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At least 199 records · Page 11Linked to original sources
PubMed1980

Characterization of RNA synthesized in isolated nuclei of herpes simplex virus type 1-infected KB cells.

Nuclei isolated from herpes simplex virus (HSV)-infected KB cells actively synthesize HSV RNA in vitro; the RNA can be hybridized with HSV DNA or nuclear RNA from HSV-infected cells. Nascent RNA molecules labeled in vivo with 32PO4 were elongated, utilizing the nuclear system to incorporate Hg-CTP at their 3' ends, and then isolated on an affinity column. Hybridization of isolated nascent RNA molecules showed that greater than 50% of them were HSV specific and that more than 25% were self-complementary.

Carcinoma↗
PubMed1996

Monensin and verapamil do not alter intracellular localisation of daunorubicin in multidrug resistant human KB cells.

The effects of monensin, verapamil and several inhibitors of membrane transport processes on the accumulation of [3H] daunorubicin by human KB-A1 cells have been investigated to determine the role of subcellular vesicular transport in the multidrug resistance phenotype. The Golgi inhibitor, brefeldin A, had no effect on drug accumulation, which suggests that vesicular transport is not a significant factor in drug resistance in these cells. KB-A1 cells were collaterally sensitive to both monensin and verapamil. Both of these compounds reduced drug efflux but did not alter subcellular distribution of daunorubicin, consistent with the view that monensin, like verapamil, acts directly on P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Reversal of multi-drug resistance in human KB cell lines by structural analogs of verapamil.

Several structural analogs of verapamil were studied for their ability to reverse multi-drug resistance (MDR) in human KB cell lines. D595, D792 and verapamil completely reversed resistance to colchicine and adriamycin. D595 and D792 had a higher reversing potency than verapamil. Devapamil, gallopamil, emopamil and D528 partially reversed MDR. The reversing potency of a drug did not correlate with its calcium antagonistic activity. No differences in reversing potency between (R)-isomers, (L)-isomers and the racemic forms were observed in the case of both verapamil and emopamil. (R)-verapamil, which has less calcium antagonistic activity and less in vivo toxicity than racemic verapamil, and D792, which has higher reversing potency and less in vivo toxicity than racemic verapamil, should be suitable for clinical applications to overcome drug resistance in cancer patients.

Cell Survival↗
PubMed1987

A Mr = 190,000 glycoprotein phosphorylated on tyrosine residues in epidermal growth factor stimulated KB cells is the product of the c-erbB-2 gene.

In human epidermoid carcinoma KB cells, a glycoprotein of Mr = 190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c-erbB-2 gene product, we have found that gp190 is the human c-erbB-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c-erbB-2 gene product on its tyrosine residues.

Epidermal Growth Factor↗
PubMed1994

Ketoconazole effectively reverses multidrug resistance in highly resistant KB cells.

The antifungal agent ketoconazole was found to overcome resistance to vinblastine and doxorubicin in multidrug resistant KB-V1 cells in vitro. These cells are several hundred-fold more resistant than the parental cell line KB-3-1. Ketoconazole had little or no effect on the parental KB-3-1 cells. The concentrations used to overcome drug resistance in vitro have already been safely used in vivo for treatment of fungal infections and in the monotherapy of hormone independent prostate carcinomas to block adrenal androgen production. Because of a possible beneficial effect of a combination of ketoconazole and a chemotherapeutic drug in multidrug resistant cancers, we examined a panel of 11 prostate carcinoma tissues for the expression of the MDR1 gene by an RNA-PCR assay. MDR1 expression was detectable, albeit at low levels, in 8 of the 11 tumors, suggesting a possible role of this gene in the drug resistance of prostate carcinomas. Our data suggest that ketoconazole might be useful in overcoming multidrug resistance in concentrations that are achievable in humans.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells.

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.

Antibodies↗
PubMed1989

Biosynthesis, processing and half-life of P-glycoprotein in a human multidrug-resistant KB cell.

