Fibroblasts

Human esophageal squamous cell carcinoma (hESCC) is a very aggressive form of cancer due to its ability to easily metastasize into proximal lymph nodes and adjacent organs. The role of the extracellular matrix (ECM) and its stromal cells in metastasis remains unclear. To better understand the effect of the ECM and fibroblast cells on esophagus cancer cell migration and invasion, we propose a biomimetic human esophagus model cultured with hESCC and human primary fibroblast cells (fibroblast). To mimic the extracellular matrix of human esophagus we use decellularized porcine esophagus matrix (DEM) to culture with hESCC and fibroblasts in static conditions. This DEM can recapitulate the human esophagus tumor microenvironment with relevant cues. This model will provide valuable information regarding esophagus cancer cell migration with respect to the heterogeneous extracellular matrix and stromal fibroblast cells. We expect to discover the mechanisms by which extracellular matrix and stromal cells affect cancer migration and invasion in vitro. Characterizing this process will provide vital insight towards the effects of fibroblasts cells on facilitating migration and invasion of esophageal cancer cells. This esophagus cancer model also provides promising potential to study drug screening and develop new strategies against esophagus metastasis.

The majority of research on drug addiction centers on dopamine (DA)- driven synaptic plasticities and how these changes ultimately lead to compulsive drug seeking. However, growing evidence supports a role of glial factors in various steps that lead to drug abuse and addiction. In this regard, significant evidence implicates glial glutamate (Glu) transporters (GLT-1) and cystine/Glu exchangers (xCT) in determining synaptic and extrasynaptic levels of Glu that support the acute and chronic actions of drugs of abuse. -lactam antibiotics have been found in rodent models to upregulate CNS GLT-1 and xCT and thereby contribute to reinstatement after chronic drug exposure and withdrawal.
Previously, the Blakely lab identified a glial expressing gene, swip-10, in Caenorhabditis elegans, whose deletion results in the hyperdominergic phenotype Swimming-Induced Paralysis (Swip), supported by Glu signalingdependent DA neuron hyperexcitability that ultimately drives oxidative stress and DA neuron degeneration. Both SWIP-10 and its putative mammalian ortholog MBLAC1 possess a highly conserved metallo -lactamase domain, and MBLAC1 has been found to bind the Glu modulating, b-lactam antibiotic ceftriaxone (Cef). Indeed, immunodepletion studies indicate that MBLAC1 may be the major highaffinity Cef-binding protein in the brain, leading to the hypothesis that MBLAC1 has a Glu modulatory role(s). Recently a functional role of MBLAC1 been proposed, involving activity as a 3’ exonuclease that processes polyA- mRNAs, including RNAs encoding cell replication-dependent histones. How this role, or others, may support the actions of MBLAC1 in the brain and the non-microbial actions of Cef to extracellular Glu homeostasis, is unclear. Recently, the Blakely lab generated Mblac1-/- mice as a tool to investigate these issues. The following work investigated the requirements of MBLAC1 in growth and the actions of Cef in mouse embryonic fibroblasts (MEFs) cultured from either Mblac1+/+ and Mblac1-/- mice. The presented data suggested that Mblac1-/- MEFs display attenuated growth and cell proliferation relative to Mblac1+/+ MEFs. For the first time, the in vitro protective actions of Cef against oxidative stress is shown to be dependent on MBLAC1. The following studies presented contribute to a definition of the role of MBLAC1 and as a Cef binding protein in native preparations, with findings that can drive models for the role of MBLAC1 in the CNS.

When infected with reovirus ST3, strain Dearing, normal and transformed human lung fibroblasts exhibit differential sensitivities. Transformed cells (WI38 VA13 2RA) are destroyed within four days. In contrast, normal cells (WI38) maintain a productive infection for as long as two months. Attempts to examine the differences between these cells included the use of cDNA subtraction and a reovirus sigma 1 protein affinity chromatography column, both of which were hampered by technical difficulties. Two-dimensional gel electrophoresis revealed patterns of differential protein expression between infected and mock-infected normal and transformed cells. Visual comparison of Coomassie blue-stained gels revealed one protein which was present in uninfected normal cells but was missing in all other samples, as well as five proteins that were present in infected and mock-infected normal cells but were missing in both transformed cell samples. Autoradiography of 35S-labeled cell samples revealed an additional eleven proteins not seen with Coomassie staining. Further characterization of these proteins should uncover the mechanism of differential cell killing by reovirus.

Increased risk for cancer in individuals with hereditary adenomatosis of the colon and rectum (ACR) has been hypothesized as resulting from a regulatory gene mutation which allows the tissue specific partial expression of a growth factor. Eight ACR and 4 normal control dermal fibroblast cell strains were grown at various concentrations of dialyzed serum protein in MCDB 104. Normal and ACR strains grew best at similar serum concentrations and grew at similar rates in low serum concentrations. Growth rates of normal and ACR strains declined similarly with increasing cell density. The equation Y = (AX/(X + B)) - CX was developed to analyze the shape of the serum dose response curves, where: Y = growth rate, X = serum protein concentration, and values of A, B, and C are constants. The A, B, and C constants for 4 normal and 4 ACR cell strains were found to be similar. It was concluded that there was no evidence for the constitutive production of a growth factor in ACR dermal fibroblasts.

Reovirus is a common virus that usually affects children; this infection causes symptoms such as respiratory and or gastrointestinal aliments. Morbidity most often occurs in impoverished countries where supportive hospitalization is not available. In the U.S. and other established countries morbidity is not an issue. When WI-38 cells are infected with reovirus the infection is either resisted by the cells or a persistent latent infection occurs. In this study, gene expression was analyzed by comparing Reovirus-infected WI-38 cells with mock infected cells. P.R.O.M(TM) analysis was performed on RNA sent to Genka Biotechnology Inc. Bioinformatics was used to analyze the data. Reovirus infection of the WI-38 cells resulted in increased mRNA levels for a number of transcription regulatory genes, and possibly more significant, decreased mRNA levels for some very important regulatory genes. These changes may be responsible for establishing the antiviral replication environment observed in these normal cells.