MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
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In vitro cytotoxicity of a series of quassinoids from Brucea javanica fruits against KB cells.
A microdilution technique was developed for the assessment of in vitro cytotoxicity against KB cells derived from a human epidermoid carcinoma of the nasopharynx. The test was used to determine the cytotoxicity of a series of quassinoids, isolated from Brucea javanica, and which have previously been shown to demonstrate activity against Plasmodium falciparum. The 50% effective dose (ED50) for 7 quassinoids tested against KB ranged from 0.008 microgram/ml for bruceantin, the most cytotoxic of the compounds tested to greater than 5 micrograms/ml for bruceolide, the least toxic tested. The activities of the quassinoids against KB did not parallel the known activities of the quassinoids against Plasmodium falciparum suggesting that the quassinoid mode of antimalarial action is not a simple cytotoxic effect and lends support to further investigation of the structure activity relationships within this group of compounds.
Antimalarials↗
Subcellular distribution of a nitroxide spin-labeled netropsin in living KB cells. Electron paramagnetic resonance and sequence specificity studies.
A nitroxide spin-labeled netropsin was studied by EPR spectroscopy with respect to its uptake and localization in living KB cells. Whereas the drug was taken up readily, there was relatively little drug in the cytoplasm, but a significant concentration of the drug in the cell nucleus. The EPR signal in the latter site corresponded to a relatively freely rotating radical. The drug exhibited good intracellular stability up to 25 hr. While a delta Tm of 24 degrees between the spin-labeled netropsin and calf thymus DNA confirmed strong binding, the absence of any DNA elongation by viscometry was consistent with nonintercalative exterior binding which was confirmed to be minor groove specific by binding of the agent to T4 DNA with a delta Tm of 17.5 degrees. The sequence specificity of the DNA binding of the spin-labeled drug was confirmed by methidiumpropyl-EDTA (MPE) footprinting on a fragment of pBR322 DNA to be very similar to that of the parent netropsin, i.e. selective for AT-rich sites, with minor differences of protection afforded by introduction of the nitroxide label.
Base Sequence↗
[Influence of the quantity of seeded cells on the fixation on glass of KB cells. Role of serum in this fixation].
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Blood↗
In vivo exposure to sodium fluoride does not modify the yield of viral tumour-induced periosteal bone nor of heterotopic bone induced by human tumour KB cells in mice.
In mice local or systemic administration of fluoride (5-25 mg NaF/kg body weight) during the proliferative phase of bone formation (up to 20 days) has no effect on the yield of bone formed either by local stimulation of periosteal membrane by Moloney sarcoma virus-induced tumour or on bone induced heterotopically by human KB cells. The lack of stimulatory activity of fluoride on rapidly induced osteogenesis in mice is in agreement with recent reports which show that fluoride is not a potent mitogen for human osteoblasts grown in vitro.
Animals↗
[Selection by means of KB cells of substances and extracts potentially active in cancer chemotherapy].
Several synthetic, semi-synthetic and natural compounds as well as plant extracts were screened for their growth inhibition activity on KB cells. The most active ones were naphthoquinones and derivatives of pyrido [4,3-b]carbazole alkaloids, with inhibition dose (ID50) less than 4 micrograms/ml. Of the crude extracts of several plants screened, Vellozia caput-ardeae showed to be the most active.
Antineoplastic Agents, Phytogenic↗
Characterization of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells.
The properties of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells were determined. The enzyme has a molecular weight of approximately 40000. Its pH optimum is at pH 4.0, it is not inhibited by ethylenediaminetetraacetate (EDTA), and it is active at temperatures up to 60 degree C. The enzyme cleaves adenovirus DNA in a stepwise manner. The limit digestion product has a molecular weight of 120000-200000. There is evidence that the cleavage reaction proceeds via an initial single-strand nick. Under the conditions tested the endonuclease did not seem to reveal a high degree of specificity as to the recognition of cleavage sites, or else the sites recognized occurred very frequently.
