Biomedical subjects

L B Chen

Publications and source records attributed to L B Chen.

At least 55 records · Page 3Linked to original sources

PubMed1997

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.

3T3 Cells↗

PubMed1997

Progressive kidney degeneration in mice lacking tensin.

Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, -/- mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell-matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell-matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.

Animals↗

PubMed1997

Measurement of apoptosis in heterogenous cell populations.

The increasing interest in programmed cell death has created the need to measure apoptosis in complex cell systems. We have combined the use of fluorescent antibodies with the Hoechst 33342/propidium iodide system in order to quantitate programmed cell death in fractions of heterogenous cell populations. Here we describe the analysis of T-cell apoptosis after ligation of the T-cell antigen receptor by superantigen in vitro and ex vivo. This technique can separate cells according to seven parameters, fluorescence caused by FITC, PE, allophycocyanin, incorporation of Hoechst 33342, PI, forward scatter, and side scatter, and it allows determination of elevated Hoechst 33342 uptake in less than 10% of the cell population.

Animals↗

PubMed1997

Multiple molecular chaperones complex with misfolded large oligomeric glycoproteins in the endoplasmic reticulum.

Thyroglobulin (Tg), the major protein secreted by thyroid epithelial cells and precursor of thyroid hormones, is a large dimeric glycoprotein with multiple disulfide bonds. The folding and assembly of this complex molecule begins in the endoplasmic reticulum (ER) and is likely to involve a variety of reactions catalyzed by molecular chaperones (Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, 22990-22995). By coimmunoprecipitation in rat thyroid cells, we were able to demonstrate that BiP, grp94, ERp72, and grp170, four proteins believed to function as specific molecular chaperones, complex with Tg during its maturation. The same complex of the four putative chaperones with Tg was observed in cells treated with tunicamycin, indicating that these four ER chaperones stably associate with Tg when it is misfolded/misassembled due to inhibition of its glycosylation. BiP, grp94, and ERp72 were also found to associate with Tg in cells in which misfolding was induced by perturbing ER calcium stores. To determine if the assembly of a complex between the four chaperones and Tg under conditions of misglycosylation was unique to the maturation of this particular secretory protein or a more general phenomenon, adenovirus-transformed rat thyroid cells that do not synthesize Tg were analyzed. In these transformed cells, the only protein these same four chaperones were found to complex with was a protein of approximately 200 kDa. This protein was subsequently identified as thrombospondin, which, like Tg, is a large oligomeric secreted glycoprotein with multiple disulfide bonds. We therefore propose that these ER chaperones complex together with a variety of large oligomeric secretory glycoproteins as they fold and assemble in the ER.

Animals↗

PubMed1997

FP-21399 blocks HIV envelope protein-mediated membrane fusion and concentrates in lymph nodes.

The identification of fusin and other chemokine receptors as coreceptors for HIV-1 has renewed the interest in agents that may prevent viral entry. Polyanionic compounds such as dextran sulfate, curdian sulfate, and suramin act on the V3 loop of the viral envelope and may prevent its interaction with fusin. These agents show activity against a wide range of HIV-1 strains, but have undesirable circulating half-life, bioavailability, and toxicity. We have developed a small molecule inhibitor of HIV-1 that has several advantages over these other agents. FP-21399 is a novel compound of the bis(disulfonaphthalene) dimethoxybenzene class that blocks entry of HIV into CD4+ cells and blocks fusion of infected and noninfected CD4+ cells. This compound only weakly inhibits binding of CD4 and gp120, at concentrations much greater than are required to block viral entry. Furthermore, FP-21399 can block the interaction between gp120 and antibodies directed against the V3 loop, but does not block binding of antibodies directed against the V4 loop. Animal studies demonstrate that FP-21399 is concentrated in lymph nodes, making it a promising compound for anti-HIV therapy. In SCID mice reconstituted with human immune cells, maintenance of HIV-1 infection was blocked by a 5-day treatment with low doses of FP-21399, suggesting that lymph node accumulation may contribute to antiviral activity. Finally, attempts to generate drug-resistant virus in cell culture resulted in only weakly resistant variants with IC90 values that are much lower than concentrations of FP-21399 found in lymph nodes.

Animals↗

PubMed1996

Loss of hIRH mRNA expression from premalignant adenomas and malignant cell lines.

We recently isolated from differential displays and subsequently cloned a human alpha-intercrine (hIRH) whose mRNA is reduced in human hepatocellular carcinomas. We now report that on Northern blots its mRNA is absent from premalignant colonic adenomas and from all of 27 human malignant cell lines (including breast, cervix, colon, duodenal, gastric, leukemia, liver, lung, melanoma, and pancreatic lines). hIRH mRNA was present in most normal human and mouse tissues and fibroblast derived cell lines but absent from leukocytes and brain. Two mRNA signals, at approximately 2 Kb and approximately 3.5 Kb, had variation in signal strength or size between tissues and species.

Adenoma↗

PubMed1996

p130CAS forms a signaling complex with the adapter protein CRKL in hematopoietic cells transformed by the BCR/ABL oncogene.

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210(BCR/ABL). CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120(CBL). Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130(CAS). p130(CAS) was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130(CAS) was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125(FAK), and paxillin constitutively associated with p130(CAS). However, in BCR/ABL transformed cells, the interaction between p130(CAS) and tensin was disrupted, while the associations between p130(CAS), p125(FAK), and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130(CAS) in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130(CAS) containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130(CAS) with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes.

Tensin is an SH2 domain-containing cytoskeletal protein that binds to and caps actin filaments. Investigation of signal transduction mechanisms associated with tensin revealed the presence of phosphoinositide 3-kinase (PI 3-kinase) activity in tensin immunoprecipitates from platelet-derived growth factor-treated cells. Association of PI 3-kinase activity with tensin was transitory, and the amount of activity was approximately 1% of the total PI 3-kinase activity found in anti-phosphotyrosine (anti-pY) immunoprecipitates. In vitro, PI 3-kinase activity associated with the SH2 domain of tensin in a platelet-derived growth factor-dependent manner. The optimal phosphopeptide binding specificity of the SH2 domain of tensin was determined to be phospho-Y (E or D), N, (I, V, or F). Synthetic phosphopeptides containing the sequence YENI could specifically block the association of PI 3-kinase activity with tensin in a dose-dependent manner. These results suggest that PI 3-kinase interacts with the cytoskeleton via the SH2 domain of tensin and may play an important role in platelet-derived growth factor-induced cytoskeletal reorganization that is concomitant with cell migration and proliferation.

Amino Acid Sequence↗

PubMed1996

Interaction of cyclin-dependent kinase 2 and the Lyn tyrosine kinase in cells treated with 1-beta-D-arabinofuranosylcytosine.

The cyclin dependent kinase 2 (Cdk2) is required for initiation and progression of DNA replication. Activation of Cdk2 involves binding to cyclin E or cyclin A and dephosphorylation of Tyr15. The present studies demonstrate that treatment of U-937 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with tyrosine phosphorylation of Cdk2 and inhibition of Cdk2 activity. The results also demonstrate that Cdk2 directly associates with the Src-like tyrosine kinase Lyn as a consequence of ara-C-treatment. Confocal microscopy studies show that Lyn is detectable in the nucleus and that it colocalises with Cdk2. Subcellular fractionation and coimmunoprecipitation studies further demonstrate nuclear binding of Lyn and Cdk2. We also show that Lyn phosphorylates Tyr15 of Cdk2 and that incubation of Lyn with Cdk2 results in inhibition of Cdk2 activity. These findings suggest that the association of Lyn and Cdk2 in ara-C-treated cells may contribute to regulation of Cdk2-dependent cell cycle checkpoints.

Antimetabolites, Antineoplastic↗

PubMed1996

Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29).

