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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 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, 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 yields two tumor suppressors, p16INK4A tumor suppressor .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 lines and in 137 patients with hematological malignancies; we used zygous or region hypermethylation a quantitative reverse -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 , p16INK4A is often inactivated by point , 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 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 , 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 or to normal expression status of the ␣ ␣ ␤ INK4A from the form transcript derived from exon 1 . The form p16 . All patients with (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 lymphoma (DLBCL) unrelated , the 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 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 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 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 pathways, respectively. 18 ARF p16INK4A is a -dependent inhibitor (CKI) specific patients. The human p14 binds directly to , to CDK4 and CDK6 and can directly block - resulting in stabilization of both p53 and MDM2, which Cip1 dependent kinase activity.3 The cyclin D/CDK4 (or 6) complex induces expression and cell-cycle arrest in both G1 16 ARF facilitates G1-phase progression toward the 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 in exon 1 are not found in tumor-derived lung, cells lack Rb, the levels of p16INK4A mRNA and protein were bladder, glioma or 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 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. Cell lines were obtained from: (A) Dr M Higashihara, First Department of Internal Medicine, University of Tokyo, (B) Dr T Nakamura, First Department of Internal Medicine, University of Tokyo, (C) Dr M Yoshida, Institute of Medical Science, University of Tokyo, (D) our lab, (E) Drs S Ogawa and H Hirai, Third Department of Internal Medicine, University of Tokyo, (F) Japanese Cancer Research Resource Bank, (G) Drs I Kubonishi and I Miyoshi, Department of Medicine, Kochi Medical School, (H) Dr Y Hayashi, Department of Pediatrics, University of Tokyo, (I) Dr K Tani, Institute of Medical Science, University of Tokyo, (J) Dr M Ogura, Aichi Cancer Hospital and Dr H Saito, First Department of Internal Medicine, Nagoya University, (K) Dr T Sato, Department of Pediatrics, Chiba University, and (L) Drs M Teramura and H Mizoguchi, Department of Hematology, Tokyo Women’s Medical School. INK4A/ARF in hematological malignancies T Taniguchi et al 1762 Clinical specimens of hematological malignancies Normal PB samples (n = 4) were obtained from healthy adult volunteers with informed consent. Normal BM aspirates We examined a total of 137 tissue specimens from 137 (n = 3) from patients with NHL, without BM involvement, patients in Japan with hematological malignancies. The speci- were obtained following acquisition of informed consent. mens had been frozen with dimethyl sulfoxide in liquid nitro- gen or in deep freezers at −80°C, until RNA preparation but some were freshly prepared. These patients were selected RNA preparation from the files of the Fourth Department of Internal Medicine, School of Medicine, University of Tokyo (Tokyo) between RNAs of cell lines were extracted from exponentially growing December 1985 and October 1998, Department of Hematol- cells by the acid guanidinium thiocyanate–phenol–chloroform ogy, Showa General Hospital (Tokyo) between March 1992 (AGPC) method,22 boiled for 1 min, measured by optic den- and February 1996, and Cancer Institute Hospital (Tokyo) sity, and stored at −80°C. Frozen cells were thawed rapidly between November 1993 and February 1996, on the basis of in a bath prewarmed at 37°C, and subsequently, RNAs of availability of frozen or fresh samples for molecular studies. these cells were extracted by the AGPC method. From freshly The proportion of malignant cells in each sample was greater prepared PB or BM, mononuclear cells (MNCs) were separ- than 70%, judged with cytosmears. Eight patients had been ated by standard Ficoll–Paque (Pharmacia Biotech, Uppsala, referred from Nagano Red Cross Hospital (Nagano), Kurobe Sweden) density-gradient centrifugation, as outlined by the City Hospital (Toyama) and Teikyo University Ichihara Hospi- manufacturer. Fresh lymph node cells were prepared by siev- tal (Chiba). Some of the specimens were obtained with written ing minced lymph nodes. RNAs of these cells were also informed consent and others were archive specimens extracted by the AGPC method. obtained at diagnostic procedures. The patients (87 men and 50 women) ranged in age from 14 to 89 years (median age: 59 years). Tissues examined cDNAs and Northern blot analysis included 69 lymph nodes, 34 bone marrow (BM) aspirates, 24 peripheral blood (PB) samples, seven pleural effusions, one The cDNA plasmids used in this study were as follows: a peritoneal effusion and two extranodal tumors. The hemato- p16INK4A (␣ form) cDNA plasmid was kindly provided by Dr logical malignancies in these patients are listed in Table 2. David Beach, Howard Hughes Medical Institute; a plasmid Among them, 93 patients (66 non-Hodgkin’s lymphomas containing the insert of the ␤ form transcript (clone 13) was (NHLs), 10 acute lymphoblastic leukemias (ALLs), four mul- kindly provided by Dr Christian-Jacques Larsen, INSERM.13 tiple myelomas (MMs), three chronic lymphocytic leukemias Probes used for Northern blot analysis are as follows: a probe (CLLs), and two prolymphocytic leukemias (PLLs), two Wald- specific to exon 1␣ of p16INK4A was a 150 bp PCR product enstro¨m’s macroglobulinemias (WMs), six adult T cell obtained by PCR using primers (P16AS206, P16S57; see Table leukemia/lymphomas (ATLs)) were included in our previous 3) from the p16INK4A cDNA plasmid. A probe specific to exon studies on cyclin D1 overexpression.20,21 Survival time was 1␤ of p14ARF was a 197 bp PCR product obtained by PCR measured from the time of pathologic diagnosis of the disease using primers (P16AS206, P16BS40; see Table 3) from clone and from the time of sampling for molecular analysis. 13. These two probes were 32P- radiolabeled with each anti-

