Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms F Tort 1 , S Herna ´ndez 1 , S Bea ` 1 , E Camacho 1 , V Ferna ´ndez 1 , M Esteller 2 , MF Fraga 2 , C Burek 3 , A Rosenwald 3 , L Herna ´ndez 1 and E Campo 1 1 Laboratory of Pathology, Hospital Clinic, Institut d’Investigacions Biome `diques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; 2 The Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Center (CNIO), Madrid, Spain; and 3 Institute of Pathology, University of Wu ¨ rzburg, Wu ¨ rzburg, Germany CHK1 gene encodes for a serine/threonine kinase involved in the regulation of cell cycle progression and DNA damage checkpoints. To determine the role of CHK1 in the pathogenesis of lymphoid neoplasms and its relationship to other DNA damage response genes, we have analyzed the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid tissues. CHK1 protein and mRNA expression levels were very low in both reactive tissues and resting lymphoid cells, whereas tumor samples showed a variable pattern of expression related to their proliferative activity. However, seven aggressive tumors showed a dissoci- ate pattern of extremely low or negative protein expression in spite of a high proliferative activity. Four of these tumors were diffuse large B-cell lymphomas (DLCLs) with concordant reduced levels of mRNA, whereas one blastoid mantle cell lymphoma (B-MCL) and two DLCLs had relatively normal levels of mRNA. No gene mutations, deletions, or hypermethylation of the promoter region were detected in any of these cases. In all these tumors ATM, CHK2, and p53 genes were wild type. These findings suggest that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations. Leukemia (2005) 19, 112–117. doi:10.1038/sj.leu.2403571 Published online 4 November 2004 Keywords: CHK1; CHK2; p53; checkpoint; lymphoma Introduction Lymphoid neoplasms are a heterogeneous group of disease entities characterized by distinctive clinical, morphologic, immunophenotypical, and genetic features. 1 Chromosomal instability is also a characteristic feature of these tumors and is associated with aggressive disease course and poor prognosis of the patients. 2–4 The molecular mechanisms involved in this genetic instability are not well known. Recent studies have shown the presence of alterations in the DNA damage response pathways in malignant lymphomas and its possible association with the genetic instability of the tumors. 2,3,5–11 CHK1 gene encodes for a serine/threonine kinase that, similarly to the functionally related CHK2, is involved in the signal transduction mechanisms of the DNA damage response pathways in response to the activation of upstream kinases. In mammals, CHK1 is mainly regulated by the ATR kinase, although it also collaborates with CHK2 in response to ionizing radiation induced-DNA damage as a downstream effector of ATM. Activated CHK1 phosphorylates and regulates different downstream targets, such as p53 and CDC25 phosphatases, promoting cell cycle arrest at G1/S and G2/M boundaries after DNA damage, and also S phase delay and inhibition of replication origin firing when DNA replication is blocked. 12 The potential role of CHK1 gene in the pathogenesis of human cancer is not well known. Different studies have shown CHK1 frameshift mutations in endometrial, colorectal, and stomach carcinomas with microsatellite instability. 13–15 However, the presence of CHK1 alterations in human lymphomas and the possible relationship with the inactivation of other elements of the DNA damage response pathways has not been previously examined. In this study we have examined CHK1 gene status and mRNA and protein expression in a series of NHLs and its relationship to the status of p53, CHK2, and ATM genes. Our results indicate that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations. Materials and methods Case selection Tumor specimens from 74 non-Hodgkin’s lymphomas (NHLs) were obtained from the Department of Pathology of the Hospital Clinic, University of Barcelona, on the basis of the availability of frozen samples for molecular studies. These tumors included 14 chronic lymphocytic leukemias (CLL), four large B-cell lympho- mas transformed from CLL (Richter’s syndrome, (RS)), 12 typical mantle cell lymphomas (T-MCL), 10 blastoid variants of mantle cell lymphomas (B-MCL), 10 follicular lymphomas (two grade 1, eight grade 2), and 24 primary diffuse large B-cell lymphomas (DLCL). The selected cases had a predominance of tumor cells with less than 5–10% normal or reactive cells. Follicular lymphomas had a higher number of reactive cells mainly of T- cell phenotype. The series of non-neoplastic lymphoid samples, including six tonsils, four reactive lymph nodes, two spleens, and six samples of normal peripheral blood lymphocytes (PBLs), were also examined. A subset of the tumors included in the study had been previously examined for CHK2, p53, and ATM gene alterations. Chromosomal imbalances had been also analyzed by comparative genomic hybridization (CGH). 2,3,6,11,16 Tumors lacking this information in which CHK1 appeared to be altered were additionally examined by CGH and for CHK2, and p53 gene alterations and protein expression as previously described. 2,3,11,16 Given that ATM gene mutations are virtually always associated with allelic deletions in mantle cell lymphoma and large B-cell lympho- mas, 6,7,17 the ATM allelic dosage was examined in these cases by quantitative PCR as described previously. 18 Received 26 March 2004; accepted 22 September 2004; Published online 4 November 2004 Correspondence: Dr E Campo, Laboratory of Pathology, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain; Fax: þ 34 93 2275717; E-mail: ecampo@clinic.ub.es Leukemia (2005) 19, 112–117 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu