Differential response of normal human fibroblasts to bombesin versus thrombin

Normal human diploid fibroblasts (WS-1 cells) were growth-arrested under serum-free conditions for 48 hr. The addition of fetal bovine serum (10% final concentration) to these cells stimulated [3H]-thymidine incorporation into DNA and phosphoinositide breakdown over nine-fold. Thrombin, at concentrations above 0.1 unit/ml (u/ml), was also effective at stimulating DNA synthesis and phosphoinositide breakdown as well as causing a rise in intracellular pH. In contrast, the peptide bombesin (concentrations ranging from 1 nM to 100 nM) stimulated phosphoinositide breakdown but did not enhance DNA synthesis or cause an increase in cytoplasmic pH. The time course of accumulation of inositol phosphates differed in response to these agents. The thrombin effect peaked rapidly and leveled off after 5 min while the bombesin effect showed a constant increase for 30 min. Serum showed an intermediate response. The different rates of inositol phosphate accumulation observed with the two growth factors is viewed as representing a difference in the mechanism of phosphoinositide turnover. The relationship between the difference in phosphoinositide turnover and the initiation of DNA synthesis is also discussed.     

Expression of TRAIL and TRAIL receptors in normal and malignant tissues

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a member of the TNF family of proteins.Tumour cells were initially found to have increased sensitivity to TRAIL compared with normal cells, raising hopes that TRAIL would prove useful as an anti-tumor agent. The production of reliable monoclonal antibodies against TRAIL and its receptors that can stain fixed specimens will allow a thorough analysis of their expression on normal and malignant tissues. Here we report the generation of monoclonal antibodies against TRAIL and its four membrane-bound receptors(TR1-4), which have been used to stain a range of normal and malignant cells, as routinely fixed specimens. Low levels of TRAIL expression were found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid. Expression of the TRAIL decoy receptors (TR3 and 4) was not as widespread as indicated by Northern blotting, suggesting that they may be less important for the control of TRAIL cytotoxicity than previously thought. TR1 and TR2 expression increases significantly in a number of malignant tissues,but in some common malignancies their expression was low, or patchy, which may limit the therapeutic role of TRAIL.Taken together, we have a panel of monoclonal antibodies that will allow a better assessment of the normal role of TRAIL and allow assessment of biopsy material, possibly allowing the identification of tumors that may be amenable to TRAIL therapy.     

Differential sensitivity to 4-hydroxynonenal for normal and malignant mesenchymal cells

Normal and malignant cells of various origin differ in their sensitivity to oxidative stress. Therefore, we used normal and malignant mesenchymal cells--human osteosarcoma cells (HOS and 143B), human fibroblasts (WI38) and two primary cultures of normal human osteoblasts to test sensitivity to reactive aldehyde 4-hydroxynonenal (HNE), known as a second messenger of free radicals and a signaling molecule. Upon HNE-treatment, decrease in cell viability (by Trypan-blue), apoptosis induction (by TiterTACS TUNEL assay), HNE-protein binding (by HNE-His ELISA) were higher in malignant than in normal cells, while glutathione content was higher in normal cells. These results indicate that HNE affects the growth of malignant mesenchymal cells more than normal and that this effect was mainly related to lower glutathione concentration and higher binding of HNE to the cellular proteins. We thus assume that HNE and GSH homeostasis play an important role in the growth regulation of normal and malignant mesenchymal cells.     

Differential susceptibility to nitric oxide-evoked apoptosis in human inflammatory cells

