Cell Line CCRF-CEM Research Articles

In vitro antitumor and antiviral activities of new benzothiazole and 1,3,4-oxadiazole-2-thione derivatives

A series of new benzothiazole derivatives 6a-h have been synthesized, in five steps, from substituted phenols via the 1,3,4-oxadiazole-2-thiones 5a-h. The in vitro antitumor activity of the compounds obtained was investigated and the benzothiazol derivatives 6d and 6e showed strong effects on leukaemia cell lines CCRF-CEM (CC50=12+/-2 micromol L(-1), 8+/-1 micromol L(-1), respectively). These compounds are leading candidates for further development. The title compounds were tested against representatives of several virus families containing single stranded RNA genomes, either positive-sense (ssRNA+), or negativesense (RNA-), and against double-stranded RNA genomes (dsRNA), as well as some Flaviviridae viruses.

Effect of noscapine and vincristine combination on demyelination and cell proliferation in vitro

Peripheral neuropathy is a common, dose-limiting side effect of vincristine, a frontline therapy for acute lymphoblastic leukemia. Combination chemotherapy that reduces the neurotoxicity without compromising the efficacy of vincristine would improve patient outcomes. We performed in vitro studies using a combination of microtubule-binding antimitotics, noscapine and vincristine. In cell cultures containing neurons, astrocytes, and oligodendrocytes, vincristine caused demyelination as shown by transmission electron microscopy. A combination of vincristine and noscapine protected against demyelination. Human acute lymphoblastic and acute myelogenous leukemia cell lines CCRF-CEM and HL-60, respectively, were used to determine the antiproliferative effect of this novel drug combination. Vincristine and noscapine decreased cell proliferation with IC50 concentrations of 1 nM and 20 µM, respectively. Analysis of dose-effect relationships using isobolograms and combination indices demonstrated that noscapine acts synergistically with vincristine. Thus, noscapine is a promising candidate for use with vincristine to decrease neurotoxicity and enhance antineoplastic effectiveness.

Protein phosphatase 2A (PP2A) has a potential role in CAPE-induced apoptosis of CCRF-CEM cells via effecting human telomerase reverse transcriptase activity

Caffeic acid phenethyl ester (CAPE) is one of the most effective components of propolis which is collected by honey bees. The aim of this study was to investigate the cytotoxic and apoptotic effects of CAPE in the CCRF-CEM cell line and to clarify the role of serine/threonine protein phosphatase 2A (PP2A) and human telomerase reverse transcriptase (hTERT) activity as an underlining mechanism of CAPE-induced apoptosis. Trypan blue dye exclusion test and XTT methods were used to evaluate the cytotoxicity and ELISA based oligonucleotide detection, which can be seen during apoptosis, was used to determine apoptosis. Acridine orange/ethidium bromide dye technique was also used to evaluate apoptosis. The cytotoxic effect of CAPE was detected in a dose and time dependent manner with the IC50 of 1 μM. ELISA and acridine orange/ethidium bromide methods have shown remarkable apoptosis at 48th hour in CAPE treated cells. To investigate the role of PP2A in CAPE-induced apoptosis of CCRF-CEM cells, we performed combination studies with CAPE and, Calyculin A and Okadaic acid, which are very well known inhibitors of PP2A, in IC20 of inhibitors and IC50 of CAPE. Combination studies revealed synergistic effect of both drugs by concomitant use. Western blot analyses of PP2A catalytic and regulatory subunits showed down-regulation of expression of PP2A catalytic subunit in CAPE treated cells at 48th hour. Since, PP2A is important in hTERT (telomerase catalytic subunit) activation and deactivation, we also performed hTERT activity in CAPE treated cells simultaneously. Treating cells with IC50 of CAPE for 96 h with the intervals of 24 h showed marked reduction of hTERT activity. The reduction of hTERT activity in CAPE treated CCRF-CEM cells was more prominent in the initial 48 h. The variation of hTERT activity in CAPE treated CCRF-CEM cells may be the reason for the protein phosphatase interaction that occurred after treatment with CAPE.

Methylation of the PTPNS1 Promoter Region in Pediatric Acute Promyelocytic Leukemia.