The biosynthesis, processing, and half-life of the drug efflux pump, P-glycoprotein, were studied in human multidrug-resistant KB (KB-C2) cells selected for resistance to colchicine. An antibody directed against a synthetic oligopeptide corresponding to the amino-acid sequence (Glu-393-Lys-408) of P-glycoprotein from human mdr1 cDNA was prepared in rabbits. With immunoblotting and immunoprecipitation, we detected a 140-170 kDa protein in KB-C2 cells but not in parental sensitive KB cells. KB-C2 cells made a 125 kDa precursor that was slowly processed (t1/2 = 45 min) to the mature form of 140-150 kDa. The processing rate of P-glycoprotein was slower than that of low-density lipoprotein receptor. We detected another 160-180 kDa smear band, which might be a completely denatured form of P-glycoprotein. With immunoblotting, a minor band of high molecular mass (greater than 500 kDa) was also detected and this form increased after the cells were treated with chemical cross-linker, 1,5-difluoro-2,4-dinitrobenzene. The half-life of P-glycoprotein was long; no significant loss of P-glycoprotein was observed within 24 h after synthesis. Cells treated with tunicamycin produced a 120 kDa form of P-glycoprotein which was no longer processed but showed stability similar to that of the mature 140-150 kDa form. Agents that reverse multidrug resistance, phorbol ester and transport substrate did not affect the stability of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1967

Electron microscopy of adenovirus 12 replication. II. High-resolution autoradiography of infected KB cells labeled with tritiated thymidine.

Incorporation of tritiated thymidine by KB cells infected with oncogenic adenovirus 12 was studied by means of high-resolution electron microscopic autoradiography. After a 1-hr pulse with tritiated thymidine, infected and control cultures were fixed at 8, 16, 24, 30, and 36 hr. Infected cultures showed a higher percentage of labeled cells. During early stages, the frequency of silver grains in the nucleus and in the nucleolus was higher in infected material. From 24 hr on, there was an inhibition of nuclear and nucleolar deoxyribonucleic acid (DNA) synthesis. At late stages, one-third of the label was located over nuclear inclusions, type II and IV, previously shown to be composed of DNA and protein, while a large majority of the remaining grains were located over the nucleoplasm. The possibility is considered, that the early increase in nuclear and nucleolar DNA synthesis produced by adeno 12 replication could in part be due to newly synthesized cellular DNA, as has been reported by others with respect to other oncogenic DNA viruses.

Adenoviridae↗
PubMed1975

"Cytoplasmic" deoxyribonucleic acid polymerase. Structure and properties of the highly purified enzyme from human KB cells.

The freshly prepared crude cytoplasmic fraction of aqueously extracted KB cells contains a single major species of DNA polymerase activity (DNA polymerase C) that sediments homogeneously in low ionic strength sucrose gradients with a peak at 10.8 S. The enzyme activity from frozen crude extracts sediments heterogeneously under these conditions with peaks at 8.4 and 10 S. In 0.45 M salt-containing gradients all of the polymerase activity is recovered as a single 6.4 S species. When purified to a specific activity of 7,300, DNA polymerase C sediments in low ionic strength gradients as a single species of 6.5 S. From combined sedimentation and gel filtration analysis, we estimate the molecular weight of the active protomeric species of the polymerase to be about 170,000. Under no conditions of ionic strength does the enzyme disaggregate to active species smaller than 6.4 to 6.5 S. Sodium dodecyl sulfate-polyacrylamide gel analysis of the most highly purified enzyme fractions reveals two major protein bands of 87,000 and 175,000 daltons, respectively. These data suggest that DNA polymerase C contains an 87,000-dalton component and permit the interpretation that the active protomer of Mr equal 170,000 may be a dimer. The purified enzyme shows maximal activity with gapped duplex DNA and has an absolute requirement for 3'-hydroxyl termini. It utilizes initiated polydeoxynucleotide templates poorly and initiated polyribonucleotide templates not at all. Although the polymerase is inhibited by PPi it has only minimal ability to promote PPi exchange (0.8% of the polymerase activity). The purified enzyme is free of endonuclease and exonuclease activities (less than or equal to 0.003% of the polymerase activity) and demonstrates no primer-template-dependent conversion of substrate dNTP to free dNMP during the polymerization reaction. Finally, DNA polymerase C does not excise misparied primer termini from a synthetic homopolymer primer-template but can utilize such termini as initiation sites, although at a very slow rate.

Cell Line↗
PubMed1976

Antiviral activity of arabinosyladenine and arabinosylhypoxanthine in herpes simplex virus-infected KB cells: selective inhibition of viral deoxyribonucleic acid synthesis in the presence of an adenosine deaminase inhibitor.

