Cultured KB Cells Research Articles

Consecutive uptake of cadmium by KB cells in culture

The uptake of cadmium ions by KB cells in culture has been analyzed by atomic absorption spectrophotometry in the course of the culture. The increase of the amount of Cd 2+ taken up into the cells corresponded to the decrease of Cd 2+ in the medium. The uptake of Cd 2+ was initiated soon after the addition and ceased to reach a stationary state after approximately 10 hr. A consecutive uptake of Cd 2+ was observed and Cd 2+ accumulated; however, the cells continued to survive.

Biological effects of unsaturated ketonucleosides on eucaryotic cells in culture—The action of 7-(3′,4′-unsaturated 2′-ketohexosyl) theophylline

This study on the effects of 7-(3',4'-unsaturated 2'-ketohexosyl) theophylline on KB cells in culture has confirmed the high cytotoxic potency of the compound. It also showed that at low doses, where no cytotoxic effect occurs, the ketonucleoside impaired DNA, RNA and protein syntheses and strongly inhibited cell multiplication. However, this action was transient, owing to the inactivation of the compound in the cell culture medium. The sensitivity of the cells to the 2'-ketonucleoside was found to depend upon the interval of time between the seeding and the addition of the compound to the culture medium—the shorter the interval of time, the more sensitive the cells. The biological effects induced by the compound were not exclusively restricted to cancer cells.

Multiple effects of glucocorticoids and hyperosmolality on isoenzyme expression in cultured KB cells.

The KB cell line, though indicted as a HeLa-contamined line, is a useful in vitro model for the study of the regulation of isoenzyme expression. KB cells produced three isoenzymic forms of alkaline phosphatase. When KB cells were grown in the presence of prednisolone and/or in hyperosmolar medium, the total enzyme activity was reduced. This change in activity was coupled with specific alterations in the proportion of each isoenzyme. The slow-moving form (identified biochemically and immunologically as the heat-stable, placental, Regan isoenzyme) was substantially increased by the steroid hormone and/or hyperosmolality. The fast-moving form [identified as the intestine-like, amnion (FL) isoenzyme] was strikingly diminished by either treatment. The intermediate form (tentatively referred to as "hybrid," inasmuch as it shared properties of the other two isoenzymes) was decreased only when KB cells were grown in hyperosmolar medium containing prednisolone. These results corroborated the notion that these stimuli cause the induction of increased levels of the heat-stable Regan alkaline phosphatase only. They also point out the necessity of performing isoenzyme analysis when one is investigating the regulation of this enzyme.

Experimental results with the combination of bleomycin plus mitomycin C.

This investigation has established the following: 1. Bleomycin, in combination with mitomycin C or other quinone-containing anticancer agents, stimulated the damage to KB cells in culture. 2. In AH66 tumor-bearing rats, the simultaneous treatments of bleomycin plus mitomycin C extend the lifespan. 3. The bleomycin-induced DNA chain breakage was enhanced by the NADPH-dependent microsomal electron transport system. The enhancement was also observed at the level of isolated nuclei and cells. Vitamin K2 and mitomycin C increased breakage at the cellular level by bleomycin and NADPH. 4. Bleomycin-Cu2+ had tendency to increase the lipid peroxidation reaction by the microsomes. However, the reaction was effectively inhibited by antioxidants. 5. Bleomycin induced aldehyde formation from DNA breakage. The formation was effectively inhibited by scavenging reactions with hydralazine hydrochloride or isoniazid. The possibility of suppressing the side effect of bleomycin was discussed in relation to TBA reactive compounds.

UV-induced alkaline labile DNA damage in human adenovirus and its repair after infection of human cells.

Abstract— UV‐induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 103 particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV‐induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.

The carbocyclic analog of cytidine, synthesis and antineoplastic activity

AbstractThe carbocyclic analog (VI) of cytidine was prepared from the carbocyclic analog (I) of uridine. The intermediate stages were a tetrabenzoyl derivative of I, the tribenzoyl derivative of the uridine analog, and the tribenzoyl 4‐chloropyrimidinone obtained from the latter derivative. The cytidine analog (VI) is active against KB cells in culture and against L1210 leukemia in mice. In the initial tests against L1210 leukemia, the highest dose (200 mg./kg./day, q.d. 1‐9), of three active doses, increased lifespan by 82% and showed no evidence of toxicity.