Signal regulatory protein-α (SIRPα) is a transmembrane receptor selectively expressed on myeloid and neuronal cells. The broadly expressed cell surface CD47 molecule acts as a major extracellular ligand. The SIRPα cytoplasmic tail contains ITIM motifs that upon CD47 binding mediate the recruitment and activation of the cytosolic tyrosine phosphatases SHP-1 and SHP-2. SIRPα signaling negatively regulates many signaling pathways leading to reduced tumor migration, survival and cell transformation. Interestingly, a reduced SIRPα expression has been found in >70% of acute myeloid leukemia (AML) patients, including APL (FAB M3). Since SIRPα-derived signals are involved in the inhibition of myeloid cell growth, reduced SIRPα expression might contribute the proliferative advantage of leukemic cells. We hypothesized that the reduced expression of SIRPα in APL might be the result of aberrant promoter hypermethylation, which could have potential relevance for innovative therapy. The aim of this study was therefore to investigate protein expression of SIRPα in pediatric APL and evaluate the methylation status of the PTPNS1 gene (encoding SIRPα). Immunocytochemical staining was performed on cryo-preserved cytospins using a commercially available antibody (Ab2971, Abcam). The leukemic cell lines THP-1, Kasumi-1 were used as positive controls. Negative controls were performed by omitting the first antibody and the inclusion of the ALL cell line CCRF-CEM. SIRPα protein expression was evaluated by two independent investigators by scoring the intensity of the staining: negative (−), low (+/−) and positive (+). Methylation specific PCR (MSP) was set up in order to study the methylation pattern of the PTPNS1 promoter in the APL cell line NB4 and 10 APL patient samples (containing >80% blasts). Two primer sets (named MSP1 and MSP2, respectively) were used which recognized different regions of the PTPNS1 promoter. SIRPα expression in THP-1 cells was high, while expression in Kasumi-1 cells was low. The lymphoblastic cell line CCRF-CEM did not express SIRPα. These findings were in agreement with results obtained by Western blotting using the same antibody. The APL cell line NB4 stained positive for SIRPα. In the APL patients, SIRPα expression was low (n=7) or absent (n=2) in 9/10 (90%) samples. Expression was high in 1 sample (10%). MSP revealed methylation in one part of the PTPNS1 promoter region. The MSP1 primer set showed both methylated and unmethylated bands in the NB4 cell line as well as in all 10 APL patient samples. No methylation was detected using the MSP2 primer set. In conclusion, we observed no to low expression of SIRPα in the majority of APL patient samples (90%). In addition, we observed methylation of the PTPNS1 promoter in APL patient samples, indicating that aberrant promoter methylation might be involved in the reduced SIRPα expression in pediatric APL. This could have important implications for future alternative treatment options for APL patients directed towards demethylation of the gene and re-expression of this inhibitory signaling protein.

Unique Resistance Mechanism to Dexamethasone by Glutathione S-Transferase M1 Involving p38 MAPK and NF-κB Pathways, Possible Prognostic Role for Childhood ALL.

Glutathione S-transferases (GSTs) are known as detoxification enzymes that catalyse the conjugation of glutathione to anticancer drugs. In addition to this activity, GSTM1, an isotype of the Mu class GSTs, has been suggested to act as a negative regulator of mitogen-activated protein kinase (MAPK) activation. About 40–60% of the population has a deficit in GSTM1 enzyme activity, and the patients possessing GSTM1 have been reported to have a greater risk of relapse in childhood acute lymphoblastic leukemia (ALL). However, the reasons for poor prognosis remain unclear. To establish our hypothesis that GSTM1-induced drug resistance was involved, GSTM1 was transfected into GSTM1-negative CCRF-CEM cell lines (hereafter, the transfectant is denoted as CEM/M1) and the drug sensitivity was compared with that of control cells (CEM/mock). CEM/M1 showed decreased sensitivity to melphalan and carmustine (relative resistance vs. CEM/mock: 1.6 and 2.1, respectively; p<0.05), but not to daunorubicin, vincristine or etoposide. Notably, sensitivity to dexamethasone (DEX) decreased to a degree of up to 8-fold (IC50:1.76 μM in CEM/M1 and 0.22 μM in CEM/mock; p<0.01). Glutathione depletion by buthionine sulfoximine abrogated the resistance of CEM/M1 to alkylating agents, but not to DEX. These results suggest that a different mechanism is involved in the resistance to DEX. To clarify the GSTM1-related DEX resistance, proapoptotic pathway via p38 MAPK was evaluated. In CEM/M1, DEX-induced apoptosis was significantly inhibited (annexin V-positive at 72hr: 27% in CEM/M1 and 57% in CEM/mock; p<0.01). Silencing of GSTM1 by siRNA restored the DEX resistance, indicating that the anti-apoptosis was caused by the expression of GSTM1. After the treatment with DEX, phosphorylation of p38 was upregulated in CEM/mock, but not in CEM/M1; and the level of Bim mRNA was higher in CEM/mock than it was in CEM/M1. These findings indicate that the overexpression of GSTM1 down-regulates the proapoptotic pathway via p38 MAPK-Bim. However, treatment with a p38 MAPK-inhibitor (SB202194) decreased the DEX-induced apoptosis in both CEM/mock and CEM/M1. The involvement in DEX-resistance seems to be partial. To investigate other mechanisms, NF-κB pathway was evaluated. The basal activity of NF-κB p50 was significantly higher in CEM/M1 than it was in CEM/mock. Inhibition of IκBα phosphorylation by BAY11-7082 increased DEX sensitivity in either cells. Of considerable importance, however, is that the increase by BAY11-7082 was prominent in CEM/M1 and the relative resistance decreased from 8 to 2-fold. It appears that the anti-apoptotic NF-κB pathway is also involved in a resistance to DEX. In conclusion, the expression of GSTM1 in CCRF-CEM caused the resistance to alkylating agents due to its catalytic activity and to DEX by its anti-apoptotic function. The latter is the first report demonstrating the relationship between the expression of GSTM1 and DEX resistance. The determination of GSTM1 expression could be a useful tool for the individualization of optimize therapy for childhood ALL.(Supported by JSPS grant C19591102 and JRFCP grant)