Adenoviruses, Human↗
[Adenylate cyclase activation in KB cells infected by Sendaï virus (author's transl)].
Adenylate cyclase activity is increased in KB cells infected by a little dose of Sendaï virus. The apparent Km for ATP substrate is decreased from 0.7 to 0.4 mM and the activity regulation is changed. This activation may be related to interactions between virus and cell membrane where adenylate cyclase is located.
Adenylyl Cyclases↗
Effects of sequence and temperature of hyperthermia and peplomycin on human pharyngeal carcinoma KB cells in vitro.
To maximize the interactive effect, we examined the time sequence of high (above 43 degrees C) or low (below 43 degrees C)-hyperthermia and peplomycin (PEP)(0.5 microgram/ml). Simultaneous or post-hyperthermic PEP treatment at 44 degrees C resulted in a slight synergistic effect with thermoenhancement ratios (TER) of 1.30 or 1.34, respectively. However, at 42 degrees C, maximal interaction was obtained (TER = 10.19) when KB cells were simultaneously heated with PEP. Furthermore, pre-treatment at 44 degrees C for 25 min or 42 degrees C for 4 h enhanced PEP cytotoxicity more than that of post-treatment at 44 degrees C for 25 min or 42 degrees C for 4 h, respectively. However, chemoenhancement ratios (CER) of pre-treatment at 44 degrees C for 25 min and at 42 degrees C for 4 h were 19.1 and 3.5, respectively, although the isothermic dose decreased the cell count to 60% in both cases. These results indicated that simultaneous or post-hyperthermic PEP treatment with high-hyperthermia, and simultaneous PEP treatment with low-hyperthermia, are the most effective means of PEP thermochemotherapy with hyperthermia.
Cell Survival↗
Production of reactive oxygen species and 8-hydroxy-2'deoxyguanosine in KB cells co-exposed to benzo[a]pyrene and UV-A radiation.
Previous studies have shown that ultraviolet (UV) A light and the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) can synergistically enhance the formation of 8-hydroxy-2'deoxyguanosine (8-OHdG) in living cells. It has been postulated that the underlying mechanism is production of reactive oxygen species (ROS) via photosensitization, but direct evidence supporting this hypothesis has been lacking. This study examined intracellular ROS production in living cells co-exposed to UV-A and BaP as well as the relationship between intracellular production of ROS and formation of 8-OHdG. KB cells were exposed to BaP for 24 h, followed by exposure to UV-A (365 nm) or UV-B (312 nm). The levels of intracellular ROS were directly measured by use of the fluorescent probe dihydrorhodamine 123 (DHR-123) in flow cytometry. Levels of 8-OHdG were measured by high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD). The results demonstrated that UV-B itself induced a much greater level of intracellular ROS than did UV-A alone under the same dose of energy (0.10 mW/cm(2), 20 min). The presence of BaP (13.3 microM) substantially increased ROS production in UV-A-treated cells (2.9-fold), but only slightly enhanced ROS production in UV-B-treated cells (1.3-fold). These results show that BaP acts mainly as a photosensitizer of UV-A, but not UV-B. Furthermore, greater intracellular ROS production was proportional to both BaP concentration and UV-A dosage. There was a linear relationship between ROS production and 8-OHdG formation in cells co-exposed to BaP and UV-A. Results of this study suggest that UV-A and BaP act synergistically to enhance ROS production and formation of 8-OHdG, resulting in increased DNA damage.
8-Hydroxy-2'-Deoxyguanosine↗
Specific detection of an interleukin 1- and tumour necrosis factor-activated beta-casein kinase in HeLa and KB cells.