Synthesis of the carbocyclic analogs of uracil nucleosides

AbstractTreatment of the required hydroxyl derivatives of cis‐3‐aminocyclopentanemethanol with 3‐ethoxyacryloyl isocyanate gave N‐(3‐ethoxyacryloyl)‐N′‐[hydroxy‐ or dihydroxy(hydroxy‐methyl)cyclopentyl]ureas. Cyclization of the ureas in dilute sulfuric acid afforded high yields of the carbocyclic analogs of uridine, 2′‐deoxyuridine, and 3′‐deoxyuridine. The uridine and 3′‐deoxyuridine analogs were also obtained in good yields by cyclizing the ureas in concentrated aqueous ammonia. None of the three analogs showed activity in tests versus KB cells in culture or L1210 leukemia in vivo.

Mechanism of action of cesalin, an antitumor protein

A protein, cesalin, isolated from Caesalpinia gilliesii is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na +, K +-ATPase (ATP phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na +, K +-ATPase. The Na +, K +-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5′-Nucleotidase and Mg ++-ATPase are not inhibited by cesalin in any cells tested.

Potential antitumor agents: A cytotoxic cardenolide from coronilla varia L

AbstractAn alcoholic extract of the seeds of Coronilla varia L. showed inhibitory activity against KB cells in culture and was fractionated through a series of partitions, column chromatography, and preparative layer chromatography to yield hyrcanoside, daphnoretin, scopoletin, and umbelliferone. Hyrcanoside was also tested in the PS mouse leukemia assay and showed borderline activity.

Actinomycin D-induced breakage of human KB cell DNA.

ACTINOMYCIN D is a potent inhibitor of RNA synthesis both in vitro and in vivo1,2. Its mode of action involves the intercalation with DNA at G-C regions and prevention of polymerase movement along the template3,4. Actinomycin D has also been shown to inhibit DNA synthesis, cell division and colony formation in mouse L cells5. We have studied the effects of this drug on the DNA of human KB cells in culture. Extensive DNA breakage was induced even at quite low concentrations of the drug.

Unscheduled DNA Synthesis after Ultraviolet Microirradiation of the Cell Nucleus

Ultraviolet (uv) microirradiation of the cell nucleus was used to study the unscheduled DNA synthesis. Xeroderma pigmentosum (XP) fibroblasts and KB cells in tissue culture were microirradiated over one or more nuclear areas of 5 μm in diameter with uv doses of the order of 10, 100, 1000, and $2000 {\rm ergs}/{\rm mm}^{2}$ . After the irradiation, the cells were labeled with ${}^{3}{\rm H}\text{-thymidine} ({}^{3}{\rm HTdR})$ for 3 hr and autoradiographed. In XP cells not in S phase, no ${}^{3}{\rm HTdR}$ uptake was detected under these conditions, but in KB cells an incorporation localized over the irradiated area(s) of the nucleus was observed. XP and KB cells in S phase during the labeling time, microirradiated with doses of the order of 10 or $100 {\rm ergs}/{\rm mm}^{2}$ , were labeled throughout the nucleus, but cells irradiated with a dose of the order of $1000 {\rm ergs}/{\rm mm}^{2}$ showed ...

Nuclear Deoxyribonucleic Acid Polymerase: PURIFICATION AND PROPERTIES OF THE HOMOGENEOUS ENZYME FROM HUMAN KB CELLS

