2 activation was prolonged in AAM cells independently of the seeding substrate. We conclude that substrate-dependent acquisition of proliferative phenotypes following repeated cycles of AAM injury correlates with modulation of the cyclin dependent kinase inhibitors, p27 and p21.

Regulation of alpha7-integrin expression in vascular smooth muscle by injury-induced atherosclerosis.

Injury of vascular smooth muscle cells (VSMCs) by allylamine (AAM) leads to phenotypic changes associated with atherogenic progression including increased proliferation, migration, and alterations in cell adhesion. In the present study, the relationship between AAM-induced vascular injury and expression of the alpha(7)-integrin subunit was investigated. The alpha(7)-mRNA and protein expression were examined using real-time RT-PCR, fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and immunoblotting. In cultured VSMCs from aortas of AAM-treated rats (70 mg/kg for 20 days), alpha(7)-mRNA levels were increased more than twofold compared with control cells. No change was seen in beta(1)-integrin expression. FACS analysis revealed increased cell surface expression of alpha(7)-protein (25 +/- 9%; *P < 0.05). AAM treatment of naive VSMCs enhanced alpha(7)-mRNA expression (2.4 +/- 0.7-fold, mean +/- SE; *P < 0.05). The increased alpha(7)-mRNA expression was attenuated by the amine oxidase inhibitor semicarbazide and the antioxidant pyrrolidine dithiocarbamate, which confirms a role for oxidative stress in modulating alpha(7)-expression. In vivo alpha(7)-mRNA and protein expression were enhanced in the aortas of AAM-treated rats. In addition, increased alpha(7)-integrin expression facilitated AAM VSMC adhesion to laminin more efficiently compared with control (51 +/- 2%; *P < 0.05). Chemical injury induced by AAM significantly enhances alpha(7)-integrin expression in VSMCs. These findings implicate for the first time the expression of alpha(7)-integrin during the response of VSMCs to vascular injury.