Interleukin 1 (IL-1) and tumour necrosis factor (TNF) activate a novel protein kinase, TIP kinase, which phosphorylates beta-casein in vitro. We have identified and purified to homogeneity a tryptic fragment of beta-casein, called T1, which was phosphorylated by TIP kinase with kinetics similar to those of the intact protein (K[m] = 27 +/- 6 microM). Phosphopeptide maps of in vitro phosphorylated T1 and beta-casein were identical, confirming that T1 contained the main phosphorylation site of the protein. T1 corresponded to residues 114 to 169 of beta-casein. It was phosphorylated by constitutively active protein kinases to a much lesser extent than beta-casein and thus constituted a specific substrate of the cytokine-activated enzyme. This made possible the detection of TIP kinase in extracts of IL-1-stimulated HeLa and KB cells, which had been hampered by high background phosphorylation when beta-casein was used as substrate. Our results show that the use of fragment T1 allows detection of low levels of TIP kinase in crude samples. They also suggest that its activation, which had previously been observed only in connective tissue cells, may be a general response of many cell types to IL-1 or TNF.
Casein Kinases↗
Reduced daunomycin accumulation in drug-sensitive and multidrug-resistant human carcinoma KB cells following phorbol ester treatment: a potential role for protein kinase C in reducing drug influx.
We have investigated the role of protein kinase C (PKC) in the multidrug resistance (MDR) phenotype of human KB carcinoma cell lines. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced daunomycin accumulation in both drug-sensitive KB-3-1 and MDR KB-C1 cells in a time-dependent manner. The inactive phorbol ester 4 alpha TPA did not reduce daunomycin accumulation, and the PKC inhibitor, Ro 31-8220, reversed the TPA effect. TPA had no effect on daunomycin efflux and did not induce Pgp expression in KB-3-1 cells or alter Pgp levels in KB-C1 cells. Linear, short-term daunomycin accumulation was reduced by pretreatment with TPA, an effect that could be reversed by Ro 31-8220. The effects of TPA on PKC subspecies localisation and downregulation were also examined. TPA initially induced translocation of PKCs alpha and delta, and to a lesser extent, PKC epsilon to the membrane fraction; 8 h after TPA treatment, differential effects on downregulation of PKCs alpha and delta were observed between cell lines, although PKC epsilon was not reduced in either cell line. We therefore propose that the TPA-induced reduction in daunomycin accumulation in KB cells is due to a PKC-mediated process, which is maintained after depletion of certain PKC subspecies or is due to activation of downregulation insensitive PKC subspecies. These results suggest that PKC may regulate drug resistance by reducing drug influx in a Pgp-independent manner in KB cells. This may represent a mechanism of drug-resistance independent of, or in addition, to, Pgp-mediated drug efflux.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
The influence of glutathione and cysteine levels on the cytotoxicity of helenanolide type sesquiterpene lactones against KB cells.
The biological activities of sesquiterpene lactones have been attributed to their reactivity with the cysteine residues of functional proteins forming covalent bonds via Michael type addition. In the present study we investigated the influence of different L-cysteine (cys) and glutathione (GSH) concentrations on the cytotoxicity of the sesquiterpene lactones (STLs) helenalin, 11alpha,13-dihydrohelenalin acetate and chamissonolide against KB cells. Due to the significantly higher reactivity of the alpha-methylene-gamma-lactone (ML) towards cys as compared with the cyclopentenone (CP) site at physiological pH, addition of 20, 50 and 100 molar equivalents of cys decreased the cytotoxicity of helenalin and chamissonolide, whereas the cytotoxicity of 11alpha,13-dihydrohelenalin acetate remained unaffected. In contrast, the influence of GSH addition on the cytotoxicity of 11alpha,13-dihydrohelenalin acetate depends on the concentration of GSH added. Concentration-effect curves obtained for chamissonolide and GSH resembled the decline in cytotoxicity after cys addition. Helenalin showed a biphasic shape of the concentration-effect curve for the 100:1 GSH/helenalin ratio resembling at higher doses the chamissonolide and in lower doses the 11alpha,13-dihydrohelenalin acetate curve at 50-fold excess. These results can be explained by the different reactivity and equilibrium conditions for thiol addition of the two reactive centers of bifunctional STLs in cellular test systems and verified a clear correlation between the different reactivity of their electrophilic centers and the observed biological effects in in-vitro cell systems.