Apoptosis of neutrophils and their subsequent phagocytosis is critical to the successful resolution of inflammation. During inflammation, activated inflammatory cells generate reactive oxygen and nitrogen species, including nitric oxide (NO) and superoxide anion (O₂^??), which rapidly combine to generate peroxynitrite (ONOO^?). NO and ONOO^? are proapoptotic in human neutrophils. This study examines the effects of NO and ONOO^? on caspase activation and mitochondrial permeability in human neutrophils and determines the ability of these species to evoke apoptosis in human monocyte-derived macrophages (MDMs). NO or ONOO^? release from donor compounds was characterized by electrochemistry and electron paramagnetic resonance. Neutrophils and MDMs isolated from the peripheral blood of healthy volunteers were exposed to NO or ONOO^? before analysis of apoptosis by caspase activation, mitochondrial permeability, and annexin V binding. Both NO and ONOO^? induced apoptosis via rapid activation of caspases 2 and 3 in neutrophils. In contrast, only ONOO^? promoted apoptosis in MDMs, whereas a variety of NO donors were ineffective at inducing apoptosis in this cell type. We propose that human macrophages are refractory to NO-stimulated apoptosis in order that they persist long enough within the inflammatory focus to phagocytose apoptotic neutrophils, thereby ensuring successful resolution of inflammation.     

Sensitivity of normal, paramalignant, and malignant human urothelial cells to inhibitors of the epidermal growth factor receptor signaling pathway

Bladder cancer evolves via the accumulation of numerous genetic alterations, with loss of p53 and p16 function representing key events in the development of malignant disease. In addition, components of the epidermal growth factor receptor (EGFR) signaling pathway are frequently overexpressed, providing potential chemotherapeutic targets. We have previously described the generation of "paramalignant" human urothelial cells with disabled p53 or p16 functions. In this study, we investigated the relative responses of normal, paramalignant, and malignant human urothelial cells to EGFR tyrosine kinase inhibitors (PD153035 and GW572016), a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) kinase (MEK) inhibitor (U0126), and a phosphatidylinositol 3-kinase inhibitor (LY294002). The proliferation of normal human urothelial cells was dependent on signaling via the EGFR and MEK pathways and was abolished reversibly by inhibitors of EGFR or downstream MEK signaling pathways. Inhibitors of phosphatidylinositol 3-kinase resulted in only transient cytostasis, which was most likely mediated via cross-talk with the MEK pathway. These responses were maintained in cells with disabled p16 function, whereas cells with loss of p53 function displayed reduced sensitivity to PD153035 and malignant cell lines were the most refractory to PD153035 and U0126. These results indicate that urothelial cells acquire insensitivity to inhibitors of EGFR signaling pathways as a result of malignant transformation. This has important implications for the use of EGFR inhibitors for bladder cancer therapy, as combination treatments with conventional chemotherapy or radiotherapy may protect normal cells and enable better selective targeting of malignant cells.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

Acetylcholinesterase in normal and malignant human cells

Acetylcholinesterase (AChE) activity was determined in normal and malignant human cell lines by histochemical methods. In normal human fibroblasts, no AChE activity could be demonstrated by any histochemical technique or substrate. Enzymic activity was observed in HT-1080 human fibrosarcoma cells, RD 2 human rhabdomyosarcoma cells, and SW 311 human colon carcinoma cells. Activity was localized around the nuclear envelope, in the cytoplasm and associated with the cortical region of most cells. The specificity of the reaction was shown through the use of specific cholinesterase inhibitors.     

Selective growth of normal adult human urothelial cells in serum-free medium

Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca²⁺ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10⁻⁶ M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca²⁺ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis. This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Reduction of the antiapoptotic protein cFLIP enhances the susceptibility of human renal cancer cells to TRAIL apoptosis

Human renal carcinoma cells (RCCs) were sensitized to the apoptotic effects of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), by treatment with cycloheximide (CHX). In contrast to a previous study, a rapid and dramatic decrease in levels of cellular FLICE (Fas-associated death domain–like IL-1–converting enzyme) inhibitory protein (cFLIP) following cycloheximide treatment was observed in all RCCs studied. The unambiguous detection of this decrease in cFLIP was dependent on the quality of the particular antibody preparation used to detect cFLIP. Cycloheximide treatment caused no major change in levels of pro-caspase-8 or cell surface expression of TRAIL receptors. Therefore, cycloheximide treatment resulted in an increase in the pro-caspase-8 to cFLIP ratio, which correlated with sensitization to TRAIL-mediated apoptosis. Furthermore, treatment of human RCCs with small interfering oligoribonucleotides (siRNA) for cFLIP caused a reduction of cFLIP protein and sensitized cells to TRAIL-mediated apoptosis. We concluded that in the presence of an intact TRAIL signaling pathway, a significant reduction of cFLIP alone is sufficient to sensitize human RCCs to TRAIL apoptosis.The publisher or recipient acknowledges the right of the US Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.     

