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PubMed1984

A variant form of beta-actin in a mutant of KB cells resistant to cytochalasin B.

Lines of KB cells resistant to cytochalasin B have been isolated and characterized. The mutant Cyt 1 exhibits increased cross-resistance to cytochalasin E. Cyt 1 cells bind less cytochalasin B than parental KB cells. Two-dimensional gel analysis shows that Cyt 1 cells carry an alteration in beta-actin. This is further confirmed by one-dimensional peptide analysis. The altered beta-actin (beta'-actin) is synthesized when poly(A)+ RNA from Cyt 1 cells is translated in a reticulocyte-cell-free translation system. These results suggest that the primary site of action of cytochalasin B on cellular motility processes is beta-actin.

Actins↗

PubMed1987

Effect of intracellular folate concentration on the modulation of 5-fluorouracil cytotoxicity by the elevation of phosphoribosylpyrophosphate in cultured human KB cells.

The effects of extracellular folate concentration on intracellular folate and phosphoribosylpyrophosphate (PRPP) levels and the cytotoxicity of methotrexate and 5-fluorouracil were studied in human KB cells grown in fetal bovine serum-supplemented Eagle's minimum essential medium, which contained standard high folic acid levels (2.3 microM) (standard or S medium), or folic acid-free serum-supplemented medium containing approximately 4 nM 5-methyltetrahydrofolate (physiological or P medium), a folate level and form more comparable to that in normal human serum. Macrocytosis and prolongation of the doubling time by 150% were observed after 5-10 serial passes in P medium, but after 10-15 serial passes, KB cells became "adapted" to P medium with return of size and doubling time to values indistinguishable from cells maintained in S medium. Cellular folate levels fell, and marked elevations in PRPP levels from 68 +/- 43 to 642 +/- 287 pmol/mg cell protein (mean +/- SD) were observed as KB cells were serially passed through P medium. Human leukemia HL-60 and K562 cells and MJY-alpha mouse mammary tumor cells serially passed in P medium also exhibited 10- to 20-fold elevations in PRPP levels. Glucose consumption, glucose decarboxylation, thymidine and adenosine specific uptake, thymidine incorporation into DNA, and 5-fluorouracil uptake were studied in KB cells with elevated and control PRPP levels. As determined by clonal assay, despite elevated PRPP levels, KB cells cultured in P medium were less sensitive to 5-fluorouracil than cells cultured in S medium unless exogenous folate was added. These data support the concept that endogenous folate levels may be inadequate for optimal 5-FU pharmacological action in KB cells with a modulated increase in PRPP levels.

Adenosine↗

PubMed1995

The activation of distinct mitogen-activated protein kinase cascades is required for the stimulation of 2-deoxyglucose uptake by interleukin-1 and insulin-like growth factor-1 in KB cells.

The uptake of 2-deoxyglucose into KB cells was stimulated about 2-fold by interleukin-1 (IL1), anisomycin or insulin-like growth factor-1 (IGF1). Stimulation by IL1 and anisomycin was prevented by SB 203580, a specific inhibitor of the mitogen-activated protein (MAP) kinase homologue termed 're-activating kinase' [RK; also known as p38, p40 and CSBP (cytokine synthesis anti-inflammatory-drug-binding protein)], but was unaffected by PD 98059, a specific inhibitor of the activation of the classical MAP kinase pathway. In contrast, the stimulation of 2-deoxyglucose uptake by IGF1 was blocked by PD 98059 and unaffected by SB 203580. Consistent with these observations, IL1 and anisomycin were potent activators of MAP kinase-activated protein (MAPKAP) kinase-2, a physiological substrate of RK, whereas IGF1 was only a very weak activator of MAPKAP kinase-2. Conversely, IGF1 was a stronger activator of p42 MAP kinase than IL1 or anisomycin. These results imply that the activation of distinct MAP kinase pathways is required for the stimulation of glucose transport by IL1/anisomycin and IGF1 in KB cells, and suggest that the combined use of SB 203580 and PD 98059 is a powerful new approach to explore the roles of different MAP kinase cascades in cell regulation.

Anisomycin↗

PubMed1984

Establishment of human KB cells resistant to 1-beta-D-arabinofuranosylcytosine, and mechanisms of cellular resistance in isolated clones.

