Transfection of metastatic capability with total genomic DNA from human and mouse metastatic tumour cell lines

Abstract. From a large series of experiments involving transfer of high molecular weight total genomic DNA from highly metastatic human and mouse tumour cell lines to other mouse tumour cell lines we have derived a few cell lines with greatly augmented metastatic properties. In one of these experiments the transfected cell line (designated AH8 Test) not only colonised the lungs but also formed secondary tumour colonies in several extrapulmonary sites including the skin, skeletal muscles, bone, liver diaphragm, spleen and heart, There were no qualitative and quantitative effects of this magnitude when we used DNA from several non-metastatic or non-tumourigenic sources.
Secondary transfection of metastatic capability with DNA obtained from a metastasis formed by one of the primary transfectant lines (AH8 Test) has also been accomplished. Concomitant transfer of human DNA through both transfection cycles in this experiment was confirmed by a variety of methods including Southern blot analysis, in situ hybridisation and polymerase chain reaction (PCR) amplification of DNA using primers recognising human-specific Alu repeat sequences.
The findings offer opportunities for the isolation of sequences programming metastatic behaviour and we have cloned and sequenced a fragment of human DNA, which has not been previously characterised, from the transfected cells.     

Predicting the effects of a high‐energy diet on fatty liver and hippocampal‐dependent memory in male rats

Objective:

In rodents, diets exceeding nutritional requirements (i.e., high‐energy diets; HED) impair hippocampal‐dependent memory. Our research suggests that the effects likely involve HED‐induced increases in liver lipids. In this experiment, rats were provided with diet choices to test whether voluntary consumption of a HED impairs spatial memory, whether differences in initial weight gain predict memory deficits, and whether increases in liver lipids are associated with the memory deficits.

Design and Methods:

Adult male Sprague‐Dawley rats were given a control diet or cafeteria‐style HED for 8 weeks. Weight gain during the first 5 days on the diet was used to divide rats into a HED‐Lean group and a HED‐Obese group. Spatial water maze memory was tested 8 weeks later and postmortem liver lipid concentrations were quantified.

Results:

Compared with the HED‐Lean and control rats, the HED‐Obese rats had impaired spatial memory and met the human diagnostic criterion of non‐alcoholic fatty liver disease (>5% liver lipids relative to liver weight). Moreover, liver lipids were correlated with memory deficits.

Conclusions:

These findings show that voluntary consumption of a HED impairs memory, that initial weight gain predicts fatty liver and memory deficits, and that fatty liver may contribute to the memory‐impairing effects of obesity.     

Appropriateness of Using Patient-Derived Xenograft Models for Pharmacologic Evaluation of Novel Therapies for Esophageal/Gastro-Esophageal Junction Cancers

The high morbidity and mortality of patients with esophageal (E) and gastro-esophageal junction (GEJ) cancers, warrants new pre-clinical models for drug testing. The utility of primary tumor xenografts (PTXGs) as pre-clinical models was assessed. Clinicopathological, immunohistochemical markers (p53, p16, Ki-67, Her-2/neu and EGFR), and global mRNA abundance profiles were evaluated to determine selection biases of samples implanted or engrafted, compared with the underlying population. Nine primary E/GEJ adenocarcinoma xenograft lines were further characterized for the spectrum and stability of gene/protein expression over passages. Seven primary esophageal adenocarcinoma xenograft lines were treated with individual or combination chemotherapy. Tumors that were implanted (n=55) in NOD/SCID mice had features suggestive of more aggressive biology than tumors that were never implanted (n=32). Of those implanted, 21/55 engrafted; engraftment was associated with poorly differentiated tumors (p=0.04) and older patients (p=0.01). Expression of immunohistochemical markers were similar between patient sample and corresponding xenograft. mRNA differences observed between patient tumors and first passage xenografts were largely due to loss of human stroma in xenografts. mRNA patterns of early vs late passage xenografts and of small vs large tumors of the same passage were similar. Complete resistance was present in 2/7 xenografts while the remaining tumors showed varying degrees of sensitivity, that remained constant across passages. Because of their ability to recapitulate primary tumor characteristics during engraftment and across serial passaging, PTXGs can be useful clinical systems for assessment of drug sensitivity of human E/GEJ cancers.     

Long‐term survival of humpback whales radio‐tagged in Alaska from 1976 through 1978

Invasive tags designed to provide information on animal movements through radio or satellite monitoring have tremendous potential for the study of whales and other cetaceans. However, to date there have been no published studies on the survival of tagged animals over periods of years or decades. Researchers from the National Marine Mammal Laboratory and the Woods Hole Oceanographic Institution tracked five humpback whales with implanted radio tags in southeastern Alaska in August 1976 and July 1977, and tracked two humpback whales in Prince William Sound, Alaska, in June 1978. All seven of these individually identified humpback whales were resighted at least 20 yr after first being tagged, and five of the seven have been observed for more than 30 yr; some of them are among the most resighted humpback whales in the North Pacific. Photos of tagging sites taken during and subsequent to tagging operations show persistent but superficial scarring and no indication of infection. These pioneering field studies demonstrated both long‐term survival of the whales and the short‐term effects of deploying radio tags, which at the time were larger and more invasive than those typically used today.     

A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?

Atomic force microscopy has rapidly become a valuable tool for quantifying the biophysical properties of single cells. The interpretation of atomic force microscopy-based indentation tests, however, is highly dependent on the use of an appropriate theoretical model of the testing configuration. In this study, a novel, thin-layer viscoelastic model for stress relaxation was developed to quantify the mechanical properties of chondrosarcoma cells in different configurations to examine the hypothesis that viscoelastic properties reflect the metastatic potential and invasiveness of the cell using three well-characterized human chondrosarcoma cell lines (JJ012, FS090, 105KC) that show increasing chondrocytic differentiation and decreasing malignancy, respectively. Single-cell stress relaxation tests were conducted at 2 h and 2 days after plating to determine cell mechanical properties in either spherical or spread morphologies and analyzed using the new theoretical model. At both time points, JJ012 cells had the lowest moduli of the cell lines examined, whereas FS090 typically had the highest. At 2 days, all cells showed an increase in stiffness and a decrease in apparent viscosity compared to the 2-h time point. Fluorescent labeling showed that the F-actin structure in spread cells was significantly different between FS090 cells and JJ012/105KC cells. Taken together with results of previous studies, these findings indicate that cell transformation and tumorigenicity are associated with a decrease in cell modulus and apparent viscosity, suggesting that cell mechanical properties may provide insight into the metastatic potential and invasiveness of a cell.     

The role of MAPK signalling pathways in the response to endoplasmic reticulum stress

Perturbations in endoplasmic reticulum (ER) homeostasis, including depletion of Ca^2 + or altered redox status, induce ER stress due to protein accumulation, misfolding and oxidation. This activates the unfolded protein response (UPR) to re-establish the balance between ER protein folding capacity and protein load, resulting in cell survival or, following chronic ER stress, promotes cell death. The mechanisms for the transition between adaptation to ER stress and ER stress-induced cell death are still being understood. However, the identification of numerous points of cross-talk between the UPR and mitogen-activated protein kinase (MAPK) signalling pathways may contribute to our understanding of the consequences of ER stress. Indeed, the MAPK signalling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses. In this article, we review UPR signalling and the activation of MAPK signalling pathways in response to ER stress. In addition, we highlight components of the UPR that are modulated in response to MAPK signalling and the consequences of this cross-talk. We also describe several diseases, including cancer, type II diabetes and retinal degeneration, where activation of the UPR and MAPK signalling contribute to disease progression and highlight potential avenues for therapeutic intervention. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.