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Objective: To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates. Methods: AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC). Results: On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all P <0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all P <0.01). Conclusion: Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.

We established spontaneously immortalized Schwann cell lines from long-term cultures of adult Fischer 344 rat dorsal root ganglia (DRG) and peripheral nerves. One of these cell lines, designated immortalized Fischer rat Schwann cells 1 (IFRS1), showed spindle-shaped morphology; immunoreactivity for S100, p75 neurotrophin receptor (p75(NTR) ), glial fibrillary acidic protein (GFAP), laminin, and vimentin; and mRNA expression of neurotrophic factors (NGF, GDNF, and CNTF), neurotrophin receptors (p75(NTR) , truncated TrkB, and TrkC), cell adhesion molecules (L1, NCAM, and N-cadherin), myelin proteins [P0, PMP22, and myelin-associated glycoprotein (MAG)], transcription factors (Krox20, Sox10, and Oct6), neuregulin-1 receptors (ErbB2 and ErbB3), and an orphan G protein-coupled receptor (Gpr126). Conditioned medium (CM) obtained from IFRS1 cells exhibited potent biological activity for the promotion of neuronal survival and neurite outgrowth of cultured adult rat DRG neurons. Furthermore, light and electron microscopic analyses revealed that IFRS1 cells were capable of myelinating neurites while in coculture with adult rat DRG neurons. These findings indicate that IFRS1 cells possess some biological properties of mature Schwann cells and that the coculture system with adult DRG neurons and IFRS1 cells can be a useful tool for the study of peripheral nerve degeneration and regeneration., (Copyright © 2011 Wiley-Liss, Inc.)

Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cell's surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between beta3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.

The molecular basis of mechanosensation in sensory neurons has yet to be defined. We found that ND-C cells, a hybrid cell line derived from neonatal rat DRG neurons, express mechanosensitive ion channels, and provide a useful expression system for testing candidate mechanosensitive ion channels. ND-C cells retain some important features of DRG neurons such as the expression of TTX-sensitive Na(+) and acid-activated currents as well as the ability to respond to mechanical stimulation with cationic currents sensitive to the analgesic peptide NMB1. ND-C cells do not respond to agonists of the 'thermoTRP' channels, suggesting that these channels are not responsible for MA currents in these cells and DRG neurons. Furthermore, transfecting ND-C cells with the candidate mechanotransducer channel TRPA1 does not increase MA current amplitudes, despite TRPA1 being functionally expressed at the plasma membrane. This correlates well with the fact that all types of MA currents can be recorded from TRPA1-negative DRG neurons.

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype., Competing Interests: The authors have declared that no competing interests exist.

Using cationic liposomes to deliver cytotoxic molecules to the tumor microvasculature is currently being developed for the treatment of cancer and other angiogenesis-related diseases. To improve on their beneficial properties, the authors have examined whether the particular cationic lipid type and lipid content employed are important factors influencing cellular interactions and formulation effects. The authors prepared different PEG (polyethylene glycol)-modified cationic liposomes (PCLs) with varying percent cationic lipid content and lipid type, and evaluated liposome size, surface charge (zeta) potential, and cellular properties in vitro. The cell lines used were human umbilical vein (HUVEC), lung microvascular (HMVEC-L and HPVE-26), coronary microvascular (HMVEC-C), dermal microvascular (HMVEC-D), and immortalized dermal microvascular (HMEC-1) endothelial cells. In vitro experiments consisted of cellular uptake and cytotoxicity studies, fluorescence-activated cell sorting (FACS) analysis, fluorescence, and transmission electron microscopic analysis. Liposome size and zeta potential analysis of five different PCLs revealed significant differences in their physicochemical properties. Some cationic lipids formed relatively toxic liposomes compared to others. The efficiency of loading chemotherapeutic drugs (doxorubicin hydrochloride, etoposide), affinity of PCLs for endothelial cells, and formulation effects varied according to cationic lipid content and the lipid type. Cellular uptake was observed in lung, dermal, and coronary endothelial cells. Heparan sulfate proteoglycans were found present on HMEC-1 cells, which may have enabled PCL uptake. In conclusion, physicochemical properties of cationic liposomes and their ability to interact with endothelial cells are important factors to consider during the early stages of formulation development for the treatment of cancer and other angiogenesis-dependent diseases.

The cerebellum is involved in complex physiological functions including motor control, sensory perception, cognition, language, and emotion. Humans and animals with prion diseases are characterized clinically by ataxia, postural abnormalities and cognitive decline. Pathology in the cerebellum affected by prions includes spongiform degeneration, neuronal loss, and gliosis. To develop an in vitro model system for studying prion biology in cerebellar cells, we established and characterized an immortal cell line (CRBL) isolated from the cerebellum of mice lacking expression of a protein involved in cell cycle arrest. The characteristics of the cells include morphological heterogeneity, rapid proliferation, serum responsiveness during growth, and a change in the number of chromosomes. CRBL cells expressed both neuronal and glial cell markers as well as a considerable level of cellular prion protein, PrP(C). Upon in vitro infection, CRBL cells exhibited selective susceptibility to prions isolated from different sources. These cells chronically propagated prions from SMB cells. Strain-specific prion infection in CRBL cells was not due to instability of the cell line, allelic variance, or mutations in the PrP gene. Molecular properties of prions derived from SMB cells were maintained in the infected CRBL cells. Our results suggest that the specific interaction between a prion strain and hosts determined the selective susceptibility of CRBL cells, which reflects the conditions in vivo. In addition to the future studies revealing cellular and molecular mechanism involved in prion pathogenesis, CRBL cells will contribute to the studies dealing with prion strain properties and host susceptibilities.

SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.

We describe the expression and in vitro activity of recombinant canstatin from stably transformed Drosophila melanogaster S2 cells. Southern blot analysis indicated that transformed S2 cells contained multiple copies of the canstatin gene in the genome. Recombinant canstatin with a molecular weight of 29kDa was secreted into the culture medium. Recombinant canstatin was purified to homogeneity using a simple one-step Ni(2+) affinity fractionation. Purified recombinant canstatin inhibited human umbilical vein endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED(50)) for recombinant canstatin expressed in stably transformed Drosophila S2 cells was approximately 0.37mug/ml. A maximum production level of 76mg/l of recombinant canstatin was obtained in a T-flask culture of Drosophila S2 cells 6 days after induction with 0.5mM CuSO(4).

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.

To clarify the effects of silica and silicates on cellular features of lymphocytes, a human T-lymphotropic virus type-1-immortalized polyclonal T-cell line, MT-2, was exposed to various concentrations of chrysotile-A, an asbestos classified as silicate. MT-2 cells underwent apoptosis in a dose- and time-dependent manner. The mitochondrial apoptotic pathway was activated during chrysotile-A-induced apoptosis of MT-2 cells, because of the phosphorylation of JNK and p38, increase of BAX and release of cytochrome-c from mitochondria to cytoplasma. In addition, anti-oxidants such as hydroxyl-radical excluders and capturers of superoxide and inhibitors of superoxide production effectively reduced the size of the apoptotic fraction in MT-2 cells cultured with chrysotile-A. These results indicate that the activation of reactive oxygen species may play a central role in asbestos-induced T-cell apoptosis, and anti-oxidants may help to prevent complications of pneumoconiosis.

Certification of continuous cell 293 culture used for cultivation of antineoplastic preparation Cancerolysin was carried out. The seeding and working banks of cells 293 were established and deposited for storage at the Vector Centre. The cells were certified in accordance with the WHO requirements. The cell 293 culture was shown to have high proliferative activity; morphology typical of the line; its karyotype and enzymogram are typical of human cells; the culture is not contaminated with bacteria, fungi, mycoplasms and viruses including oncogenic ones; it has high virus-producing activity; it preserves stability of all the biological properties in long-term cultivation. The seeding and working cell banks were recommended for the use in production of drugs for the treatment of oncologic patients.

Cell proliferation rate and 3H-thymidine labeling index of "young" (i. e. harvested in 3 days after subcultivation) cultured Chinese hamster cells (B11 dii-FAF28 line) have been determined in growth medium conditioned by the same cells for various periods of time during their growth and subsequent "stationary phase aging" (medium of different "age"). Cells were serially cultured in Eagle's medium with 10 % bovine serum. The experiment was conducted as follows. The "young" cells were seeded in Carrel's flasks (4500 cells/cm2) with fresh growth medium and placed at 37 degreesC. At definite time intervals, media from 3 randomly selected flasks were filtrated and stored in small glass flasks at 4 degreesC. The cells from all 3 flasks were collected by trypsin treatment and counted with hemocytometer. During the period of 26 day cultivation we collected a set of media of different "age" corresponding to certain points of the growth and "stationary phase aging" curve of the culture. Then, the "young" cells in fresh medium were seeded into tissue culture plates with cover slips placed into wells of the plates (26,600 cells/cm2) and grown at 37degreesC, 5 % CO2 for 2 h. At this point, the medium was replaced with media of different "age". 22 h later (i. e. on the first day after seeding) cell density was evaluated microscopically in all the wells. On the next day (i. e. in 2 days after seeding) 3H-thymidine was added to every well to final concentration 1.85 x 10(4) Bq/ml. After next 24 h (i. e. in 3 days after seeding) cell density was counted again, and the medium was removed. The cover slips were rinsed with Hank's solution and air-dried. Autoradiography was performed in standard manner by photoemulsion exposing for 5 days and subsequent developing in amidol developer. The relative number of nuclei with 10 and more "grains" was revealed microscopically. Based on the obtained results, two basic parameters were evaluated for every "age" medium: 1) cell proliferation activity index calculated as log2 (N3/N1), where N1 - cell density on the first day after seeding, and N3 - the same parameter on the third day after seeding; 2) cell labeling index calculated as percentage of cells with nuclei labeled by 3H-thymidine during incubation from 2nd to 3rd day of cultivation. These two indexes for cell growth in different "age" media appeared to be highly correlating (R = 0.85). Besides, it was found that the observed "age-related" diminishing of ability of the growth media of different "age" to stimulate proliferation of "young" cells cannot completely explain the "stationary phase aging" phenomenon (in particular, even for the "oldest" medium cell labeling index was 65 %). We conclude that the phenomenon is based on exactly intrinsic changes of cells, most likely on molecular level, though environmental effects cannot be entirely excluded. The authors are grateful to the Russian Basic Research Foundation for support (grants 03-04-49030 and 00-04-48049).

