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Expression of 38-kD cell-surface glycoprotein in transformed human keratinocyte cell lines, basal cell carcinomas, and epithelial germs.

In this study, we attempted to identify and characterize transformation-induced cell-surface glycoproteins of human keratinocytes. Therefore, we first searched for glycoproteins which are significantly elevated in human keratinocytes after transformation and immortalization by SV40 virus and which are also found at high levels in keratinocytic cell lines derived from squamous cell carcinomas of the skin. Out of at least 80 different cell-surface antigenic systems of human tumor cells, only three glycoproteins showed elevated expression in transformed keratinocytes. Among these, a 38-kD glycoprotein (gp 38) was highly increased in all transformed keratinocyte cell lines tested, but was not elevated in transformed fibroblasts. The expression of gp 38 was further characterized in normal epidermis and in its benign and malignant hyperproliferative disorders: gp 38 was generally not expressed in normal epidermis and in benign hyperproliferative disorders. In contrast, strong and homogeneous reactivity was found in solid and fibrosing basal cell carcinomas whereas no or low reactivity was detected in squamous cell carcinomas and in those parts of BCC revealing keratotic differentiation. Interestingly, high expression of gp 38 was also found in primary epithelial germs of fetal skin, secondary germ cells of the telogenic hair follicle and secretory tubules of sweat glands. The immunohistologic data suggest that gp 38 is preferentially expressed by epidermal cells which lack squamous and pilosebaceous differentiation.

Basal Cell Carcinoma↗

Interleukin-4 and interleukin-13 enhance CCL26 production in a human keratinocyte cell line, HaCaT cells.

Eotaxin-2/CCL24 and eotaxin-3/CCL26 are CC chemokines and their receptor, CC chemokine receptor 3 is preferentially expressed on eosinophils. It was reported that vascular endothelial cells and dermal fibroblasts produced CCL26. However, the regulation of CCL24 and CCL26 production in keratinocytes has not been well documented. We investigated the expression and production of CCL24 and CCL26 in the human keratinocyte cell line, HaCaT cells. Reverse transcription and polymerase chain reaction was performed using these cells and Enzyme-linked immunosorbent assay was carried out using supernatant of these cells. The production of CCL24 in HaCaT cells was slightly enhanced by IL-4 and that of CCL26 was strongly enhanced by IL-4 and IL-13. Furthermore, TNF-alpha generated a synergistic effect on IL-4 enhanced CCL26 production. Dexamethasone, IFN-gamma and the p38 mitogen-activated protein kinase inhibitor SB202190 inhibited IL-4 enhanced CCL26 production. IL-4 enhanced production of CCL26 was inhibited by leflunomide and JAK inhibitor 1, but not by JAK3 inhibitor, which indicates that it is mediated by JAK1-STAT6-dependent pathway. This result also strongly suggests the involvement of the type 2 IL-4 receptor in IL-4 enhanced production of CCL26. These results suggest that keratinocytes are involved in the migration of CC chemokine receptor 3 positive cells such as eosinophils in a Th2-dominant situation like atopic dermatitis.

Cell Line, Tumor↗

[Palmitic acid induces apoptosis in human hepatoma cell line, HepG2 cells].

OBJECTIVE: To investigate the effects of palmitic acid (PA) on human hepatocytes and its mechanism. METHODS: We administered a mimic hyperlipidemia condition of 0.2-0.4 mmol/L PA to human hepatoma cell line, HepG2 cells. Cell viability was determined by Trypan blue staining. Cell cycle and early apoptosis were determined by propidium iodide and/or Annexin V staining, and the levels of Bcl-2 and Bax were analyzed by flow cytometry. RESULTS: An inhibition of cell growth was observed at a dose- and time-dependent manner in HepG2 cells after the treatment of PA. An apoptosis with appearance of sub-G1 fraction determined by cell cycle analysis significantly increased after the treatment of PA for 4 days. Bcl-2 level slightly decreased; in contrast, Bax level elevated markedly, which resulted in a significant decrease of Bcl-2/Bax ratio. CONCLUSION: PA may induce cell death on hepatocytes via mitochondria-mediated apoptosis by reducing the level of Bcl-2/Bax.

Apoptosis↗

Down-regulation of Bcl-2 and Bcl-xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death.

