Isolation and preliminary characterisation of DNA-protein complexes from the mitochondria of Saccharomyces cerevisiae

Four independent rat L6 myoblast cell lines have been selected in a single step for resistance to the cytotoxic effects of the lectin concanavalin A (conA). In contrast to parental wild-type myoblast lines, all of the variant clones are unable to undergo normal cellular differentiation to form multinucleated myotubes or biochemical differentiation to produce an increase in the specific activity of the muscle-specific enzyme, creatine phosphokinase (CPK). The correlation between lectin resistance and loss of fusion potential is very tight; clonal variation studies show that there is less than a 2.8×10^?8 chance that the two are not directly related. Membrane preparations from the conA-resistant myoblast lines incorporate significantly less GDP-[¹⁴C]mannose into the lipid intermediates of protein glycosylation than preparations from parental wild-type cells. Also, conversion of mannose label to fucose occurs in myoblasts and this pathway is more active in conA-resistant cells than wild-type cells. Reduced binding of labelled conA to the cell surfaces of variant myoblasts was observed which may result from alterations to membrane glycoprotein receptors. These studies suggest that mannosylated glycoproteins of the cell surface play a role in the development of the myotubes from myoblasts. Lectin-resistant myoblasts should be useful model systems for investigating what appears to be a pleiotropic mutation affecting the myogenesis process through membrane modifications.     

Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library

RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.     

The effects of concanavalin A and other agents on protein degradation and migration of non-histone proteins (NHP) to the nucleus in lymphocytes

Concanavalin A (conA) inhibits the degradation of [3H]leucine-labeled cellular proteins of human lymphocytes. The lectin also stimulates the migration of non-histone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H]actinomycin D [(3H]AD). Decreased protein breakdown and increased migration of NHP are parallel events, i.e. both changes occur as a function of the lectin concentration and display a similar time course, suggesting that these events could be related. Similar effects are observed with fluoride, chloroquine and iodoacetate: these agents simultaneously decrease proteolysis and increase the nuclear level of NHP, associated with increased cellular [3H]AD binding. Fractionation of the acidic NHP according to pH 2.5-6.5 shows that proteins with a high degree of degradation in unstimulated cells correspond to proteins with a high degree of migration in conA-stimulated cells. A similar correlation was observed in fluoride-treated lymphocytes. conA, fluoride and iodoacetate decrease cellular [3H]chloroquine [(3H]CQ) accumulation, indicating a lysosomotropic effect. These and previously reported data suggest, but do not prove that conA inhibits degradation of cellular proteins via the lysosomal pathway. Ammonium chloride, methylamine and sodium azide also inhibit proteolysis and increase cellular [3H]AD binding; however, their effects are weak. On the basis of these observations it appears that lysosomal degradation and migration of NHP to the nucleus are linked; however, the mechanism of the linkage is unknown.     

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 °C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 °C, even though it took a longer time to show the same effect. The effect of 10 μg/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 °C. Ninety percent of this effect was abolished by 10 mM of α-methyl-d-mannoside. The effect was completely nullified by 100 mM. At 4 °C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.     

Changes in cell surface glycoproteins on non-differentiating L6 rat myoblasts selected for resistance to concanavalin A

Four independently selected conA-resistant, non-differentiating rat L6 myoblast cell lines and their parental wild-type populations were examined for cell surface alterations. [³H]conA-binding studies indicated that the variant myoblasts bound significantly less lectin than wild-type cells at 4 and at 37 °C. Scatchard analysis revealed two general types of binding sites (high and low affinity sites) on wild-type cells; the variants appeared to be deficient in the high affinity sites. These changes in conA binding probably play an important role in determining the conA-resistant phenotype. Lectin-binding results could be significantly modified by altering the composition of the serum in the growth medium used to culture myoblasts prior to performing binding experiments, suggesting the existence of productive and non-productive lectin-binding sites on the cell surface. SDS slab gel electrophoresis of [³H]mannose-labelled surface membranes prepared from variant and wild-type cells showed that several glycoproteins of the conA-resistant myoblasts were defective in mannosylation. The conA-binding abilities of a pronase digest of one of these altered regions from variant separations, with a molecular weight of 44 500 D, was found to contain glycopeptides with reduced affinity for the lectin, supporting the idea that variant membranes are deficient in a set of high affinity lectin-binding sites. Studies on [GDP-¹⁴C]-mannose incorporation into lipid by membranes from variant and wild-type myoblasts indicated that the biosynthetic lesion likely involved a mannosyl transferase enzyme directly, rather than a lack of free dolichol-PO₄. These studies link conA resistance, cell surface glycoprotein alterations, and defective mannosyl transferase activity with the inability to carry out normal cellular differentiation to form multinucleated myotubes.     

ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS : Effects on Mitochondria In Vivo

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a₃ show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.     

