Selection of a dexamethasone-resistant H-4-IIE-C3 rat hepatoma tissue-culture line

Summary Exposure of H-4-IIE-C3 rat hepatoma cell cultures to the synthetic glucocorticoid, dexamethasone, results in an inhibition of cellular proliferation which is not the result of steroid-induced cytolysis. A significant decrease in both the rat of DNA synthesis and DNA content precedes, a detectable effect on cell number. Continuous culture of H-4-IIE-C3 cells in medium containing 10⁻⁵ m dexamethasone results in the selection of a steroid-resistant cell population that has the growth characteristics of unselected sensitive cultures and shows normal steroid induction of tyrosine transaminase. Selection is a slow process requiring 24 to 36 months to obtain, a phenotypically stable resitant cell line, and can be subdivided into three phases—a sensitive phase, adaptation and resistance. A comparison of the karyotypes of unselected and resistant cultures shows that the selection process enriches for a dexamethasone-resistant subpopulation. This work was supported by Special Grant No. 716 from the California Division of the American Cancer Society, and a grant-in-aid from the San Diego State University Foundation.     

A new mechanism for steroid unresponsiveness: loss of nuclear binding activity of a steroid hormone receptor

The glucocorticoid, dexamethasone, binds to the specific cytosol receptors of a steroid-resistant mouse lymphoma cell line with the same affinity as to the receptors of the steroid-responsive parental cells. In the sensitive cells, the receptor-steroid complex translocates to the nucleus, whereas in the resistant cells nuclear transfer is greatly diminished. “Activated” receptor-dexamethasone complex from sensitive cells binds to isolated nuclei from both sensitive and resistant cell types, whereas the complex from the resistant cells binds to neither nuclei. Furthermore, the activated complex from sensitive cells binds to isolated homologous and heterologous DNA, whereas the complex from the resistant cells displays greatly reduced binding activity, implying that DNA plays a significant role in nuclear binding. These results suggest that the normal glucocorticoid receptor has two active domains: one for steroid binding, and the other for interaction with nuclear acceptor sites. The resistant cells described in this paper contain a receptor apparently defective in the latter activity.     

Properties of mer HeLa cells sensitive or resistant to the cytotoxic effects of MNU; effects on DNA synthesis and of post treatment with caffeine

A line of HeLa cells was shown to be particularly sensitive to N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but not to variety of other cytotoxic agents. A resistant line (designated HeLa/A22), was derived by treating Hela cells repeatedly with MNU. Both the sensitive (HeLa) and resistant (Hela/A22) cells have a mer⁻ phenotype based both on their reduced rates of loss of O⁶-methylguanine (O⁶-MeG) from DNA and their low levels of the enzyme O⁶-methylguanine methyltransferase (MT). HeLa cells are therfore sensitive to unrepaired O⁶-MeG in DNA while the Hela/A22 cells are resistant to unexcised O⁶-MeG and thus the A22 cells have the mer⁻ rem⁺ phentype. MNU produced an imediate dose-dependent inhibition of DNA synthesis in cultures of both sensitive resistant cells which increased with time until about 4 h after treatment. DNA synthesis then recovered to near control rates in both sensitive and resistant cells before then exhibiting a progressive decrease after 24 h. DNA synthesis was more depressed at these late times after treatment in cultures of sensitive cells than in those of similarly-treated resistant cells. DNA synthesis remained depressed in sensitive cells but recovered 3 days after treatment in resistant cells.

Post treatment of incubation of MNU-treated HeLa cells with caffeine did not increase the toxic action of MNU. In contrast, post treatment of the resistant HeLa/A22 cells with caffeine resulted in a dramatic increase in the toxic effects of a higher equitoxic dose of MNU. The depressed rate of DNA synthesis observed in both cell lines after doses of MNU was partially reversed by post treatment with caffeine in both sensitive and resistant cells. These observations can be interpreted in terms of the effects of caffeine on DNA replication in treated cells.     