In this paper we have explored the role of different kinase pathways of signal transduction in proliferation control of E1A + Ras transformants, using specific inhibitors of MAP-kinases ERK, JNK, p38 and PI3-kinase. According to our data, suppression of signalling cascades driven by RI3K only arrested proliferation of E1A + Ras cells, while suppression of either MAP-kinase did not lead to noticeable antiproliferative effect. We have shown that suppression of RI3K with LY294002 gave rise to accumulation of cyclin-dependent kinase inhibitor p27(KiP1) but not p21(Waf1). Accumulation of p27(KiP1) in LY294002-treated E1A + Ras cells was accompanied by a decrease in Cyclin E-Cdk2 and Cyclin A-Cdk2 activity, which caused diminution of Rb phosphorylation and strengthening of E2F-Rb binding. Binding of E2F with hypophosphorylated Rb resulted in inhibition of E2F activity and reduction of E2F-regulated gene transcription, these genes being necessary for S-phase entry and DNA synthesis. Thus, RI3K--Akt cascade plays the key role in maintenance of autonomous proliferation of cells transformed with E1A and cHa-ras oncogenes. Inhibition of PI3K leads to p27(Kip1) accumulation and cell cycle arrest, consequently.

Background: Two features of the progression from organ-confined to metastatic prostate cancer are dysregulation of the androgen receptor (AR) and a decrease in insulin-like growth factor-type-I receptor (IGF-IR) expression. The purpose of this study was to determine the effect of changes in IGF-IR expression on AR activity., Methods: M12 human prostate cells were stably transfected with an AR expression construct to produce the M12-AR parental (PAR) cell line. PAR cells were implanted orthotopically into nude mice and M12-AR primary (PRI) cell lines were derived from intraprostatic tumors and metastatic cell lines (MET) were derived from PRI tumors that had metastasized to diaphragm or lung., Results: Tumor formation in the prostate by PAR cells was decreased significantly compared to M12 controls. PAR, PRI, and MET cells expressed equivalent amounts of AR protein; however, IGF-IR expression was increased significantly in PAR and PRI cells. IGF-IR expression decreased in MET lines to the levels seen in M12 control cells. IGF-I significantly enhanced dihydrotestosterone (DHT)-stimulated, but not basal, AR transcriptional activity in PRI cells. In MET cells, IGF-I significantly suppressed DHT-stimulated transcriptional activity. In MET cells in which the IGF-IR was re-expressed from a retroviral vector, the effects of DHT and IGF-I on AR activity were similar to those seen in PRI cells., Conclusions: This study demonstrates that the changes in IGF-IR expression exhibited by this model of metastatic progression cause significant alterations in AR signaling and suggest that this interaction may be an important aspect of the changes seen in AR function in disease progression in vivo., (2004 Wiley-Liss, Inc.)

The Epstein-Barr-transformed B lymphoblastoid cell lines, LCL, which express antigens, are potential antigen-presenting cells (APCs) for the induction of cytotoxic T lymphocytes in vitro. However, transfecting LCL with subsequent selection by antibiotics is notoriously difficult because the plating efficiencies of LCL are reported to be 1% or less. Therefore, this study investigated the optimal conditions for increasing the transduction efficiency of a recombinant adenovirus to LCL for use as a source of APCs. The transduction efficiencies were < 13% (SD +/- 2.13) at a multiplicity of infection (MOI) of 100, while it was increased to 28% (SD +/- 9.43) at an MOI of 1000. Moreover, its efficiencies to LCL that expressed the coxsackie adenovirus receptor were increased to 60% (SD +/- 6.35) at an MOI of 1000, and were further increased to 70% (SD +/- 4.56) when combined with the centrifugal method. The cationic liposome or anionic polymer had no effect on the transduction efficiency when compared to that of the centrifugal method. These results may be used as a convenient source of target cells for a CTL assay and/or autologous APCs for the induction of the in vitro CTL responses that are specific to viral and tumor antigens.

Background and Aims: Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting., Methods: First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T-and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics., Results: The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intra-cellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1., Conclusions: This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.

We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33 degrees C, but on shift to the nonpermissive temperature (39 degrees C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39 degrees C show increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated alpha-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.

Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.

Background: The mesenchymal-epithelial conversion of metanephric mesenchyme (MM) in the formation of nephronic tubules has long served as a paradigm for inductive signaling in morphogenesis. However, the mechanisms underlying this differentiation have remained an enigma due to insufficient numbers of primary mesenchymal cells that must be isolated manually from animal embryos. To overcome this problem, we have established a conditionally immortalized cell line, the rat-inducible metanephric mesenchyme (RIMM-18) by transfection of primary mesenchymal cells with a vector, encoding an estradiol-dependent E1A-ER fusion protein., Methods: Reverse transcription-polymerase chain reaction (RT-PCR), luciferase reporter assay, electrophoretic mobility shift assay, immunocytochemical, and immunohistochemical stainings were used to characterize the established cell line., Results: We demonstrate that in the presence of estradiol, the RIMM-18 cell line proliferates continuously, maintaining mesenchymal characteristics for over 40 passages. These cells are vimentin-positive and cytokeratin-negative. Under inductive conditions in the absence of estradiol, they are responsive to a number of cytokines, which are established inducers of mesenchymal cells in vivo and in vitro [i.e., fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and transforming growth factor-beta 2 (TGF-beta 2)]. We show the presence in RIMM-18 cells of specific protein markers and functionally active signaling pathways required for induction of tubule formation in MM. Furthermore, induced RIMM-18 cells change morphology, acquiring epithelial-like features, and begin to express epithelial markers (e.g., E-cadherin, cytokeratin, gamma-glutamyl-transpeptidase, and secreted frizzled-related protein 2 (sFRP2)., Conclusion: This preliminary characterization of the RIMM-18 cell line suggests that it will be useful in the study of biochemical and molecular mechanisms of nephronic development and, possibly, of some types of renal cancer such as Wilms' tumor, which caricatures the normal process of kidney development.

Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.

During sex differentiation, androgens are essential for development of the male genital tract. The Wolffian duct is an androgen-sensitive target tissue that develops into the epididymis, vas deferens, and seminal vesicle. The present study aimed to identify androgen-regulated proteins that are involved in development of Wolffian duct-derived structures. We have used male mouse embryos transgenic for temperature-sensitive simian virus 40 large tumor antigen at 18 d of gestation, to generate immortalized mouse fetal vas deferens (MFVD) parental and clonal cell lines. The MFVD parental and clonal cell lines express androgen receptor protein and show features of Wolffian duct mesenchymal cells. Clonal cell line MFVD A6 was selected for proteomic analysis and cultured in the absence or presence of androgens. Subsequently, two-dimensional gel electrophoresis was performed on total cell lysates. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and two androgen-regulated proteins were identified as mElfin and CArG-binding factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal F-actin. Both proteins appeared to be regulated by androgens at the posttranslational level, possibly involving phosphorylation. Posttranslational modification of mElfin and CBF-A by androgens may be associated with a cytoskeletal change that is involved in androgen-regulated gene expression.

The current study was undertaken to analyse growth and differentiation-related functions of normal keratinocytes (NOK) and an SV40T-immortalized keratinocyte line (SVpgC2a) from buccal mucosa, viewing the latter cell line as a model of a dysplastic epithelium. Morphological and immunohistochemical assessments of organotypic epithelia generated from 10 or 17 d of culture showed three- to five-fold higher apoptotic and proliferative activity in SVpgC2a relative to NOK. Conditions with or without serum (up to 10%) did not significantly influence these parameters in NOK whereas serum supported proliferation of SVpgC2a. Both cell types showed basal expression of collagen IV and laminin 1, indicating basal lamina, as well as vimentin, indicating an activated, proliferative state. Reduced expression of keratin, including the non-keratinizing marker K13, was seen in SVpgC2a. Assessment of proliferative monolayer cultures by microarray showed that NOK transcribed tissue-specific keratins, but also the epidermal keratin K2a, several simple epithelial keratins and low levels of hair keratins. SVpgC2a transcribed keratins seen in epithelial dysplasia, and K2a and hair keratins, albeit at low level. Overall, the results implied aberrant apoptosis, proliferation and keratin expression in the immortalized state of SVpgC2a. Comparison of NOK and SVpgC2a under identical culture conditions may serve to model the progression from a normal to a pre-neoplastic state of buccal epithelium.

Background & Aims: The availability of in vitro expandable human hepatocytes would greatly advance liver-directed cell therapies. Therefore, we examined whether human fetal hepatocytes are amenable to telomerase-mediated immortalization without inducing a transformed phenotype and disrupting their differentiation potential. Telomerase is a ribonucleoprotein that plays a pivotal role in maintaining telomere length and chromosome stability. Human somatic cells, including hepatocytes, exhibit no telomerase activity. Consequently, their telomeres progressively shorten with each cell cycle until critically short telomeres trigger replicative senescence., Methods: The catalytic subunit, telomerase reverse transcriptase, was expressed in human fetal hepatocytes. Transduced cells were characterized for telomerase activity, telomere length, proliferative capacity, hepatocellular functions, oncogenicity, and their in vivo maturation potential., Results: The expression of human telomerase reverse transcriptase restored telomerase activity in human fetal hepatocytes. Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression., Conclusions: Reconstitution of telomerase activity induces indefinite replication in human fetal hepatocytes and offers unique opportunities for examining basic biologic mechanisms and for considering development of stable cell lines for liver-directed therapies.

Aim: To develop conditionally immortalized gastric mucosal cell lines that show distinct types of cell differentiation from transgenic mice harboring temperature-sensitive simian virus 40 (tsSV40) large T antigen., Methods: Gastric mucosal cells from the transgenic mice were cultured at a permissive temperature (33 degrees C), and proliferative cells were then cloned by colony formation., Results: Eight gastric cell lines showed epithelial-like morphology and grew at 33 degrees C. Three different types of the cell lines have been established: (1) MGE12-1, MGE3-2, and MGE509 cells expressing mRNAs for pit cell markers (gastric mucin and cathepsin E); (2) MGE02, MGE503, and MGE511 cells expressing mRNAs for pit and zymogenic (pepsinogen F) cell markers, and (3) MGE507 and MGE727 cells expressing mRNAs for pit, zymogenic, and parietal (H,K-ATPase alpha-subunit) cell markers. Moreover, the TaqMan assay showed that mRNA levels of mucin, H,K-ATPase alpha-subunit, and pepsinogen F were influenced by nonpermissive temperature (39 degrees C) in MGE503 and MGE727 cells., Conclusion: These gastric epithelial cell lines seem to reflect different stages of development of gastric mucosal cells., (Copyright 2003 S. Karger AG, Basel)