Externalization of the endogenous intracellular lectin of a cellular slime mold

Intracellular purpurin, the endogenous lectin of Dictyostelium purpureum, has previously been shown to be externalized upon exposure of the cells to anti-purpurin IgG. Externalization of additional purpurin was presumably the consequence of cross-linking of the purpurin molecules already on the cell surface by the IgG, since binding univalent anti-purpurin Fab to the cell surface did not have this effect. In the present report we show that multivalent glycoconjugates that interact with purpurin—including asialo-bovine submaxillary mucin, a bacterial galactan, and bovine albumin derivatized with multiple chains of either lactose or lacto-N-neotetraose—all elicit the externalization of intracellular purpurin. Some of the externalized lectin is bound to the cell surface and some is found in the medium, presumably bound to the glycoconjugates. A similar effect is produced by exposure of the cells to high concentrations of purified purpurin. Several plant lectins, including conA, also have some effect, whereas others are inactive. Since cells in late stages of aggregation have about three times as much cell surface purpurin as those in early stages, this externalization reaction may have significance late in development. The results suggest that intracellular purpurin may be released in response to cross-linking of the endogenous cell surface glycoconjugates that are: (1) already bound to endogenous purpurin; (2) capable of binding purpurin; (3) capable of binding certain other lectins. The intracellular lectin may be a reserve pool, that functions only upon externalization.     

In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

On the mechanisms of inhibition of lymphocyte mitogenesis by high conA concentrations

ConA at high concentrations inhibits lymphocyte mitogenesis. Previous studies have shown that inhibitory conA concentrations do not inhibit the acquisition of responsiveness to interleukin-2 (IL-2) when excessive conA is removed. To analyse further the problem of high-dose inhibition by conA, we determined whether inhibition of mitogenesis is related to inhibition of IL-2 production or, alternatively, whether factor production is intact, but the cells are rendered incapable of responding to the factor. ConA stimulates IL2 production at concentrations that are inhibitory to mitogenesis of human lymphocytes. IL-2 was assayed both in a murine cytotoxic T cell line and human memory cells. The response of IL-2-dependent cells to IL-2-containing medium was, on the other hand, inhibited by conA in a dose-dependent fashion. One mechanism whereby high conA concentrations inhibit mitogenesis is by rendering cells resistant to IL-2, possibly via extensive cross-linking of cell surface sites.     

Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands

The anticancer properties of two new fluorescent platinum(II) compounds, cis-[Pt(A9opy)Cl₂] and cis-[Pt(A9pyp)(dmso)Cl₂] are described. These compounds are highly active against several human tumor cell lines, including human ovarian carcinoma sensitive and cisplatin-resistant cell lines (A2780 and A2780R). To study the cellular processing of these new compounds, a series of in vitro studies have been performed, including the investigation of intracellular platinum accumulation and DNA-platination experiments in A2780 and A2780R cells. Compared to cisplatin, both compounds are accumulated highly in both sensitive and resistant cell lines, and more platinum has been found to bind to the nuclear DNA. Interestingly, cis-[Pt(A9opy)Cl₂] shows high accumulation and DNA adduct formation in the resistant cell line A2780R, as compared to the sensitive counterpart A2780 cell line. This suggests that cis-[Pt(A9opy)Cl₂] is able to overcome some of the well-known resistance mechanisms in this cell line, such as decreased cellular uptake and increased DNA repair.     

Characterization of a CHO variant in respect to alkylating agent-induced biological effects and DNA repair

From the Chinese hamster ovary line CHO-9 a resistant variant, Cl 3, was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cl 3 cells were much more resistant to the cytotoxic effects of MNNG (D10 of 1.8 microgram/ml MNNG as compared to 0.23 microgram/ml for parental line) and other methylating N-nitroso compounds, but they had the same sensitivity to various other alkylating agents. MNNG was equally effective in sensitive parent line and resistant variant in inducing sister-chromatid exchanges (SCEs) and mutations to 6-thioguanine resistance. The increased resistance of Cl 3 was not due to reduced cellular uptake of MNNG, to a more efficient repair of methylated purine bases, or to differences in MNNG-induced inhibition of DNA synthesis. It is concluded that the resistant variant has some unknown tolerance mechanism which alters the cytotoxic, but not the SCE- and mutation-inducing effects of methylating N-nitroso compounds.     

佛波酯能改变细胞表面糖蛋白N－糖链的结构

利用标记Ｎ－糖链的凝集素亲和层析法研究了佛波醇肉桂酸乙酸酯（ＰＭＡ）对人肝癌细胞ＳＭＭＣ－７７２１表面糖蛋白上Ｎ－糖链结构的影响,发现１００ｎｍｏｌ／Ｌ的ＰＭＡ处理５天后,可使细胞表面Ｎ－糖链中高甘露糖型和杂合型以及四天线、Ｃ２Ｃ２,６三天线复杂型的比例增高,而二天线复杂型降低。此结果与我们曾报道的视黄酸（ＲＡ）和双丁配环磷酸腺苷（ｄｂ－ｃＡＭＰ）对该细胞表面Ｎ－糖链的影响相反。因ＲＡ和ｄｂ－ｃＡＭＰ是ＳＭＭＣ－７７２１细胞的分化诱导剂,可抑制细胞生长;而ＰＭＡ是该细胞的增殖促进剂,故细胞表面Ｎ－糖链的变化与细胞的分化和增殖密切相关。     

Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

Modification of Glycosylation Mediates the Invasive Properties of Murine Hepatocarcinoma Cell Lines to Lymph Nodes

Among the various posttranslational modification reactions, glycosylation is the most common, and nearly 50% of all known proteins are thought to be glycosylated. In fact, changes in glycosylation readily occur in carcinogenesis, invasion and metastasis. This report investigated the modification of glycosylation mediated the invasive properties of Hca-F and Hca-P murine hepatocarcinoma cell lines, which have high, low metastatic potential in the lymph nodes, respectively. Analysis revealed that the N-glycan composition profiling, expression of glycogenes and lectin binding profiling were different in Hca-F cells, as compared to those in Hca-P cells. Further analysis of the N-glycan regulation by tunicamycin (TM) application or PNGase F treatment in Hca-F cells showed partial inhibition of N-glycan glycosylation and decreased invasion both in vitro and in vivo. We targeted glycogene ST6GAL1, which was expressed differently in Hca-F and Hca-P cells, and regulated the expression of ST6GAL1. The altered levels of ST6GAL1 were also responsible for changed invasive properties of Hca-F and Hca-P cells both in vitro and in vivo. These findings indicate a role for glycosylation modification as a mediator of tumor lymphatic metastasis, with its altered expression causing an invasive ability differentially.     

Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines.

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.     

Characterization of a camptothecin-resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication.

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin-resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565-5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose-response relationship and in its time course for DNA synthesis. Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125-fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conferring camptothecin-resistance on the enzyme does not affect its functional efficiency in this system.     

Butyrate effects on normal and adapted hepatoma cells: morphological response and implications for vectoral cholesterol transport

The cell line 4IC6, adapted for growth in 6 mM sodium butyrate from Hepatoma Tissue Culture cells [R. Chalkley, and A. Shires (1985) J. Biol. Chem. 260, 7698-7704], exhibits a fourfold increase in histone acetate turnover. The 4IC6 cells were about 25 times more resistant to butyrate relative to the parental cell line as measured by cloning efficiency. This line also maintains a flatter and more extended morphology when growing in the presence of 6 mM sodium butyrate relative to the parental line. Both cell lines maintain similar intracellular butyrate levels and incorporate [1-14C]butyrate into lipids to similar extents when incubated in medium containing high levels of the fatty acid. These results show that 4IC6 cells have not attained butyrate resistance through acquiring the ability to metabolize butyrate more efficiently or in a significantly different manner when compared with the parental cell line. The membrane lipid composition was nearly identical between the two cell types. Thus the different morphologies exhibited by each cell line were not a consequence of altered membrane lipid composition. The resistant line, 4IC6, maintains about 10-fold higher cholesterol ester levels and half the level of triglycerides found in the parental line. The butyrate-resistant cells also synthesize cholesterol at about a 1.8-fold higher rate than do the parental cells. This difference in de novo synthesis is reflected by a difference of a similar factor in the amount of radioactive cholesterol the two cell lines accumulate over 12 generations. These results are discussed with respect to models for equilibration of serum lipoprotein-derived and newly synthesized cholesterol.     

Changes in plasma membrane glycoproteins during differentiation of an established myoblast cell line and a non-fusing α-amanitin-resistant mutant

The glycoproteins of purified plasma membranes from mononucleated myoblasts and from myotubes of the L₆ line were characterized according to their apparent molecular weight (MW) and to their ability to bind concanavalin A (conA). We identified 25 proteins in membranes from mononucleated myoblasts and fused myotubes which specifically bound the lectin. Comparison with the pattern of membrane glycoproteins of a non-fusing mutant allowed us to identify developmentally regulated changes in the accumulation of 8 proteins with an apparent MW of 160, 80, 60, 51.5, 43, 40, 38, and 27 Kilodalton (kD), and changes in the glycosylation of six others which migrate at 215, 150, 135, 90, 85, and 32 kD. Two of them (160 and 38 kD) appeared at fusion, whereas the 51.5 kD band could not be identified in plasma membrane from myotubes. As conA inhibits fusion of myoblasts, it is suggested that at least some of these proteins might be involved in this process.     

Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine

Isogenic variants resistant to alkylating agents have been isolated from the human lymphoblast cell line TK6. The cell lines may be divided into four classes on the basis of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The sensitive TK6 parental line shows a 37% survival after 45-min exposure to 0.04 microM MNNG; the three classes of more resistant mutants show 37% survival after 45-min exposure to 2 microM (MF lines), 6 microM (MT lines), and greater than or equal to 10 microM (MX line) MNNG. A representative MF line, MF1, is resistant to both killing and mutation by MNNG or N-methyl-N-nitrosourea. An MT clone, MT1, is highly resistant to killing but hypermutable by MNNG. The MT1 line, like the parental TK6, does not remove O6-methylguanine adducts from the DNA. Our data are consistent with the hypothesis that the MT1 line possesses a nonexcision pathway of defense against killing by alkylating agents. Rather than preventing alkylation of DNA or removing alkylated adducts, the MT1 cells appear to be tolerant of the adducts that are not removed from the DNA.