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Pharmacology and resistance mechanisms of nucleoside analogues and topoisomerase II interactive agents: studies on human leukemia cells with a focus on cross-resistance
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The purpose of this thesis was to elucidate mechanisms of action and resistance of clinically relevant nucleoside analogues and topoisomerase interactive agents in human leukemia cell lines and leukernia cells isolated from peripheral blood of leukemia patients. Interactions and cross-resistance patterns of these different cytotoxic drug families were also studied since these drugs are usually administrated in combination in the clinic.

Two novel nucleoside analogues, clofarabine (2-chloro-2'-arabino-fluoro 2'-deoxyadenosine, CAFdA) and nelarabine (9-ß-D-arabinofuranosylguanine, AraG) were studied regarding cellular activation and mechanisms of resistance. Compared to cladribine (2-chloro- 2'-deoxyadenosine, CdA), CAFdA was more effective due to better stability and more efficient phosphorylation by deoxycytidine kinase (dCK). The mechanism of resistance to CAFdA was decreased activity of dCK. The most important mechanism contributing to resistance to AraG seems to be the deficiency of the activating enzymes dCK and deoxyguanosine kinase (dGK), as measured by enzyme activity assays, Western blotting, and real-time polymerase chain reaction. Unexpected cross-resistance between topoisomerase interactive agents and nucleoside analogues was identified in CEM and MOLT-4 cell lines developed for resistance to etoposide (VP) and AraG, respectively, by means of a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide viability assay. Resistance to VP was due to a decrease in the activity and amount of topoisomerase TI. Major cause of resistance to the nucleoside analogues CdA and AraC was metabolic alterations producing increased activity of 5'-nucleotidase and higher level of endogenous deoxycytidine triphosphate. The AraG-resistant cells showed also classical multidrug resistance (MDR) phenomena. The accumulation and cytotoxicity of daunorubicin (Dnr) were studied in AraG-resistant cells and in response to the resistance modifiers, such as cyclosporin A. The level of mdr1 mRNA and its product, P-glycoprotein, was increased. The topoisomerase interactive agent, idarubicin (Ida), a semisynthetic derivative of Dnr, was more effective in inducing apoptosis as determined by the Annexin V -FITC method, and Ida-resistant cells did not show any classical MDR phenomena.

Thus, these studies suggest that anticancer agents from the same class of cytostatics could have important differences in effectivity and mechanisms of resistance. These results confirm the possibility of coexpression of multiple mechanisms of resistance in human leukemic cells, which have been selected by exposure to a single-dmg. The generally assumed lack of crossresistance between nucleoside analogues and topoisomerase interactive agents is questionable.

The rationale for combination therapy should be based on biological properties and cross-resistance analyses of the included drugs.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 75 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 698
Keyword [en]
Nucleoside analogue, cross-resistance, multidrug resistance, deoxycytidine kinase, leukemia, anthracyclines, P-glycoprotein
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27518Local ID: 12174ISBN: 91-7373-140-4 (print)OAI: oai:DiVA.org:liu-27518DiVA: diva2:248070
Public defence
2001-11-16, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-11-02Bibliographically approved
List of papers
1. Biochemical Pharmacology and Resistance to 2-Chloro-2′-arabino-fluoro-2′-deoxyadenosine, a Novel Analogue of Cladribine in Human Leukemic Cells
Open this publication in new window or tab >>Biochemical Pharmacology and Resistance to 2-Chloro-2′-arabino-fluoro-2′-deoxyadenosine, a Novel Analogue of Cladribine in Human Leukemic Cells
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1999 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 5, no 9, 2438-2444 p.Article in journal (Refereed) Published
Abstract [en]

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine (CAFdA) — a fluorinated analogue of cladribine [2-chloro-2′-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability — in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5′-triphosphate (CAFdATP) was also longer than that for CdA 5′-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27106 (URN)11753 (Local ID)11753 (Archive number)11753 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide
Open this publication in new window or tab >>Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide
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2001 (English)In: British Journal of Haematology, ISSN 0007-1048, E-ISSN 1365-2141, Vol. 113, no 2, 339-346 p.Article in journal (Refereed) Published
Abstract [en]

Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP. The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIα (P < 0·001) and β protein (P < 0·026), formed substantially lower amounts of the topo II–DNA complex, and had a markedly decreased level of Fas (CD95/APO-1)-ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P-glycoprotein, multidrug resistance-associated protein and the lung resistance-related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara-C, was the same for resistant and wild-type cells. However, there was an increase in the activity of the cytosolic 5′-nucleotidases (5′-NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0·001) for the high Km and 134% (P < 0·331) for the low Km 5′-NT in resistant cells. The high Km 5′-NT is probably responsible for the decreased amount of the active metabolite CdA 5′-triphosphate [40% decreased (P < 0·045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0·001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara-C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5′-NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide-degrading enzymes and DNA topoisomerases, which may be altered in cross-resistant cells.

Keyword
etoposide, cladribine, CD95, deoxycytidine kinase, 5′-nucleotidase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26977 (URN)10.1046/j.1365-2141.2001.02751.x (DOI)11611 (Local ID)11611 (Archive number)11611 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. On the mechanism of 9-ß-D-arabinofuroanosylguanine resistance in human leukemic cells
Open this publication in new window or tab >>On the mechanism of 9-ß-D-arabinofuroanosylguanine resistance in human leukemic cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The present study was undertaken to elucidate the mechanism of resistance to a relatively new nucleoside, 9-ß-D-arabinofuranosylguanine (Ara-G), which is highly toxic to T-cell malignancies. In order to do this we generated two stable resistant cell lines, MOLT-4/AraG500 (100-fold resistant) and MOLT-4/Ara-G900 (180-fold resistant). The presumed limiting step in the activation of Ara-G is catalyzed by both deoxycytidine kinase (dCK) and the mitochondrial deoxyguanosine kinase (dGK). Cross-resistance was noted to analogues such as cytosine arabinoside (Ara-C), cladribine, and fludarabine, but not to difluorodeoxycytidine. HPLC measurements of intracellular triphosphates of Ara-C and Ara-G showed that resistant cells generated significantly lower levels of metabolites. The activity of dGK (MOLT-4/AraG500, 79%, and MOLT-4/Ara-G900, 83%) and dCK (MOLT-4/Ara-G500, 54%, and MOLT- 4/Ara-G900, 73%), as well as protein and mRNA levels were significantly reduced in the resistant cell lines compared to the wild type. The resistant cells also showed decreased activity as well as decreased mRNA expression of the cytosolic 5'-nucleotidase (5'-NT), compared to the wild type. The reduced levels of enzyme activity in the resistant cells may be a consequence of reduced numbers of chromosomes carrying the genes for dGK, dCK and 5 'NT. Unexpectedly, the resistant cells showed higher activity of the mitochondrial enzyme thymidine kinase 2, probably resulting from down-regulation of dGK. No mutations were found in the dCK gene ofMOLT-4/Ara-G500 but in 2 of 13 clones ofMOLT-4/Ara-G900, we found two point mutations, one T to C and other A to G, which gave rise to changes in the amino acid sequence and affected the catalytic activity. The intracellular dNTP levels were determined and a nearly unchanged dCTP pool was observed in the resistant cells, while the other dNTP pools were significantly reduced compared to those in the wild type. These results show that altered activity of the deoxyribonucleoside kinases confer resistance to Ara-G in these cell lines.