Dexamethasone-induced apoptosis in acute lymphoblastic leukemia involves differential regulation of Bcl-2 family members

The mechanism of glucocorticoid -induced apoptosis is not fully understood and early predictive assays based on apoptotic markers for clinical outcome in acute lymphoblastic leukemia (ALL) are scarce. The aim of this study was to characterize the involvement of Bcl-2 family members and caspase activation in dexamethasone(Dex)-induced apoptosis in ALL. Primary childhood ALL samples, the pre-B ALL cell line RS(4;11), and the T-ALL cell line CCRF-CEM were used. The involvement of Bcl-2 family members was evaluated by flow cytometry, immunocytochemistry, and western and northern blotting. Apoptosis was analyzed by annexin V and TMRE staining. Caspase activity was evaluated by a fluorometric assay. Dex induced significant down-regulation of the anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, differential activation of the pro-apoptotic Bak and Bax, loss of Delta psi m and cytochrome c release. Dex-induced apoptosis also involved early activation of caspases 2 and -3. Inhibition of caspase activity did not, however, protect against Dex-induced Bak/Bax activation, loss of Delta psi m or cell death. In 12 primary ALL samples Dex-induced apoptosis was associated with activation of Bax (p=0.045) and down-regulation of Bcl-2 (p=0.016) and/or Bcl-xL (p=0.004). Furthermore, ex vivo Dex-sensitivity was associated with an early treatment response to polychemotherapy (p=0.026). The differential regulation of pro- and anti-apoptotic Bcl-2 family members appears to be a key event in the execution of Dex-induced apoptosis in ALL cell lines, and also indicates a role for these proteins in primary ALL cells.

Differentially expressed nuclear proteins in human CCRF‐CEM, HL‐60, MEC‐1 and Raji cells correlate with cellular properties

The human cell lines CCRF-CEM (T-cell acute lymphocytic leukemia), HL-60 (acute myeloid leukemia), MEC-1 (B-cell chronic lymphocytic leukemia) and Raji (Burkitt's B-cell lymphoma) have been analysed for differences in their nuclear proteomes. Using 2-D DIGE, 55 nuclear proteins have been identified that are differentially expressed (p<0.025) between the four cell lines, including proteins associated with transcription, proliferation, DNA repair and apoptosis. Of these 55 proteins, 22 were over-expressed in just one cell line, and four were down-regulated in one cell line. Proteins uniquely over-expressed between myeloid and lymphoid cell lines include those that may have use as markers for diagnosis, disease progression and B-cell maturation and differentiation. Expression of various proliferation-associated nuclear proteins correlated with relative growth rates of the cell lines, giving these proteins potential diagnostic applications for distinction of chronic versus acute subtypes of haematological malignancies. Identification of these differentially expressed nuclear proteins should facilitate elucidation of the molecular mechanisms underlying leukocyte differentiation and transformation to leukemias and lymphomas. The nuclear expression profiles should enable classification of subtypes of leukemia, and identify potential nuclear protein targets for development of diagnostic and therapeutic strategies.

Crystal structure of recombinant human stromal cell‐derived factor‐1α

Crystal structure of recombinant human stromal cell‐derived factor‐1α

Synthesis of N-(5,7-diamino-3-phenyl-quinoxalin-2-yl)-3,4,5-substituted anilines and N-[4[(5,7-diamino-3-phenylquinoxalin-2-yl)amino]benzoyl]- l-glutamic acid diethyl ester: Evaluation of in vitro anti-cancer and anti-folate activities

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI 50 < 0.01 μM) and the ovarian cancer cell line OVCAR-4 (GI 50 = 0.03 μM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.

2,3,5-Substituted tetrahydrofurans as cancer chemopreventives. Part 1: Synthesis and anti-cancer activities of 5-hydroxymethyl-2,3-diaryl-tetrahydro-furan-3-ols

The allylation of appropriate benzoin in presence of indium metal followed by m-CPBA mediated cyclization gave 5-hydroxymethyl-2,3-diaryl-tetrahydro-furan-3-ols. Investigations on 59 human tumor cell lines of these compounds identify four compounds exhibiting significant growth inhibition of tumor cells at particular cell lines. Compound 12 is very specific toward CCRF-CEM and SR cell lines of leukemia.

Gene Dosage Is Not Responsible for the Upregulation of MRP1 Gene Expression in Adult Leukemia Patients

Upregulation of multidrug resistance-associated protein (MRP1) gene has been detected in many in vitro systems and could be the basis of the drug resistance phenotype in vivo. Increase in gene dosage and overexpression are two major mechanisms for increasing MRP1 expression level. In many drug resistant cell lines, MRP1 gene amplification has been detected. However, it is not yet known whether gene amplification plays a role in inducing the multidrug resistance phenotype clinically. To establish whether MRP1 gene copy number is a common feature of the upregulation of MRP1 expression in cancer patients, we studied the MRP1 gene copy number in leukemia patients by fluorescent in situ hybridization (FISH) and real-time PCR. This involved determination of the MRP1 gene copy number and mRNA level in the peripheral blood of 52 adult leukemic patients and ten healthy volunteers. The leukemic CCRF-CEM cell line (drug sensitive) and its drug-resistant subline CCRF-E1000, which has MRP1 overexpression, were used as controls. The MRP1 gene copy number in CCRF-CEM was normal but increased significantly in CCRF-E1000 cell line. However, in the presence or absence of MRP1 overexpression, increase in gene dosage was not detected in patients. Our data suggest that the increase in MRP1 gene dosage observed in resistant cell lines is not responsible for the upregulation of MRP1 expression in leukemic patients.

In Vitro Antitumoral Activity of Baculovirus-expressed Chimeric Recombinant Anti-CD4 Antibody 13B8.2 on T-cell Lymphomas

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule. The recombinant IgG1 antibody 13B8.2 was previously shown to inhibit HIV-1 replication and to abrogate the one-way mixed-lymphocyte reaction and block proliferation of CD3-stimulated peripheral blood CD4 lymphocytes from healthy donors. Before testing this recombinant anti-CD4 antibody in in vivo preclinical trials, in vitro mechanisms of action of rIgG1 13B8.2 were assessed using various CD4 T-cell lymphomas. The baculovirus-expressed rIgG1 13B8.2 antibody led to 14% to 40% proliferation inhibition of the lymphoblastic leukaemia-derived SUP-T1, the acute T lymphoma-derived CCRF-CEM and Jurkat, and the cutaneous T-Cell lymphoma-derived HUT-78 cell lines, but it did not affect the cell cycle nor induce cell apoptosis. rIgG1 antibody 13B8.2 bound the C1q fraction, leading to 9% to 17% complement-mediated lysis of the HUT-78, H9, Sup-T1, and the CCRF-CEM cell lines. No correlation was observed between cell sensitivity to rIgG1 13B8.2-triggered complement-dependent lysis and CD35-, CD46-, CD55-, and CD59-surface expression on T lymphoma cells. Using fluorescence-activated cell sorter analysis, the antibody was shown to bind to FcgammaRI/CD64-transfected IIA1.6, FcgammaRII/CD32-transfected CDw32L, and FcgammaRIII/CD16-transfected Jurkat CD16 cell lines. In correlation with these findings, rIgG1 13B8.2 induced 11% to 31% antibody-dependent cell-mediated cytotoxicity of the CCRF-CEM, SUP-T1, A2.01 CD4, and Jurkat cell lines. These convincing results on the activity of the recombinant chimeric anti-CD4 antibody 13B8.2 have led us to perform in vivo preclinical study in a murine xenograft model of CD4 lymphomas.