Anti-alpha 4 integrin mAb coprecipitated CD81 (TAPA-1), a 25-kDa cell surface protein, from various alpha 4 beta1 -positive hemopoietic cell lines, including Molt4, Jurkat, Ramos, and alpha 4-transfected K562 (KX4C4) cells. In reciprocal experiments, the integrin alpha 4 beta 1 (VLA4, CD49d/CD29) could be reprecipitated from CD81 immunoprecipitates. Anti-alpha 4 integrin mAb also coprecipitated CD81 from the alpha 4 beta 7-positive B cell line RPMI 8866. In contrast, no CD81 was identified in alpha 2 beta 1, alpha 5 beta 1, or alpha L beta 2 immunoprecipitates. Abs to other members of the transmembrane-4 superfamily, including CD53, CD63, and CD82, also coprecipitated alpha 4 beta 1. As shown by confocal microscopy, CD81 and CD82 colocalized with alpha 4 beta 1 in cell surface clusters. The cytoplasmic domain of the alpha 4 integrin was not necessary for alpha 4 beta 1/CD81 association, nor was the association influenced by divalent cations, EDTA, integrin-activating mAb, or alpha 4 subunit cleavage. Notably, two independent alpha 4 adhesion-deficient mutants (D346E and D408E) were deficient in their ability to associate with CD81. Thus, CD81 and other transmembrane-4 superfamily members may participate in functionally relevant interactions with alpha 4 beta 1 and other integrins.

Antibodies, Monoclonal↗

PubMed1996

Nuclear localization of nucleoside diphosphate kinase type B (nm23-H2) in cultured cells.

Nucleoside diphosphate (NDP) kinases are metabolic enzymes found ubiquitously in cells. Recently, two known human isoforms of NDP kinase (A and B), identical to the protein products of the genes nm23-H1 and nm23-H2, respectively, have been implicated in cancer metastasis and transcriptional regulation. To date, NDP kinase has been studied extensively in tissue sections and its cellular localization was described as being cytoplasmic. However, the recently discovered role of the nm23-H2 gene product in transcriptional activation of the c-myc proto-oncogene also suggests a nuclear localization of the protein. In this study, we used isoform-specific antibodies against NDPK-B to examine the subcellular localization of the nm23-H2 gene product. The cytoplasmic fluorescence is intense and homogeneous with pronounced labeling in the centromere region. The distribution of NDPK-B in interphase nuclei exhibits a pattern of numerous uniformly dispersed fine dots with reduced staining of the nucleoli. To further characterize the nuclear localization of NDPK-B, in situ sequential extraction of nuclear components was performed. Brief exposure to Triton X-100 and subsequent treatment with RNase A do not change the nuclear staining pattern of NDPK-B. In contrast, treatment of Triton X-100-permeabilized nuclei with DNase I results in a significant loss of fluorescence. In mitotic prophase cells, the protein segregates from forming chromosomes and reappears in newly formed daughter nuclei after cell division. Taken together, the results indicate an association of NDPK-B with chromatin in interphase nuclei, supporting its proposed role in transcription.

Animals↗

PubMed1996

Nuclear signaling induced by ionizing radiation involves colocalization of the activated p56/p53lyn tyrosine kinase with p34cdc2.

The Src-like protein-tyrosine kinase p56/p53lyn associates with cell membranes and transduces signals from activated cell surface receptors. In the present work, cell fractionation and confocal microscopy studies demonstrate expression of Lyn in the nucleus. We also demonstrate that exposure of intact cells to ionizing radiation is associated with selective activation of nuclear Lyn. Similar findings have been obtained following irradiation of purified nuclei. Immunoprecipitation studies of nuclear lysates demonstrate radiation-induced binding of Lyn to p34cdc2. Nuclear colocalization of Lyn with Cdc2 has been confirmed by confocal microscopy. Other studies with glutathione S-transferase-Lyn fusion proteins demonstrate that the binding of Lyn to nuclear Cdc2 is associated with inhibition of Cdc2 activity. These findings suggest that the association of activated Lyn with Cdc2 in the nucleus may contribute to regulation of a DNA damage-dependent premitotic checkpoint.