Toxicity of prostaglandins A1 and A2 for cells in culture

Summary Prostaglandins A₁ and A₂, in concentrations near 3×10⁻⁵ M, produced striking toxicity to muscle, skin and liver cells in culture. Prostaglandins E and F_2α were much less active in this regard. Toxicity could be measured by reduction in viable cell number, protein and DNA synthesis in the cultures. The sensitivity of cultured cells was related to their age and population density. Dense cultures were sensitive early, in the first 2 days, and resistant after they manifested confluent growth. Sparse cultures remained sensitive later while they continued DNA synthesis and active cell division. It is hypothesized that the prostaglandin A effect is related to active cell division and DNA synthesis. Supported by USPHS NIAMDD 1RO1 AM 14650 and the Muscular Dystrophy Associations of America, Inc.     

Temporal relationships between DNA metabolism and the growth-inhibitory response produced by dexamethasone in rat glioma cell cultures

Summary The temporal relationships between aspects of DNA metabolism and the suppression of cell proliferation were investigated in rat glioma (strain C6) monolayer cultures exposed to 10μM dexamethasone. Cell densities (cell number per cm²), rates of DNA synthesis (dpm of [³H]thymidine incorporated per μg DNA per min), and cellular DNA (μg DNA per cm²) were measured daily in control and dexamethasone-treated cultures over a 3-day period. The percentage of cells in metaphase and the proportion of metaphases containing >2n(42) chromosomes also were determined in control and treated cultures. When log-phase C6 cultures were exposed to dexamethasone (day 0), cell densities were not significantly different from controls by day 1. Cell proliferation ceased thereafter in dexamethasone-treated cultures, whereas control cell populations continued to proliferate at log-phaserates. In contrast, cellular DNA increased exponentially in control and treated cultures over the 3-day period. On days 0 and 1, control and treated cells each contained 6 pg DNA. By day 3, the DNA content per treated cell increased to >20 pg; control cells each contained 10 pg DNA. The rates of DNA synthesis in the treated cultures did not differ significantly from controls on days 1 and 2. However, the rate in the treated cultures decreased significantly on day 3, one day after cell proliferation ceased. On day 2, the percentage of cells found in metaphase in the treated cultures was 0.32% compared to 0.64% in control cultures. By day 3, these percentages decreased to 0.20% and 0.22%, respectively. However, the proportion of metaphases containing >42 chromosomes increased 1.5-fold in the treated cultures relative to controls. These results indicate that nonproliferating dexamethasone-treated cells contain elevated amounts of DNA. Thus dexamethasone action appears to arrest the cell cycle at any point between the completion of DNA replication and mitosis. A preliminary report of this work was presented on June 8, 1977, at the 28th Annual Meeting of the Tissue Culture Association in New Orleans, Louisiana. This investigation was supported in part by grants from Merck Sharp & Dohme Research Laboratories, West Point, Pa., the American Cancer Society (IN-113), and NIH (AM 18719).     

Glucocorticoids inhibit trypsin-induced DNA release from phytohemagglutinin-stimulated blood lymphocytes.

Trypsin-induced DNA release from phytohemagglutinin (PHA)-stimulated human lymphocytes is inhibited by different glucocorticosteroid compounds at low pharmacologic concentrations, in a dose-dependent manner, and in order of the known anti-inflammatory potency of the different preparations. In contrast, PHA-stimulated cell growth is 100- to 1000-fold less sensitive to inhibition by the same glucocorticoids. Nonglucocorticoid steroids have little effect on either DNA release or cell growth except at high concentrations. Inhibition of DNA release appears to be mediated through glucocorticoid receptors since progesterone, which is ineffective alone, competitively inhibits the effect of dexamethasone. The glucocorticoid effect on DNA release is tightly coupled to the initial, PHA-induced stimulus. Glucocorticoids are maximally effective when added to cultures 1 hr before PHA. When added 6 hr after PHA, their effect is minimal or absent, even though they are then continuously present until DNA release is measured 5 days later. Lymphocytes from certain donors in these studies were resistant to glucocorticoids; these individuals all had allergies, including asthma, allergic rhinitis, and bee sting hypersensitivity.     