Synthesis, antimycobacterial and antitumor activities of new (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl N,N-disubstituted dithiocarbamate/O-alkyldithiocarbonate derivatives

Reaction of 2-chloromethylsaccharin with substituted potassium dithiocarbamates and substituted potassium dithiocarbonates furnished (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl N,N-disubstituted dithiocarbamates (4–15) and (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl O-alkyldithiocarbonates (16–20). The new derivatives were evaluated for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compounds 4–13, 15, and 16–20 described herein showed moderate to good inhibitory activity. In particular, seven analogs 4, 5, 6, 13, and 7, 8, and 12 exhibited excellent MIC values of 1.56 and 0.78μg/mL, respectively. Compounds 4, 5, 10, 12, 13, and 16 were selected and screened for antitumor activity. Among the tested compounds, 4 and 5 were found to be cytotoxic, especially against leukemia cell lines CCRF-CEM, HL-60(TB), RPMI-8226, and SR with log10GI50 values lower than −6.69, and against non-small cell lung cancer NCI-H522 cell line with log10GI50 values lower than −6.31. Compound 10 was cytotoxic against leukemia cell line HL-60(TB), whereas 16 displayed favorable cytotoxicity against ovarian cancer cell line OVCAR-3 with log10GI50 values of −6.31 and −7.45, respectively.

Synthesis and in vitro anti-tumor activity of N-{1-[(3-thioxo-5,6-dihydroimidazo[2,1- c][1,2,4]thiadiazol-7-ylthio)thiocarbonyl]-2-imidazolidene}arylsulfonamides

A series of N-{1-[(3-thioxo-5,6-dihydroimidazo[2,1- c][1,2,4]thiadiazol-7-ylthio)thiocarbonyl]-2-imidazolidene}arylsulfonamides ( 2a– z) was obtained by reacting 6,7-dihydro-1 H-imidazo[2,1- c][1,2,4]thiadiazol-3-thione ( 1) with arylsulfonyl chlorides. The relationships between structure and anti-tumor activity revealed that compound 2o with p-Cl substituent at the phenyl ring was most active (−log GI 50 > 8.00, −log TGI = 7.66) and was found to exhibit high selectivity toward the leukemia CCRF-CEM cell line (Δ f = 3.08 and 3.31, respectively).

Chromosome microdissection identifies genomic amplifications associated with drug resistance in a leukemia cell line: an approach to understanding drug resistance in cancer

A significant problem encountered in the treatment of cancer patients is that cancer cells often evolve resistance to chemotherapeutic agents. One of the mechanisms responsible for drug resistance is gene amplification. The study of the behavior of genes conferring drug resistance is very important to determine future treatments for cancer patients that will minimize the effect of gene amplification. One of the best methods to investigate this phenomenon is to use chromosome microdissection to directly access the amplified gene or genes. In the present study, chromosome microdissection and fluorescent in-situ hybridization (FISH) were applied for the identification of genes residing in a homogeneously staining region (HSR) in drug-resistant cell sublines developed by treatment of the T-cell leukemia cell line CCRF-CEM with increasing levels of the anthracycline, epirubicin. We have demonstrated that the selection by epirubicin actually elevated the level of the multidrug resistance-associated protein (MRP1) gene. We argue that the breakage fusion bridge (B-F-B) cycle offers a plausible explanation for this amplification. The DNA prepared from the amplified regions by chromosome microdissection provides a resource for future investigations looking for the possible presence of novel genes contributing to drug resistance.

Synthesis of a novel series of 6-chloro-1,4,2-benzodithiazine 1,1-dioxide derivatives with potential biological activity

A novel series of 6-chloro-1,4,2-benzodithiazine 1,1-dioxide derivatives 2-19 with alkyl, aryl or het-eroaryl substituents at position 3 have been synthesized by the reaction of 4-chloro-2-mercaptobenzenesul-fonamides with aldehydes, aldehyde acetals or acid anhydrides. 6-Chloro-3-(2-hydroxyphenyl)-7-methyl-2,3-dihydro-l,4,2-benzodithiazine 1,1-dioxide (7) exhibited remarkable activity on the leukemia CCRF-CEM cell line (GI50<10 nM) and moderate activity against the other 49 human tumor cell lines derived from nine different cancer type.