Inhibition of TACE activity enhances the susceptibility of myeloma cells to TRAIL

Background

TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. However, the susceptibility of MM cells to TRAIL is largely low in most of MM cells by yet largely unknown mechanisms. Because TNF-α converting enzyme (TACE) can cleave some TNF receptor family members, in the present study we explored the roles of proteolytic modulation by TACE in TRAIL receptor expression and TRAIL-mediated cytotoxicity in MM cells.

Methodology/Principal Findings

MM cells preferentially expressed death receptor 4 (DR4) but not DR5 on their surface along with TACE. Conditioned media from RPMI8226 and U266 cells contained a soluble form of DR4. The DR4 levels in these conditioned media were reduced by TACE inhibition by the TACE inhibitor TAPI-0 as well as TACE siRNA. Conversely, the TACE inhibition restored surface levels of DR4 but not DR5 in these cells without affecting DR4 mRNA levels. The TACE inhibition was able to restore cell surface DR4 expression in MM cells even in the presence of bone marrow stromal cells or osteoclasts, and enhanced the cytotoxic effects of recombinant TRAIL and an agonistic antibody against DR4 on MM cells.

Conclusions/Significance

These results demonstrate that MM cells post-translationally down-modulate the cell surface expression of DR4 through ectodomain shedding by endogenous TACE, and that TACE inhibition is able to restore cell surface DR4 levels and the susceptibility of MM cells to TRAIL or an agonistic antibody against DR4. Thus, TACE may protect MM cells from TRAIL-mediated death through down-modulation of cell-surface DR4. It can be envisaged that TACE inhibition augments clinical efficacy of TRAIL-based immunotherapy against MM, which eventually becomes resistant to the present therapeutic modalities.     

TRAIL/TRAIL receptor system and susceptibility to multiple sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10⁻⁴, OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10⁻⁵, OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.     

Differential susceptibility of cultured neural cells to the human coronavirus OC43.

By using cell-type-specific markers and neural cultures derived from various areas of the nervous system, it has been possible to identify various interactions between OC43 virus and mouse oligodendrocytes, neurons, astrocytes, and fibroblasts. Neurons derived from dorsal root ganglia produced viral antigen and infectious virus. Astrocytes and fibroblasts both produced viral antigen but not infectious virus. Oligodendrocytes produced neither infectious virus nor viral antigen. Human embryo brain cells, including astrocytes, were susceptible to OC43 infection but did not produce infectious virus.     

Differential sensitivity of normal and transformed human cells to reovirus infection.

Normal and simian virus 40-transformed WI-38 cells exhibited a differential sensitivity to infection with type 3 reovirus. A progressive decrease in viability began 24 to 36 h after infection of transformed cells terminating in complete lysis of cultures by 96 h. Normal cells were productively infected and continued to produce and release virus for as long as 14 days after infection, but exhibited no detectable cytopathology. Inhibition of cellular DNA synthesis began 15 to 18 h after infection in transformed cells before development of cytopathology. No inhibition of DNA synthesis was detected in infected normal cells. No significant differences were noted in the adsorption or early replication characteristics of reovirus in normal and transformed cells. Virus replication and host cell DNA synthesis in normal and transformed human cells were compared to reovirus-infected L-929 mouse fibroblast cells.     

