K-Balb Cells Research Articles

CycloSal-dRFIB, a Thymidine Mimetic, Thymidine Kinase by-Pass Nucleoside Prodrug: Radioiodination, in vitro Cellular Uptake and Biodistribution in Murine Models

Cyclo-(3-methylsaligenyl)-5-O-[1-(2,4-difluoro-5-[125I]iodophenyl)-2-deoxy-β-D-ribofuranosyl]phosphate (cycloSal- dRF[125I]IB) was radioiodinated with sodium [125I]iodide via copper-catalyzed isotope exchange in 48% radiochemical yield. cycloSal-dRF[125I]IB was found to be incorporated into the cytoplasmic nucleic acid and mitochondrial fractions of murine KBALB and K-STK (engineered to express HSV-1 thymidine kinase) cells in cell culture. Uptake was greater than that for either the corresponding nucleoside dRF[125I]IB or [125I]IUdR. These in vitro studies support a mechanism of metabolic activation to the free nucleotide, thereby effecting TK-bypass. Pharmacokinetic studies in rats reflect a complex interplay of tissue depot effects, hepatobiliary recycling, and metabolism. Biodistribution studies in tumor- bearing mice provide further evidence for lipophilic depot effects and hepatobiliary recirculation, with no evidence for active (metabolic) accumulation in any tissue.

Glucose Controls Cathepsin Expression in Ras-Transformed Fibroblasts

Overexpression and altered trafficking of cathepsins have been associated with the malignant properties of tumors and transformed cells. A characteristic phenotype of transformed cells is also a profound deviation in their metabolism (aerobic glycolysis, glutaminolysis) which enables them to adapt to extreme nutritional conditions. However, whether the altered metabolism may change the expression of proteinases involved in malignancy has not been determined. Herein we present evidences in Kirsten-virus-transformed 3T3 fibroblasts (KBALB) thatd-glucose selectively increases active forms of cathepsins L, B, and D, without altering other lysosomal nonproteolytic hydrolases (β-d-glucosaminidase, acid phosphatase, β-d-glucuronidase, and β-d-galactosidase).d-Glucose did not modify mRNA levels for cathepsin B or L and did not affect secretion of pro-cathepsin L. However,d-glucose enhanced strongly the amount of the mature forms of cathepsins B and L, without altering their preferential distribution to light endosomal fractions. Induction byd-glucose of intracellular mature cathepsins B and L required a high growth density of KBALB cells and was reproduced in BALB/3T3 fibroblasts stably transfected with a constitutively activated form of Ras.d-Glucose induction of active cathepsins however was not observed in nontransformed BALB/3T3.d-Mannose, in contrast to nonmetabolized sugars (d-galactose, orl-glucose), caused a similar increase in lysosomal cathepsin activities in dense KBALB cells. Thed-glucose analogue, 3-O-methyl-d-glucose, which is transported but not further metabolized, did not reproduce thed-glucose effects. Our findings indicate that, dependent on the nutrient supply and as a consequence of their altered metabolism, transformed cells may modulate the production of active proteinases implicated in malignant progression.

Formation of a G-tetrad and higher order structures correlates with biological activity of the RelA (NF-kappaB p65) 'antisense' oligodeoxynucleotide.

We have examined the behavior of the phosphorothioate antisense Rel A (NF-kappaB p65) oligodeoxynucleotide (oligo) and related molecules. Because of the presence of a G-tetrad near its 5'terminus, this molecule is capable of forming tetraplexes and other higher order structures in a temperature and time dependent manner. The G-tetrad in the phosphodiester congener is protected from methylation by dimethylsulfate when the oligomer is 3'-phosphorylated. However, this protection is completely lost when it is 5'phosphorylated, indicating that the formation of at least some higher order structures has been blocked. In addition, we also prevented tetraplex formation by substitution of 7-deazaguanosine (7-DG) for guanosine at several positions within and outside of the tetrad. This substitution retains Watson-Crick base pair hybridization but prevents Hoogsteen base-pair interactions. When murine K-Balb cells were treated with 20microM antisense RelA oligo, complete blockade of nuclear translocation of RelA was observed. However, this effect was virtually entirely abrogated in most cases by 7-DG substitution within the tetrad, but retained when the substitution was made 3' to the tetrad. The AS RelA-induced downregulation of Sp-1 activity behaved similarly after 7-DG substitution. Thus, the parent phosphorothioate AS RelA molecule cannot be a Watson-Crick antisense agent. However, these conclusions cannot be extrapolated to other G-tetrad containing oligomers and each must be evaluated individually.