The functions of the antiapoptotic proteins Bcl-2 and Bcl-xL were examined in glioblastoma cells. Expression of both Bcl-2 and Bcl-xL were found to be elevated in protein lysates from seven early passage cell lines derived from human glioblastoma tumors compared with non-neoplastic glial cells. Down-regulation of both bcl-2 and bcl-xL expression in glioblastoma cell lines U87 and NS008 with bcl-2/bcl-xL bispecific antisense oligonucleotide resulted in spontaneous cell death. The mechanism of cell death was partially caspase-dependent. Executioner caspase 6 and caspase 7, but not caspase 3, were involved in apoptosis induced by bcl-2/bcl-xL antisense treatment. Interestingly, western blots failed to demonstrate expression of caspase 3 in two of the seven glioblastoma cell lines examined. The data support the hypothesis that Bcl-2 and Bcl-xL are important in preventing cell death in glioblastoma cells. It also suggests that there are functional pathways capable of successful completion of caspase-dependent cell death in gliomas. These findings support a potential role of bcl-2/bcl-xL bispecifc antisense oligonucleotide therapy as a treatment strategy to enhance caspase-dependent cell death in patients with glioblastoma.

Caspases↗

Apolipoprotein E uptake and low-density lipoprotein receptor-related protein expression by the NTera2/D1 cell line: a cell culture model of relevance for late-onset Alzheimer's disease.

Apolipoprotein E has been shown to be a risk factor for late-onset Alzheimer's disease, with the apolipoprotein epsilon 4 allele conferring the risk. Apolipoprotein E is found in neurofibrillary tangles and senile plaques, the pathological characteristics of Alzheimer's disease. To date there is no direct evidence that human neurons can take up exogenous apolipoprotein E, which is necessary if apolipoprotein E is involved in the formation of neurofibrillary tangles. To examine apolipoprotein E uptake we employed the human NTera2/D1 cell line, which can be induced by retinoic acid to differentiate into postmitotic NTera2-N neurons, which have the characteristics and morphology of human central nervous system neurons. We defined the cell line as genotype apolipoprotein epsilon 3/3 and demonstrated that the cells do not synthesize apolipoprotein E but can take up and internalize exogenous recombinant apolipoprotein E3. We also confirmed the expression of the low-density lipoprotein receptor-related protein, a known receptor for apolipoprotein E. The NTera2/D1 cell line therefore provides a useful human cell model for examining the effects of other apolipoprotein E isoforms with a view to defining intraneuronal interactions of apolipoprotein E.

Alzheimer Disease↗

Comparison of the transcriptional activity of the long terminal repeats of simian immunodeficiency viruses SIVmac251 and SIVmac239 in T-cell lines and macrophage cell lines.

The U3 regions of the long terminal repeats (LTRs) of simian immunodeficiency viruses SIVmac251 and SIVmac239 were analyzed for basal transcriptional activity and for interaction with cellular factors in the T-cell line HUT-78 and the monocyte/macrophage cell line U937. A number of 5' deletions and mutations were made in the U3 regions of the two LTRs, and these constructs were placed upstream of a plasmid containing the bacterial chloramphenicol acetyltransferase reporter gene. The nucleotide sequences between -225 and +18 were sufficient to maintain full transcriptional activity of both LTRs in HUT-78 and U937 cells. Nucleotide sequence analysis revealed several differences between SIVmac251 and SIVmac239 within this region. Analysis of deletion mutants revealed that an additional removal of bases, from -124 to -225, had little effect on the transcriptional activity of the clone 239 LTR, whereas this deletion resulted in a significant reduction of activity in the clone 251 LTR. DNase protection assays using nuclear extracts from HUT-78 and U937 cells showed that bases within this region bound cellular factors. In addition, the NF-kappa B site was protected in DNase assays with HUT-78 cells and 12-O-tetradecanoylphorbol-13-acetate-treated U937 cells. An additional DNase footprint was detected in SIVmac239, at -52 to -38, just upstream of the TATA box. This site overlaps the 3' half of the 3'-most Sp-1 site and is downstream of 11 bases that are found in SIVmac239 but not SIVmac251. Thus, differences in the sequences in the U3 region of the LTRs of SIVmac251 and SIVmac239 have been identified which appear to alter the transcriptional activity of these promoters as well as changing the interaction of cellular proteins with sequences in the LTRs.

Base Sequence↗

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium.

Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.

Adipose Tissue↗

A novel, rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+ monocytes within 48 hours of in vitro culture.

AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factoralpha (TNFalpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and prostaglandin E(2) (PGE(2)). One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocyte-derived dendritic cells within 48 h of in vitro culture (FastDC). METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/L L-glutamine, 100 microg/mL gentamicin, 1 000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFalpha (1 000 U/mL), IL-1beta (10 ng/mL), IL-6 (10 ng/mL) and PGE(2) (1 microg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/L polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritomas. The FastDCs and novel dendritomas were immunostained with anti-CD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS). Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy. RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas. CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.

Carcinoma, Hepatocellular↗

VEGF inhibition and cytotoxic effect of aplidin in leukemia cell lines and cells from acute myeloid leukemia.

BACKGROUND: Aplidine (APL) is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that is under clinical phase II development. In contrast to the lack of bone marrow toxicity reported in phase I/II studies, it has been shown to induce cytotoxicity at very low concentration against lymphoblastic leukemia blast, as well as having an impact in the vascular endothelial growth factor (VEGF)/VEGF receptor 1 loop. PATIENTS AND METHODS: To confirm these findings we investigated APL-related VEGF inhibition and its cytotoxic effect on myeloid leukemic cells lines (K-562, HEL and HL60) and fresh leukemia blasts derived from 30 patients with acute myeloid leukemia (AML). The conventional active 4-demetoxi-daunorubicin (idarubicin; IDA) was included as a positive control. RESULTS: APL was found to be significantly (P<0.001) more active than IDA in obtaining 50% growth-inhibition in K-562, HEL and HL60 cell lines. Results obtained with AML blast cells were super imposible. ID(50) ranged from 0.024 to 0.610 microM for IDA (0.200+/-0.176) and from 0.001 to 0.108 microM for APL (0.020+/-0.031). Annexin V tests and cell cycle analysis performed on cell lines confirmed the stronger citotoxic capability of APL as apoptotic inducer and as a G(1) blocker. The inhibitory effects of APL on VEGF release and secretion have been confirmed by ELISA tests performed on HEL: the VEGF concentration in cell surnatant was reduced from 169 to 36 pg/ml after 24 h of exposure to a pharmacological concentration of APL. CONCLUSIONS: APL harbors a strong in vitro antileukemic activity at a concentration achievable in patients at non-myelotoxic doses. Our data also support the notion of an impact on VEGF secretion. Clinical studies with this new marine-derived compound in relapsed/resistant leukemia are underway.

Acute Disease↗

Transient expression of M-CSF is important for osteoclast-like cell differentiation in a monocytic leukemia cell line.

Cells of U937, a human monocytic leukemia cell line, differentiate into macrophages by treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA), whereas cells treated with 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] continue to grow without undergoing differentiation. When U937 cells were successively treated with TPA and 1,25-(OH)2D3, tartrate-resistant acid phosphatase-positive multinucleated cells appeared at 5 days after the treatment. These osteoclast-like cells released a soluble form of 45Ca from 45Ca-labeled bone particles. These cells were not formed when the order of treatment with TPA and 1,25-(OH)2D3 was reversed. Use of either dexamethasone or interferon-gamma (IFN-gamma) was effective in inhibiting the formation of these osteoclast-like cells. The expression of c-src, c-fms, and macrophage colony stimulating factor (M-CSF) was induced by TPA treatment; however, TPA-induced M-CSF gene transcription was attenuated by the subsequent addition of 1,25-(OH)2D3. Furthermore, both dexamethasone and IFN-gamma impaired the attenuation of M-CSF expression, suggesting that the transient expression of M-CSF may be important for the formation of osteoclast-like cells.

Acid Phosphatase↗

Mechanisms responsible for size differences between hemopoietic colonies: an analysis using a CSF-dependent hemopoietic cell line.

Cells of the colony stimulating factor (CSF)-dependent continuous hemopoietic cell line 32Dcl-3 generate strikingly different large, medium and small colonies in semisolid cultures. Reculture of cells from such colonies showed that all colony types contained clonogenic cells able again to form colonies of all three size types. Clone mapping showed that late initiation of colony formation did not account for the final size difference between colonies, a conclusion confirmed by mapping studies on normal CSF-stimulated murine granulocyte-macrophage colonies. Clonogenic cells within 32 D Clone 3 (32Dcl-3) colonies varied in quantitative responsiveness to CSF stimulation. This responsiveness was generally lower than that of the initiating clonogenic cell and was higher with cells from small colonies than with cells from corresponding large colonies. The data eliminated differences in intrinsic proliferative potential, cellular hierarchy, cell cycle times or responsiveness to CSF stimulation as an adequate explanation for colony size differences. Colony size differences may possibly arise from random differences in the sequence of self-generative versus differentiative divisions in clonogenic cells in developing clones.