The E1 region of adenovirus (Ad) type 5 is capable of transforming cells. According to current concepts, the Ad E1B 55 kDa (E1B 55K) protein enables transformed cells to grow by constantly binding and inactivating the p53 tumour suppressor protein. To test this model, the transcriptional activity of p53 was determined in Ad E1-transformed cells. Surprisingly, it was found that a p53-responsive promoter is highly active in Ad E1-transformed cells and further activated only 3- to 4-fold (compared to 200-fold in p53(-/-) cells) by exogenously expressed p53 or p53mt24-28, a p53 mutant that is transcriptionally active but unable to bind the E1B 55K. On the other hand, the transient overexpression of E1B 55K led to a strong downregulation of a p53-responsive promoter relative to its baseline activity in Ad E1-transformed cells but not in p53(-/-) cells. COS-7 cells, transformed by simian virus 40 (SV40), also showed constitutive p53 activity, whereas HeLa cells, transformed with oncogenic human papillomavirus, did not. Upon stable transfection, Ad E1-transformed cells but not p53(-/-) cells gave rise to colonies that expressed exogenous p53 or p53mt24-28 but, nonetheless, grew at near-wild-type rates. It is proposed that E1B 55K or the SV40 tumour antigen are saturated by the p53 protein, which accumulates in virus-transformed cells, leaving a proportion of active p53 molecules. The transformation of cells by the Ad E1 genes confers permissiveness for active p53, conceivably by inactivating the relevant products of p53 target genes that would otherwise prevent cell growth. Thus, Ad-transformed cells contain and tolerate active p53.

Loss of heterozygosity in two in vitro-immortalized human oral keratinocyte cell lines was analysed by polymerase chain reaction using 42 polymorphic microsatellite markers on chromosomes 4, 6, 7 and 15. These chromosomes are regarded as candidates for harbouring genes involved in the immortalization of human cells or tumour-suppressor genes in several tumours, including oral cancers, and karyotypic analysis has revealed that both cell lines have non-random alterations in these chromosomes. No allele losses were detected at any informative loci on chromosomes 4 and 6 in the cell lines, including genomic regions adjacent to putative human tumour-suppressor genes and putative senescence genes. When analysed for loss of heterozygosity on chromosomes 7 and 15, allele losses common to both cell lines were detected in the regions at 7q11.2, 7q21.1-21.3 and 7q31.1. High frequencies of loss of heterozygosity on chromosome 7q in at least two distinct regions, particularly centred around 7q31, are observed in a variety of tumours, including oral squamous-cell carcinoma, suggesting that multiple genes involved in immortalization of these cell lines might be present on chromosome 7q.

To identify the underlying mechanisms that limit the mitotic potential of normal somatic cells, we have undertaken a high resolution differential proteomic analysis aimed at identifying proteins that were differentially expressed upon replicative senescence. Since replicative senescence in heterogeneous primary fibroblast cultures is asynchronous, we analysed a group of conditionally immortalized rat embryo fibroblast cell lines that have previously been shown to undergo synchronous senescence upon inactivation of SV40 tsA58 T antigen. This identified 43 spots that were differentially expressed in these cell lines. Comparison of the identity of these features with those identified in a complimentary independent differential proteomic analysis of replicative senescence, directly in primary rat embryo fibroblasts upon serial passaging, identified nine features that were in common between the two studies even though they had been conducted entirely separately. None of these proteins have previously been recognized to be involved with replicative senescence. Thus, they represent novel starting points for elucidating the underlying mechanism that regulates the finite mitotic life span of somatic cells and how it can be overcome in cancer cells.

Central nervous system progenitor cells that are self-renewing in culture and also differentiate under controlled conditions are potentially useful for developmental studies and for cell-based therapies. We characterized growth and plasticity properties and gene expression in a rat mesencephalic cell line, AF5, that was immortalized with an N-terminal fragment of SV40 large T (T155g). For over 150 population doublings in culture, the growth rate of AF5 cells remained steady, the cells remained responsive to bFGF, and telomerase activity and telomere lengths were unchanged. While karyotype analyses revealed some chromosomal abnormalities, these were also unchanged over time; additionally, no mutations in p53 gene sequences were found, and wild-type p53 activation was normal. AF5 cells produced PDGF, TGFbeta1, TGFbeta2, and bFGF. Similar to primary progenitor cells, AF5 cells retained their plasticity in culture; they could be propagated in an undifferentiated state as "neurospheres" in serum-free media or as adherent cultures in serum-containing media, and they differentiated when allowed to become confluent. Adherent subconfluent actively growing cultures expressed a marker for immature neurons, nestin, while few cells expressed the mature neuronal cell marker betaIII-tubulin. Confluent cultures ceased growing, developed differentiated morphologies, contained few or no nestin-expressing cells, and acquired betaIII-tubulin expression. Global gene expression was examined using a 15,000 gene microarray, comparing exponential growth with and without bFGF stimulation, and the differentiated state. The AF5 cell line exhibited stable genetic and growth properties over extended periods of time, while retaining the ability to differentiate in vitro. These data suggest that the AF5 cell line may be useful as an in vitro model system for studies of neural differentiation., ((c) 2002 Elsevier Science (USA).)

