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Long-term biological effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) were examined with human epidermoid carcinoma KB cells. EGF inhibited the growth of KB cells in both serum-containing and serum-free synthetic media by reducing the growth rate and by lowering the saturation density. The cells cultured with EGF showed relatively high motility and grew dispersely as single cells, whereas the cells cultured in the absence of EGF grew in clusters. Although TGF-beta itself did not inhibit the growth of KB cells, it augmented the growth inhibition by EGF. TGF-beta also affected the cell morphology. In the presence of TGF-beta, the cells became flattened and actin stress fibers were well developed compared to those cultured in its absence. The effects of EGF on growth, cell motility, and cell morphology were reversible. Tyrosine phosphorylation of EGF receptors was continuously observed for at least 50 h in the presence of EGF. TGF-beta did not increase the phosphorylation induced by EGF. These results suggested that signals continuously transmitted through EGF receptors caused the changes in cell growth and morphology and that TGF-beta did not act on the cells by modulating binding of EGF to its receptors or activation of the receptor kinase. In contrast to EGF and TGF-beta, neither insulin nor IGF-I affected cell morphology or growth, although KB cells express their receptors and the receptor kinases were also continuously activated during exposure of the cells to insulin or IGF-I.
We have demonstrated the presence of two DNA polymerases in KB cells and studied the variation of their activities in a synchronous cell population. During the cell cycle we observed in nuclei, only one DNA dependent DNA polymerase, the 3.4 S or minipolymerase, and similarly in the cytoplasm only one enzyme, the 8.3 S or maxipolymerase. The former shows preference for native DNA and the latter for denatured DNA. Their Mg++ and K+ requirements are different and their pH optima are 8.5 and 7 for nuclear polymerase and cytoplasmic polymerase respectively. The cytoplasmic polymerase activity remains stable from one cell cycle to the other with each cell reconstituting its stock at the start of the following cycle (G1 and early S phases). On the contrary nuclear activity decreases in G2, M and early G1, then increases to a maximum in the middle of the S phase. This fluctuation in enzyme activity could be due to degradation, transfer to the cytoplasm or the association of the enzyme with the chromatin and/or the nuclear membrane after completion of DNA synthesis. Our results do not permit us to choose between these three hypotheses. However their significance is discussed in the light of the results obtained by some authors who, on the contrary, have tended to minimise the role of the minipolymerase in DNA duplication, whereas we, from our findings, ascribe a preponderant role to this enzyme. The cytoplasmic maxipolymerase (8.3 S) may simply be a storage form of the enzyme from which minipolymerase can be formed as needed.
Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.
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Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.
KB-A1 and KB-A10 are 2 multi-drug-resistant cell lines which are 100- and 1,000-fold resistant to Adriamycin, respectively. We have examined the expression of P-glycoprotein at the molecular and cellular levels in these human carcinoma cells. Both MDR cell lines, when compared to the parental KB-3-1, show characteristic increases in mdr 1 gene copy number, an increase in mdr 1 mRNA expression, a corresponding increase in transcription rate and a consequent over-expression of P-glycoprotein. However, the more highly resistant KB-A10 cells have a lower gene copy number, express less mdr 1 mRNA and contain less P-glycoprotein than the A1 cell line. To determine whether higher levels of cellular resistance were attributable to enhanced efficacy of P-glycoprotein or to other cellular regulatory mechanisms, we examined other major cellular properties known to be associated with the mdr phenotype. Both the KB-A1 and KB-A10 lines exhibit similar increases in protein kinase C activity as compared to the drug-sensitive parent. In addition, neither glutathione-S-transferase nor topoisomerase II activities account for enhanced resistance of the KB-A10 cells. The above observations are contrary to the premise that the level of drug resistance is necessarily proportional to expression of P-glycoprotein or to other common factors thought to participate in drug insensitivity; consequently, new mechanisms of resistance must be in operation in these cells.
Several uracil and cytosine nucleoside analogues with 2-hydroxyethoxymethyl, 2-aminoethoxymethyl or 1,3-dihydroxypropoxymethyl side chains were synthesized and evaluated for cytostatic (L5178y mouse lymphoma cells) and antiviral (herpes simplex virus type 1 infected KB cells) activity. Two compounds exhibited antiherpesvirus activity. These were 1-(2'-hydroxyethoxymethyl)-5-aza-cytosine and 1-(1',3'-dihydroxypropoxymethyl)-5-iodouracil. The MIC values were 25.8 and 325.7 micrograms/ml, respectively.
KB cell strains which are resistant to the lethal effects of diphtheria toxin were isolated from the toxin-sensitive KB cell line. For one such strain, KB-R2, the concentration of diphtheria toxin required to cause a 50% reduction of incorporation of amino acids into protein was more than 2,000 times that required in the sensitive KB-S cell. The KB-R2 strain was cloned by a single-cell isolation method to yield uniform strains with an even greater resistance to toxin. Resistance is a stable, inheritable property, providing a good genetic marker, and appears to be related to some alteration in the cell membrane. Preliminary studies showed that these cell strains are resistant to poliovirus and vesicular stomatitis virus as well as to toxin.
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The permeability transition pore (PTP) is a mitochondrial inner membrane Ca(2+)-sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca(2+)-induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.