Proliferation control in cloned normal and malignant human cells

A method for cloning of a variety of normal and malignant human cells in culture is described. Palladium is precipitated on agarose through masks prefabricated by a photolithographic technique. Practically any pattern can be produced where cells exclusively settle and multiply as ‘miniclones’ on the metal ‘islands’. This communication establishes

1. 1. that ‘miniclones’ settle and multiply with the same efficiency as cells of a mass population, thus constituting an unbiased cell sample

2. 2. that certain malignant lines cannot be studied with the unmodified technique suitable for normal glia-like cells because of excessive translocation between islands

3. 3. that this can be circumvented by the interposition of a thin agar overlay between the cells and the fluid medium. Proliferation of normal glia-like cells was inhibited on palladium islands in a cell density-dependent way. On sufficiently small islands even single cells could be prevented from dividing. Circles of about 3 000 μm² prevented multiplication of about 50% of single cells otherwise capable of division, suggesting that cell-to-cell contacts may not be necessary for proliferation inhibition of normal cells.

     

Differential susceptibility of human primary aortic and coronary artery vascular cells to RNA interference

BackgroundRNAi technology is a promising tool for gene therapy of vascular disease. However, the biological heterogeneity between endothelial (EC) and vascular smooth muscle cells (SMC) and within different vascular beds make them differentially susceptible to siRNA. This is further complicated by the task of choosing the right transfection reagent that leads to consistent gene silencing across all cell types with minimal toxicity. The goal of this study was to investigate the intrinsic RNAi susceptibility of primary human aortic and coronary artery endothelial and vascular smooth muscle cells (AoEC, CoEC, AoSMC and CoSMC) using adherent cell cytometry.MethodsCells were seeded at a density of 5000 cells/well of a 96well plate. Twenty four hours later cells were transfected with either non-targeting unlabeled control siRNA (50 nM), or non-targeting red fluorescence labeled siRNA (siGLO Red, 5 or 50 nM) using no transfection reagent, HiPerFect or Lipofectamine RNAiMAX. Hoechst nuclei stain was used to label cells for counting. For data analysis an adherent cell cytometer, Celigo was used.ResultsRed fluorescence counts were normalized to the cell count. EC displayed a higher susceptibility towards siRNA delivery than SMC from the corresponding artery. CoSMC were more susceptible than AoSMC. In all cell types RNAiMAX was more potent compared to HiPerFect or no transfection reagent. However, after 24 h, RNAiMAX led to a significant cell loss in both AoEC and CoEC. None of the other transfection conditions led to a significant cell loss.ConclusionThis study confirms our prior observation that EC are more susceptible to siRNA than SMC based on intracellular siRNA delivery. RNAiMax treatment led to significant cell loss in AoEC and CoEC, but not in the SMC populations. Additionally, this study is the first to demonstrate that coronary SMC are more susceptible to siRNA than aortic SMC.     

Differential susceptibility to human serum byAeromonas spp.

A total of 91 strains ofAeromonas (A. hydrophila, A. sobria, andA. caviae) of clinical origin were challenged in vitro against pooled human serum. A majority of the isolates ofA. hydrophila andA. sobria were resistant to the bactericidal activity of human serum, as opposed to the more serum-sensitiveA. caviae species. This difference in serum sensitivity may potentially explain the greater association of the former species with bacteremia and invasive disease.     

Homer1 regulates the susceptibility to TRAIL

TRAIL is an apoptotic cell death-inducing ligand that belongs to a TNF superfamily. To identify the regulators that govern the susceptibility to TRAIL, TRAIL-resistant HeLa (TR) cells were established by repeatedly treating HeLa cells with TRAIL. Here we showed that scaffolding protein Homer1 plays a decisive role in regulating the apoptotic susceptibility to TRAIL. TR cells showing the normal susceptibility to FasL and chemotherapeutic agent etoposide expressed the lower protein levels of Homer1 than parental HeLa cells. They showed the delayed activation of caspases-8, Bid cleavage and Bax translocation to mitochondria in response to TRAIL. Reconstitution of Homer1 expression in TR cells significantly restored the susceptibility to TRAIL. In addition, knock-down of Homer1 using interfering shRNA in parental HeLa cells lost the susceptibility to TRAIL. Together, our data indicate that Homer1 plays a critical role in determining the apoptotic susceptibility to TRAIL.