Animals↗

Distinct pathways of apoptosis triggered by FTY720, etoposide, and anti-Fas antibody in human T-lymphoma cell line (Jurkat cells).

2-amino-2-[2-(4-octylphenyl)ethyl] propane-1,3-diol hydrochloride (FTY720), a synthetic product derived from a metabolite of Isaria sinclairii, has been demonstrated to have a potent immunosuppressive activity that induces apoptotic cell death in T cells and several other cell lines. In this study, using the human T-lymphoma cell line, Jurkat cells, we investigated the apoptotic signal transduction mediated by FTY720, in particular comparing its role on the cleavage of caspases, with that mediated by etoposide or anti-Fas antibody. All of these agents cleaved caspases, inducing their active form in the affected cells. Pretreatment with a broad caspase inhibitor [benzyloxycarbonyl-Val-Ala-Asp-(Ome) fluoromethyl ketone] markedly decreased the incidence of apoptotic cells induced by FTY720, etoposide, and anti-Fas antibody, through the abrogation of cleavage of Bid, poly(ADP-ribose) polymerase, and caspases 3, 8, and 9. The overexpression of Bcl-2 gene prevented FTY720- and etoposide-mediated apoptosis, but not Fas-mediated apoptosis. In addition, mitochondria were demonstrated to play a critical role in FTY720-triggered cell death, suggesting that this drug has a potent anticancer activity.

Antibodies↗

E-cadherin expression during the acidic FGF-induced dispersion of a rat bladder carcinoma cell line.

Cell dissociation and acquisition of cell motility are major events in morphogenesis, wound repair, and cancer invasion and metastasis. We have used the NBT-II bladder carcinoma cell line as a model system to study the mechanisms of these events. Upon exposure to acidic fibroblast growth factor (aFGF), NBT-II cells undergo morphological changes that resemble those described in epithelial-mesenchymal transitions, i.e., dissociation of some or all polygonal epithelial cells and their transformation into motile, fibroblastic-like cells. The disruption of intercellular contacts, which accompanies cell dissociation and acquisition of motility, is correlated with a redistribution of E-cadherin, a Ca(2+)-dependent cell adhesion molecule, over the entire cell surface and within the cytoplasm. However, these modifications are not accompanied by a reduction of the intercellular adhesiveness or a loss of E-cadherin expression. Moreover, the formation of intercellular contacts between fibroblastic-like NBT-II cells results in the relocation of epithelial cadherin (E-cadherin) immunoreactivity on lateral membranes, but is not sufficient to abrogate cell motility. Finally, the overexpression of E-cadherin by NBT-II cells stably transfected with a plasmid containing the mouse E-cadherin cDNA does not impair the scattering effect of aFGF, indicating that high levels of E-cadherin expression do not prevent cells from disrupting their intercellular connections. Altogether, these results suggest that the scattering activity of aFGF is not mediated by direct modulations of E-cadherin expression.

Animals↗

Monitoring the apoptotic process induced by oxidized low-density lipoprotein in Jurkat T-lymphoblast and U937 monocytic human cell lines.

Cell death is a major event in the pathophysiology of atherosclerosis. Oxidized low-density lipoprotein (Ox-LDL), which plays a key role in the atherogenesis, has a powerful cytotoxic effect and causes necrosis or apoptosis of different types of cells. In the present work we studied the mechanism of cell death in two model systems: T lymphocytes and monocytes cell line, exposed to Ox-LDL. Ox-LDL, but not native low-density lipoprotein (LDL), was found to be cytotoxic to both cell types in a dose and time dependent manner. Apoptotic cell death was analyzed by evaluating cell size, nucleus DNA content and plasma membrane asymmetry. Early cytoplasmic condensation resulting from cell shrinkage was measured by monitoring fluorescence polarization (FP) of fluorescein labeled cells. The radical scavenger superoxide dismutase (SOD), in a time- and dose-dependent manner, reduced the apoptotic effect of Ox-LDL. Hyperpolarization of fluorescein-labeled cells preceded the appearance of phosphatidylserine (PS) on the plasma membrane. This sensitive parameter for early apoptosis detected different cell death kinetics, as well as varying sensitivity to the inhibitory effect of SOD in monocytes and lymphocytes. Such data suggest that reactive oxygen species generation are involved in Ox-LDL-induced apoptosis and that monocytes are more susceptible to cell death triggered by oxidative stress.