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Cyclin B1 plays a critical role in regulating cell-cycle progression from G2 through M phase (including exit from M phase). In this study, we investigated the relationship between taxol-induced M-phase arrest, disruption of the cyclin B1-regulation pathway and apoptosis in KB cells. Continuous exposure of KB cells to 0.5 microg/ml taxol caused mitotic arrest and >90% cell death at 48 hr. Mitotic blockade peaked at 24 hr, with 68% of cells in mitosis at that time compared with 3% at baseline, and decreased thereafter. Apoptosis assessed by morphological changes and DNA ladder fragmentation was a later event, peaking at 48 hr (later time points were not studied). Taxol also caused an increase in cyclin B1 accumulation, as assessed by Western blot analysis, and stimulated cyclin B1-dependent kinase. Cyclin B1 accumulation and kinase stimulation peaked at 12 and 24 hr, respectively, at which times they were 5-fold and 90-fold higher than in control untreated cells. These effects decreased thereafter. All taxol-induced cellular effects were abrogated by the protein and RNA synthesis inhibitors cycloheximide and actinomycin D. In contrast, the endonuclease inhibitors aurintricarboxilic acid and zinc markedly inhibited taxol-induced DNA ladder fragmentation without altering taxol-induced cell-cycle arrest, cyclin B1 accumulation, activation of cyclin B1 kinase activity and cytotoxicity. We conclude that taxol-induced stimulation of cyclin B1-dependent kinase activity parallels mitotic arrest, is more pronounced than mitotic arrest and precedes the induction of programmed cell death.
Because of its unique DNA-cleaving and strand-passing activities, topoisomerase II is involved in many aspects of DNA metabolism, including replication, transcription, recombination, and repair. The cytotoxic potential of topoisomerase II-targeted drugs, such as etoposide, is related to their ability to stabilize covalently linked enzyme-DNA complexes, which are intermediates in the enzyme's catalytic cycle. Epidermal growth factor receptor is expressed on the cell surface of the majority of squamous cell carcinomas, and epidermal growth factor binding is known to stimulate a number of cellular transduction pathways, including tyrosine kinase, protein kinase C, and phospholipase C. Because topoisomerase II is a proliferation-dependent protein and has been shown to be a high-affinity substrate for many of these cellular transduction pathways, the effects of epidermal growth factor on cellular regulation and sensitivity to etoposide were studied with the human oral cavity squamous cell line, KB. Topoisomerase II catalytic activity was rapidly and transiently inhibited after the addition of epidermal growth factor to the cellular growth media. Western blot on nuclear extracts did not demonstrate alterations in topoisomerase II polypeptide levels to account for changes in catalytic activity. Epidermal growth factor treatment also led to the formation of stabilized, covalently linked enzyme-DNA complexes. Furthermore, epidermal growth factor-induced, topoisomerase II-mediated DNA strand breaks were additive to those induced by etoposide. This study indicates that epidermal growth factor specifically regulates the catalytic and DNA-cleaving activities of topoisomerase II in KB cells. This may direct clinical strategies for circumventing the intrinsic cellular resistance to chemotherapy commonly observed in squamous cell carcinomas of the head and neck.
DNA polymerase activities in uninfected KB cells or KB cells infected with adenovirus type 5 (Ad5) were compared by chromatography on DNA-cellulose and DEAE-cellulose and by isoelectric focusing. On DNA cellulose three components were found both in infected and in uninfected cells. The major component eluted at 0.15 M NaC1 and contained DNA polymerase alpha. Two minor components were found, one which did not bind to DNA-cellulose and one which bound strongly. This latter component contained DNA polymerase beta as characterized by DEAE-cellulose chromatography and sedimentation studies. No difference in properties between uninfected or Ad5-infected KB cells was found for the beta-polymerase. DEAE-cellulose chromatography of DNA polymerase alpha revealed the presence of two activities eluting at 0.11 and 0.13 M NaC1 designates as alphaI and alphaII, respectively. In Ad5-infected cells alphaII was the major component. In uninfected, stationary cells alphaI was the major component and alphaII was only detectable as a shoulder in the elution profile. However, fast growing, uninfected cells gave a similar pattern as Ad5-infected cells. These results indicate that the observed change of the DNA polymerase pattern after infection with Ad5 is related to the level of DNA synthesis and not to the induction of a viral enzyme.
The uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.
Effects of adriamycin on cardiomyocytes and KB cells have been examined. Adriamycin inhibited DNA and RNA synthesis in both rat myocardial cells and KB cells. Protein synthesis was also inhibited but at a much higher dosage. In myocardiocytes, protein synthesis was dose-dependent with inhibition being almost complete at 100 microM. In contrast, inhibition of protein synthesis in KB cells leveled off at 50%.
Cytopathic effects of adenovirus-infected human cells consist of rounding and detachment from the substrate at late times postinfection. It is not known, however, whether any changes in cell adhesion are induced by adenoviruses before cytopathic effects become evident. We show here that attachment and spreading of human adenovirus type 2 (Ad2)-infected KB cells on fibronectin (FN) are, unexpectedly, promoted at intermediate times postinfection. The promotion of spreading by Ad2 is not only on fibronectin but also on laminin and vitronectin. In contrast, Ad2 or Ad5 mutants defective in the E1b 19-kDa function did not lead to a promotion in cell attachment but they led to a promotion in cell spreading. Inhibition of spreading of Ad2-infected KB cells on FN by the peptide Gly-Arg-Gly-Asp-Ser-Pro is partial, although it is complete in uninfected KB cells. We found, however, that Ad2 infection does not significantly change the levels of expression of beta 1 integrin mRNA and FN receptor polypeptides. Our results thus suggest that adenovirus infection leads to a promotion in adhesion through influencing integrin molecules without altering the quantity and/or through activation in the cytoskeleton or some other pathway.