Although cAMP response element binding protein (CREB)- and extracellular-regulated protein kinase (ERK)-mediated pathways have been linked to each other in neuronal differentiation, involvement of these in hippocampal neuronal cell line has not been defined. Using an immortalized hippocampal cell line, HiB5, we have tried a pairing of forskolin with KCl depolarization, which acts as an ERK and CREB kinase activator in hippocampal neurons, to investigate if an activation of ERK and phosphorylation of CREB at the critical regulatory site, serine 133 might be coupled in differentiation. Differentiation toward a neuronal phenotype was synergistically and markedly increased by the pairing of forskolin and KCl depolarization. The synergistic effect was accompanied by an increase in phosphorylation of CREB Ser-133, but not phosphorylation of ERK, and was not inhibited by MEK inhibitor, PD98059. These findings indicate that phosphorylation of the transcriptional factor CREB may function to facilitate differentiation of HiB5 cells.

This unit presents methods for isolating clonal, neural-derived cell lines. One approach for isolating such neural cell lines involves a replication-deficient retrovirus encoding a specific oncogene and a selectable marker which are used to infect dissociated CNS cells dissected at a developmental stage at which the cell population of interest has not undergone its terminal mitotic division. Also presented is a method for cloning by limiting dilution, which may be necessary to obtain a pure population of cells. Following growth under appropriate selection conditions, clones are isolated and tested for their ability to differentiate with the desired phenotypic properties. A method is also provided for coating tissue culture dishes, which is necessary for successful culture of CNS neurons.

One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells., (Copyright 2000 Academic Press.)

The stoichiometry of the p16INK4A and p15INK4B proteins bound to the cyclin D-CDK4/6 complex regulates the entry of cells into the G1 phase of the cell cycle. Thus, their level of expression is essential in maintaining regulated cell growth. In several tumors, deletion of these genes has been reported and, more recently, promoter methylation has been suggested as an alternative mechanism to decrease the expression of these cell cycle inhibitor proteins. Here, we studied the methylation status and the integrity of the p16INK4A and p15INK4B genes in 8 chronically HTLV-I-infected T cell lines and in ex vivo cells from 14 ATLL patients. Deletion of the locus carrying both genes was not found in the HTLV-I-infected T cell lines but was found in seven of eight acute ATLL cases and in none of the PBMCs from the chronic cases or the affected lymph nodes of the lymphoma type. In contrast, partial or complete methylation of one or both genes was found only in chronically HTLV-I T cells. Thus, HTLV-I infection targets the p16INK4A and p15INK4B loci both in vitro and in vivo, although the mechanisms may differ.

Can science discover some secrets of Greek mythology? In the case of Prometheus, we can now suppose that his amazing hepatic regeneration was caused by a peptide growth factor called hepatocyte growth factor (HGF). Increasing evidence indicates that HGF acts as a multifunctional cytokine on different cell types. This review addresses the molecular mechanisms that are responsible for the pleiotropic effects of HGF. HGF binds with high affinity to its specific tyrosine kinase receptor c-met, thereby stimulating not only cell proliferation and differentiation, but also cell migration and tumorigenesis. The three fundamental principles of medicine-prevention, diagnosis, and therapy-may be benefited by the rational use of HGF. In renal tubular cells, HGF induces mitogenic and morphogenetic responses. In animal models of toxic or ischemic acute renal failure, HGF acts in a renotropic and nephroprotective manner. HGF expression is rapidly up-regulated in the remnant kidney of nephrectomized rats, inducing compensatory growth. In a mouse model of chronic renal disease, HGF inhibits the progression of tubulointerstitial fibrosis and kidney dysfunction. Increased HGF mRNA transcripts were detected in mesenchymal and tubular epithelial cells of rejecting kidney. In transplanted patients, elevated HGF levels may indicate renal rejection. When HGF is considered as a therapeutic agent in human medicine, for example, to stimulate kidney regeneration after acute injury, strategies need to be developed to stimulate cell regeneration and differentiation without an induction of tumorigenesis.

Background: This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine beta-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag)., Results: KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37 degrees C. In contrast, at the permissive temperature of 33 degrees C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37 degrees C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode beta-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37 degrees C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells., Conclusion: KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland.

The neurofilament L/68 protein (NF-L/68) gene is expressed in the immature Schwann cell phenotype but suppressed after myelin-formation. We have investigated conditions which regulate the activity of the NF-L/68 promoter in green fluorescent protein reporter constructs expressed in the immortal rat Schwann cell strain SCL4.1/F7 in coculture with neurons. Constructs expressed in a plasmid vector containing both the full-length promoter and the 3' proximal 107 bp sequence which includes the cyclic AMP response element (CRE), were active in SCL4.1/F7 cells, but were suppressed as the cells underwent spontaneous growth-arrest. Interaction of SCL4.1/F7 with axons accelerated downregulation of expression from both constructs, however expression of the full-length promoter continued in some cells until the onset of myelin-formation. Expression of the NFL/68 construct recommenced when demyelination was induced in culture by exposure to human sera from patients with paraproteinemic gammopathy. We have demonstrated a method to study the regulation of gene expression patterns in single Schwann cells interacting with neurons and shown that different promoter regions may be controlled by axon-related and -unrelated factors.