A subline of human KB cells that was resistant to 1-beta-D-arabinofuranosylcytosine (ara-C) was established by continuous exposure of the cells to increasing concentrations of ara-C. Thirteen resistant clones were isolated from the resistant subline (KB/ara-C). KB/ara-C showed 1,300-fold higher resistance than the parent KB cells to ara-C; the most resistant clones, clones 7 and 10, showed 1,330-fold higher resistance. In the absence of ara-C, the resistance of the parent KB/ara-C cells was stable for at least 14 weeks, whereas that of clone 7 was stable for 10 weeks, but was slightly less after 14 weeks. The ara-C kinase and ara-C deaminase activities of the 13 clones and the cellular uptake of ara-C by several clones were measured. In general the clones showed decreased deoxycytidine kinase activity and decreased cellular uptake of ara-C. Most clones had higher cytidine deaminase activity than KB cells, but some had activity similar to that of the KB cells. A clear inverse relationship was found between the ara-C sensitivity of the clones and their kinase activity, but not their deaminase activity or their ara-C uptake. These results clearly demonstrate that a major mechanism of ara-C resistance of these human KB cells was a decrease in the activity of the ara-C activating enzyme deoxycytidine kinase. The parent KB/ara-C cells showed no clear cross-resistance to various antitumor agents other than an ara-C derivative, including metabolic inhibitors, alkylating agents, DNA binders and mitotic spindle poisons.

Antineoplastic Agents↗

PubMed1977

Biochemical study of KB-cell receptor for adenovirus.

Three different approaches were used in an attempt to characterize the KB-cell receptor for adenovirus: affinity chromatography, immunoadsorption and cross-linking with a cleavable bifunctional reagent. The first system used an affinity gel consisting of adenovirus-fibre projection linked to Sepharose matrix by an intermediate bis(aminopropyl)amine arm, the amino groups of the fibre ligand being preserved by prior citraconylation. The second system consisted of adenovirus complete penton capsomere attached to anti-(penton base) antibody and cross-linked to polyacrylamide particles with glutaraldehyde. In this latter affinity model, the penton-fibre projection was appropriately oriented outwards, as in the virus particle. Both affinity systems permitted isolation from a KB-cell plasma-membrane extract of fibre-binding and penton-fibre-binding protein material, which inhibited adenovirus attachment. The penton-immunoadsorbent appeared more efficient and more specific than the affinity column of fibre-bis(aminopropyl)amino-Sepharose gel in specific activity of inhibition of adenovirus attachment. The third method consisted of reversibly cross-linking KB-cell receptor proteins with adenovirus particles by means of a cleavable di-imidoester and isolation of the complexes by sucrose-density-gradient centrifugation. Polypeptide analysis on sodium dodecyl sulphate/polyacrylamide gel of labelled KB-cell surface proteins, selected by the different procedures, showed that three major protein subunits of 78000, 42000 and 34000mol.wt. were common to the three selection systems. A possible model for the structure and function of the KB-cell receptor for adenovirus is discussed.

Adenoviruses, Human↗

PubMed1988

Functional alterations in beta'-actin from a KB cell mutant resistant to cytochalasin B.

We recently described the isolation of mutant KB cells (Cyt 1 cells) resistant to the cytotoxic effect of cytochalasin B (CB). This mutant carried an altered beta-actin; i.e., beta'-actin (Toyama, S., and S. Toyama. 1984. Cell. 37:609-614). In the present study, we have examined the functional properties of actin in Cyt 1 cells. Our results showed that increased resistance of Cyt 1 cells to CB was reflected in altered properties of beta'-actin itself. This was shown directly by two findings. First, the polymerization of beta'-actin was more resistant than that of beta- or gamma-actin to the multiple effects of CB. Second, beta'-actin bound less CB than beta- or gamma-actin. The functional alteration of beta'-actin in Cyt 1 cells was further supported by the observation that, although treatment of KB cells with CB increased the pool of unpolymerized actin, the same treatment did not affect the pool of unpolymerized actin in Cyt 1 cells, and that microfilaments of Cyt 1 cells were more resistant to the disrupting action of CB than those of KB cells. These results strongly suggest that the primary site of action of CB on cell motility processes is actin.

Actin Cytoskeleton↗

PubMed1981

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.

Animals↗

PubMed1975

Glycosphingolipids of human KB cells grown in monolayer, suspension, and synchronized cultures.

