Effect of polyamine deficiency on proteins involved in Okazaki fragment maturation Veronica M. Johansson a, * , Maria Falck Miniotis a , Cecilia Hegardt b , Go ¨ran Jo ¨nsson b , Johan Staaf b , Pia S.H. Berntsson a , Stina M. Oredsson a , Kersti Alm a a Department of Cell and Organism Biology, Lund University, Helgonava ¨gen 3B, SE-223 62 Lund, Sweden b Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden Abstract Polyamine depletion causes S phase prolongation, and earlier studies indicate that the elongation step of DNA replication is affected. This led us to investigate the effects of polyamine depletion on enzymes crucial for Okazaki fragment maturation in the two breast cancer cell lines MCF- 7 and L56Br-C1. In MCF-7 cells, treatment with N 1 ,N 11 -diethylnorspermine (DENSPM) causes S phase prolongation. In L56Br-C1 cells the prolongation is followed by massive apoptosis. In the present study we show that L56Br-C1 cells have substantially lower basal expressions of two Okazaki fragment maturation key proteins, DNA ligase I and FEN1, than MCF-7 cells. Thus, these two proteins might be promising markers for prediction of polyamine depletion sensitivity, something that can be useful for cancer treatment with polyamine analogues. DENSPM treatment affects the cellular distribution of FEN1 in L56Br-C1 cells, but not in MCF-7 cells, implying that FEN1 is affected by or involved in DENSPM-induced apoptosis. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: DNA ligase I; FEN1; human breast cancer; DENSPM; CGP 48664 1. Introduction DNA is replicated simultaneously at the two template strands in eukaryotic cells. The leading strand is synthesized continuously, whereas the lagging strand is synthesized discontinuously. The discontinuous synthesis on the lagging strand is achieved by the joining of multiple short DNA fragments, around 100e150 nucleotides long, designated Okazaki fragments. The situation on the lagging strand is far more complex than that on the leading strand and at least 23 different proteins are involved (Hubscher and Seo, 2001). The replication process on the lagging strand is initiated with the synthesis of an RNA primer, which is extended with a DNA primer by DNA polymerase a (pol a). Since pol a is incapable of proofreading, the primer must later be replaced (MacNeill, 2001). Further elongation takes place when the clamp loader replication factor C (RF-C) displaces pol a and loads the sliding clamp proliferating cell nuclear antigen (PCNA). DNA polymerase d (pol d) binds to RF-C and PCNA and is responsible for DNA synthesis. When pol d reaches the 5 0 -end of the next Okazaki fragment, a flap is formed by displacement of the segment containing the RNA primer together with the DNA primer synthesized by pol a. There are several proposed models of how this is accomplished. According to one model, DNA2 removes the RNA primer and Flap endo- nuclease 1 (FEN1) then removes the DNA synthesized by pol Abbreviations: AdoMetDC, S-adenosylmethionine decarboxylase; BASE, BioArray Software Environment; BAC, bacterial artificial chromosome; CGP 48664, 4-amidinoindan-1-one 2 0 -amidinohydrazone; DENSPM, N 1 ,N 11 -dieth- ylnorspermine; ECL, enhanced chemiluminescence; FCM, flow cytometry; FCS, fetal calf serum; FEN1, flap endonuclease 1; HRP, horseradish peroxi- dase; NIM, nuclear isolation medium; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline with Tween 20; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; pol a, DNA polymerase a; pol d, DNA poly- merase d; RF-C, replication factor C; RNase A, ribonuclease A type II; SSAT, spermidine/spermine N 1 -acetyltransferase. * Corresponding author. Tel.: þ46 46 222 8201; fax: þ46 46 222 4539. E-mail address: veronica.johansson@cob.lu.se (V.M. Johansson). 1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.08.018 Cell Biology International 32 (2008) 1467e1477 www.elsevier.com/locate/cellbi