Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Early hCG-induced desensitization in Leydig cells.

The early mechanism of hCG induced down regulation of its own receptor as well as steroidogenesis refractoriness of rat Leydig cells to gonadotropin stimulation have been investigated. A single injection of 5, 12, 25, 50 and 100 IU of hCG in rats induced within 8 hours, Leydig cells desensitization. However, apparent receptor loss was significantly lower only in the rats who received 50 and 100 IU of hCG. Cycloheximide inhibits hCG-induced receptors loss but had no effect on hCG-induced desensitization. The most likely explanation for desensitization in the presence of binding sites and a normal adenylate cyclase, is a defective coupling between the receptor sites and the catalytic subunit.     

Antimetabolic activities of 2-fluoro-L-histidine.

2-Fluoro-L-Histidine inhibits protein synthesis in various cell cultures, as measured by ³H-leucine incorporation. This histidine analog also inhibits the cytopathogenicity of a number of RNA and DNA viruses in primary and continuous cell cultures; it blocks the transformation of normal mouse (MO) cells by murine sarcoma virus, and partially suppresses the release of murine leukemia virus by a continuously infected mouse cell line (JLSV5). In human skin fibroblasts, it reduces the interferon-inducing capacity of poly(I)·poly(C). Inhibition of cell protein synthesis may be the common cause of the various effects. 4-Fluoro-L-histidine is essentially inert in all of the test systems examined.     

Effect of High-Intensity Interval Training on Cardiac Apoptosis Markers in Methamphetamine-Dependent Rats

Chronic methamphetamine use increases apoptosis, leading to heart failure and sudden cardiac death. Previous studies have shown the importance of high-intensity interval training (HIIT) in reducing indices of cardiac tissue apoptosis in different patients, but in the field of sports science, the molecular mechanisms of apoptosis in methamphetamine-dependent rats are still unclear. The present article aimed to investigate the changes in cardiac apoptosis markers in methamphetamine-dependent rats in response to HIIT. Left ventricular tissue was used to evaluate caspase-3, melusin, FAK, and IQGAP1 gene expression. Rats were divided into four groups: sham, methamphetamine (METH), METH-control, and METH-HIIT. METH was injected for 21 days and then the METH-HIIT group performed HIIT for 8 weeks at 5 sessions per week. The METH groups showed increased caspase-3 gene expression and decreased melusin, FAK, and IQGAP1 when compared to the sham group. METH-HIIT showed decreased caspase-3 and increased melusin and FAK gene expression compared with the METH and METH-control groups. The IQGAP1 gene was higher in METH-HIIT when compared with METH, while no difference was observed between METH-HIIT and METH-control. Twenty-one days of METH exposure increased apoptosis markers in rat cardiac tissue; however, HIIT might have a protective effect, as shown by the apoptosis markers.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

Gap junction remodeling and altered connexin43 expression in the failing human heart

Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analyis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies.     

Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival

Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.     

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13-16% increase in maximal Ca²⁺-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca²⁺ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca²⁺] transients and increased time to peak force. Analysis of force and Ca²⁺ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca²⁺ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca²⁺ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.