Studies have been carried out on the glycosphingolipids of human KB cells grown in monolayer and suspension culture, and by synchronization of the latter with a double thymidine (2mM) block. Glycosphingolipids were identified tentatively by thin layer chromatography, gas-liquid chromatography, and combined gas-liquid chromatography and mass spectrometry. The predominant gangliosides in the these cells were AcNeu-Gal-Glc-Cer and AcNeu-Gal-GalNAc-Gal-(AcNeu). Glc-Cer. Theprincipal neutral glycosphingolipids were Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer. Incubation of KB cells (grown in monolayer and subsequently in suspension culture) for 48 hours with D-[1-14Clgalactose resulted in appreciable incorporation of radioactivity into all of the principal glycosphingolipids of these cells. These experiments confirmed that KB cells are capable of synthesizing their constituent glycosphingolipids. KB cells grown in suspension culture showed A 2- to 3-fold increase inthe concentration of Glc-Cer, Gal-Glc-Cer, GalNAc-Gal-Gal-Glc-Cer, and AcNeu-Gal1NAc-Gal-Gal-Glc-Cer, and AcNeu-Gal-Ga1NAcGal-(AcNeu)-Glc-Cer. Thus, the occurrence of tissue culture-dependent changes in the level of glycosphingolipids is demonstrated. Perhaps messages governing the synthesis of glycosphingolipids are translated earlier in thecell cycle under certain conditions of growth and are affected by cell-cell contact and cell adhesion.

Carbon Radioisotopes↗

PubMed1991

Increased phosphorylation of DNA topoisomerase II in etoposide-resistant mutants of human cancer KB cells.

We have isolated two etoposide (VP16)-resistant cell lines, KB/VP-1 and KB/VP-2, from human cancer KB cells after stepwise exposure to increasing doses of VP16. KB/VP-1 and KB/VP-2 showed 30- and 50-fold higher resistance to VP16 and also 20- and 30-fold higher resistance to teniposide than the parent cell line. Furthermore, both resistant cell lines showed more than 2-fold cross-resistance to Adriamycin and daunomycin than KB cells. The levels of accumulation and outward transport of radioactive VP16 were similar in KB/VP-1, KB/VP-2, and KB. The activity of nuclear extracts of DNA topoisomerase II for both KB/VP-1 and KB/VP-2 assayed by decatenation of kinetoplast DNA was consistently similar to that of KB. However, in both immunoblot assay with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA, cellular levels of topoisomerase II in both resistant cell lines were less than one-tenth the level in KB. The cellular levels of DNA topoisomerase I, however, were similar between the mutants and their parent. A quantitative precipitation assay of covalent DNA-topoisomerase II complexes showed greatly reduced VP16-induced cleavages of 3'-32P-DNA by nuclear extracts of KB/VP-1 or KB/VP-2 cells in comparison with KB cells. The relative specific phosphorylation of DNA topoisomerase II was about 14- to 18-fold higher in the mutants than in the parental cells. Phosphoamino acid analysis of DNA topoisomerase II showed that serine was the phosphorylated amino acid in all three cell lines, KB, KB/VP-1, and KB/VP-2. These data suggest that reduced expression of DNA-topoisomerase II might account for the acquired VP16 resistance and reduced VP16-induced cleavages of DNA-topoisomerase II complexes in both VP16-resistant variants.

Carcinoma, Squamous Cell↗

PubMed1999

Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.

We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.

Apoptosis↗

PubMed2005

DNA damaging drugs-induced down-regulation of Bcl-2 is essential for induction of apoptosis in high-risk HPV-positive HEp-2 and KB cells.

DNA damaging chemotherapeutic agents like carboplatin (Carb) and 5-fluorouracil (5-FU), whose effects are mediated through diverse intracellular targets, induce apoptosis in various cancer cells including human papillomavirus (HPV) positive HEp-2 and KB cells. The present work reports the involvement of Bcl-2 in response to the exposure of HEp-2 and KB cells to Carb or 5-FU. We demonstrate that both these drugs are potent inducers of apoptosis. Apoptosis was preceded by decrease in Bcl-2 protein level accompanied by caspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage without altering Bax expression. Further analysis revealed down-regulation of Bcl-2 mRNA as well as protein in drugs treated cells. Ectopic expression of Bcl-2 protected cells against drugs mediated DNA damage-induced apoptosis. Overall, data indicates that genotoxic stress leads to down-regulation of Bcl-2 in HEp-2 and KB cells, which plays a decisive role in the outcome of stress in these cells.