Abstract We have purified the small nuclear DNA polymerase from cultured KB cells to ≥95% homogeneity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel slabs demonstrates that the polymerase is comprised of a single polypeptide chain of molecular weight 43,000, and this size is in agreement with that of the polymerase activity as determined by gel filtration. Although the enzyme carries out a conventional polymerization reaction, incorporating complementary deoxynucleoside triphosphates at 3'-hydroxyl termini with concomitant stoichiometric release of PPi, it cannot be demonstrated to carry out PPi exchange. The polymerase is devoid of exonuclease activities, particularly the 3'→5'-exonuclease activity, as tested by the most sensitive available assays. However, the enzyme does exhibit prominent primer-template-dependent conversion of deoxynucleoside triphosphate to deoxynucleoside monophosphate. We propose that this reaction represents an independent polymerase-associated activity that is unrelated to 3'→5'-exonuclease, in contrast to the model that has been developed from studies with prokaryotic polymerases. Finally, the KB polymerase is unable to excise mismatched primer termini from a synthetic homopolymer primer-template, but rather can utilize such termini as functional initiation sites for polymerization.

Method for tissue culture evaluation of the cytoxic activity of drugs active through the formation of metabolites

A “tissue culture” method for testing the cytotoxic activity of compounds active through the formation of metabolites is described and cyclophosphamide (CPA), which is known to exert its effect only after hepatic biotransformation, has been chosen as “model” substance. A monolayer of cultured KB cells is treated for one hour with CPA at 37°C, in the presence of different amounts of microsomal enzymes ( 105,000 × g fraction) and their co-factors and at the end of the incubation the medium is removed and changed. A dose-effect relation is observed by measuring the inhibition of growth or the destruction of the KB cells after 24 hr. The significance of this procedure for screening purposes or for studying the possible cytotoxicity of unstable metabolites is outlined.

Melinacidins, a new family of antibiotics.

Melinacidin is a crystalline mixture of closely related antibacterial agents produced by Acrostalagmus cinnabarinus var. melinacidinus. Melinacidin inhibits a variety of Gram-positive bacteria in vitro but is found to be toxic and ineffective in the treatment of experimental bacterial infections in mice. Melinacidin which inhibits the growth of KB and L-1210 cells in tissue culture appears to belong to the "3, 6-epidithiadiketopiperazine" group of antibiotics.

Does cycloheximide interfere with protein degradation?

The presence of the protein synthesis inhibitor cycloheximide does not affect the rate of general protein degradation (turnover), in growing as well as in resting cultured KB cells.

Radiosensitization of KB Cells in Vitro by Hydrazine Derivatives

Acid hydrazides have been found to have apparent sensitizing activity on the effects of 300-kV x-rays on survival of KB cells in culture. "Active" compounds, as measured by survival modification factor at a single x-ray dose, include the hydrazides of the following acids: acetic, succinic, malic, aconitic, 2,5-pyridine-dicarboxylic, 4,5-imidazole-carboxylic, and 1,1,3,3-propanetetracarboxylic. Twelve other hydrazides, some closely similar structurally, were without activity. Radiation dose-response experiments with the first four listed hydrazides have shown significant, though not extreme, effects on slopes and dose-modifying factors. If an active hydrazide is added to cells within 15 minutes after irradiation, the effects on survival are equivalent to those observed when the compound is added one-half hour prior to irradiation and remain present through irradiation. It is thus strongly indicated that these compounds affect a recovery process, or that radiation sensitizes the cells to otherwise nontoxic ...

Daunomycin Nephrosis with Special Reference to Cross-Striated Fibrils in Glomeruli

Daunomycin, a glycosidic antibiotic isolated from cultures of Streptomyces peucetius, consists of a chromophor (daunomycinone) linked to an amino sugar (daunosamine). It is inhibitory to the growth of He La and KB cells in culture, and has antitumor activity in rodents and man. Daunomycin complexes with DNA and has been shown to inhibit both DNA and RNA synthesis.Rats given a single intravenous injection of 20 mg/kg (ca LD50)rapidly develop severe bone marrow depression, and damage to lymphoid tissues and intestinal crypts. Recovery from the initial toxicity to proliferative tissues takes place within several days but shortly, thereafter, a nephrotic syndrome develops. The renal phase of intoxication is manifested by proteinuria, hypoalbuminemia, elevation of α1 globulin, anarsarca, hyperlipemia, and hypercholesterolemia.

