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L B Chen

Publications and source records attributed to L B Chen.

At least 91 records · Page 5Linked to original sources
PubMed1993

Nucleotide and deduced amino acid sequence of human ribosomal protein L18.

Ribosomal protein L18 mRNA is overexpressed in human colorectal cancer compared to normal colon tissue. We report the nucleotide sequence of human L18 cDNA derived from a normal colon source. There were no mutational changes in segments of L18 cDNA derived from two tumor sources. The L18 cDNA was 690 base pairs long and predicts a single open reading frame of 564 nucleotides, encoding 188 amino acids with a M(r) = 21,621, it is homologous to rat L18 and Xenopus laevis L14.

Amino Acid Sequence↗
PubMed1993

Gastric and hepatocellular carcinomas do not overexpress the same ribosomal protein messenger RNAs as colonic carcinoma.

The levels of a number of ribosomal protein mRNAs are reported to be increased in human colon cancer. We have assessed whether selected ribosomal protein mRNAs are overexpressed in other gastrointestinal malignancies, namely gastric and hepatocellular carcinomas. Subtracted complementary DNA libraries were generated from paired samples of human (a) colorectal carcinoma minus adjacent normal colonic mucosa and (b) hepatocellular carcinoma minus adjacent normal liver. Screening of approximately 3% of these library clones determined that ribosomal protein mRNAs encoding L18 and L37 (not previously reported) and P0 and S6 were overexpressed in one or the other library. Their complementary DNA inserts were then used as probes to evaluate their expression in a larger number of paired tumor/normal surgical samples of human colonic, gastric, and hepatocellular carcinomas, by Northern hybridization. The mRNA signal was greater in the colonic carcinoma than in paired adjacent normal colonic mucosa in 38 of 42 cases for P0 [tumor/normal (T/N) ratio = 3.0 +/- 0.3, mean +/- SE, P < 0.001] (G. F. Barnard, R. J. Staniunas, S. Bao, K. Mafune, J. L. Gollan, G. D. Steele, Jr., and L. B. Chen, Cancer Res., 52: 3067-3072, 1992), in 25 of 28 cases for L18 (T/N ratio = 3.7 +/- 0.5, P < 0.001), in 27 of 28 cases for L37 (T/N ratio = 5.3 +/- 0.4, P < 0.001), and in 24 of 28 cases for S6 (T/N ratio = 3.1 +/- 0.5, P < 0.01). The level of mRNA overexpression of L18 and S6 did not correlate with the Dukes' stage of disease. In hepatocellular carcinoma samples, using the same four ribosomal protein complementary DNA probes, only P0 mRNA was significantly increased (T/N ratio = 2.8 +/- 0.4, n = 6, P = 0.047). In gastric carcinoma samples, none of these mRNAs was increased (mean T/N ratios = 0.9-1.2, n = 6). Therefore, gastric and hepatocellular carcinomas do not overexpress the same ribosomal protein mRNAs as do colonic carcinoma.

Base Sequence↗
PubMed1993

Ubiquitin-ribosomal protein S27a gene overexpressed in human colorectal carcinoma is an early growth response gene.

One of the extension proteins on the carboxy terminus of ubiquitin was reported as the ribosomal protein S27a. We have cloned a gene which encodes this ubiquitin hybrid protein from a complementary DNA library of a human colon carcinoma cell line. Northern blot analysis of surgical specimens from colon cancer patients showed that these messenger RNA levels were higher in tumor tissue than in adjacent normal mucosa. Furthermore, to investigate the role of this novel ubiquitin hybrid gene in cellular growth control, the responsiveness of this gene to serum growth factors was examined. Within 30 min after serum or 12-O-tetradecanoylphorbol-13-acetate stimulation, its messenger RNA expression in rat fibroblast cells (Rat 1) was increased. Nuclear runoff transcription studies showed that the kinetics of induction of this gene is almost identical to that of protooncogene c-jun or c-fos, the known early growth response genes. Thus, this ubiquitin hybrid gene appears to be a novel early growth response gene overexpressed in human colon cancer and warrants further studies in the pathogenesis of colorectal carcinoma.

Animals↗
PubMed1993

Cytotoxic effect of thiacarbocyanine dyes on human colon carcinoma cells and inhibition of bovine heart mitochondrial NADH-ubiquinone reductase activity via a rotenone-type mechanism by two of the dyes.

