Modulation of Ara-C Cytotoxicity by Coadministration with Antisignalling Drugs in HL60 and Ara-C-Resistant HL60/Ara-C Cells
Ara-C (cytosine arabinoside) is one of the most effective drugs in the treatment of acute myeloid leukemia (AML). Ara-C is phosphorylated to Ara-CTP (cytosine arabinoside triphosphate) which inhibits DNA polymerase and induces DNA strand breaks after incorporation into DNA. There is increasing evidence that ara-C also affects some cellular signal transduction pathways; it induces transcription/expression of c-fos, c-jun, NF kappaB, protein kinase C δ, mitogen-activated protein kinase and cyclin E and downregulates cyclin-dependent-kinase-2 and retinoblastoma protein phosphorylation. These events may contribute to ara-C cytotoxicity. The antisignalling drugs all-trans retinoic acid (ATRA), staurosporine, quercetin and bryostatin-1 have recently been shown to enhance ara-C cytotoxicity.
We chose 7 different antisignalling agents [ATRA and the kinase inhibitors quercetin, genistein, CGP 52411, tyrphostin A48, nordihydroguaiaretic acid (NDGA) and staurosporine] and compared their ara-C sensitising potencies with those of hydroxyurea (HU), arabinosyl-2-fluoroadenine (F-Ara-A) and 2-chlorodeoxyadenosine (2-CdA), agents currently in use as “second line treatment” in resistant and relapsed AML. Cytotoxicity was assessed by the tetrazolium (MTT) assay in HL60 cells and a newly derived ara-C-resistant subline (HL60/ara-C) in at least three separate experiments. Supraadditive cytotoxicity was found for combinations containing ATRA, CGP 52411, tyrphostin A48 and HU in both cell lines, for 2-CdA, staurosporine and NDGA in HL60, and for F-Ara-A in HL60/ara-C cells. Quercetin and genistein did not sensitise cells against ara-C.
To elucidate the mechanism of sensitisation in HL60 cells we studied the influence of the modulators of ara-C cytotoxicity on cellular markers of apoptosis. After 4h-coincubation we measured cell size (volume), DNA loss by flow cytometry (sub-G1 peak) and DNA fragmentation by conventional gel electrophoresis. Tyrphostin A48, NDGA, ATRA and HU increased ara-C-induced apoptosis, whereas staurosporine did not affect it. CGP 52411 decreased the effect of ara-C on apoptotic indicators after 4h-, but no longer after 12h-coincubation.
The results suggest that antisignalling drugs such as ATRA, CGP 52411, tyrphostin A48, staurosporine and NDGA may be efficacious alternatives to the already clinically applied ara-C modulators. Among the clinically used ara-C modulators, HU sensitised more potently against ara-C than F-Ara-A and 2-CdA.
KeywordsAcute Myeloid Leukemia HL60 Cell Cytosine Arabinoside Human Myeloid Leukemia Cell Relapse Acute Myeloid Leukemia
Unable to display preview. Download preview PDF.