Cell line-dependent changes in sensitivity to mitomycin C by 1-methyl-3-isobutylxanthine is due to an altered intracellular dose of the anticancer drug and/or to changes in DT-diaphorase activity.

Sensitization by 1-methyl-3-isobutylxanthine (MIX), a potent inhibitor of cAMP phosphodiesterase, of cellular sensitivity to mitomycin C (MC) was tested using various cell lines. Sensitization was seen with CHO cells and Balb/c 3T3 cells, but a decrease in sensitivity was seen with HeLa, K-balb and other cell lines. To measure the extent of crosslinks produced by MC, we used an alkaline elution assay. The extent of crosslinks was increased by MIX in CHO cells and decreased in K-balb cells. These changes in extent are well reflected by changes in sensitivity to MC in both cell lines. In CHO cells, an intracellular dose of MC was increased by MIX with no change in the rate of uptake or efflux of MC. There was a slight decrease in the dose in HeLa or K-balb cells. Among the enzymes which engage in the reductive activation of MC, we tested DT-diaphorase and NADPH-cytochrome P450 reductase using CHO, HeLa and K-balb cells. MIX had almost no effect on the activity of NADPH-cytochrome P450 in each cell line, whereas it suppressed the activity of DT-diaphorase, significantly in HeLa and K-balb cells, and slightly in CHO cells. All these facts indicate that MIX caused an increase in the extent of crosslinks via an increase in the intracellular dose of MC in CHO cells, and that these increases may lead to the sensitization. On the other hand, the decrease in the sensitivity shown in HeLa and K-balb cells may be due to the suppression of DT-diaphorase activity and/or to a decrease in MC dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional characterization of murine cell lines expressing high, intermediate, or negative levels of surface receptors for interferon-gamma.

Using radioiodinated murine interferon-gamma (IFN-gamma), various cell lines were analyzed for their capacity to bind IFN-gamma and to respond to its biological activity as measured by activation of the 2',5'-oligoadenylate (2-5A) synthetase, induction of the antiviral activity, and inhibition of [3H]thymidine uptake. A T-cell lymphoma, EL-4, was found to express relatively high number of specific membrane receptors for IFN-gamma (60,000/cell) with a dissociation constant of around 9 X 10(-10) M. By contrast, a fibroblast cell line, K-BALB, transformed by the Kirstein murine sarcoma virus, was found negative for binding of radioiodinated IFN-gamma, even in the presence of high ligand concentrations. Other cell lines displayed numbers of receptors varying between 650 to 20,000 binding sites/cell with KD ranging between 5 X 10(-9) to 9 X 10(-10) M. When incubated with EL-4 expressing high levels of specific receptors, IFN-gamma caused stimulation of cell growth, but not resistance to viral (vesicular stomatitis virus) infection and expression of 2-5A synthetase. By contrast, when L1210 and TS/A cells, expressing intermediate levels of specific receptors, were incubated with IFN-gamma, inhibition of cell growth, induction of antiviral resistance and activation of 2-5A synthetase was observed. Finally, K-BALB cells, lacking specific receptors for IFN-gamma, were completely unsensitive to its biological activity. The present system could represent a useful model for the characterization of the interaction of IFN-gamma with target cells at molecular levels.