Determining DNA strand breakage from embryogenic cell cultures of a conifer species using the single-cell gel electrophoresis assay

Although a routine procedure to detect mutagenesis by DNA strand breakage in animal cells, the single-cell gel electrophoresis (“comet”) assay is difficult to apply in plant material due to constraints in obtaining suitable nucleoids (formed by DNA trapped in the agarose matrix after the cell lysis process) in either quality or quantity. A suitable protocol is described for the first time to perform the comet assay in conifer somatic embryogenic cultures by determining total DNA strand breakage in protoplasts, after having failed to acquire nuclei by standard mechanical techniques. The results show that protoplasts obtained from embryogenic cultures of the Norway spruce (Picea abies) are suitable to be lysed and surveyed for DNA damage through the standard alkaline version of the comet assay. Several common comet metrics were compared and all were found suitable for analysis, with the percentage of DNA in the comets' tail (constituted by DNA fragments that migrated during electrophoresis), given by the proportion between tail fluorescence intensity and total nucleoid intensity, being simplest and the most sensitive to compare between control and hydrogen peroxide-treated cells. The established procedures may be useful, for instance, for a comparative evaluation of somatic embryogenesis protocols and selection of less damaging treatments for clonal propagation or for mutagenesis-related studies with conifer cell cultures.     

Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities

Dexamethasone inhibited the basal and EGF-stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid-specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU-38486 in a concentration-dependent manner. Dexamethasone acted by decreasing the rate of entry into S-phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half-maximal effect occurring 11 hr after the addition of dexamethasone. Densely populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densely populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.     

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10^?6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 (<3 × 10^?9). The receptors of both cell lines have the same affinity for dexamethasone (K_d = 1.3 ± 0.3 × 10^?8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10^?6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r^?,+), but in only one functional copy (r^+/?) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.     

Apoptosis of human T-cells: induction by glucocorticoids or surface receptor ligation in vitro and ex vivo.

Activated T-cells are susceptible to induction of apoptosis or programmed cell death in response to ligation of several cell surface structures, including CD2, CD3, and CD95/Fas. These mechanisms may be important in the regulation of immune responses and in prevention of autoimmunity. We used flow cytometric quantitation of DNA strand breaks to detect T-cells committed to programmed cell death. Activated human peripheral blood T-lymphocytes, and freshly isolated human thymocytes underwent apoptosis when exposed to dexamethasone or to monoclonal antibodies directed at CD2 or CD3. Interleukin-2 reduced spontaneous or dexamethasone-induced apoptosis, but augmented apoptosis due to ligation of CD2. A neutralizing anti-Fas antibody reduced the amount of DNA strand breakage, not only in T-cells exposed to antibodies to CD2 or CD3, but also in dexamethasone-treated cultures. In vivo activated T-cells, from inflammatory synovial fluids, were sensitive to immediate induction of DNA strand breaks without prior in vitro activation by lectin and IL-2. Taken together, the results indicated that: 1. Human lymphocytes, like murine thymocytes, are sensitive to glucocorticoid-induced apoptosis, as well as to programmed cell death triggered through surface receptors; 2. The effects of IL-2 on T-cell apoptosis depend on the apoptotic stimulus; 3. Fas/Fas ligand interactions may be relevant for both membrane receptor and glucocorticoid-induced cell death; and 4. Induction of T-cell apoptosis may be important in therapeutic effects of glucocorticoids in human disease.     

Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells

Dexamethasone is capable of directing osteoblastic differentiation of bone marrow stromal cells (BMSCs) in vitro, but its effects are not lineage-specific, and sustained exposure has been shown to down-regulate collagen synthesis and induce maturation of an adipocyte subpopulation within BMSC cultures. Such side effects might be reduced if dexamethasone is applied in a regimented manner, but the discrete steps in osteoblastic maturation that are stimulated by dexamethasone are not known. To examine this, dexamethasone was added to medium to initiate differentiation of rat BMSCs cultures and then removed after a varying number of days. Cell layers were analyzed for cell number, rate of collagen synthesis, expression of osteocalcin (OC), bone sialoprotein (BSP) and lipoprotein lipase (LpL), and matrix mineralization. Withdrawal of dexamethasone at 3 and 10 days was found to enhance cell number relative to continuous exposure, but did not affect to decrease collagen synthesis slightly. Late markers of osteoblastic differentiation, BSP expression and matrix mineralization, were also sensitive to dexamethasone and increased systematically with exposure while LpL systematically decreased. These results indicate that dexamethasone acts at both early and late stages to direct proliferative osteoprogenitor cells toward terminal maturation.     

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3–4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage in the of VP-16 was, in part, related to a 2–3-fold decrease in both the amount and activity of topisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors.

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

A possible role for altered poly(adenosine diphosphoribose)-synthesis in the sensitivity of human head and neck squamous carcinoma cells to ionizing radiation

Cytotoxicity, extent of DNA double-strand breaks, and stimulation of poly(adenosine diphosphoribose)-synthesis were measured in two established human head and neck squamous carcinoma cell lines (183A and 1483) following x-irradiation. The 1483 cell line was 15-fold more resistant to x-ray-mediated cytotoxicity than was the 183A cell line. X-ray-mediated DNA strand cleavage also differed in these two cell lines with the absolute frequency of DNA double-strand breaks in the sensitive cells 183A cells being twice that in the resistant 1483 cell line. No detectable stimulation of poly(adenosine diphosphoribose)-synthesis was measured in the sensitive 183A cells whereas a marked increase in incorporation of [3H]-nicotinamide adenine dinucleotide was readily detected following x-irradiation of the resistant 1483 cells. These findings suggest a possible role of altered poly(adenosine diphosphoribose)-synthesis in the sensitivity of human head and neck squamous carcinoma cells to ionizing radiation.     

Apoptotic resistance exhibited by dexamethasone-resistant murine 7TD1 cells is controlled independently of interleukin-6 triggered signaling

Interleukin-6 (IL6)-mediated signaling is known to play a role in pathogenesis and resistance in several cancers like multiple myeloma (MM). In this report we used the IL6-dependent 7TD1 murine B-cell hybridoma as an in vitro model to study the interactions between IL6-signaling pathways and the development of dexamethasone resistance. Though in initial stages, 7TD1 cells grew IL6-dependent and were sensitive to dexamethasone-induced apoptosis, chronic exposure to dexamethasone led to a dexamethasone-resistant phenotype (7TD1-Dxm) that grew independent of exogenous IL6. While IL6-mediated JAK/STAT3 and PI3K/AKT signaling was important for proliferation of both cell lines, as shown in proliferation assays using the respective pathway inhibitors, AG490 and LY294002, the resistant cells were insensitive to induction of apoptosis using the same. STAT3 was constitutively phosphorylated in resistant cells and inhibition of its dimerization induced apoptosis but did not alter their insensitivity to dexamethasone. Our results suggest a role of entities downstream of IL6-mediated JAK/STAT3 signaling in development of dexamethasone resistance by 7TD1-Dxm cells.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. V. Biochemical characterization of a strain (ebony) that is UV- and X-ray sensitive and deficient in photorepair