Biochemical effects of U-12241, an antibiotic that binds to DNA

U-12241 inhibited the growth of KB cells in cell culture 50% at a concentration of 4 mμ/ml. In KB cells, the antibiotic preferentially inhibited incorporation of labelled cytidine into RNA as compared with incorporation of precursors into DNA or protein. RNA polymerase activity of KB cells was more sensitive to the antibiotic than DNA polymerase activity. U-12241 was postulated to act by binding to the ade nine or thymine moiety of DNA, thus inhibiting DNA-directed RNA synthesis. U-12241 had no effect on tryptophan stimulation of tryptophan pyrrolase, but induction by hydrocortisone was inhibited markedly.

Biochemical and biological studies with tubercidin (7-deaza-adenosine), 7-deazainosine and certain nucleotide derivatives of tubercidin

Various biological and biochemical properties of tubercidin (Tu), tubercidin-5′-phosphate (pTu), the methyl ester of tubercidin-5′-phosphate (MepTu), tubercidin-3′,5′-cyclic phosphate, several dinucleoside phosphates of tubercidin, and the chemical deamination product of Tu, 7-deazainosine (7-DI), including whole animal toxicity and drug distribution, were compared. All compounds containing Tu were much more cytotoxic to KB cells in culture than was 7-DI (ID 50's 0.006-0.05 vs. 0.52 μg/ml) both in the resting and growing phases of cell metabolism. No evidence of conversion of MepTu to Tu or pTu was obtained after incubation in vitro with KB or red blood cells, whereas pTu was partially converted to Tu under the same conditions by KB cells. Tu and pTu were absorbed into and retained by blood cells when incubated in vitro (90–99% absorbed) to a much greater extent than were MepTu or 7-DI (5 and 60%). Tu inhibited DNA, RNA and protein synthesis by KB cells after short-term exposure to 4 or 20 μg/ml in vitro whereas MepTu showed very little effect on macromolecule synthesis even at 60 μg/ml under the same test conditions. Tu and pTu were quite inhibitory to Penicillium oxalicum in vitro whereas MepTu and 7-DI were inactive at identical concentrations. In mice, Tu and pTu were more toxic than MepTu or 7-DI, both on an acute and subacute basis. Both Tu and pTu were toxic orally whereas MepTu was not at the same dosage. While 7-DI showed very high serum (40 μg/ml) and urine levels (4000 μg/ml) after a single I.V. dose of 25 mg/kg in the dog, Tu showed low activity and recovery of biologically active material in the urine (0.25% for Tu, 25% for 7-DI). Considerably more tritiated material with biological activity was found in the urine of dogs which had received MepTu (25% of dose recovered as 3H, 13.7% as bioactivity). The mobilities on paper or silica gel of the excretion products in urine from dogs receiving Tu or MepTu differed, particularly on the bioautogram. Tu, MepTu and 7-DI were studied for their effects on hydrocortisone and tryptophan-induced tryptophan pyrrolase synthesis in rat liver. In these experiments, MepTu and 7-DI inhibited hydrocortisone induced enzyme synthesis at doses of 43 and 32 mg/kg, respectively (20 μmoles/rat). Tu was active at 16 mg/kg (10 μmoles/rat). Inhibitory activity was also observed when the compounds were administered 24 hr before steroid. At drug dosages that clearly inhibited the hydrocortisone induced stimulation, Tu and MepTu did not affect the tryptophan induced increase while 7-DI was inhibitory. The tubercidin derivatives did not stimulate aged TP preparations. Reactions of adenine or tubercidin derivatives with hematin, as determined by difference spectroscopy, were not consistent with TP stimulatory activities. These studies demonstrate several unique biological and biochemical effects for MepTu which could not have been predicted on the basis of its relatively simple chemical structure.

975 Another quick screening test for surfactants: Benarde, M. A. & Kuchler, R. J. (1964). Quantitative effects of alkyl benzene sulfonate (ABS) on KB cells in tissue culture. Proc. Soc. exp. Biol. Med.116, 781

975 Another quick screening test for surfactants: Benarde, M. A. & Kuchler, R. J. (1964). Quantitative effects of alkyl benzene sulfonate (ABS) on KB cells in tissue culture. Proc. Soc. exp. Biol. Med.116, 781