Antimetabolites, Antineoplastic↗

PubMed1972

Ribonucleotide reductase activity of synchronized KB cells infected with herpes simplex virus.

The replication of herpes simplex virus (HSV) is unimpeded in KB cells which have been blocked in their capacity to synthesize deoxyribonucleic acid (DNA) by high levels of thymidine (TdR). Studies showed that the presence of excess TdR did not prevent host or viral DNA replication in HSV-infected cells. In fact, more cellular DNA was synthesized in infected TdR-blocked cells than in uninfected TdR-blocked cells. This implies that the event which relieved the TdR block was not specific for viral DNA synthesis but allowed some cellular DNA synthesis to occur. These results suggested that HSV has a means to insure a pool of deoxycytidylate derivatives for DNA replication in the presence of excess TdR. We postulated that a viral-induced ribonucleotide reductase was present in the cell after infection which was not inhibited by thymidine triphosphate (TTP). Accordingly, comparable studies of the ribonucleotide reductase found in infected and uninfected KB cells were made. We established conditions that would permit the study of viral-induced enzymes in logarithmically growing KB cells. A twofold stimulation in reductase activity was observed by 3 hr after HSV-infection. Reductase activity in extracts taken from infected cells was less sensitive to inhibition by exogenous (TTP) than the enzyme activity present in uninfected cells. In fact, the enzyme extracted from infected cells functioned at 60% capacity even in the presence of 2 mm TTP. These results support the idea that a viral-induced ribonucleotide reductase is present after HSV infection of KB cells and that this enzyme is relatively insensitive to inhibition by exogenous TTP.

Carbon Isotopes↗

PubMed1984

Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport.

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.

Actins↗

PubMed1992

Enhanced sensitivity to G418 of human KB cells adapted to certain media and sera.

Sensitivity to the neomycin derivative G418 was determined for a human cell line, KB 3-1, that had been adapted to six different combinations of media and sera. The results indicate that while the plating efficiency is similar for all conditions, the susceptibility to G418 can differ markedly depending on the particular combination of media and sera used. This suggests that in experiments using neomycin resistance as a selectable marker, conditions may be found where the amount of G418 required for selection and maintenance of transfected cell lines can be reduced, providing a significant savings.

Blood↗

PubMed1982

DNA polymerase-alpha. Common polypeptide core structure of three enzyme forms from human KB cells.

Aqueous extracts of exponentially growing human KB cells contain three quantitatively significant forms of DNA polymerase-alpha activity. The predominant form (about 70% of total cellular activity), which is recovered from the cytoplasm, has been previously purified to near-homogeneity and structurally characterized (Fisher, P. A., and Korn, D. (1977) J. Biol. Chem. 252, 6528-6535). The other two polymerase forms include a minor cytoplasmic activity (about 20% of total cellular activity), DNA polymerase-alpha'; and a species that is tightly associated with detergent-purified nuclei (about 10% of total cellular activity), nuclear DNA polymerase-alpha. We have now purified both minor enzyme species to near-homogeneity and show that they are physically and enzymologically similar to the predominant cytoplasmic species. All three purified forms of KB polymerase-alpha behave as physically homogeneous 7 S species with minimum protomer molecular weights of about 140,000. By examining in vitro 14C-labeled enzyme fractions by high resolution sodium dodecyl sulfate-polyacrylamide gel fluorography, we demonstrate that the three purified species display a similar subunit structure composed of a quartet of polypeptides with molecular masses of 70,000, 65,000, 59,000, and 55,000 daltons, respectively. Extended fluorographic exposures of the gels fail to reveal the presence of any polypeptides larger than 70,000 daltons. It is our present interpretation, based on these and our previous observations, that the four polypeptides reflect microheterogeneity involving two primary species, and we do not yet know how they may associate to form the catalytically active protomer of 140,000 molecular weight. From arithmetical considerations, it is plausible that one or more pairwise combinations of them might suffice to generate the 7 S polymerase activity. These results thus corroborate the principal conclusions of our previous study of the structure of the KB cell cytoplasmic polymerase. In addition, they provide the first comparative analysis of the structure of multiple, operationally distinguishable polymerase-alpha forms obtained from a single tissue, as well as the first description of the purification and structural characterization of a nuclear DNA polymerase-alpha species from any source.