We investigated larval sensitivity to UV and repair of UV- and X-ray-induced lesions in the DNA of the ebony strain compared to a wild-type strain (Canton S). The ebony strain was previously characterized as being more sensitive to UV-induced killing of embryos than Canton S. Also the ebony strain is more sensitive to X-rays for induction of larval killing, dominant lethals and recessive lethals. In this paper it is demonstrated that (1) ebony larvae are more sensitive to killing by UV and less proficient in photoreactivation (PR) ability than Canton S larvae; (2) the ebony strain has a defect in PR repair of endonuclease-sensitive sites induced in the DNA of primary cell cultures by UV irradiation; (3) the ebony strain has a defect in the repair of single-strand breaks induced in the DNA by X-rays (again in primary cell cultures), at least early on in the repair incubation. A rough localization of the UV sensitivity and the PR ability is presented and the possible relevance of the biochemical to the genetic results is discussed.     

Stimulation of DNA synthesis by transient exposure of cell cultures to TPA or polypeptide mitogens: Induction of competence or incomplete removal?

A transient exposure of cell cultures to 12-0-tetradecanoyl-phorbol-13-acetate (TPA) is sufficient to stimulate DNA synthesis during a subsequent incubation in TPA-free medium. We show that (1) a substantial fraction of TPA remains bound to cultures following a transient exposure to TPA and thorough washing, (2) the ability of TPA to induce DNA synthesis is a function of the amount of TPA bound to cell cultures irrespective of whether it is incubated continuously with cultures or transiently exposed to cultures under various conditions, and that (3) a transient exposure of cultures to phorbol-12-13-dibuytrate (PDB), a mitogenic phorbol ester which binds reversibly to cell cultures, does not stimulate DNA synthesis during a subsequent incubation in PDB-free medium. Therefore the persisting effects of TPA are due to it binding to cultures in a manner resistant to washing and not due to the induction of a stable cellular change prerequisite for mitogenesis. Further, we show that certain combinations of polypeptide growth factors induce DNA synthesis in the absence of any such stable cellular change. Evidence is also presented that the persisting effects on DNA synthesis following transient exposure of cultures to other polypeptide growth factors (e.g., platelet-derived growth factor) reflect tenacious binding rather than induction of a lasting biological event.     

The role of DNA repair in resistance of L1210 cells to isomeric 1,2-diaminocyclohexaneplatinum complexes and ultraviolet irradiation

Resistance to cisplatin in several murine leukemia L1210 cell lines is due to enhanced DNA repair. Other platinum complexes, particularly those containing 1,2-diaminocyclohexane (DACH) are of interest as they effectively kill both sensitive (L1210/0) and cisplatin-resistant (L1210/DDP) cell lines. An L1210/DACH cell line has been developed that is preferentially resistant to DACH-Pt complexes. In the current experiments, we investigated the role that DNA repair has in resistance to DACH-Pt compounds. The DACH ligand exists in 3 isomeric forms which exhibit markedly different activities in the various resistant cell lines. Generally, R,R-DACH-Pt was the most effective isomer. DNA repair was assayed by host-cell reactivation of platinated pRSVcat. DNA damage induced by all the isomeric DACH-Pt-SO4 complexes markedly reduced CAT expression in sensitive L1210/0 cells. One adduct per transcribed strand of the cat gene inhibited CAT expression demonstrating that the sensitive cells exhibited no detectable DNA repair. All the resistant cell lines reactivated the plasmid DNA whether damaged with cisplatin or any of the 3 DACH-Pt isomers. Therefore, resistance to both cisplatin and DACH-Pt appears to be mediated by enhanced DNA repair, but the level of reactivation of the transfected plasmid did not correlate with the toxicity of each analogue. These results suggest that some additional event(s) is responsible for the substrate specificity of repair of genomic DNA. These resistant cell lines also exhibited resistance to UV irradiation but this was much less than, and did not correlate with the degree of resistance to either cisplatin or DACH-Pt. However, there was a good correlation between resistance to UV irradiation and reactivation of UV-damaged plasmid DNA. This enhanced reactivation suggests that enhanced repair may be the sole reason for the resistance to UV irradiation.