CDC2 Protein Kinase↗

PubMed1996

Two differentially expressed genes in normal human prostate tissue and in carcinoma.

Alterations in transcriptional control may contribute directly to carcinogenesis. Using the differential display technique in prostate cancer cells compared to normal prostate epithelial cells, we identified a down-regulated gene and an up-regulated gene in cancer cells. The down-regulated gene encodes human epithelial tropomyosin (TMe1), a member of the family of actin filament-binding proteins. The up-regulated gene encodes cytochrome c oxidase subunit VIc (COSVIc), a protein of the respiration chain in the mitochondrial inner membrane. The differential display pattern was confirmed by Northern hybridization in both prostate tissue and cell lines. In situ hybridization of malignant prostate epithelial tissue using a digoxigenin-labeled antisense riboprobe detected strong staining for mRNA of COSVIc, as opposed to very weak staining in normal prostate epithelium. The expression pattern of COSVIc may be a useful marker for studying the alteration of energy metabolism in cancer cells and for the diagnosis of prostate cancer.

Base Sequence↗

PubMed1996

Analysis of MHC class II presentation of particulate antigens of B lymphocytes.

To generate Ab responses to most protein Ags, B cells must first degrade proteins in endocytic compartments and then display antigenic peptides bound to MHC class II molecules. T helper lymphocytes recognize these complexes and stimulate the B cell to synthesize Ab. Although Ab play a key role in host defense against bacteria, it is believed that B cells are incapable of internalizing particulate Ags. However, we find that B lymphoblastoid cell lines and LPS-activated B lymphocytes can present particulate Ag up to 10(5)-fold more efficiently compared with soluble Ag. Moreover, particulate Ags are presented efficiently by unstimulated B cells when they bind to surface Ig. In comparison to B cells, macrophages in general presented particulate Ags 10- to 1000-fold more efficiently and could also present Ag from particles of a much wider range of sizes. We document by ultrastructural and immunofluorescence analysis that B lymphoblastoid cell lines bind and internalize these particles. The internalization and presentation of the particulate Ag is inhibited by cytochalasin B. In contrast, a similar morphologic analysis of normal lymphocytes demonstrated that while Ag beads are bound to the cell surface, they are internalized only rarely. These results suggest there may be both surface and intracellular pathways for the presentation of particulate Ags by B cells. Interestingly, for both macrophages and B cells, the epitopes generated from particulate and soluble Ags were not identical quantitatively or qualitatively, indicating that there are differences in how these forms of Ag are processed and presented.

Amino Acid Sequence↗

PubMed1996

MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation.

MKT-077 (formerly known as FJ-776) is a newly synthesized, highly water-soluble ( > 200 mg/ml) rhodacyanine dye that exhibits significant antitumor activity in a variety of model systems. In culture, MKT-077 inhibits the growth of five human cancer cell lines (colon carcinoma CX-1, breast carcinoma MCF-7, pancreatic carcinoma (CRL 1420, bladder transitional cell carcinoma EJ, and melanoma LOX) but not monkey kidney CV-1, an indicator cell line for normal epithelial cells. In nude mice, MKT-077 inhibits the growth of s.c. implanted human renal carcinoma A498 and human prostate carcinoma DU145 and prolongs the survival of mice bearing i.p. implanted human melanoma LOX (tumor:control = 344%). Subcellular localization indicates that MKT-077 is taken up and retained by mitochondria, and flow cytometric analysis suggests that CX-1 cells take up MKT-077 to a much greater extent than CV-1 cells. Quantitation of MKT-077 uptake by ethanol extraction shows that CX-1 cells accumulate 65-fold more MKT-077 than do CV-1 cells. MKT-077 is the first delocalized lipophilic cation with a favorable pharmacological and toxicological profile in preclinical studies. MKT-077 is now being investigated in Phase I clinical trials.

Animals↗

PubMed1996

Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077.