Five lipophilic-cationic thiacarbocyanine compounds differing in the side chains (methyl-S13, ethyl-S23, propyl-S33, butyl-S43, and pentyl-S53) and a related thiadicarbocyanine compound with ethyl side chains (S25) exhibited a selective cytotoxic effect on human colon carcinoma cells compared to green monkey kidney epithelial cells. The inhibitory concentration for 50% inhibition of growth (IC50) for the carcinoma cells ranged from 13 nM for S13 and S23 to 160 nM for S25. The carcinoma cells were 4- to 100-fold more sensitive than the normal cells. Two of the five compounds, S13 and S23, selectively inhibited NADH oxidase activity with bovine heart submitochondrial particles (SMP). There was no discernable inhibitory effect by the other three thiacarbocyanine compounds on electron transport chain activity. The primary site of inhibition within the respiratory chain for S13 and S23 appeared to be the NADH to coenzyme Q portion of the mitochondrial electron transport chain. Artificial electron acceptors for this segment of respiratory chain were used to localize the inhibitory site. Using SMP, both S13 and S23 inhibited reduction of menadione, duroquinone, and coenzyme Q. Using purified complex I (NADH-ubiquinone reductase) (EC 1.6.99.3), S13 slightly inhibited reduction of juglone, duroquinone, and coenzyme Q, whereas S23 had no effect on any of the substrates. When rotenone-saturated SMP were used, the inhibitory effects of S13, but not S23, on the reduction of menadione were abolished, as was the inhibitory effect of S13 on coenzyme Q reduction when rotenone-insensitive complex I was used as the source of the enzyme. These results suggest that (1) S13 and S23 inhibition of NADH-ubiquinone reductase activity is enhanced by the membrane environment of the enzyme, and (2) the inhibition appears to be in part akin to the inhibiting mode of rotenone.

Animals↗
PubMed1993

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1993

The significance of carbonic anhydrase expression in human colorectal cancer.

BACKGROUND: Carbonic anhydrase isoenzymes I and II are present in normal colorectal mucosa. This study aimed to determine if carbonic anhydrase is present in colorectal cancer and what is its potential clinical significance. METHODS: The messenger RNA (mRNA) and protein expression of carbonic anhydrase isoenzyme I were analyzed in fresh specimens of colorectal adenocarcinoma by Northern and Western blots, respectively. The immunohistochemical expression was subsequently studied in a larger number of formalin-fixed surgical specimens. RESULTS: All of 30 normal colon samples had a strong RNA hybridization signal. Only 4 of 39 paired colorectal cancer and none of 9 normal liver samples had detectable levels of carbonic anhydrase mRNA. Isoenzyme I protein expression showed similar results. In a separate group of patients, immunohistochemical studies showed that 16 of 96 colorectal tumors had positive staining cells. All positive tumors were well or moderately differentiated carcinomas (P < 0.05). When analyzed retrospectively, immunoreactive cases were more likely to be in a group with a good outcome (P < 0.01) and to lack vascular invasion (P < 0.01) than negative cases. CONCLUSIONS: The majority of colorectal cancers do not express carbonic anhydrase isoenzyme I. The presence of any isoenzyme I-positive immunoreactive cancer cells may be associated with a more favorable outcome in colorectal cancer.

Adult↗
PubMed1993

13C- and 31P-NMR studies of human colon cancer in-vitro and in-vivo.

