CCRF-CEM Human Lymphoblastic Leukemia Cells Research Articles

Synthesis and Antifolate Activity of New Pyrrolo[2,3‐d]pyrimidine and Thieno[2,3‐d]pyrimidine Inhibitors of Dihydrofolate Reductase.

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Synthesis and antifolate activity of new pyrrolo[2,3‐d]pyrimidine and thieno[2,3‐d]pyrimidine inhibitors of dihydrofolate reductase

AbstractThree previously undescribed dihydrofolate reductase (DHFR) inhibitors, Nα‐[4‐[N‐[(2,4‐diaminopyrrolo[2,3‐d]pyrimidin‐5‐yl)methyl]amino]benzoyl]‐Nδ‐hemiphthaloyl‐L‐ornithine (7), Nα‐ [4‐ [N‐[(2,4‐diaminothieno[2,3‐d]pyrimidin‐5‐yl)methyl]amino]benzoyl]‐ Nδ‐hemiphthaloyl‐L‐ornithine (8), and N‐[4‐[N‐[(2,4‐diaminothieno[2,3‐d]pyrimidin‐5‐yl)methyl]amino]benzoyl]‐L‐glutamic acid (12), were synthesized and their antifolate activity was assessed. The ability of 7 and 8 to bind to DHFR and inhibit the growth of CCRF‐CEM human lymphoblastic leukemia cells in culture were dramatically reduced in comparison with the corresponding pteridine analogue, Nα‐(4‐amino‐4‐deoxypteroyl)‐Nδ‐hemiphmaloyl‐L‐ornithine (1, PT523). In a similar manner, the antifolate activity of 12 was markedly reduced in comparison with that of the corresponding glutamate analogue, aminopterin (5, AMT). In contrast, 7, 8, and 12 all displayed excellent affinity for the reduced folate carrier (RFC) of CCRF‐CEM cells as measured by a standard competitive influx assay. Lack of a consistent correlation between the results of the growth inhibition assays and those of the DHFR and RFC binding assays results suggest that additional factors also play a role in the antifolate activity of these compounds.

Analysis of in vivo and in vitro DNA strand breaks from trihalomethane exposure

BackgroundEpidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water.MethodsDNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB.ResultsCCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs.ConclusionCCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.

Synthesis, Antifolate, and Antitumor Activities of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines

Classical and nonclassical isosteric C8-N9 bridged analogues of the multitargeted antifolate LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and as antitumor and antiopportunistic infection agents. The syntheses of the analogues were accomplished by reductive amination of the appropriate anilines with 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (28) followed by saponification of the ethyl esters, for the classical analogue 6. The N9-methyl analogues were obtained from the N9-H precursors by reductive methylation. In general, the nonclassical compounds 7-17 were similar in potency to TMP against Toxoplasma gondii DHFR, with selectivity ratios greater than 38 and 21 for 11 and 16, respectively. These compounds were poor inhibitors of Pneumocystis carinii DHFR and rat liver DHFR. The nonclassical analogues were also inactive against TS. The classical analogue 6 was a marginal inhibitor of isolated human TS (IC50 = 46 microM) and of human DHFR (IC50 = 10 microM), however, it was a potent inhibitor of the growth of two human head and neck squamous cell carcinoma cell lines and of CCRF-CEM human lymphoblastic leukemia cells in culture and was similar to LY231514 against ZR-75-1 human breast carcinoma cell line. Evaluation of 6 against MTX-resistant sublines indicated that DHFR is not the major target of 6. Metabolite protection studies of the growth inhibitory activity of 6 suggest that TS is a major target of this drug and that polyglutamyl forms of 6 may serve as the intracellular TS inhibitors. These studies also suggest that 6 has a site of action in addition to sites in the folate pathway.