Anti-Inflammatory Agents, Non-Steroidal↗
Regulation by phorbol ester and protein kinase C inhibitors, and by a protein phosphatase inhibitor (okadaic acid), of P-glycoprotein phosphorylation and relationship to drug accumulation in multidrug-resistant human KB cells.
Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells. This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation. In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid. Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro. The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells. Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of [3H]vinblastine. These compounds did not significantly alter [3H]vinblastine levels in drug-sensitive KB-3 cells. These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Deoxyribonucleic acid synthesis in synchronized mammalian KB cells infected with herpes simplex virus.
We examined the patterns of host cell and virus deoxyribonucleic acid (DNA) synthesis in synchronized cultures of KB cells infected at different stages of the cell cycle with herpes simplex virus (HSV). We found that the initiation of HSV DNA synthesis, we well as the production of new infectious virus, is independent of the S, G1, and G2 phases of the mitotic cycle of the host cell. This is in contrast to data previously found with equine abortion virus. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. This eliminates the need for separation of viral and cellular DNA by isopycnic centrifugation in CsCl. We found that HSV DNA synthesis was initiated between 2 to 3 hr after infection. The rate of DNA synthesis increased rapidly, reaching a maximum 4 hr after infection, and decreased to 50% of maximum by 8 hr. Evidence is also presented which suggests that HSV infection can inhibit both the ongoing synthesis of host DNA as well as the initiation of the S phase.
Carcinoma↗
Effects of VIP and VIP-DAP on proliferation and lipid peroxidation metabolism in human KB cells.
In the present study, we have utilized the transglutaminase (TGase) enzyme to modify the primary structure of VIP with diaminopropane (DAP) at the level of the Gln16. We have investigated the conformational stability of VIP and VIP-DAP in solution by limited proteolysis experiments. The VIP-DAP appears to be more resistant to the proteolytic attack of trypsin, thus indicating that the derivatization in position 16 is able to stabilize the structure of the peptide. However, we have studied their role in cell cycle modulation and antioxidant activity in the oropharyngeal epidermoid carcinoma KB cells.
Cell Line, Tumor↗
Characterization of the ATP-dependent LTC4 transporter in cisplatin-resistant human KB cells.
An active efflux pump for cis-diamminedichloroplatinum(II) (cisplatin) has been identified in cisplatin-resistant KCP-4 cells isolated from human epidermoid carcinoma KB-3-1 cells. The adenosine triphosphate(ATP)-dependent transport of leukotriene C4 (LTC4), an endogenous substrate for the glutathione S-conjugate export pump(GS-X pump), has been found in membrane vesicles prepared from KCP-4 cells. Multidrug resistance-associated protein (MRP) has also been identified as an ATP-dependent LTC4 transporter. To examine whether the GS-X pump expressed in KCP-4 cells in MRP, we investigated the expression of MRP in KCP-4 cells and compared the LTC4 transporting activity of GS-X pump expressed in KCP-4 cells with that of MRP. The level of MRP gene expression in KCP-4 cells was low and similar to that in KB-3-1 cells. MRP was not detected in membrane vesicles prepared from KB-3-1 and KCP-4 cells by immunoblot analysis. The ATP-dependent transport of LTC4 in KCP-4 and C-A120 vesicles showed saturable kinetics with an apparent Km of 0.18 microM and 0.25 microM, respectively. [3H]LTC4 transport in KCP-4 vesicles was more inhibited by 2,4-dinitrophenyl-S-glutathione(DNP-SG), bis-(glutathionato)-platinum(II) (GS-platinum) complex and glutathione disulfide(GS-SG) and less by LTD4 compared with that in C-A120 vesicles. The character of the LTC4 transporter expressed in KCP-4 vesicles is similar but not identical to that of MRP. Our results suggest that a glutathione S-conjugate export pump which is different from MRP exists in cisplatin-resistant KCP-4 cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA.
A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.
Carrier Proteins↗