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 microM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of induction may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA.

Enhancement of endogenous xenotropic murine retrovirus expression by tuftsin.

The exposure of a high-passage clone of Kirsten sarcoma virus transformed Balb/c (K-Balb) mouse cells to tuftsin (Thr-Lys-Pro-Arg) enhanced the expression of endogenous xenotropic retrovirus. The tetrapeptide increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion (0.001-1000 micrograms/ml). Increased virus expression could be achieved during short-term incubations (3-4 hr), with maximum enhancement occurring over longer time periods (16-18 hr). The enhancement of virus expression by tuftsin was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp 70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. Tuftsin stimulated DNA, RNA, and protein synthesis in K-Balb cells during 16-hr incubations. Increased cellular proliferation was also seen at various time periods. The effects observed using K-Balb cells offer an opportunity to study the modulation of gene expression by tuftsin in a fibroblast culture system.

A new tumor promoter, dihydroteleocidin B, enhances cell growth and the production of murine leukemia virus by fibroblasts.

The effects of tumor promoters on the expression of murine leukemia virus (MuLV) were studied in tissue culture. Dihydroteleocidin B, an indole alkaloid, recently found to be a tumor promoter, enhanced not only the production of Moloney MuLV (M-MuLV) by a mouse fibroblast cell line, C3H2K, persistently infected with M-MuLV, but also growth on the C3H2K cells. The production of infectious M-MuLV by M-MuLV-infected C3H2K cells that had been treated with dihydroteleocidin B for 1-7 days was four or five times higher than that of control cells. C3H2K cells grew faster and became stationary at higher cell densities in the presence of dihydroteleocidin B than in its absence. The tumor-promoting phorbol ester phorbol-12, 13-didecanoate and 12-O-tetradecanoylphorbol-13-acetate also enhanced the production of M-MuLV, but their effects were not so strong as that of dihydroteleocidin B. These tumor promoters, however, did not induce production of endogenous MuLV in C3H2K or K-BALB cells.

Interferon induced by UV-irradiated murine cytomegalovirus decreases type C virus expression in BALB/c cells treated by 5-iodo-deoxyuridine or cycloheximide.

UV-irradiated mouse cytomegalovirus (MCMV) activates at low incidence (2.0 X 10(-4) - 8.0 X 10(-4)) a xenotropic type C virus in Kirsten sarcoma virus-transformed nonproducer BALB 3T3 (KBALB) cells. When KBALB cells are treated with UV-MCMV and subsequently with potent chemical inducers, such as 5-Iododeoxyuridine (IdUrd) or cycloheximide, the activation frequencies of type C virus are significantly lower than in controls exposed to the chemical inducers alone: 60% for IdUrd and 44% for cycloheximide. This decrease of activation appears to be mediated by endogenous interferon (IFN) induced by UV-MCMV in KBALB cells, as could be proven by neutralization of the inhibitory activity. Indeed, after exposure of UV-MCMV-infected cells to anti-IFN gamma globulin, the level of C type virus increases, reaching values obtained with IdUrd or cycloheximide in the absence of UV-MCMV. The inhibitory activity associated with UV-MCMV is dose-dependent and the xenotropic type C virus appears to be more sensitive than the ecotropic (N-tropic) retrovirus of BALB/c cells.

5-Azacytidine induction of mouse endogenous type C virus and suppression of DNA methylation.

5-Azacytidine was found to induce the expression of BALB:virus-1 and BALB:virus-2 from K-BALB cells and ecotropic endogenous virus from AKR2B cells. Efficiency of the induction was high and comparable to that by 5-bromodeoxyuridine. The level of methylcytosine in newly synthesized DNA was drastically decreased when K-BALB cells were treated with 5-azacytidine. There was an inverse relationship between the level of DNA modification and the frequency of virus expression.