Apoptosis↗

Ligation of MHC class I induces apoptosis in human pre-B cell lines, in promyelocytic cell lines and in CD40-stimulated mature B cells.

A murine mAb (BAL-1) was previously shown to induce apoptosis when cross-linked on the cell surface of different B acute lymphocytic leukemia (ALL) and pro-myelocytic cell lines. The present study shows that BAL-1 specifically recognizes the MHC class I (MHC-I). The apoptotic response was not dependent on the epitope specificity, since other anti-MHC-I antibodies, reacting with different monomorphic determinants of the alpha chain or beta 2-microglobulin, also induced apoptosis in these cells. However, external cross-linking of antibodies was strictly required for the apoptotic effect. Among cells originating from mature peripheral blood B cells, anti-CD40-stimulated cells were susceptible to anti-MHC-I-induced apoptosis, whereas B cells activated with Staphylococcus aureus Cowan I (SAC) or with the superantigen staphylococcal enterotoxin A (SEA) were non-responsive. Mature SEA-activated T cells were also resistant to MHC-I-induced apoptosis. In situ terminal deoxynucleotidyl transferase staining of apoptotic cells at various stages during MHC-I-induced cell death revealed that apoptosis occurred predominantly in the G2/M phase of the cell cycle, with the first apoptotic cells appearing after approximately 12 h of incubation. These results suggest a role for MHC-I-mediated apoptosis during differentiation and activation of certain hematopoietic cells.

Animals↗

The rBAT gene is responsible for L-cystine uptake via the b0,(+)-like amino acid transport system in a "renal proximal tubular" cell line (OK cells).

Several studies have shown that the cRNA of human, rabbit, or rat rBAT induces in Xenopus oocytes sodium-independent, high affinity uptake of L-cystine via a system b0,(+)-like amino acid exchanger. We have shown that mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-425; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from oocytes, no other expression system has been used for transfection of functional rBAT activity. Furthermore, the b0,(+)-like transport activity has not been clearly described in the kidney or intestine. Here, we report that a "proximal tubular-like" cell line derived from opossum kidney (OK cells) expresses an rBAT transcript. Poly(A)+ RNA from OK cells induced by system b0,(+)-like transport activity in oocytes. This was hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase chain reaction-amplified cDNA fragment (approximately 700 base pairs) from OK cell RNA corresponds to an rBAT protein fragment 65-69% identical to those from human, rabbit and rat kidneys. We have also examined transport of l-cystine in OK cells and found characteristics very similar to the amino acid exchanger activity induced by rBAT cRNA in oocytes. Uptake of L-cystine was of high affinity, sodium-independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the same specificity as rBAT-induced transport activity in oocytes. Furthermore, it was localized to the apical pole of confluent OK cells. To demonstrate that the rBAT protein is functionally related to this transport activity, we have transfected OK cells with human rBAT antisense and sense sequences. Transfection with rBAT antisense, but not with rBAT sense, resulted in the specific reduction of rBAT mRNA expression and b0,(+)-like transport activity. These results demonstrate that rBAT is functionally related to the L-cystine uptake via system b0,(+)-like in the apical pole of the renal OK cell line.

Amino Acid Sequence↗

Retinoids and the control of growth/death decisions in human neuroblastoma cell lines.

Cell proliferation, the balance between mitosis and apoptosis is the result of the continuous integration of a number of different signal transduction pathways stimulated in a cell at any given point in its life. Neuroblastoma cells regulate the switch between mitosis and death, according both to intrinsic factors and extrinsic factors, such as growth factor withdrawal and action of the vitamin A derivative, retinoic acid. In this review, we describe the balance of some factors regulating growth and death of human neuroblastoma cells in vitro. These dynamic studies are necessarily-performed on cell lines, which offer controlled conditions enabling the disection of the complex stimuli mediating survival and growth (IGF, trk, BDNF) and death (transglutaminase, free radicals, Bcl-2). Although the conclusions drawn may therefore not be directly applicable to tumour cells in vivo, the results herein discussed are of sufficient significance to warrant in vivo relevance.

Cell Death↗

Bilirubin- and light induced cell death in a murine lymphoma cell line.

Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate.

Animals↗