Human T Cell Leukemia Virus Type I-Infected Patients with Hashimoto’s Thyroiditis and Graves’ Disease

Autoimmune thyroid diseases have been reported to be associated with human T cell leukemia virus type I (HTLV-I) infection. HTLV-I proviral load is related to the development of HTLV-I-associated myelopathy/tropical spastic paraparesis and has also been shown to be elevated in the peripheral blood of HTLV-I-infected patients with uveitis, arthritis, and connective tissue disease. The objective of the study was to evaluate the proviral load in HTLV-I-infected patients with Hashimoto's thyroiditis (HT) or Graves' disease (GD) and ascertain the ability of HTLV-I to infect thyroid cells. A quantitative real-time PCR assay was developed to measure the proviral load of HTLV-I in peripheral blood mononuclear cells from 26 HTLV-I-infected patients with HT, eight HTLV-I-infected patients with GD, or 38 asymptomatic HTLV-I carriers. Rat FRTL-5 thyroid cells were cocultured with HTLV-I-infected T cell line MT-2 or uninfected T cell line CCRF-CEM. After coculture with T cell lines, changes in Tax and cytokine mRNA expression were studied by RT-PCR. HTLV-I proviral load was significantly higher in the peripheral blood of patients with HT and GD than asymptomatic HTLV-I carriers. In the peripheral blood from HTLV-I-infected patients with HT, HTLV-I proviral load did not correlate with the thyroid peroxidase antibody or thyroglobulin antibody titer. After coculture with MT-2 cells, FRTL-5 cells expressed HTLV-I-specific Tax mRNA. These cocultured FRTL-5 cells with MT-2 cells expressed IL-6 mRNA and proliferated more actively than those cocultured with CCRF-CEM cells. Our findings suggest the role of the retrovirus in the development of autoimmune thyroid diseases in HTLV-I-infected patients.

Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster

The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.

Are telomeres a specific target for mutagenic attack by cytostatics in neoplastic cells?

Damage to telomeres induced by cytostatic therapy theoretically could generate telomere shortening and, subsequently, induce an additional genomic instability in neoplastic cells. Model experiments were carried out to examine this hypothesis. Cells of the T-ALL derived cell line CCRF-CEM were exposed to various different concentrations of Bleomycin (BLM) or Mitomycin C (MMC) for various times. Telomere lengths of metaphase chromosomes of the exposed cells were compared with those without this exposure (controls). In addition, telomerase activity was determined with a TRAP assay under the given conditions using the BLM experiments as a model. Although slight changes of total telomere length could be found in single experiments, the differences between exposed and non-exposed cells were not significant. Also, a considerable telomerase activity was shown which, however, did not substantially differ between exposed and non-exposed cells. From these data it may be concluded that, at least in the examined cell line, telomeres are not a preferential target for this kind of mutagenic attack.

Expression of DNA repair gene Ku80 in lymphoid neoplasm

Ku, a heterodimer of KU70 and Ku80 that binds to double-strand DNA breaks (DSBs) and activates the catalytic subunit (DNA-PKcs) when DNA is bound, is essential in DSB repair and V(D)J recombination. Ku80 is a putative tumor suppressor gene that might play an important role in drug resistance. Our aim was to determine the role of Ku80 in lymphoid malignancy. Competitive reverse transcription-polymerase chain reaction assays were performed and the expression levels of Ku80 were measured in normal peripheral blood mononuclear cells (n = 9) and malignant cells from 25 patients with acute lymphoblastic leukemia (ALL) (14 children, 11 adults), and chronic lymphoproliferative disorders (n = 6). The Ku80 transcripts were sequencing for the possibility of mutation. No mutation or Ku80 variant at the RNA level was seen in any patient samples or in the Raji or CCRF-CEM cell lines. In Ku80 expression, 8.8-, 1.9-, and 6.2-fold mean increases were seen in adult, pediatric ALL, and chronic lymphoid malignancies compared with the control. The Ku80 was significantly higher in adult than in pediatric ALL (P = 0.02). The amount of Ku80 expression in ALL was moderately correlated with peripheral white blood cell counts, but not with Ki67 labeling index. High Ku80 expressers (higher than the mean of all patients with ALL) tended to respond poorly to therapy: Only 22% of high Ku80 expressers achieved durable complete remission compared to 62% of low expressers. Our study suggests that Ku80 might contribute to generally poor prognoses in adult ALL.