Increased Expression of Endogenous Xenotropic Murine Retrovirus by Treatment with the Tetrapeptides, Tuftsin and Kentsin

Enhancement of endogenous xenotropic virus expression has been found upon treatment with tetrapeptides of a high-passage clone of Balb/c (K-Balb) mouse cells transformed with Kirsten sarcoma virus. Tuftsin (Thr-Lys-Pro-Arg) and kentsin (Thr-Pro-Arg-Lys) increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion. The enhancement of virus expression by the tetrapeptides was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. The effects observed using K-Balb cells offer an opportunity to study the many biological effects of these peptides in a fibroblast culture system.

Effects of aphidicolin on retrovirus induction by halogenated pyrimidines

Aphidicolin, a specific inhibitor of eukaryotic DNA polymerase-α, suppressed virus induction from K-Balb cells by halogenated pyrimidines. Virus induction, detected by virus-associated RNA-dependent DNA polymerase activity, was reduced to 23% at 0.1 μg/ml of aphidicolin and to 3%n at 0.5 μg/ml. A similar degree of inhibition by aphidicolin was observed in DNA synthesis in vivo and [ 125I]iododeoxyuridine incorporation into the host DNA fraction. The inhibition of virus induction was observed when the cells were treated with iododeoxyuridine and aphidicolin simultaneously. These results suggest that DNA polymerase-a participates in the process of virus induction by halogenated pyrimidines.

Radiation Induction of Endogenous Type C Virus From Mouse Cells Transformed In Vitro by Murine Sarcoma Virus<xref ref-type="fn" rid="fn2">2</xref>

Cell lines from AKR and BALB/c mouse embryos were compared for their sensitivity to X-ray induction of endogenous type C virus. K-Balb cells, a Balb/3T3 cell line nonproductively transformed by Kirsten murine sarcoma virus, were found to be sensitive to X-irradiation. At a dose as low as 50 R, X-rays induced virus expression in K-Balb cells, and the induction frequency increased with increasing dose of X-rays up to 400 R. Among two classes of inducible endogenous viruses carried by K-Balb cells, only Balb:virus-2 was activated by X-irradiation, whereas both Balb:virus-1 and Balb:virus-2 were activated after the cells were treated with 5-iodo-2'-deoxyuridine. UV light and 4-nitroquinoline 1-oxide were also shown to induce virus expression in K-Balb cells. The virus-induction frequency for these physical and chemical carcinogens was much lower (approximately 3 times 10(-4)) than that for 5-bromo-2'-deoxyuridine (approximately 1 times 10(-1)).

Effect of Antipain on Radiation Induction of Endogenous Type-C Virus from Mouse Cells <i>in vitro</i>

Xenotropic murine type-C virus was induced after X-ray or UV light irradiation of K-Balb cells. The induction was markedly suppressed by postirradiation treatment of the cells with antipain, a protease inhibitor of microbial origin. Virus expression was unaffected when the cells were treated with halogenated pyrimidines.

Isolation of Thioguanine Resistant Variants of K-BALB Cells Non-inducible for Type C Viruses by 5-Iododeoxyuridine

Cones of thioguanine resistant K-BALB mouse cells wereisolated which were inducible for endogenous type C virus synthesis by cycloheximide and dexamethsone, but not 5-iododeoxyuridine. A comparison of the number of foci formed on NRK and SC-I cells suggested that the xenotropic virus was suppressed. The variants were not defective in the incorporation of thymidine or iododeoxyuridine or deficient in thymidine kinase, but were deficient in hypoxanthine-guanine phosphoribosyltransferase and the incorporation of hypoxanthine into nucleic acid. Because these cells are blocked at some point in the expression of endogenous virus, they may prove useful in establishing the steps involved in chemical activation of virus synthesis.