Role of N-glycosylation in the structure and function of the methotrexate membrane transporter from CCRF-CEM human lymphoblastic leukemia cells

The carrier protein for methotrexate and tetrahydrofolate cofactors (GP-MTX) in CCRF-CEM human lymphoblastic leukemia cells is a 117kDa glycoprotein containing both N- and O-linked oligosaccharides (Matherly et al., J Biol Chem 267: 23253–23260, 1992). Tunicamycin, an inhibitor of N-glycosylation, was used to investigate the roles of asparagine-linked oligosaccharides in the structure, intracellular routing, and transport function of GP-MTX. Tunicamycin was growth inhibitory toward CCRF-CEM cells ( ic 50 = 0.80 μ g/mL ) and caused a potent suppression of [ 3H]mannose incorporation into nascent glycoproteins. From 1–3 μg/mL, inhibition of [ 3H]mannose incorporation was 66–87%, exceeding that for [ 35S]methionine incorporation by 2 to 4-fold. Tunicamycin (1 and 2 μg/mL) exposures decreased the median molecular masses of GP-MTX on immunoblots (to 82 and 67 kDa, respectively) and were accompanied by reduced maximal rates of methotrexate uptake (31 and 37%, respectively, of control levels). Conversely, the K t values for methotrexate binding to the transporter were unaffected by tunicamycin treatments. The effects of tunicamycin on methotrexate influx closely correlated with lower levels of immunoreactive GP-MTX in plasma membranes and specific [ 3H]methotrexate binding to intact cells, suggesting that the transport effect was due to decreased numbers of carrier proteins at the membrane surface. The reduced molecular mass values for GP-MTX, which accompanied tunicamycin exposures, were further decreased (to 55 and 50 kDa at 1 and 2 μg/mL, respectively) by digestions with N-glycanase. Hence, despite the large loss of N-glycan from GP-MTX in tunicamycin-treated cells, residual core oligosccharides remained. The sizes of hypoglycosylated GP-MTX following both treatments were similar to that of the functionally homologous methotrexate membrane carrier previously identified in L1210 murine leukemia cells.

Characterization of transport-mediated methotrexate resistance in human tumor cells with antibodies to the membrane carrier for methotrexate and tetrahydrofolate cofactors

An earlier report (Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420-3426) described a K562 human erythroleukemia line (K562.4CF), characterized by an elevated uptake capacity for methotrexate (MTX) and 5-formyltetrahydrofolate, and the identification of a highly glycosylated membrane transporter (GP-MTX) by radioaffinity labeling with N-hydroxysuccinimide [3H] methotrexate. In the present study, radioaffinity-labeled GP-MTX from K562.4CF cells was isolated by Ricinus communis agglutinin I-agarose affinity chromatography, coupled with gel filtration and preparative electrophoresis. Antiserum to the purified, radio-labeled protein was raised in a rabbit and screened by immunoblot analysis of K562.4CF plasma membrane proteins or purified GP-MTX. The antiserum detected a broad GP-MTX band centered at 92 kDa on 7.5% gels. On 4-10% gels, the apparent molecular mass for GP-MTX shifted to 99 kDa. Antiserum specificity was established by quantitatively converting the immunoreactive glycoprotein in plasma membrane homogenates to its N- and O-deglycosylated forms with N- and O-glycanases, respectively. Whereas the methotrexate uptake capacity of K562.4CF cells was elevated 6.1-fold over parental cells, the GP-MTX content on immunoblots was increased approximately 3-fold. For two methotrexate-resistant K562 lines (33- and 70-fold), decreased drug uptake (28 and 18% of parental levels) closely correlated with their reduced GP-MTX contents. A GP-MTX isoform was also detected on immunoblots of membrane proteins from CCRF-CEM human lymphoblastic leukemia cells. With a transport-impaired CCRF-CEM line (13% of wild type uptake), an aberrant electrophoretic migration for GP-MTX was observed, establishing the presence of structural modifications in the transport protein. These structural differences were independent of carrier glycosylation since they were detected following the glycosidase treatments. These findings implicate important roles for distinct carrier-specific alterations in the expression of diminished drug transport in methotrexate-resistant human tumor cells.

Biochemical factors affecting the tightness of 5-fluorodeoxyuridylate binding to human thymidylate synthetase

Ligand binding studies of thymidylate synthetase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyltransferase, EC 2.1.1.45) isolated from CCRF-CEM human lymphoblastic leukemia cells were conducted to examine the mechanism of 5-fluorodeoxyuridylate (FdUMP) binding to the human enzyme in the presence of L-1-(+)-5,10-methylenetetrahydrofolate (5,10-CH 2H 4PteGlu) and to assess biochemical factors which could account for decreased binding of FdUMP by the enzyme in cells and tissues. Scatchard plots showed one class of binding sites for FdUMP with an apparent dissociation constant ( K D ) of 3.1 × 10 −11 M in the absence, and 3.5 × 10 −10 M in the presence, of 80 mM potassium phosphate (P i). The observed rate constant for FdUMP association ( k on obs) was dependent on the 5,10-CH 2H 4PteGlu concentration and attained a maximal value of 1.7 × 10 8M −1 min −1 at a concentration of ca. 12μM 5,10-CH 2H 4PteGlu. Increasing the concentration of 5,10-CH 2H 4PteGlu decreased the apparent rate constant of FdUMP dissociation ( k off obs), although FdUMP had no effect on the rate of 5,10-CH 2H 4PteGlu dissociation. These studies showed that CCRF-CEM thymidylate synthetase has an ordered mechanism of ligand binding and release, with the nucleotide binding first and dissociating last. Incubation of the enzyme-FdUMP-5,10-CH 2H 4PteGlu ternary complex with the substrate dUMP resulted in renewal of enzyme activity at about the same rate as that of FdUMP release. Nucleotides, deoxyuridine (dUrd), and polyoxyanions decreased the rate of FdUMP association but had no significant effect on the rate of FdUMP dissociation. dUMP was the most potent inhibitor of FdUMP binding found, with a binding constant determined from competition experiments of 0.36 μM compared to a Michaelis constant of 2.8 μM. The binding constant for p i was determined similarly to be 10 mM. The effects of dUMP plus P i, on decreasing the rate of FdUMP association were additive, whereas the combined effects of dUMP and sub-optimal concentrations of 5,10-CH 2H 4PteGlu were not additive, but multiplicative or greater. The levels of dUMP, P i, and 5,10-CH 2H 4PteGlu that reportedly are present in some cells and tissues, theoretically could increase the K D to more than 10 −6M. These results could account for the coexistence of substantial levels of FdUMP and unbound thymidylate synthetase found in some cells and tissues.

Biochemical determinants of tumor sensitivity to 5-fluorouracil: ultrasensitive methods for the determination of 5-fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthetase.

Techniques have been developed to measure FdUMP, the active metabolite of 5-FUra; thymidylate synthetase (TMP synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45), the target enzyme for this antimetabolite; and dUMP, the substrate that competes with FdUMP for binding to TMP synthetase. As little as 0.02 pmol of FdUMP can be quantitated with a competitive ligand binding assay by using homogeneous Lactobacillus casei/MTX TMP synthetase as a binding protein. A new binding assay for TMP synthetase allows detection of 0.005 pmol of enzyme. The quantitative enzymatic conversion of dUMP to [methyl-(14)C]-TMP using 5,10-methylene[(14)C]tetrahydrofolate by pure L. casei TMP synthetase is used as an assay for dUMP with a sensitivity of 10 pmol. Cultured CCRF-CEM human lymphoblastic leukemia cells formed high levels of FdUMP (2.6 nmol per 10(9) cells) within 11 hr after exposure to 30 muM 5-FUra. Tumor cell TMP synthetase levels dropped, and then free FdUMP appeared. The intracellular dUMP pool was low (2-5 nmol per 10(9) cells) in logarithmically growing cultures of several tumor cell lines but expanded rapidly in CCRF-CEM cells on exposure to 5-FUra after enzyme levels decreased. The levels of dUMP found after exposure to 5-FUra are sufficient to severely retard inhibition of TMP synthetase by FdUMP.The methods described are sufficiently sensitive to allow these biochemical parameters of 5-FUra action to be measured in cell culture or in needle biopsy samples of human tumors.