- 2.Plunkett W, Iacoboni S, Keating MJ (1986) Cellular pharmacology and optimal therapeutic concentrations of 1–13-D-arabinofuranosylcytosine 5’-triphosphate in leukemic blasts during treatment of refractory leukemia with high-dose 1ß-D-arabinofuranosylcytosine. Scand J Haematol 34 (Suppl. 44): 51–59Google Scholar
- 4.Zühlsdorf M, Vormoor J, Boos J: Cytosine arabinoside resistance in childhood leukemia. Submitted.Google Scholar
- 9.Teofili L, Pie relli L, lovino MS, Leone G, Scambia G, de Vincenzo R, Benedetti-Panici P, Menichella G, Macri E, Piantello M, Ranelletti FO, Larocca LM (1992) The combination of quercetin and cytosine arabinoside synergistically inhibits leukemic cell growth. Leukemia Res 16: 497–503Google Scholar
- 10.Grant S, Turner AJ, Bartimole TM, Nelms PA, Joe VC, Jarvis WD (1994) Modulation of 1-[3-Darabinofuranosyl] cytosine-induced apoptosis in human myeloid leukemia cells by staurosporine and other pharmacological inhibitors of protein kinase C. Oncology Res 6: 87–99Google Scholar
- 11.Grant S, Jarvis WD, Swerdlow PS, Turner AJ, Traylor RS, Wallace HJ, Lin PS, Pettit GR, Gewirtz DA (1992) Potentiation of the activity of 113-D-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: Association with enhanced fragmentation of mature DNA. Cancer Res 52: 6270–6278PubMedGoogle Scholar
- 12.Jarvis WD, Povirk LF, Turner AJ, Traylor RS, Gewirtz DA, Pettit GR, Grant S (1994) Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[ß-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem Pharmacol 47: 839–852PubMedGoogle Scholar
- 16.Kharbanda S, Emoto Y, Kisaki H, Saleem A, Kufe D (1994) 1-ß-D-arabinofuranosylcytosine activates serine/threonine protein kinases and cjun gene expression in phorbol ester-resistant myeloid leukemia cells. Mol Pharmacol 46: 67–72Google Scholar
- 18.Yuan ZM, Kharbanda S, Kufe D (1995) 1–0-Darabinofuranosylcytosine activates tyrosine phosphorylation of p34cdc2 and its association with the Src-like p56/p53’ kinase in human myeloid leukemia cells. Biochem 34: 1058–1063Google Scholar
- 21.Traxler P, Lydon N (1995) Recent advances in protein tyrosine kinase inhibitors. Drugs of the Future 20: 1261–1274Google Scholar
- 23.Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinase. JBC 262: 5592–5595Google Scholar
- 24.Meggio F, Donella Deana A, Ruzzene M, Brunati AM, Cesaro L, Guerra B, Meyer T, Mett H, Fabbro D, Furet P, Dobrowolska B, Pinna LA (1995) Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur J Biochem 234: 317–322PubMedCrossRefGoogle Scholar
- 25.Domin J, Higgins T, Rozengurt E (1994) Preferential inhibition of platelet-derived growth-factor ( PDGF)-stimulated DNA synthesis and protein tyrosine phosphorylation by nordihydroguaiaretic acid. JBC 269: 8260–8267Google Scholar
- 28.Mosmann T: Rapid colorimetric assay for cellular growth and survival (1983) Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63Google Scholar
- 31.Pagliacci MC, Spinozzi F, Migliorati G, Fumi G, Smacchia M, Grignani F, Riccardi C, Nicoletti I (1993) Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival. Eur J Cancer 29A: 1573–1577CrossRefGoogle Scholar
- 32.Gorczyka W, Gong J, Ardelt B, Traganos F, Darzynkievicz Z (1992) The cell cycle related changes in susceptibility of HL60 cells to apoptosis induced by antitumour agents. Cancer Res 52: 6270–6278Google Scholar
- 34.Ormerod MG (1990) Flow cytometry–a practical approach ( Ormerod ). Oxford University Press: pp 69–87Google Scholar
- 36.Donehower RC (1992) An overview of the clinical experience with hydroxyurea. Sem Oncol 19: 11–19Google Scholar
- 37.Chen LL, Gansbacher B, Gilboa E, Taetle R, Oval J, Hibbs MS, Huang CK, Clawson ML, Bilgrami S, Schlessinger J et al. (1993) Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoid acid-induced granulocytic differentiation. Cell Growth Differ 4: 769–776PubMedGoogle Scholar
- 39.Curtis JE, Messner HA, Minden MD, Tritchler DL, McCulloch EA (1989) Improved maintenance therapy for acute myelogenous leukemia (AML) using a retinoic acid ( RA) containing regimen. Proc Am Assoc Cancer Res 30: 60–66Google Scholar
- 43.Beerenbaum MC (1989) What is synergy? Pharmacol Rev 41: 93–141Google Scholar