Synthesis of type C virus particles from murine-cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone

Previous studies have shown that in certain cell systems dexamethasone may enhance the production of type C viruses. Conversely, interferon has been shown to inhibit their production. Both appear to exert their influence late in the viral replication cycle rather than on the synthesis of viral-specific RNA. In this report dexamethasone and interferon have been used to study some aspects of the mechanisms involved in the synthesis of type C viruses in murine K-BALB cells following induction of virus production by iododeoxyuridine. Interferon inhibited production of xenotropic type C virus induced by iododeoxyuridine from K-BALB cells both in the absence and presence of dexamethasone, but it did not affect production of N-tropic type C virus. Exposure of the cells to interferon for longer than 12 h was required for maximum effect. Two types of inhibitory effects were observed: one diminished by dexamethasone when the steroid was added 24 h after interferon removal, and the second resistant to dexamethasone. The concentration of intracellular group-specific antigen was diminshed after interferon and increased after dexamethasone exposure. When induced cells were treated with both interferon and dexamethasone, the intracellular group-specific protein concentration was slightly increased, but virus production was reduced 10-fold compared with induced cells treated with dexamethasone alone. We conclude that interferon and dexamethasone may affect both the synthesis of viral proteins and the assembly or release of virus particles and that dexamethasone can partially nullify the inhibitory activity of interferon. The results also support previous conclusions that the regulatory mechanisms for synthesis of viral proteins and for the release of viral particles may differ and that controls for xenotropic and ecotropic virus formation may not be identical.

A comparison of membrane glycoconjugates from mouse cells transformed by murine and primate RNA sarcoma viruses

Kirsten murine sarcoma virus (Ki-MSV) transformed Balb/eT3 mouse cells (K-Balb) were found to have altered membrane glycoconjugates compared to normal Balb/3T3 cells. There were reduced amounts of mono- and disialogangliosides, GM1 and GD1a, and activity of the specific galactosyltransferase required for synthesis of these gangliosides was reduced to between 0 and 18.5% of normal in the several K-Balb clones examined. When fucose-labeled glycopeptides derived from the surfaces of Balb/3T3 and K-Balb cells were compared by gel filtration chromatography, the glycopeptides from the transformed cells were enriched in earlier eluting components. These differences were also observed when the glycopeptides were derived from the entire cell and were diminished when the surface or cellular glycopeptides from Balb/3T3 and K-Balb were digested with neuraminidase prior to chromatographic analysis. Changes in these membrane sialoglycolipids and sialoglycopeptides were not influenced by Rauscher leukemia virus infection. In marked contrast, these changes in membrane glycoconjugates were not observed in Wooley monkey sarcoma virus (WSV) transformed Balb/3T3 cells (W-Balb). Although W-Balb cells like K-Balb were transformed by tissue culture criteria, their ganglioside composition, galactosyltransferase activity, and glycopeptide patterns were similar to normal Balb/3T3. These findings have potential implications concerning the role of these complex carbohydrates in the phenotypic alterations of transformed cells.

Surface Antigens of Mammalian Sarcoma Virus-Transformed Nonproducer Cells

In vitro, clonal BALB/3T3 mouse cells were transformed into nonproductive sarcoma cells by Kirsten murine sarcoma virus. The resulting cells are called K-BALB. Similarly, the Kirsten sarcoma virus and woolly monkey sarcoma virus transformed normal rat embryonic kidney (NRK) cells to nonproductive sarcoma cells K-NRK and W-NRK, respectively. The presence of sarcoma cell surface antigen (SCSA) on these three different cell lines has been examined by immunoelectron microscopy using the rabbit anti-K-BALB, anti-K-NRK, and anti-W-NRK sera preabsorbed with normal embryonic rat cells, NRK cells, and BALB/3T3 cells. Four different specific sarcoma cell surface antigens have been demonstrated: SCSAa, an antigen specific to the surface of W-NRK cells; SCSAb, a surface antigen common to K-NRK and W-NRK cells; SCSAc, a surface antigen common to K-BALB and W-NRK cells; and SCSAd, a surface antigen broadly reactive with the rabbit antisera to W-NRK, K-NRK, and K-BALB.