Effect of methotrexate on the leukemic cell cycle in mouse ascitic leukemia L1210]

In cells of L1210 ascite leukemia cells, methotrexate inhibited H3-thymidine incorporation, blocked shortly (during 4 hours) the G1 leads to S transition, and did not affect cells in G2-phase or in the late S phase. Almost half a cell population was degenerated and cells in S- and G1-phases were affected in equal proportion. This may suggest that methotrexate is not S-phase specific for cells of leukemia L1210. A simultaneous administration of vinblastine increases the antitumour effect of methotrexate. Cells in G2-phase constitute, presumably, a significant proportion of cells recovered after methotrexate administration. A comparison of the data obtained with literature evidence shows that in the sensitive (leukemia L1210) and resistant (acute mieloid leukemia of man) forms of leukemia, methotrexate affects cells that are in S-phase, whereas cells being in G1-phase are affected only when the sensitive tumours are treated.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Structural differences between sensitive and resistant L1210 cells

The main structural differences between sensitive L1210 mouse leukaemic cells and their multidrug resistant counterpart, obtained by adaptation of the parental cell line to vincristine (VCR), concern the size and shape of the cells, their surface properties and changes in organelles involved in proteosynthesis and transport of substances. The resistant cells are larger with higher density of microvilli. In light and electron micrographs containing a group of cells, cells were found to be closer to each other in L1210/VCR cells than in L1210 cells. This difference in cell aggregation suggests different surface properties which could be visualised by decreased staining of L1210/VCR cell surface coat (glycocalyx) with a polycationic dye ruthenium red. A decrease in surface to volume ratio as a consequence of increased cell size in resistant cells is compensated by proliferation of villi and cytoplasmic protrusions of the cell surface. L1210/VCR cells were further distinguished by higher amount of euchromatin, increase in density of rough endoplasmic reticulum, more developed Golgi apparatus and aggregation of free ribosomes into tetrameric and pentameric polyribosomes. These structural changes may be interpreted as a sign of increase in proteosynthesis and transport of substances.     

An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.     

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.     

Immunologic heterogeneity of dihydrofolate reductase from methotrexate sensitive and resistant L1210 leukemia cells

Dihydrofolate reductase from L1210 leukemia cells which are sensitive and resistant to methotrexate has the same physical and kinetic properties and immunoreactivity with a guinea pig antiserum raised to the enzyme purified from the methotrexate resistant strain. However, a chicken antiserum to dihydrofolate reductase from methotrexate sensitive L1210 cells has greater affinity for the homologous enzyme than for the enzyme from the MTX resistant cells indicating that there is some antigenic difference in these molecules.     

Effects of acivicin and dichloroallyl lawsone upon pyrimidine biosynthesis in mouse L1210 leukemia cells

Acivicin (NSC 163501) and dichloroallyl lawsone (NSC 126771) are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, we have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 microliter of an extract of cells grown in the presence of [14C]bicarbonate. Addition of acivicin (25 microM) to mouse L1210 leukemia cells causes severe depletion in the cellular levels of CTP and GTP, accumulation of uridine nucleotides, and abrupt but transient increases in the concentrations of the early intermediates of both the pyrimidine and purine pathways. Addition of dichloroallyl lawsone (25 microM) results in a rapid depletion of uridine and cytidine nucleotides; carbamyl aspartate and dihydroorotate accumulate to high levels in an equilibrium ratio of 20.5:1, and orotate, orotidine, and UMP increase transiently before decreasing to levels approaching their original steady states. The predominant inhibitory effects of acivicin are upon the reactions UTP----CTP and XMP----GMP, but there is also an initial transient activation of both the pyrimidine and purine pathways by acivicin. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP----UDP initially followed by potent inhibition of dihydroorotate----orotate.     

Alternol inhibits proliferation and induces apoptosis in mouse lymphocyte leukemia (L1210) cells

Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz–Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27^Kip1. Furthermore, the stimulatory effect of LKB1 on p27^Kip1 promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27^Kip1.     

Binding and cross-linking of recombinant mouse interferon-gamma to receptors in mouse leukemic L1210 cells; interferon-gamma internalization and receptor down-regulation

Recombinant E. coli-derived murine IFN-gamma (Mu-rIFN-gamma; 5 X 10(7) U/mg) was radiolabeled with 125I by the chloramine-T method without loss of its antiviral activity. The 125I-Mu-rIFN-gamma showed specific binding to L1210 cells. Scatchard analysis indicates about 4000 binding sites per cell and an apparent Kd of 5 X 10(-10)M. Binding of 125I-Mu-rIFN-gamma to cells was inhibited by both natural (glycosylated) and rIFN-gamma, but not by IFN-alpha/beta. Receptor-bound 125I-Mu-rIFN-gamma was rapidly internalized when incubation temperature was raised from 4 degrees C to 37 degrees C. On internalization, almost no IFN-gamma degradation was observed during 16 hr incubation. 125I-Mu-rIFN-gamma binding capacity decreased in cells preincubated with low doses of unlabeled Mu-rIFN-gamma, but not with IFN-alpha/beta. This receptor down-regulation was dose-dependent: 90% reduction of 125I-Mu-rIFN-gamma binding was observed after preincubation with 100 U/ml. After removal of IFN-gamma from the culture medium, the binding capacity increased with time. However, reappearance of receptor was completely blocked by cycloheximide or tunicamycin, suggesting that re-expression of receptors is not due to recycling but to the synthesis of new receptors, and that the receptor is probably a glycoprotein. Cross-linking of 125I-Mu-rIFN-gamma to surface L1210 cell proteins by using bifunctional agents yielded a predominant complex of m.w. 110,000 +/- 5000. Thus, assuming a bimolecular complex, the m.w. of the receptor or receptor subunit would be close to 95,000 +/- 5000. The formation of such a complex appeared highly specific on the basis of the following criteria: it could be inhibited by the addition of Mu-rIFN-gamma but not by Mu-rIFN-alpha/beta, it was not obtained in cells pretreated with IFN-gamma to induce down-regulation of IFN-gamma receptors, and it was also identified in the IFN-alpha/beta-resistant L1210R cell line, known to be sensitive to IFN-gamma and which we have recently shown to express IFN-gamma receptors.     

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.     

Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells.

By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.     

An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress

Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.     

Comparative cytotoxic and antitumoral effects of ellipticine derivatives on mouse L 1210 leukemia.

Twelve derivatives of the antitumoral alkaloid ellipticine (E) and ellipticinium were assayed in vitro on cultured L 1210 cells. These drugs possess varying abilities to decrease the cell growth rate in a 1--1000-fold range. Some of them have a highly cytotoxic effect in the 10(-8)--10(-6) M range. Non-specific intracellular damages are produced: multilobation of nuclei, occurrence of numerous lipid granules, diminution of the size and increase in the number of mitochondrial profiles and several modifications of the internal architecture of mitochondria. 2-Methyl-9-hydroxyellipticinium (2-CH3-9-OHE) was submitted to a bioassay; it inactivates the tumorigenic potency of the cells exposed to it, when they are grafted back into mice in the same dose range which reduces in vitro the growth rate of the cells. A fairly good correlation holds between the in vitro and in vivo (antitumor effect) assays, offering a possible prescreening test for a cheaper and rapid evaluation of chemotherapeutic activity of these compounds. The results stress again the importance of the 9-hydroxy substitution in these series for improving the anticancer efficiency. The nature of the biochemical target of E and derivatives is discussed according to our data.