We report comparative 31P-NMR studies in-vivo and in-vitro of the human adenocarcinoma cell line HCT-116 in a high-density, perfused microcarrier culture and as a tumour from the same cell line grown in three different immune-suppressed animal models (NIH triple deficient, Nude, SCID). The phosphate metabolite ratios, pHNMR and intracellular free magnesium, derived from the 31P-NMR spectra, were compared for the in-vivo and in-vitro systems. Results obtained with HCT-116 cells on microcarrier beads are quantitatively similar to that of small (122 mm3), tumours in-vivo derived from the same cell line in any of the immune-suppressed animal systems studied. This suggests that in-vitro microcarrier cell culture serves as a useful model system for deriving information about metabolism of small, tumours in-vivo. It offers the additional advantages of allowing for precise control of substrate milieu, perfusion and oxygenation. The microcarrier system was also used to measure flux through glycolysis and the pentose cycle. In particular, we measured glucose utilization and the production of lactate, alanine, glutamine and glycogen in proton-decoupled 13C-NMR experiments following administration of [1-13C]glucose. We found that (63% +/- 6%) of the glucose utilized was released as [3-13C] lactate in the presence of oxygen, indicating that the HCT-116 cells have a high level of aerobic glycolysis. Serial labelling experiments with [1-13C] glucose and [6-13C] glucose reveal that at least (11.6% +/- 1.3%) of the glucose utilized enters the pentose cycle. We determined that (6.9% +/- 1.2%) of the glucose utilized is recycled to glucose via the pentose cycle while (4.7% +/- 1.4%) of the glucose utilized enters the pentose cycle to form lactate. The high rate of recycling via the pentose cycle suggests that a significant fraction of cellular NADPH is generated by the pentose cycle as opposed to generation by the malate-pyruvate shuttle.

Adenocarcinoma↗
PubMed1993

Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells.

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) approximately 33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) approximately 22,000 and approximately 11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of approximately 11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.

Amino Acid Sequence↗
PubMed1993

The prognostic significance of proliferating cell nuclear antigen in clinical gastric cancer.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is an intranuclear protein that is closely linked to the cell cycle. This antigen can be detected in formalin-fixed specimens. We studied the expression of PCNA in primary gastric cancer to identify its significance as a prognostic factor. METHODS: The avidin-biotin-peroxidase complex method was used for PCNA staining in sections from 138 patients with primary gastric cancer. All sections were formalin fixed and paraffin embedded. Two different sections were examined in each case. RESULTS: The PCNA labeling index (PCNA-positive cells/1000 cells x 100) varied from 7.0% to 59.7%. There were significant differences in tumor size, morphologic type, depth of tumor invasion, lymphatic permeation, vascular permeation, and lymph node metastasis between the high (> or = 33.4) and low (< 33.4) PCNA labeling index groups. The 5-year survival rates of the high and low PCNA labeling index groups were 5.8% and 66.2%, respectively, a significant difference (p < 0.001). Multivariate analysis showed that the PCNA labeling index was an independent prognostic factor for gastric cancer. CONCLUSIONS: Because PCNA immunostaining can be done in routine formalin-fixed paraffin-embedded sections, this could be a powerful tool for providing information about the prognosis of patients with gastric cancer.

Antigens, Neoplasm↗
PubMed1993

Prothymosin-alpha mRNA expression correlates with that of c-myc in human colon cancer.

Prothymosin alpha (PT-alpha) is a nuclear protein involved in cell proliferation. Transcription of PT-alpha has been reported to be regulated by the c-myc gene in vitro. We identified PT-alpha as being overexpressed in a human colon cancer minus normal mucosa subtraction cDNA library. Northern blot (messenger RNA) analysis showed that both PT-alpha and c-myc genes were overexpressed in human colorectal cancers compared with adjacent normal tissues. Immunohistochemical studies for PT-alpha and c-myc supported these findings. There was no correlation between PT-alpha or c-myc messenger RNA expression and Dukes' stage of colorectal cancer; or between either of these two and actin messenger RNA expression. There was, however, a significant correlation between the PT-alpha expression and c-myc expression (P < 0.001). These findings support the hypothesis that PT-alpha gene transcription may be associated with, and possibly under the control of, the c-myc gene in human colorectal cancers.

Blotting, Northern↗
PubMed1992

Increased expression of human ribosomal phosphoprotein P0 messenger RNA in hepatocellular carcinoma and colon carcinoma.

