Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients

The appearance of asparaginase-resistant acute lymphoblastic leukemia (ALL) in transformed cell lines has been correlated with increased expression of asparagine synthetase (ASNS). Recent measurements using mRNA-based assays have raised doubts, however, as to the importance of ASNS protein in the cellular mechanisms that confer drug resistance upon the leukemic cells. Studies aimed at determining the concentration of ASNS protein in human leukemias are therefore needed to resolve this issue. A mass spectrometry (MS)-based procedure is presented for the direct quantification of ASNS protein concentration in complex sample mixtures. This assay is able to distinguish samples from transformed cell lines that express ASNS over a wide dynamic range of concentration. Importantly, this method directly detects ASNS protein, the functional entity that may be synthesizing sufficient asparagine to render leukemia cells resistant to asparaginase-treatment. We also report the successful use of this MS method, which has lower limits of detection and quantification of 30 and 100 attomoles, respectively, for the first direct measurements of ASNS protein concentrations in four patient blast samples.     

Glutathione biosynthesis in murine L5178Y lymphoma cells

The pyruvate dehydrogenase complex from pea leaf mitochondria was rapidly deactivated in the presence of 50 to 200 μm ATP. The deactivation of the complex requires Mg²⁺ as shown by EDTA inhibition of deactivation. Deactivation was inhibited by 0.1 to 1 mm pyruvate or dichloroacetate. Activation required 10 mM Mg²⁺ or Mn²⁺ but Ca²⁺ and K⁺ had no effect. Activation was inhibited by the phosphatase inhibitor, F^?. Autoradiograms of nondissociating electrophoresis gel, crossed immunoelectrophoresis gels, and dissociating sodium dodecyl sulfate electrophoresis gels of the complex showed that one protein is labeled. Labeling of this protein is prevented by Mg²⁺, pyruvate, and dichloroacetate. The pyruvate dehydrogenase complex was isolated in a partially deactivated state and reactivation required exogenous Mg²⁺ and was inhibited by F^?. These results are taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylation) catalyzed by a kinase and phosphatase. Isolation of the complex in a partially deactivated (phosphorylated) state suggests a physiologically significant role for this regulatory mechanism.     

Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli

Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.     

Inhibition of calcium and glucose uptake by murine leukemia L5178Y cells treated with antiserum

Studies were performed to determine whether decreases in transport of calcium and glucose might be among the earliest changes triggered by the antigen-antibody reactions occurring on the cell surface of murine leukemia L5178Y cells after treatment with rabbit antisera. After treatment with antisera, in the absence of complement, these cells exhibited a decreased uptake of ⁴⁵Ca, 2-deoxy[³H]glucose, and 3-0-methyl[³H]glucose. These changes occurred rapidly, within 2 minutes after the addition of antiserum, in contrast to the previously reported inhibitory effects of antiserum on DNA, RNA, and protein synthesis, which became demonstrable only after 4 to 8 hours. The kinetics of uptake of the radioactive substrates was biphasic, with a very rapid initial uptake followed by less rapid linear uptake. The precise mechanism of cell growth inhibition remains to be elucidated, but one of the initial effects of antiserum treatment may be a perturbation at the cell membrane such that transport of specific nutrients is decreased, resulting in the observed effects on macromolecular synthesis.     

Ultrastructural localization of actin in nuclear matrices from mouse leukemia L5178Y cells

We examined the distribution of actin in isolated nuclear matrices from mouse leukemia L5178Y cells using an anti-actin antibody and protein A-conjugated colloidal gold particles. Before immunogold staining, we partially digested the surface lamina of the nuclear matrix with trypsin (Nakayasu and Ueda, Exp. Cell Res. 143, 55-62, 1983) to allow penetration of the gold particles into the nuclear matrix. Trypsin digestion slightly modified the internal structure of the nuclear matrix, but did not affect the actin content in the nuclear matrix nor the reactivity of actin with the antibody. Many colloidal gold particles were present along fibrogranular structures in the nuclear matrix. The results reported here confirm the existence of actin in the interior of the nuclear matrices of L5178Y cells.     

cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells

The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.     

Mutation induction by very low dose rate gamma rays in cultured mouse leukemia cells L5178Y

Induction of cell killing and mutation to 6-thioguanine resistance was studied in growing mouse leukemia cells in culture following gamma rays at dose rates of 30 Gy/h, 20 cGy/h, and 6.3 mGy/h, i.e., acute, low dose rate, and very low dose rate irradiation. A marked increase was observed in the cell survival with decreasing dose rate; no reduction in the surviving fraction was detected after irradiation at 6.3 mGy/h until a total dose of 4 Gy. Similarly, the induced mutation frequency decreased after low dose rate irradiation compared to acute irradiation. However, the frequency after irradiation at 6.3 mGy/h was unexpectedly high and remained at a level which was intermediate between acute and low dose rate irradiation. No appreciable changes were observed in the responses to acute gamma rays (in terms of cell killing and mutation induction) in the cells which had experienced very low dose rate irradiation.     

Methyl methanesulphonate mutagenesis in L5178Y mouse lymphoma cells.

The alkylating agent MMS was toxic to mouse lymphoma L5178Y cells and decreased their growth rate. A dose-dependent induction of thioguanine- and thymidine- but not ouabain-resistant variants was observed. The prolonged period for expression of thioguanine-resistant variants observed with other mutagens was also found in these studies. A comparison of MMS and EMS showed that MMS on a molar basis was approximately 10 times more toxic than EMS. With mutation, however, when evaluated at equal levels of cell killing MMS and EMS induced the same number of thymidine-resistant variants. For thioguanine-resistant variants MMS was approximately 10-fold less efficient than EMS, while for ouabain-resistance MMS, unlike EMBS, produced no variants at all. The ouabain results were further compared with positive results obtained using a modified Luria--Delbrück fluctuation test.     