We recently developed an immortalized osteoclast (OCL) precursor cell line that forms large numbers of OCLs. This cell line was derived from mice doubly transgenic for bcl-X(L) and large T antigen that was targeted to cells in the OCL lineage (bcl-X(L)/Tag cells). We have now characterized these cells in terms of their surface and enzymatic phenotype, responsiveness to osteotropic factors, and differentiation potential. The bcl-X(L)/Tag cells expressed interleukin-1 receptors 1 and 2, gelatinase B (MMP9), as well as Mac-1, CD16/CD32 (Fcgamma receptors), CD45.2 (common leukocyte marker), CD86 (costimulatory molecule expressed on B cells, follicular dendritic cells, and thymic epithelium), major histocompatibility complex I, and nonspecific esterase when cocultured with MC3T3E1 cells. However, they did not express the antigens for F4/80 (mature macrophage/dendritic cell marker) by immunostaining. Treatment of bcl-X(L)/Tag cells, cocultured with MC3T3E 1 cells, with the combination of 1,25-dihydroxyvitamin D3 and dexamethasone induced high levels of OCL formation. The bcl-X(L)/Tag cells formed large numbers of OCLs when cultured with RANK ligand and macrophage colony-stimulating factor in the absence of feeder cells. In the absence of RANK ligand and a feeder cell layer, 100% of the cells differentiated into F4/80-positive cells. However, neither PTH nor PTH-related protein enhanced OCL formation by bcl-X(L)/Tag cells even when they were cocultured with primary osteoblasts, suggesting that they differ from primary mouse bone marrow cells in their responsiveness to PTH/PTH-related protein. Thus, bcl-X(L)/Tag cells have many of the properties of primary mouse OCL precursors and should be very useful for studies of OCL differentiation and divergence of OCL precursors from the macrophage lineage.

Bovine cell lines of the monocyte-Mphi lineage were tested for surface marker expression and were characterized with respect to functions. Cell lines tested encompassed an SV40-transformed cell line (Bo-Mac), a spontaneously emerging monocytoid cell line (M617), and T. annulata-transformed lines derived from bovine Mphi. All lines failed to express surface markers expressed by 1 degrees Mphi, with the exception of CD44, WC9 and the DH59 myleoid cell marker. T. annulata-derived lines expressed, in addition, CD45 and MHC-class-II molecules. Except for nonspecific esterase staining, none of the typical macrophage functions were expressed by any of the cell lines. These included phagocytosis of opsonized E. coli bacteria and of IgG-treated erythrocytes, eliciting of an oxidative burst, the ability to express type-I-interferon (IFN) and to respond to lipopolysaccharide, as determined by four different effector functions (nitric oxide synthesis, tumor necrosis factor (TNF) secretion, IFN production and procoagulant activity upregulation). When transformation induced by T. annulata was reversed by chemical elimination of the parasite, cells ceased to proliferate but started to acquire some of the phenotypic characteristics of Mphi. This suggests that regardless of their origin, exponentially growing bovine cells of the monocyte-Mphi lineage poorly represent a lineage-specific phenotype and should be used with caution in immunological studies.

We review recent data on senescence and immortalization of human B-lymphoblastoid cell lines (LCLs) transformed by the Epstein-Barr virus (EBV). Although EBV-transformed LCLs are generally believed to be immortalized, a series of recent studies, including ours, provided strong evidence that they are mostly mortal and have non-malignant properties, except for a small proportion of LCLs that are immortalized by developing a strong telomerase activity. A large proportion of mortal LCLs have exceptionally long lifespans. Some of them have a lifespan over 150 population-doubling levels, keeping a relatively constant telomere length in spite of the absence of a detectable telomerase activity, suggesting that they maintain telomeres by a pathway other than that using telomerase. Here we propose a model of an alternative pathway to maintain telomeres of such long-lived mortal LCLs by exploiting extra-chromosomal telomere repeat DNA, which was recently found by us.

The renal proximal tubule (PT) is a major site for a complete tissue renin-angiotensin system (RAS) and produces endogenous angiotensin II (ANG II). The present studies demonstrate autocrine RAS feedback in a line of origin-defective SV40 plasmid transformed immortalized rat PT cells (IRPTC) designated as line 93-p-2-1, which are highly differentiated and express all RAS components. Receptor competition assays and Southern blot following RT-PCR demonstrated that these IRPTC express AT1 and AT2 angiotensin receptor subtypes. Autocrine RAS feedback was examined following exposure to ANG II (10(-8) M), and it was noted that angiotensinogen mRNA increases significantly by 1 h and remains elevated through 24 h. The AT1 blocker losartan prevents this increase. Moreover, ANG II upregulates expression of ANG II receptor mRNA (both AT1 and AT2). Thus the present studies demonstrate positive ANG II feedback with angiotensinogen and ANG II receptors in PTC, suggesting that the main site of such intrarenal feedback in vivo is within PT. ANG II secreted by line 93-p-2-1 is increased by isoproterenol, suggesting beta-adrenergic regulation in IRPTC.