Synthesis of mono- and bifunctional .alpha.-methylene lactone systems as potential tumor inhibitors

Synthetic nono- and bifunctional alpha-methylene lactone derivatives including deoxyvernolepin and kihydrodeoxyvernolepin were tested as inhibitors of the growth of CCRF-CEM human lymphoblastic leukemia cells in culture. The range of ID-50 values for compounds 1-7 (ca. 10(-5)-10(-6)M) was roughly comparable to the doses observed earlier in the CCRF-CEM cell system with synthetic alpha-methylene-gamma-butyrolactones. Of significance is that dihydrodeoxyvernolepin and deoxyvernolepin were at least an order of magnitude more active than natural vernolepin.

Nucleosides. 1. 9-(3'-Alkyl-3'-deoxy-.beta.-D-ribofuranosyl)adenines as lipophilic analogs of cordycepin. Synthesis and preliminary biological studies

A series of lipophilic 9-(3'-alkyl-3'-deoxy-beta-D-ribofuranosyl)adenines of increasing chain length was synthesized from the corresponding branched sugars via titanium chloride catalyzed ribosylation of chloromercuri-6-benzamidopurine. Enhanced growth inhibitory activity was observed against (CCRF-CEM human lymphoblastic leukemia cells in culture as the length of the alkyl side chain in the sugar and the resultant lipophilic character of the nucleoside were increased. Experiments involving incorporation of radiolabeled uridine, thymidine, and leucine revealed that in contrast to cordycepin (1) the 3'-n-butyl and 3'-n-hexyl analogues 5 and 6 markedly inhibit not only RNA synthesis but DNA and protein synthesis as well.

Methotrexate analogs. 4.7-Methyl derivatives of methotrexate and dichloromethotrexate. A new synthesis and some biological studies.

The title compounds were synthesized with the aim of obtaining methotrexate and dichloromethotrexate analogs resistant to the catabolic action of hepatic aldehyde. oxidase, an enzyme known to cause significant conversion of dichloromethotrexate into an inactive 7-hydroxy metabolite in man as well as experimental animals. On the basis of antibacterial assays against Streptococcus faecium ATCC 8043, mammalian cell culture experiments involving use of P388 murine lymphatic leukemia cells and CCRF-CEM human lymphoblastic leukemia cells, and in vivo experimental antitumor evaluation against L1210 lymphatic leukemia in the mouse, it appears that biological activity is severely compromised by 7-methyl substitution. Inhibition studies with purified dihydrofolate reductase derived from Lactobacillus casei ATCC 7469 and also the L1210-FRR tumor suggest that the lack of in uitro and in uiuo activity of these analogs is due to impaired enzyme binding. 3’,5’-Dichloromethotrexate (1, DCM),2,3 a well-known folate antagonist evaluated several years ago as a clinical antileukemic agent in man435 on the basis of promising early trials against rodent tumors,6.7 has been shown to be an active substrate for the enzyme aldehyde oxidase (aldehyde:oxygen oxidoreductase, E.C. 1.2.3.1) in rodentss.9 as well as in man.10 In this respect, DCM differs significantly from methotrexate (2, MTX), which is extensively hydroxylated at the 7 position in the rabbit and guinea pig11J2 but not appreciably in the mouse12J3 or in man.14,15 Present in the supernatant fraction of liver homogenates from diverse mammalian species, aldehyde oxidase is a molybdoflavoprotein enzyme similar to xanthine oxidase in its ability to hydroxylate a variety of N-heterocyclic substrates,16-18 but its precise physiologic role is not fully elucidated.19 As with MTX,20,21 the action of aldehyde oxidase on DCM results in the formation of a metabolite devoid of activity against dihydrofolate reductase.22 This metabolite has been identified on the basis of spectral evidence and chemical degradation as the 7-hydroxy derivative.23 Thus, while not a significant metabolic event with MTX in man, hepatic “detoxification” via 7hydroxylation is viewed as an important aspect of the pharmacology of DCM along with other phenomena such as binding to plasma proteins and ease of transport across cell membranes. These considerations prompted us to undertake the synthesis of a DCM analog in which the vulnerable 7 position is blocked. The 7-methyl derivative 3 appeared to be a logical candidate for this purpose, and the synthesis of this compound forms the subject of the present report.