Genetic inactivation of the Fanconi anemia gene FANCCidentified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

Palagyi Andreas, Neveling Kornelia, Plinninger Ursula, Ziesch Andreas, Targosz Bianca-Sabrina, Denk, Ochs Stephanie, Rizzani Antonia, Meier Daniel, Thasler, Hanenberg Helmut, De, Bassermann Florian, Schäfer Claus, Göke Burkhard, Schindler Detlev, Gallmeier Eike

Description

Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored. Results A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC , FANCF , or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type. Conclusions As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2010
• Nombre de lectures: 101
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Palagyi et al.  Molecular Cancer 2010, 9 :127 http://www.molecular-canc er.com/content/9/1/127

R E S E A R C H  Open Access R G es e ear n ch etic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents Andreas Palagyi 1 , Kornelia Neveling 2 , Ursula Plinninger 1 , Andreas Ziesch 1 , Bianca-Sabrina Targosz 3 , Gerald U Denk 1 , Stephanie Ochs 1 , Antonia Rizzani 1 , Daniel Meier 2 , Wolfgang E Thasler 4 , Helmut Hanenberg 5 , Enrico N De Toni 1 , Florian Bassermann 3 , Claus Schäfer 1 , Burkhard Göke 1 , Detlev Schindler 2 and Eike Gallmeier* 1

Background of 13 FA genes, all of which have now been identified [2]. Fanconi anemia (FA) is a rare recessive disorder, charac- The FA genes appear to act in a common pathway of terized by congenital skeletal abnormalities, progressive DNA damage signalling and DNA remodelling, distal bone marrow failure and an increased cancer susceptibil- parts of which interact with regulators of cell cycle con- ity [1]. The disease is caused by bi-allelic mutations in one trol and DNA repair, especially the repair of DNA inter- strand-crosslinks. * Correspondence: [e-mail] There are three consecutive compartments of the FA 1 Department of Medicine II, Camp us Grosshadern, Ludwig-Maximilians- pathway [3]. The proximal compartment consists of eight Unive Full list r   s o i f t   y a , ut M h a o r r c i h n i f o o n r i m ni a s ti tr o a n s i s s e     a 1 v 5 a , i   l 8 a 1 bl 3 e 7 a 7 t   t M h u e n e i n c d h   , o G f e t r he m a a r n ti y cle FA proteins (A, B, C, E, F, G, L, and M), which form a 2010 Palagyi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative C ommons © Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestri cted use, distribution, and reproduction in any medium, provided the original work is properly cited.