Search PubMed⌕ Search

Biomedical subjects

L B Chen

Publications and source records attributed to L B Chen.

At least 73 records · Page 4Linked to original sources
PubMed1995

Ubiquitin fusion proteins are overexpressed in colon cancer but not in gastric cancer.

A cDNA clone (AF3) encoding the ubiquitin A gene 52 amino acid extension fusion protein (UbA52) was isolated from a subtracted cDNA library of human colorectal carcinoma minus adjacent normal mucosa. In Northern hybridization the mRNA signal for UbA52 was greater in surgical samples of colonic carcinoma (T) than in paired adjacent normal (N) tissues in 24 of 29 cases (T/N = 3.4 +/- 0.5, P < 0.01). An oligonucleotide probe specific for only the 52 amino acid extension confirmed the overexpression of UbA52. In contrast, there was no overexpression of UbA52 mRNA in gastric cancer samples (n = 7, T/N = 1.0 +/- 0.3). The mRNA of several ribosomal proteins, and of another ubiquitin A gene fusion protein, UbA80, with an 80 amino acid extension of ribosomal protein S27a, have been reported to be over-expressed in colon cancer, but not as yet at the protein level. Using rabbit antisera to the ribosomal protein component S27a we demonstrate over-expression of S27a at the protein level in colonic (n = 5), but not gastric (n = 6) carcinomas. Therefore it is likely that both UbA80 and UbA52 are overexpressed in colon cancer, but not in gastric cancer.

Base Sequence↗
PubMed1995

CRKL links p210BCR/ABL with paxillin in chronic myelogenous leukemia cells.

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and induces growth factor independence of hematopoietic cell lines in tissue culture. p210BCR/ABL is localized at least in part to the cytoskeleton, and has been shown to interact directly with actin filaments through an actin binding domain located in the C-terminus of ABL. CML cells have reduced adhesion to some extracellular matrix components but the mechanism of this phenomenon is unknown. In this study we examined tyrosine phosphorylation of focal adhesion proteins in cells expressing p210BCR/ABL. An interleukin-3 (IL-3)-dependent cell line, 32Dc13, was transformed with a BCR/ABL cDNA, and the patterns of localization, expression, and tyrosine phosphorylation of focal adhesion proteins were compared among untransformed 32Dc13 cells with and without IL-3 stimulation and BCR/ABL-transformed 32Dc13 cells. Of the focal adhesion proteins examined, only paxillin exhibited tyrosine phosphorylation in response to IL-3; while in cells transformed by p210BCR/ABL, paxillin, vinculin, p125FAK, talin and tensin were constitutively tyrosine phosphorylated. IL-3 induced a transient association between paxillin and vinculin, while in BCR/ABL-transformed cells, several proteins coimmunoprecipitated with paxillin, including vinculin, p125FAK, talin and tensin. Pseudopodia enriched in focal adhesion proteins were transiently detected in 32Dc13 cells in response to IL-3, but constitutively detected in cells expressing p210BCR/ABL. p210BCR/ABL protein was also found concentrated in punctate structures adjacent to the cell membrane in myeloid cell lines, which often contained vinculin and paxillin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Photosensitization by 3,3'-dihexyloxacarbocyanine iodide: specific disruption of microtubules and inactivation of organelle motility.

Photodynamic therapy is a useful new direction for cancer treatment. However, relatively little is currently known about the cellular targets and processes underlying the efficacy of these therapies. In this study, we report evidence of specific photosensitization of a novel intracellular target, cytoskeletal microtubules, that has great importance for cancer treatment. Photosensitization destroys microtubules, halts intracellular organelle motility processes, and leads to rapid cell death. We have examined the cell biological effects of photosensitization with the carbocyanine dye 3,3'-dihexyloxacarbocyanine iodide, which concentrates in mitochondria and the endoplasmic reticulum. Exposure of stained CV-1 kidney epithelial cells to as little as 30-120 s standard fluorescence excitation light caused disruption of the interphase microtubule network and complete inhibition of motility of the endoplasmic reticulum and all phase-contrast visible organelles, as specific effects of dye photoexcitation. Photoexcitation of rhodamine 123 or Hoechst produced neither of these effects. Furthermore, 3,3'-dihexyloxacarbocyanine iodide-mediated photodamage was specific to microtubules; other elements of the cytoskeleton, including vimentin intermediate filaments and actin stress fibers, were unaffected. We have reproduced the photoinactivation of microtubules in vitro with purified microtubule proteins.

Animals↗
PubMed1995

Removal of carcinoma cells from contaminated bone marrow using the lipophilic cation rhodamine 123.