Carcinoma↗

PubMed1965

Effects of arginine deprivation, ultraviolet radiation, and x-radiation on cultured KB cells. A cytochemical and ultrastructural study.

CULTURED KB CELLS (DERIVED FROM A HUMAN ORAL CARCINOMA) GROWN IN MONOLAYERS WERE INJURED BY ONE OF THREE AGENTS: starvation by arginine deprivation or treatment with high doses of either ultraviolet radiation or x-radiation. The different agents produced changes in nucleolar structure and varying accumulations of triglyceride and glycogen. All three agents produced an increase in number and size of lysosomes. These were studied in acid phosphatase preparations, viewed by both light and electron microscopy, and, occasionally, in vital dye, esterase, and aryl sulfatase preparations. Ultrastructurally, alterations in lysosomes suggested that "residual bodies" developed in a variety of ways, i.e., from the endoplasmic reticulum, multivesicular bodies, or autophagic vacuoles. Following all three agents the endoplasmic reticulum assumed the form of "rough" or "smooth" whorls, and, after two of the agents, arginine deprivation or ultraviolet radiation, it acquired cytochemically demonstrable acid phosphatase activity. Near connections between the endoplasmic reticulum and lysosomes raise the possibility that in KB cells, at least when injured, the endoplasmic reticulum is involved in the formation of lysosomes and the transport of acid phosphatase to them.

Acid Phosphatase↗

PubMed2000

Modulation of c-jun and c-fos transcription by UVB and UVA radiations in human dermal fibroblasts and KB cells.

We have previously demonstrated that the oxidizing component of ultraviolet-A (UVA) plays a central role in the activation of the nuclear oncogene and transcription factor, c-fos, in cultured human skin fibroblasts. We have now shown that expression of both c-jun and c-fos (AP-1) family of transcription factors is modulated by short and long wavelength solar ultraviolet (UV) radiation in human fibroblasts and human KB cells. UVA radiation activated c-jun and c-fos in both fibroblasts and KB cells, whereas ultraviolet-B (UVB) radiation activates such oncogenes only in KB cells. Moreover, decreasing the intracellular levels of reducing equivalents in human fibroblasts by glutathione (GSH) depletion lowered the UVA dose threshold for c-jun and c-fos activation several-fold and greatly amplified the UVA-mediated activation of such genes. A more modest effect was observed in GSH-depleted KB cells. In both GSH-depleted fibroblasts and KB cells, UVB radiation failed to amplify c-jun and c-fos activation indicating that the oxidative component of UVB plays a minor role in the modulation of such oncogene expression. These findings clearly indicate that both c-jun and c-fos are activated by the oxidizing component of UVA radiation in human fibroblasts and KB cells, while UVB-mediated modulation seems to be restricted to human epithelial cells and does not involve oxidizing intermediates.

Cell Line↗

PubMed1988

Epidermal growth factor-dependent growth of human KB cells in a defined medium and altered growth factor requirements of KB mutants resistant to EGF-Pseudomonas exotoxin conjugates.

A serum-free culture system was established for human KB carcinoma (HeLa) cells that consisted of a chemically defined medium and several growth factors including epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and ethanolamine. EGF and insulin showed the greatest effects on the growth rate of KB cells. Insulin-like growth factor I (IGF-I) at the same concentration as insulin stimulated cell growth less than insulin. Transferrin, hydrocortisone, or ethanolamine had no growth-stimulatory effects alone but were stimulatory when combined with EGF and/or insulin. Transforming growth factor-beta inhibited growth and triiodothyronine stimulated growth. The growth factor requirements were established for several KB mutants with low EGF receptor levels that had been selected for resistance to a conjugate of EGF with Pseudomonas exotoxin (EGF-PE). Three of five KB mutants did not respond to EGF; two other mutants responded to a lesser extent than the parental KB cells. Four mutants had a reduced response to insulin and responded to T3; one mutant (ET-30) responded to neither. These results indicate that KB cells selected for EGF-PE resistance have lost their growth response to EGF and illustrate the usefulness of serum-free medium for studying the growth factor requirements of mutants with altered receptor levels.

ADP Ribose Transferases↗