Leukemia (1999) 13, 1760–1769 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Expression of p16INK4A and p14ARF in hematological malignancies T Taniguchi1, N Chikatsu1, S Takahashi2, A Fujita3, K Uchimaru4, S Asano5, T Fujita1 and T Motokura1 1Fourth Department of Internal Medicine, University of Tokyo, School of Medicine; 2Division of Clinical Oncology, Cancer Chemotherapy Center, Cancer Institute Hospital; 3Department of Hematology, Showa General Hospital; 4Third Department of Internal Medicine, Teikyo University, School of Medicine; and 5Department of Hematology/Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan The INK4A/ARF locus yields two tumor suppressors, p16INK4A tumor suppressor genes.10,11 In human hematological malig- ARF and p14 , and is frequently deleted in human tumors. We nancies, their inactivation occurs mainly by means of homo- studied their mRNA expressions in 41 hematopoietic cell lines and in 137 patients with hematological malignancies; we used zygous deletion or promoter region hypermethylation a quantitative reverse transcription-PCR assay. Normal periph- (reviewed in Ref. 12). In tumors such as pancreatic adenocar- eral bloods, bone marrow and lymph nodes expressed little or cinomas, esophageal squamous cell carcinomas and familial undetectable p16INK4A and p14ARF mRNAs, which were readily melanomas, p16INK4A is often inactivated by point mutation, detected in 12 and 17 of 41 cell lines, respectively. Patients with 12 INK4A which is not the case in hematological malignancies. On hematological malignancies frequently lacked p16 INK4C INK4D ARF the other hand, genetic aberrations of p18 or p19 expression (60/137) and lost p14 expression less frequently 12 (19/137, 13.9%). Almost all patients without p14ARF expression are rare in human tumors. lacked p16INK4A expression, which may correspond to deletions The INK4A/ARF locus yields two transcripts derived from of the INK4A/ARF locus. Undetectable p16INK4A expression with alternative first exons, exon 1␣ and exon 1␤, each of which p14ARF expression in 41 patients may correspond to p16INK4A is joined to sequences in exon 2.13,14 p16INK4A is translated promoter methylation or to normal expression status of the ␣ ␣ ␤ INK4A from the form transcript derived from exon 1 . The form p16 gene. All patients with follicular lymphoma (FL), mye- ␤ loma or acute myeloid leukemia (AML) expressed p14ARF while transcript that has an initiation codon in exon 1 encodes an nine of 23 patients with diffuse large B cell lymphoma (DLBCL) unrelated protein, the reading frame of which differs from that lost p14ARF expression. Patients with ALL, AML or blast crisis of p16INK4A; it is designated ARF, derived from an alternative of chronic myelogenous leukemia expressed abundant p16INK4A reading frame protein.14 Ectopic expression of mouse p19ARF mRNAs more frequently than patients with other diseases in the nucleus of rodent fibroblasts induces G1 and G2 phase Ͻ ARF (12/33 vs 6/104, P 0.01). Patients with FL and high p14 14 expression had a significantly shorter survival time while sur- arrest. Transfection of human ARF cDNA induces marked vival for patients with DLBCL and increased p14ARF expression growth inhibition in head and neck squamous cell carcinoma tended to be longer. These observations indicate that p16INK4A cell lines and HeLa cells with nonfunctional Rb,15 while and p14ARF expression is differentially affected among hemato- growth suppression by p16INK4A requires functional Rb.5,6 logical malignancies and that not only inactivation but also Thus, ARF is thought to function negatively on cell-cycle pro- increased expression may have clinical significance. INK4A Keywords: INK4A; ARF; leukemia; lymphoma; RT-PCR; prognosis gression, in a manner different from p16 . Human ARF protein, predicted to be 13902 Da, is referred to as p14ARF.16 ARF is also a candidate tumor suppressor, because mice lacking p19ARF develop tumors and mouse embryo fibroblasts Introduction lacking p19ARF are transformed by oncogenic Ha-ras alone.17 Furthermore, in patients with T cell acute lymphoblastic leu- The human INK4A/ARF locus located on chromosome 9p21 ARF attracts the attention of many oncologists, because it encodes kemia (T-ALL) and rearranged alleles of this region, p14 INK4A encoding exons are always disrupted or deleted, whereas two different candidate tumor suppressors, p16 and INK4A INK4B ARF 1,2 p16 and p15 encoding exons are spared in some p14 , which affect Rb and p53 pathways, respectively. 