To search for differentially expressed gene products in selected cancers of endodermal origin, cDNA libraries derived from mRNA in human hepatocellular carcinoma and adjacent grossly normal tissue were generated. From these parent libraries, subtracted cDNA libraries of tumor minus normal and normal minus tumor tissues were constructed. After screening these subtracted libraries by +/- hybridization, a cDNA clone that is overexpressed in hepatocellular carcinoma and encodes the human acidic ribosomal phosphoprotein P0 (P0) was identified. We then evaluated the expression of this phosphoprotein P0 in human colon carcinoma samples. Surgical specimens of primary tumors and liver metastases were examined by Northern hybridization of total RNA with one of 2 32P-labeled P0 probes. The mRNA level of the P0 was greater in primary colon carcinoma than in paired adjacent normal colonic epithelium in 36 of 38 cases; the mean tumor/normal ratio was 2.7 (range, up to 13). The tumor/normal ratio, when plotted against the Dukes' stage of disease, gave evidence for increasing P0 expression with increasing stage of colon carcinoma (P = 0.02). In all 8 cases of paired colon carcinoma metastatic to liver and 2 cases of paired primary hepatocellular carcinoma, the P0 mRNA level was greater in tumor than in adjacent normal liver tissue. The mean tumor/normal ratio was 4.0 (range, up to 11) for the colon cancers metastatic to liver and 4.2 for the primary hepatocellular carcinoma samples. These findings support a common increased expression of selected gene products in different tumors of endodermal origin and suggest that increased P0 expression, in line with certain other ribosomal proteins, may be associated with human colorectal cancer progression and biological aggressiveness.

Blotting, Northern↗
PubMed1992

Differences in messenger RNA expression of carcinoembryonic antigen in surgical specimens of colorectal carcinoma.

Carcinoembryonic antigen (CEA) is the most widely used tumor marker for colorectal cancer. Plasma CEA levels have been variably associated with prognosis. Since plasma CEA level is multifactorial, CEA gene expression in tumors may provide one precise mechanism to evaluate its functional role. This study evaluated CEA expression at the messenger RNA (mRNA) level in 22 human colorectal carcinomas and their adjacent normal mucosae by Northern blot hybridization using a 32P-labeled CEA probe (a loop-domain specific cDNA, LV7). Both tumor and normal mucosa displayed three mRNA species of 4.0, 3.6, and 3.0 kb in length. The expression of 3.6-kb mRNA which encodes for CEA was dominant and it was correlated with another 4.0-kb CEA mRNA expression. The expression of 3.0-kb mRNA which encodes for nonspecific cross-reacting antigen was weak and not detectable in 8 of 22 colon tumors and 12 of 22 normal colon mucosae. In only one tumor, a 4.5-kb mRNA (which might encode for a new family member of CEA) was expressed. A two- to fourfold higher expression of CEA mRNA (3.6 kb) was observed in 11 of 22 colorectal tumors (2 of 9 proximal colon tumors and 9 of 14 rectosigmoid tumors) when compared with morphologically normal adjacent mucosae. Preoperative plasma CEA levels and Dukes' staging had no correlation with this CEA mRNA expression. CEA mRNA did not appear to correlate with metastasis because its expression in the primary colon cancers with metastases (Dukes' stage D tumor) was not always increased. These data also imply that factors other than mRNA expression in tumor might be important in regulating plasma CEA levels.

Aged↗
PubMed1992

Ultrastructural study on extrahepatic infection of duck hepatitis B virus in ducks.

Kidney and pancreas tissues from congenitally infected Ma ducks with duck hepatitis B virus (DHBV) were examined by immunohistochemical staining technique and electron microscopy. The immunostaining showed that DHBV antigen was localized in the cytoplasm of tubular epithelial cells of kidney and acinar cells of pancreas of infected ducks. Under electron microscope, we found that complete and incomplete DHBV particles existed in the dilated cisternae of rough endoplasmic reticulum of both the cells. The size of complete and incomplete virus particles was nearly uniform in both the cells, 50-65 nm and 40-50 nm in diameter respectively. Therefore, extrahepatic infection and replication of DHBV were directly demonstrated ultrastructurally.

Animals↗
PubMed1991

Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential.

The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aqueous buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized positive charge. It formed under favorable conditions a concentration-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission maximum at 527 nm and J-aggregates at 590 nm. Increasing concentrations of JC-1 above a certain concentration caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained constant. The membrane potential of energized mitochondria (negative inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was found to be pH independent within the physiological pH range of 7.15-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.

Animals↗
PubMed1991

Presence of an SH2 domain in the actin-binding protein tensin.

The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

Actins↗
PubMed1991

Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1.

By using a potential-dependent J-aggregate-forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1), we find that membrane potentials across mitochondria in a living cell can be heterogeneous. Remarkably, even within a long contiguous mitochondrion, regional heterogeneity in membrane potentials appears to be possible.

Animals↗