Confluent monolayers of primary endothelial cells display a high viability and an apparently constant cell density. However upon prolonged cultivation the monolayer degenerates with increasing numbers of senescent cells finally representing the whole culture. Recently we showed that lack of hemodynamic forces induces apoptosis in organ cultures as well as in confluent monolayers of human umbilical cord vein endothelial cells (HUVEC). The apoptosis started at a low level and was counteracted by a continuous proliferation of the remaining cells. Here we show that the induction of apoptosis by lack of hemodynamic forces is a general characteristic of vascular endothelial cells, valid for endothelial cells from various organs and species: human umbilical cord vein endothelial cells (HUVEC), human microvascular placental endothelial cells (HPEC) and bovine aorta endothelial cells (BAEC). Furthermore apoptosis due to the lack of hemodynamic forces can also be induced in various endothelial cell lines: EA.hy 926 derived from HUVEC and PBMEC-A1 derived from PBMEC. However degeneration of confluent monolayers does not occur with these cell lines even in monolayers kept for several weeks. This indicates that the degeneration of normal endothelial cell monolayers is caused by depletion of the proliferation potential of the endothelial cells.

After performing the miracles that takes us from conception to birth, and then to sexual maturation and adulthood, natural selection was unable to favor the development of a more elementary mechanism that would simply maintain those earlier miracles forever. The manifestations of this failure are called aging. Because few feral animals age, evolution could not have favored a genetic program for age changes. Natural selection favors animals that are most likely to become reproductively successful by developing better survival strategies and greater reserve capacity in vital systems to better escape predation, disease, accidents, and environmental extremes. Natural selection diminishes after reproductive success because the species will not benefit from members favored for greater longevity. The level of physiological reserve remaining after reproductive maturity determines longevity and evolves incidental to the selection process that acts on earlier developmental events. Physiological reserve does not renew at the same rate that it incurs losses because molecular disorder increases at a rate greater than the capacity for repair. These are age changes, and they increase vulnerability to predation, accidents, or disease. Failure to distinguish aging from disease has not only blurred our efforts to understand the fundamental biology of aging, but it has profound political and economic consequences that compromise the field of biogerontology. Changes attributable to disease, or pathological change, can be distinguished from age changes for at least four important reasons. Unlike any known disease, (1) age changes occur in every human given sufficient time, (2) age changes cross virtually all species barriers, (3) no disease afflicts all members of a species only after the age of reproductive success, and (4) aging occurs in all feral animals subsequently protected by humans, even when that species probably has not experienced aging for thousands or millions of years. The resolution of age-associated diseases will not advance our knowledge of aging, just as the resolution of the diseases of childhood did not advance our knowledge of childhood development. We have failed to convey that greater support must be given to a question that is rarely posed. It is a question that is applicable to all age-associated diseases, and its resolution will also advance our fundamental knowledge of aging: "Why are old cells more vulnerable to pathology and disease than are young cells?" During the first half of this century it was believed that because cultured normal cells were immortal, aging must be caused by extra-cellular events. Thirty-five years ago we overthrough this dogma when we found that normal cells do have a limited capacity to divide, and that age changes can occur intracellularly. We also observed that only abnormal or cancer cells are immortal. Normal cells are mortal because telomeres shorten at each division. Immortal cancer cells express the enzyme telomerase that prevents shortening. Recently, it was discovered that when the catalytic subunit of the telomerase gene is inserted into normal cells they become immortal.

Anoikis is a form of cell death that occurs when cells are denied attachment to the extra-cellular matrix. Using p6 cells, that are 3T3 cells overexpressing the type 1 insulin-like growth factor receptor (IGF-IR), we show that these cells undergo apoptosis when seeded on polyHEMA plates in serum-free medium (SFM). IGF-I protects p6 cells from anoikis, without inducing mitogenesis or DNA synthesis. In the surviving p6 cells in suspension cultures, the focal adhesion kinase (FAK) is tyrosyl phosphorylated by IGF-I, although this phosphorylation occurs only after several hours. The importance of FAK in protection from anoikis is confirmed by v-src-transformed R-cells, in which FAK is constitutively phosphorylated, that survive even in SFM. Surviving cells, whether p6 or v-src transformed, tend to form large cell aggregates, whose appearance precedes the phosphorylation of FAK. These and other findings suggest that FAK phosphorylation in the case of IGF-I is a mediated effect rather than a direct one. When p6 cells are plated on polyHEMA dishes, IGF-I induces cell aggregation and this aggregation correlates with survival and the eventual phosphorylation of FAK.

The EGF-like family of proteins, such as epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), amphiregulin (AR), betacellulin (BTC), cripto-1 (CR-1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL-5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL-5H2 cell line, overexpressing erbB-2, Kimol cells, transformed by the wild-type K-ras and A6 clone, transformed by a temperature sensitive K-ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF-like growth factors and corresponding receptors (erbB-2, erbB-3, and erbB-4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL-5 cells, both K-ras and erbB-2-transformed cells expressed elevated levels of erbB-2 receptor. Moreover, both K-ras transformed cells, Kimol and A6 cells, but no FRTL-5H2 cells, were found able to express also high levels of erbB-4 receptor and HRG/NDF ligand. Treatment of K-ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB-2 overexpressing FRTL-5 cells, in K-ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB-2/erbB-4 receptors in an autocrine fashion.