The antiviral activity of the fraudulent nucleoside arabinosyladenine (ara-A) against herpes simplex virus (HSV) type 1 was increased nearly 20-fold by the adenosine deaminase inhibitor, coformycin. The combination of ara-A plus coformycin was 90 times more potent in blocking HSV replication than was arabinosylhypoxanthine (ara-H). In suspension culture both drugs were more active than they were in monolayer culture. Deoxyribonucleic acid (DNA) synthesis also was inhibited by the nucleosides. Depending upon the species of DNA examined, ara-A was 8 to 15 times more active in the presence of coformycin, and the combination was 35 to 70 times more potent than ara-H. Both drugs inhibited total DNA synthesis to the same extent in uninfected and HSV-infected KB cells. In contrast, viral DNA synthesis was three to six times more susceptible to inhibition than was cellular DNA synthesis. Inhibition of viral DNA synthesis was more pronounced in suspension culture than in monolayer culture. However, the method of cell propagation did not alter the degree to which the drugs inhibited DNA synthesis in uninfected KB cells. An index has been derived to quantitate the extent of the selective inhibition of viral or cellular DNA synthesis. Fifty percent inhibitory concentrations of a drug were calculated for uninfected KB DNA synthesis and viral DNA synthesis and expressed as a ratio. The logarithm of this ratio was termed the selective index and was positive if viral DNA synthesis was inhibited preferentially or negative if uninfected KB DNA synthesis was more strongly inhibited. Data from experiments performed in monolayer culture gave positive selective index values of 0.3, 0.5, and 0.4 for ara-A plus coformycin, ara-A, and ara-H, respectively. Values of 0.7 and 0.6 were obtained from suspension culture data for ara-A plus coformycin and ara-H, respectively. Considered collectively, the data presented in this communication establish that coformycin increased the potency of ara-A but did not increase its selectivity.

Adenosine Deaminase Inhibitors↗
PubMed1998

Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells.

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective increases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1-D clone demonstrated a significant resistance to dNTP inhibition of RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.

Clone Cells↗
PubMed1988

Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. Essential sites of src-related growth factor receptor kinases.

We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.

Amino Acid Sequence↗
PubMed2003

Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells.

Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.

Actin Cytoskeleton↗
PubMed1980

Transfection of KB cells by polyethylene glycol-induced fusion with erythrocyte ghosts containing adenovirus type 2 DNA.

Cell ghosts were formed by hypotonic haemolysis and subsequent isotonic re-sealing of human erythrocytes in the presence of adenovirus type 2 DNA. The ghosts entrapped virus DNA with an efficiency which depended on the salt concentration employed during haemolysis and on the concentration of the DNA itself. The entrapped DNA was largely protected from digestion by deoxyribonuclease I and could be recovered intact by phenol extraction. Erythrocyte ghosts containing 32P-adenovirus type 2 DNA were fused with KB cells during brief treatment with polyethylene glycol (PEG). Following fusion, counts equivalent to an average of 25 molecules of labelled DNA were micro-injected and transported to each KB cell nucleus. Less than 1/4 as many DNA counts were recovered from nuclei when PEG treatment was omitted. A direct immunofluorescent assay demonstrated virus replication in some cells following their fusion with DNA-containing ghosts. The efficiency of transfection was considerably lower than that expected from the large number of successfully micro-injected DNA molecules. This suggests that most of the micro-injected DNA molecules were degraded before a successful infection could be completed.

Adenoviruses, Human↗
PubMed2003

Reversal of cancer multidrug resistance by tea polyphenol in KB cells.

Tea polyphenols, (-)-epigallocatechin gallate in particular, were examined for their modulating effects on the drug resistance KB-A-1 cells and drug sensitive KB-3-1 cells. Both KB-3-1 and KB-A-1 cells were equally sensitive to tea polyphenol and (-)-epigallocatechin gallate. When 10 microgram/ml (-)-epigallocatechin gallate or 40 microgram/ml tea polyphenol were present simultaneously with doxorubicin, the IC50 of doxorubicin on KB-A-1 cells decreased from 10.3 +/- 0.9 microgram/ml to 4.2 +/- 0.2 or 2.0 +/- 0.1 microgram/ml. Tea polyphenol and (-)-epigallocatechin gallate enhanced the cytotoxicity of doxorubicin on KB-A-1 cells by 5.2 and 2.5 times, respectively, but did not show a modulating effect on KB-3-1 cells. Both tea polyphenol and (-)-epigallocatechin gallate showed reversal effects on the multidrug resistance phenotype.

Camellia sinensis↗
PubMed1992

Functional and immunological distinction between insulin-like growth factor I receptor subtypes in KB cells.

Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.

Blotting, Western↗
PubMed1984

Subcellular distribution of spin-labeled bithiazoles and bleomycin in living KB cells: an ESR study.

Spin-labeled derivatives of bithiazole and bleomycin were studied with respect to their uptake and localization in KB cells in vivo. It was found that the amidification of the C-terminal carboxylic group of bleomycinic acid was essential for the penetration of the probes in the cells and that the subcellular localization depended on the number and spacing of positively charged groups.

Bleomycin↗