We investigated the mitochondrial toxicity of the lipophilic cation, MKT-077, and the role of mitochondria in selective malignant cell killing by this compound by examining the effect of MKT-077 on mitochondrial structure and function in treated cells and in isolated organelles. Results of this study demonstrate changes in mitochondrial ultrastructure that are induced by MKT-077 treatment in carcinoma cells but not in similarly treated normal epithelial cells. In addition, MKT-077 was found to inhibit respiratory activity in isolated intact mitochondria and electron transport activity in freeze-thawed mitochondrial membrane fragments in a dose-dependent manner. The concentration of MKT-077 necessary to obtain half-maximal inhibition of ADP-stimulated respiration was approximately 4-fold greater in mitochondria isolated from cells of the normal epithelial cell line, CV-1 (15 micrograms MKT-077/mg protein), as compared to the human colon carcinoma cell line, CX-1 (4 micrograms MKT-077/mg protein). Further, the data show a selective loss of mitochondrial DNA in CX-1 and CRL1420 cells (carcinoma) but not CV-1 cells (normal epithelial) treated with 3 microgram/ml MKT-077 for up to 3 days. Under the same conditions, nuclear DNA was unaffected in all three cell lines. The sensitivity of the cell lines tested to mitochondrial damage by MKT-077 correlates well with their sensitivity to cytotoxicity by MKT-077. These results demonstrate selective mitochondrial damage by MKT-077 at the cellular, biochemical, and molecular levels and suggest that selective effects on mitochondrial structure and function may provide a basis for the selective malignant cell killing exhibited by this compound.

Animals↗

PubMed1996

In vivo administration of MKT-077 causes partial yet reversible impairment of mitochondrial function.

The effects of in vivo administration of a pharmacologically toxic dose of the lipophilic cationic compound, MKT-077, were investigated in selected vital organs of the rat. MKT-077 (15 mg/kg body weight), administered by bolus i.v. injection every day for 5 days, did not detectably influence rat heart and kidney mitochondrial respiration. Although the same dosage of MKT-077 significantly decreased respiratory rates in rat liver mitochondria relative to untreated controls, complete recovery was evident within 3 days following drug withdrawal. Whereas the mitochondrial DNA of rat kidney and liver appeared to be unaffected by MKT-077 treatment, levels of heart mtDNA were noticeably less than control levels in the immediate interval following drug administration. However, this latter effect was partially reversed as early as 10 days following treatment and completely reversed within a 30-day posttreatment period. These results strongly suggest that a pharmacologically toxic dose of MKT-077 minimally affects the overall functional integrity of mitochondria in such critical, although highly vulnerable, tissues as the heart, liver, and kidney.

Animals↗

PubMed1996

Anticarcinoma activity of a novel drug, 3-ethyl-3'-methyl-thiatelluracarbocyanine iodide (Te), a tellurium-containing cyanine targeted at mitochondria.

Lipophilic cationic compounds such as rhodamine 123, AA1, and dequalinium chloride have been reported to constitute a new class of anticarcinoma agents based on their selective localization, accumulation, and retention within the mitochondria of certain carcinoma cells. After screening more than 1000 lipophilic cationic compounds in clonogenic assays, we found that a tellurium-containing cyanine, 3-ethyl-3'-methyl-thiatelluracarbocyanine iodide (Te), exhibits significant anticarcinoma activity. In vitro testing showed that Te was 64-fold more toxic to the carcinoma cell line CX-1 than to the normal epithelial cell line CV-1. In vivo testing showed that Te significantly prolonged the survival of mice implanted with tumors. For C57BL x DBA/2 F1 mice implanted with the mouse bladder carcinoma cell line MB49, the treated:control (T:C) ratio ranged from 250 to 268%. For Swiss nu/nu mice implanted with the human melanoma cell line LOX, the T:C ratio ranged from 176 to 270%. For Swiss nu/nu mice implanted with the human ovarian tumor cell line OVCA-III, the T:C ratio was 344%. These anticarcinoma activities warrant further investigation of Te as a potential anticarcinoma agent.

Animals↗