Monitoring the gene expression profiles of doxorubicin-resistant acute myelocytic leukemia cells by DNA microarray analysis

Acquired drug-resistance phenotype is a key factor in the relapse of patients suffering hematological malignancies. In order to investigate the genes involved in drug resistance, a human leukemia cell line that is resistant to doxorubicin, an anthracycline anticancer agent (AML-2/DX100), was selected and its gene expression profile was analyzed using a cDNA microarray. A number of genes were differentially expressed in the AML-2/DX100 cells, compared with the wild type (AML-2/WT). Pro-apoptotic genes such as TNFSF7 and p21 (Cip1/Waf1) were significantly down-regulated, whereas the IKBKB, PCNA, stathmin 1, MCM5, MMP-2 and MRP1 genes, which are involved in anti-apoptotic or cell cycle progression, were over-expressed. The AML-2/DX100 cells were also resistant to other anticancer drugs, including daunorubicin and camptothecin, and the expression levels of the differentially regulated genes such as STMN1, MMP-2 and CTSG, were constantly maintained. This suggests that the deregulated genes obtained from the DNA microarray analysis in a cell line model of drug resistance might contribute to the acquired drug resistance after chronic exposure.     

Specificity of transport of bleomycin and cobalt-bleomycin in L5178Y cells

The mechanism of transport of [³H]peplomycin (PEP), a new member of bleomycin group antibiotics, was studied in cultured L5178Y mouse leukemic cells. Cobalt ions enhanced the uptake of PEP, but Cu, Zn, Fe(II) and Fe(III) had no effect. The initial rate of uptake of cobalt chelated PEP [PEP(Co)] was several times higher than that of free or Cu-chelated PEP and was temperature independent. A double reciprocal plot of the data demonstrated both saturable (K_m = 4.5 μM, V_max = 1.3 × 10^?18 mole/min/cell) and non-saturable components of the uptake of PEP(Co). The saturable component was inhibited specifically by cobalt chelated bleomycin analogs. PEP-chelates with metals other than cobalt, such as PEP(Cu) were metabolically unstable. These results suggest that bleomycin enters into cells as a metal chelate through a specific transport site.     

Trifluorothymidine resistance and colony size in L5178Y/TK +/- cells treated with methyl methanesulfonate

L5178Y/TK +/- cells treated with methyl methanesulfonate (MMS) were allowed to recover for 0,48,96,144, or 240 hours, and were then plated in soft-agar medium containing trifluorothymidine (TFT). Dose-dependent and consistent increases in the frequency of TFTR cells were observed after each of the 48-240-hour expression periods through the counting of predominantly large, mutant colonies. Size distributions of soft-agar colonies from either MMS-treated or control cells were bimodal in the presence, and unimodal in the absence, of TFT. An increase of small, presumptive TFTR colonies with either increasing MMS concentration or decreasing recovery time was probably a manifestation of chemical toxicity, for a similar increase in small-colony number was observed in the absence of TFT when cells were cloned immediately after MMS treatment, when no induced mutants were yet detectable. Recloning experiments with 22 small-colony-derived cell lines revealed that, with one exception, small-colony morphology was not a heritable trait. While all large- and some small-colony-derived stocks from MMS-treated cells were of the phenotypically stable TK-/- type; spontaneous small TFTR colonies generally were not, their occurrence being directly correlated with serum concentration. No aneuploidy was evident in MMS-treated cell lines several generations after isolation as small TFTR colonies. These results suggest that delayed MMS cytotoxicity in TK +/- cells can temporarily produce increased physiological resistance to TFT in some cells, giving rise to secondary populations of small-colony TFTR variants.     

Schedule dependence of the lethal effects of 6-azauridine and cytosine arabinoside on murine leukemia cels (L5178Y) in culture

The antineoplastic drugs 6-azauridine and cytosine arabinoside exhibit a supra-additive lethal effect on murine L5178Y lymphoblasts if exposure to 6-azauridine precedes exposure to cytosine arabinoside; an additive effect is seen if cytosine arabinoside precedes 6-azauridine, while a sub-additive effect is obtained when the two drugs are present simultaneously. The potentiation of the effect of cytosine arabinoside by 6-azauridine increases for $\text{2}\text{1}\text{2}\text{hours}$ following the removal of the 6-azauridine from the culture.     

Evidence for caffeine-sensitive damage in methylazoxymethanol acetate-treated L5178Y cells.

The effects of methylazoxymethanol (MAM) acetate on colony survival, cell proliferation and DNA synthesis of murine lymphoma L5178Y cells are studied. Decreased sensitivity and immediate depression of cell proliferation and DNA synthesis were found in L5178Y cells in contrast to the reports on HeLa cells. Pre-labelling with 5-bromodeoxyuridine (BUdR) did not enhance significantly the carcinogen-induced cell lethality. Post-treatment with caffeine greatly enhanced cell lethality and depression of cell proliferation. These effects of caffeine were diminished when the cells had passed through two generations following the MAM acetate treatment. Experiments with synchronized cells showed that the action of caffeine was located primarily in S phase following the MAM acetate-treatment. These results strongly suggest that in L5178Y cells, MAM acetate induces damage, which is repaired by a mechanism analogous to post-replication repair of UV light-induced damage.     

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.