Autologous bone marrow transplants for solid tumor treatment are severely limited by the potential presence of residual cancer cells in the reinfused bone marrow and can lead to future tumor recurrence. This article presents a novel method of removing carcinoma cells from bone marrow with contaminating cancer cells. This method is based on our previous studies demonstrating that carcinoma cells have a higher uptake of lipophilic cations such as rhodamine 123 than their normal epithelial counterparts. When the relative differences in rhodamine 123 uptake are quantified, carcinoma cell lines demonstrated a 7.4-21 times greater uptake of rhodamine 123 than normal mouse bone marrow cells. More important, when normal bone marrow cells and carcinoma cell lines are mixed to simulate carcinoma-contaminated bone marrow, individual cell populations continue to exhibit characteristic and identifiable relative differences (10-20 times) in rhodamine 123 uptake. Differential sorting of bone marrow/carcinoma cell mixtures with respect to rhodamine 123 fluorescence intensity resulted in the removal of 95-99% of the "contaminating carcinoma cells." The recovered bone marrow cells were fully viable as ascertained by their ability to form splenic colonies. In our preliminary experiments, sorted bone marrow cells transplanted into lethally irradiated C57BL6 mice allowed the mice to survive for more than 8 months. In light of these promising results, we propose that lipophilic cations may play a role in the purification of autologous bone marrow used in transplants for patients with advanced solid tumors.

Animals↗
PubMed1994

Several endoplasmic reticulum stress proteins, including ERp72, interact with thyroglobulin during its maturation.

We have previously demonstrated that several endoplasmic reticulum (ER) proteins, including BiP, ERp72, grp94, and protein disulfide isomerase, bind to a denatured thyroglobulin (Tg) affinity column and can be specifically eluted by ATP (Nigam, S.K., Goldberg, A.L., Ho, S., Rohde, M.F., Bush, K.T., and Sherman, M.Y. (1994) J. Biol. Chem. 269, 1744-1749). Using chemical cross-linking, we now demonstrate that BiP, ERp72, and grp94 associate with Tg in two types of cultured thyroid cells, FRTL-5 and PCC13. Whereas BiP could be coimmunoprecipitated with anti-Tg antibodies in the absence of cross-linking, only trace amounts of ERp72 and grp94 were coimmunoprecipitated. Likewise, in both cell types, anti-BiP antibodies were able to coimmunoprecipitate Tg in the absence of cross-linking, though ERp72 and grp94 were only minimally present. Coprecipitation of BiP and Tg was abolished when ATP and Mg2+ were added to cell lysates. In contrast, after cross-linking, there was a large increase in the amount of ERp72 and grp94 that coimmunoprecipitated with anti-Tg antibodies, although there was only a slight increase in BiP. Similarly, in cross-linked lysates, grp94 and ERp72 were also coimmunoprecipitated with anti-BiP antibodies. An apparently novel 200-kDa protein was also consistently immunoprecipitated by anti-BiP antibodies in both cell types. In addition, anti-ERp72 antibodies coimmunoprecipitated Tg, BiP, and grp94 only after cross-linking. Analysis of uncross-linked and cross-linked samples by sucrose density gradient centrifugation confirmed that Tg, BiP, grp94, and ERp72 are present together in high molecular weight complexes only after treatment of cells with cross-linking reagent. These results suggest that ERp72, as well as BiP and grp94, function as molecular chaperones in the maturation of Tg, potentially as part of a macromolecular complex.

Adenosine Triphosphate↗
PubMed1994

Molecular cloning of chick cardiac muscle tensin. Full-length cDNA sequence, expression, and characterization.

Here we describe the molecular cloning of 7.1-kilobase cDNA encoding chick cardiac muscle tensin. It contains an open reading frame of 1,744 amino acid (aa) residues. Sequence analysis reveals that, in addition to the previously noted SH2 domain (Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., Roberts, T. M., An, Q., and Chen, L. B. (1991) Science 252, 712-715), tensin contains virtually all of the known sequence (362 aa) of insertin, an actin-capping protein that allows actin monomer to be "inserted" (Schroer, E., and Wegner, A. (1985) Eur. J. Biochem. 153, 515-520). Moreover, tensin shares partial homology with actin (46.7% identity in 30 aa), beta-spectrin's actin-binding consensus (40% identity in 26 aa), BCR (40% identity in 25 aa), catenin alpha (35% identity in 45 aa), synapsin Ia (25.6% identity in 156 aa), IL-3 receptor (20.2% identity in 384 aa), and IL-2/EPO receptors (14% identity in 20 aa). Recombinant full-length tensin, tagged with an influenza-derived epitope, was over-expressed by a baculovirus system and purified to apparent homogeneity. It migrates as a 200-kDa protein in SDS-polyacrylamide gel electrophoresis, similar to the native tensin. The structure of the tensin molecule has been characterized by light scattering, electron microscopy, and gel filtration. Nine monoclonal antibodies recognizing different regions of tensin have been prepared and characterized. The epitope-tagged recombinant tensin gene was subcloned into a pRcCMV vector and transfected into NIH 3T3 cells. Immunofluorescence stainings with monoclonal antibodies specific for chick tensin (not cross-reactive with mouse tensin) showed that the expressed protein is indeed localized at focal contacts, as that of native tensin.