Keyword
Leukemia, 9-ß-D-arabinofuroanosylguanine, deoxycytidine kinase, deoxyguanosine kinase, resistance and cytotoxicity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80283 (URN)
Available from: 2012-08-23 Created: 2012-08-23 Last updated: 2012-08-23Bibliographically approved
4. Altered expression of multidrug resistance (mDR1) gene in cells selected for resistance to 9-ß-D-Arabinofuranosylguanine
Open this publication in new window or tab >>Altered expression of multidrug resistance (mDR1) gene in cells selected for resistance to 9-ß-D-Arabinofuranosylguanine
(English)Manuscript (preprint) (Other academic)
Abstract [en]

9-ß-D-Arabinofuranosylguanine (AraG) is an important and relatively new guanine nucleoside analogue that is highly toxic to T-cell maliguancies. Two resistant sublines with low activity of nucleoside phosphorylating enzymes, deoxycytidine kinase (dCK) (50-70% reduction) and deoxyguanosine kinase (dGK, 70-80% reduction), were generated from a MOLT-4 cell line with stepwise-increasing concentrations of AraG. As expected, the resistant sublines were highly cross-resistance to analogues that are activated by dCK and dGK such as cytosine arabinoside, cladribine, and fludarabine. Surprisingly, the resistant cells were siguificantly less sensitive to anthracyclines and podophyllotoxin derivatives resulting from decreased cellular drug accumulation restored by cyclosporin A, as determined by calcein uptake and flow cytometry studies. To clarify the mechanisms of this resistance, the gene expression of various multidrug resistance proteins, e.g. the multidrug resistance gene (mdr1), multidrng resistance-associated protein, lung resistance-associated protein and topoisomerase IIα or IIß were investigated. We found that the resistant cells overexpressed the mdr1 gene, as assessed by a real-time PCR, and contained higher levels of P-glycoprotein, as assessed by western blotting. The expression of other multidrug resistance proteins was not affected in the AraG-resistant sublines. These fmdings may be useful in the clinical trials of AraG singly and in combination with anthracyclines and related agents.

Keyword
leukaemia, 9-ß-D-arabinofuranosylguanine, multidrug resistance, cytotoxicity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80287 (URN)
Available from: 2012-08-23 Created: 2012-08-23 Last updated: 2012-08-23Bibliographically approved
5. Comparison of idarubicin and daunorubicin regarding intracellular uptake, induction of apoptosis, and resistance
Open this publication in new window or tab >>Comparison of idarubicin and daunorubicin regarding intracellular uptake, induction of apoptosis, and resistance
2002 (English)In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 178, no 2, 141-149 p.Article in journal (Refereed) Published
Abstract [en]

Anthracycline antibiotics are widely used as anticancer agents. Idarubicin (4-demethoxydaunorubicin; Ida), a semisynthetic derivative of daunorubicin (Dnr) is more potent than the parent compound in vitro and in vivo. The equitoxic dose of Ida in patients is about one-fourth of that of Dnr. We compared these drugs regarding cytotoxicity, apoptosis induction, and resistance mechanisms in human leukaemic cell lines. Cytotoxicity was studied by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and drug-induced apoptosis by means of the Annexin V–fluorescein isothiocyanate method at similar intracellular concentrations (extracellular concentrations of 0.35 μM for Ida and 1 μM for Dnr). Ida was at least twice as potent as Dnr in MOLT-4, HL60, CEM, and K562 cell lines. It took 8 h for Ida to induce approximately 20% apoptosis, but at least 22 h for Dnr to reach 20% apoptosis at identical intracellular concentration. Ida induces a faster and higher apoptosis rate compared with Dnr. The human chronic myelogenous leukaemia cell line (K562) was selected for resistance to Dnr and Ida with and without verapamil (Ver). Continuous incubation with Dnr, but not with Ida, led to an increased mdr1 gene expression as assessed by real-time PCR. The development of mdr1 gene expression in Dnr-resistant cells could be reversed by the presence of Ver. Ver also reversed the cytotoxicity to Dnr, but not to Ida, in K562/Dnr cells. The results show that Ida is more effective than Dnr in inducing apoptosis and that there are differences in resistance mechanisms between the drugs.

Keyword
Idarubicin, Daunorubicin, Apoptosis, Intracellular accumulation, MOLT-4, Cytotoxicity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27006 (URN)10.1016/S0304-3835(01)00824-2 (DOI)11642 (Local ID)11642 (Archive number)11642 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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