18 ARF p16INK4A is a cyclin-dependent kinase inhibitor (CKI) specific patients. The human p14 binds directly to MDM2, to CDK4 and CDK6 and can directly block cyclin D- resulting in stabilization of both p53 and MDM2, which Cip1 dependent kinase activity.3 The cyclin D/CDK4 (or 6) complex induces p21 expression and cell-cycle arrest in both G1 16 ARF facilitates G1-phase progression toward the S phase by phos- and G2/M. In contrast, p14 is negatively regulated by phorylating and thus inactivating the Rb protein (pRb).4 There- wild-type p53 expression, resulting in a negative feedback 16,19 ARF fore, the upregulated expression of p16INK4A causes G1-phase loop. p14 can be inactivated by homozygous 18 19 arrest and function is dependent on normal Rb.5,6 In contrast, deletion, and promoter region hypermethylation, while ␤ transcription of p16INK4A is repressed by Rb function.7 Once mutations in exon 1 are not found in tumor-derived lung, cells lack Rb, the levels of p16INK4A mRNA and protein were bladder, glioma or melanoma cell lines or in primary T-ALL elevated without growth arrest.8,9 The aberrantly high cells.18 expression of p16INK4A was evident in tumors without func- DNA alterations and methylation status of the p16INK4A gene tional Rb.3,7 p16INK4A is a member of the INK4 family that has in hematological malignancies have been frequently exam- three other structurally related members, p15INK4B, p18INK4C ined.12 However, expression of p16INK4A and especially and p19INK4D. Among them, INK4A and INK4B located on p14ARF in primary hematological malignancies has not been 9p21 just next to the INK4A/ARF locus are often inactivated described in detail. We investigated the expression of p16INK4A in human malignancies and are considered to be candidate and p14ARF in primary hematological malignancies and hema- topoietic cell lines using a quantitative reverse transcription- polymerase chain reaction (RT-PCR) assay. We found that INK4A/ARF expression was often altered and differs among Correspondence: T Motokura, Fourth Department of Internal Medi- cine, University of Tokyo, School of Medicine, 3-28-6 Mejirodai, hematological malignancies. Patients with follicular lym- ARF Bunkyo-ku, Tokyo 112-8688, Japan; Fax: 81–3–3943–3102 phoma (FL) and increased p14 expression are likely to have Received 2 April 1999; accepted 13 July 1999 a poor prognosis. INK4A/ARF in hematological malignancies T Taniguchi et al 1761 Materials and methods RPMI1640 medium (GibcoBRL Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum Cell lines (Bio Whittaker, Walkersville, MD, USA) and 60 mg/l kanamy- cin (Meiji Seika Kaisha, Tokyo, Japan) at 37°C in a humidified Cell lines used in this study are shown in Table 1. These cell atmosphere with 5% CO2. Cultures for FLAM-76 and SP-49 lines, except for FLAM-76 and SP-49, were passaged in cells required additional interleukin-6 (10 ng/ml) and 5% fetal bovine serum, respectively. Table 1 p16INK4A and p14ARF mRNA expressions in hematopoietic cell lines Cell line p16INK4A mRNA p14ARF mRNA pRB p16 p53a Source genome a Northern RT-PCR Northern RT-PCR status (unit) (unit) Lymphoid cell lines non-B non-T Reh − 0 − 0 + del NR A B cells Nalm-6 − 0 − 0 + NR w B SMS-SB − 0sm0+ NR NR B LBW-2 − 0 + 3.8 + NR NR B BALL-1 − 0 − 0 + re NR C Namalwa − 0 + 39 + met mt D Ramos − 0 + 32 + met mt B HS-sultan − 0 + 49 + met mtb F HA − 0 − 0 + del NR E IM-9 − 0.036 + 1.1 − pmet NR E SP-49c − 0 − 0 + NR NR G FLAM-76c + 64 + 70 + NR NR G T cells P30/Ohkubo − 0 − 0 + del NR F CEM − 0sm0+ del mt C RPMI-8402 − 0 − 0 + del NR H HPB-ALL − 0 − 0 + NR NR E KOPT-K1 + 0.63 ab, sm 0 + met, mt w H MOLT-3 − 0 − 0 + del NR C Jurkat − 0 − 0 + del mt C MOLT-4 − 0sm0+ re mt, w I MOLT-16 − 0 − 0 + del mt E PEER − 0.016 sm 0 + re mt H SKW-3 − 0 ab, sm 0 + re NR E A3/Kawakami − 0 + 13 + wmtE HTLV-1 MT-1 − 0.026 + 1.6 + NR mt C infected MT-2 − 0.069 + 0.46 + wwC HUT102 − 0 + 4 + wwC Myeloid cell lines KG-1 − 0.21 − 0.025 + met mt F K562 − 0 − 0 + del mt F KCL-22 − 0.52 − 0.04 + wmtD HL-60 − 1.2 + 12 + mt del F THP-1 − 0 − 0 + del mt F U937 − 0 + 16 + met mt D JK-1 sm 0 sm 0 + del NR I HEL − 0 − 0 + del NR F MEG-01s + 16 + 13 − NR NR J MEG-01 + 23 + 18 + deld mt J CMK − 0 − 0 + wreK CMK11–5 − 0 − 0 + NR NR K Meg-J − 0 − 0 + del NR L MOLM-1 − 0.045 − 0.066 + wNRE aData from other studies (see Results and Discussion for references). bHS-sultan is a derivative of Jijoye, which has mutated p53. cCell lines with cyclin D1 overexpression. dIn MEG-01 cells, homozygous deletion of p16 gene is reported, but another group reported p16 expression (see Discussion for references). ab, aberrant size transcript; del, deletion; met, methylated; mt, mutation; NR, not reported; pmet, partially methylated; re, gene rearrange- ment; w, wild type.