3T3 Cells↗
PubMed1994

Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain.

Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species.

Amino Acid Sequence↗
PubMed1994

Inhibition of energy metabolism alters the processing of amyloid precursor protein and induces a potentially amyloidogenic derivative.

The cellular mechanisms which lead to the generation and pathological deposition of beta amyloid in Alzheimer's disease are unknown. In this report we describe the proteolytic processing of the amyloid precursor protein (APP) to an 11.5-kDa COOH-terminal derivative which contains the full-length beta amyloid sequence. This processing step normally occurs at low levels in parallel with APP maturation in the secretory pathway. Inhibition of oxidative energy metabolism by sodium azide or the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone increased the proteolysis of APP to the 11.5-kDa derivative by about 80-fold with accumulation of this APP derivative in the Golgi complex. Agents which inhibit protein transport in the secretory pathway, including monensin and brefeldin A, also increased the production of the 11.5-kDa derivative. Inhibition of APP maturation demonstrated that the 11.5-kDa derivative could be produced by proteolysis of immature APP. These results demonstrate that APP processing to potentially amyloidogenic COOH-terminal derivatives occurs in either the endoplasmic reticulum or Golgi complex and can be modulated by the state of cellular energy metabolism. Deficits in oxidative energy metabolism have recently been found in the cerebral cortex of patients with Alzheimer's disease. These findings raise the possibility that energy-related metabolic stress may lead to altered metabolism of APP and contribute to amyloidosis in Alzheimer's disease.

Adenosine Triphosphate↗
PubMed1994

AA1, a newly synthesized monovalent lipophilic cation, expresses potent in vivo antitumor activity.

Certain lipophilic cations have been reported to display anticarcinoma activities because of their selective uptake and retention by mitochondria of cancer cells. Thus, these agents may comprise a unique class of agents directed against carcinoma. After screening more than 1000 lipophilic cations, we found that the monovalent lipophilic cation, 2,6-bis(4-amino-phenyl)-4-[4-(dimethylamino)phenyl]thiopyrylium chloride (AA1), displayed remarkable anticarcinoma activity both in vitro and in vivo. Unlike most other lipophilic cations, AA1 is stable and displays minimal light sensitivity. In vitro testing showed that AA1 was 10 times more toxic to the carcinoma cell line CX-1 than to the normal epithelial cell line CV-1. In vivo animal experiments showed that AA1 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 ratio was 344%. For Swiss nu/nu mice implanted i.p. with the human melanoma cell line, LOX, the treated:control ratio was 341%. The most significant observation was obtained with Swiss nu/nu mice that were implanted i.p. with the human ovarian cell line, OVCAR-III. The treated:control ratio in this situation was greater than 450%. In all these tumor models, AA1 produced minimal toxicities. AA1 exhibited little inhibition of electron transport in isolated rat liver mitochondria; however, it inhibited mitochondrial ATPase with 50% inhibitory concentration of 6 microM. Compared with previously reported anticarcinoma lipophilic cations such as rhodamine 123 and dequalinium chloride, AA1 appeared to display more effective in vivo anticarcinoma activity. Thus, AA1 could be considered for further clinical development as a candidate for anticarcinoma chemotherapy.

Adenocarcinoma↗
PubMed1994

Tensin: a potential link between the cytoskeleton and signal transduction.

Cytoskeletal proteins provide the structural foundation that allows cells to exist in a highly organized manner. Recent evidence suggests that certain cytoskeletal proteins not only maintain structural integrity, but might also be associated with signal transduction and suppression of tumorigenesis. Since the time of the discovery of tensin, a fair amount of data has been gathered which supports the notion that tensin is one such protein possessing these characteristics. In this review, we discuss recent studies that: (1) elucidate a role for tensin in maintenance of cellular structure and signal transduction; (2) implicate tensin as the anchor for actin filaments at the focal adhesion; (3) describe the phosphorylation of tensin; (4) describe potential targets for its Src homology region 2 domain; (5) describe the association between tensin and the nuclear protein p130; and (6) demonstrate that increased tensin expression in a cell line appears to reduce its transformation potential.

Actin Cytoskeleton↗
PubMed1994

Interactions of tensin with actin and identification of its three distinct actin-binding domains.

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

Actin Cytoskeleton↗
PubMed1994

HIV antigen-reactive T cells detected by antigen-induced interferon gamma secretion.

The T cell repertoire to human immunodeficiency virus (HIV) was studied in HIV-infected patients of different clinical stages by the detection and enumeration of cells that secreted interferon gamma (IFN-gamma) in short-term cultures of blood mononuclear cells after stimulation in vitro with the HIV recombinant antigens pB1, p121, p24-15, gp160bac, and the HIV V3 loop peptide. T cell reactivities to cytomegalovirus (CMV) and Mycobacterium tuberculosis-purified protein derivative (PPD) were examined in parallel. Among 29 patients with HIV infection, 48% had blood cells recognizing one or more of the five HIV antigens. The mean numbers of HIV antigen-reactive T cells varied between 1/approximately 6000 blood cells for pB1 and 1/approximately 20,000 cells for p24-15. None of the five HIV antigens studied was identified as an immunodominant T cell epitope in HIV infection. T cells from 20% of the patients responded to all five HIV antigens in parallel, but the antigen preferentially recognized varied from patient to patient. Those with more advanced disease had a tendency to lower numbers of HIV antigen-reactive T cells. Most HIV-infected patients had both CMV- and PPD-reactive T cells, but numbers were significantly lower in more advanced disease. It should be possible to adopt the present method to evaluate fine specificities of the T cell repertoire to other antigens and to study the involvement of other cytokines besides IFN-gamma, for example, the Th2 cell-related cytokine interleukin 4.

Adult↗
PubMed1994

[Transposing the great trochanter bone flap pedicaled with the deep branch of the medial circumflex artery in repair of necrotic femoral head].

The transposition of the greater trochanter bone flap pedicled with the deep branch of the medial circumflex artery was designed as a new therapeutic method for the treatment of early ischemic necrosis of the femoral head in adults. Bone flap was successfully used in 15 limbs of 13 patients. Applied anatomy, operative indications and procedure of the bone flap was introduced.

Adult↗
PubMed1993

Quantitative differential effects of rhodamine 123 on normal cells and human colon cancer cells by magnetic resonance spectroscopy.

Rhodamine 123 is a lipophilic cationic compound that is selectively taken up by cancer cell mitochondria. This compound is toxic to epithelial cancer cells in vitro and displays significant anticancer activity in vivo. However, the mechanism of action of rhodamine 123 in intact, actively metabolizing cell preparations is unknown. We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to quantitatively characterize how rhodamine 123 affects the energetics of human colon cancer cells (HCT-116) and spontaneously immortalized normal epithelial cells (CV-1). Rhodamine 123 differentially altered the phosphorus and glucose metabolism of HCT-116 and CV-1 cells. 31P-nuclear magnetic resonance detected mitochondrial poisoning in the HCT-116 human colon cancer cell line in its early stages after selective uptake of rhodamine 123. When we compared administration of rhodamine 123 and [1-C13]glucose to administration of [1-C13]glucose alone in the HCT-116 cells, we noted a marked decrease in intracellular pH to 6.7 +/- 0.06 (mean +/- SD) units, a 2.2-fold increase in lactate production, and a 1.8-fold increase in glucose consumption after 10 h. In addition, we found a 2-fold rise in intracellular free magnesium 12 h after rhodamine 123 administration. These results suggest that when rhodamine 123 inhibits mitochondrial ATP production, it initially stimulates cytoplasmic glycolysis in an attempt to maintain cellular energy demands. The marked fall in intracellular pH and rise in intracellular free magnesium after administration of rhodamine 123 may inhibit activity of several glycolytic enzymes: this effect would inhibit cytoplasmic ATP generation and interfere with multiple cell enzymatic processes, leading to cell death. The CV-1 cells showed no change in intracellular pH, intracellular free magnesium, or magnesium-bound ATP levels over the 24-h period following rhodamine 123 administration. Rhodamine 123 also failed to alter glucose utilization and lactate production levels significantly in the CV-1 cells. These results prove the usefulness of 31P- and 13C-nuclear magnetic resonance spectroscopy for quantifying differing effects of rhodamine 123 on the high energy phosphate metabolism and glucose metabolism of HCT-116 and CV-1 cells.

Adenosine Triphosphate↗