Table 2 Lack of p16INK4A and p14ARF expression in hematological malignancies

Disease No. of Lacking expression of patients p16INK4A p14ARF Both p16INK4A only p14ARF only

Non-Hodgkin’s lymphoma 76 41 12 12 29 0 B cell diffuse large 23 15 9 9 6 0 small lymphocytic 2 1 0 0 1 0 follicular 30 13 0 0 13 0 mantle cell 4 (4) 1 (1) 1 (1) 1 (1) 0 (0) 0 (0) lymphoplasmacytoid 2 (2) 2 (2) 1 (1) 1 (1) 1 (1) 0 (0) Burkitt’s 2 2 1 1 1 0 others 7 4 0 0 4 0 T cell 4 3 0 0 3 0 Unclassified 2 0 0 0 0 0 Multiple myeloma/PCL/WM 12 (6) 7 (3) 0 (0) 0 (0) 7 (3) 0 (0) CLL/PLLa 8 (3) 3 (3) 1 (1) 1 (1) 2 (2) 0 (0) ALL/LBLb 13 3 3 2 1 1 Acute myeloid leukemia 12 1 0 0 1 0 CML-BC 8 2 2 2 0 0 Adult T cell lymphoma/leukemia 8 3 1 1 2 0 Total 137 (15) 60 (9) 19 (3) 18 (3) 42 (6) 1 (0)

aOne patient with T-CLL is included and expressed both genes. bFour patients with T-ALL are included and expressed both genes. PCL, plasma cell leukemia; WM, Waldenstro¨m’s macroglobulinemia; CLL, chronic lymphocytic leukemia; PLL, prolymphocytic leukemia; CML-BC, blast crisis of chronic myelogenous leukemia; ALL, acute lymphoblastic leukemia; LBL, lymphoblastic lymphoma. Numbers of cyclin D1-overexpressing patients are given in parentheses. INK4A/ARF in hematological malignancies T Taniguchi et al 1763 Table 3 Primers used for PCR amplification

Name Sequence Primer length (bp)

P16AS206 5Ј-TGCCCATCATCATGACCTGG-3Ј 20 P16S57 5Ј-GGAGCAGCATGGAGCCTT-3Ј 18 P16BS40 5Ј-TTCTTGGTGACCCTCCGGATT-3Ј 21 BA299S 5Ј-GCACCACACCTTCTACAATG-3Ј 20 BA538ASP16AS 5Ј-TGCCCATCATCATGACCTGGTAGATGGGCACAGTGTGGGT-3Ј 40

sense PCR primer and Klenow fragment (Takara, Kyoto, Japan). A ␤-actin probe was a human ␤-actin 0.63-kb cDNA fragment, which was 32P-radiolabeled using a random primer labeling (Takara). An aliquot (10 ␮g/lane) of each total RNA was separated on a formaldehyde-agarose gel and blotted on to nitrocellulose membrane. The membrane was hybridized with each 32P-labeled probe as described,20 washed in high stringency of 0.1–0.2 × SSC and 0.1% sodium dodecyl sulfate (SDS) at 65°C and autoradiographed at −80°C with an inten- sifying screen.

RT-PCR

cDNA was synthesized with oligo(dT)15 from total RNA, as described.20 An aliquot (1 ␮l) of cDNA (20 ng RNA equivalent) was placed in 20 ␮lof1× PCR buffer (10 mm Tris- ␮ HCl, 50 mm KCl, 1.5 mm MgCl2, pH 8.3) with 200 m each deoxyribonucleoside triphosphate (dNTP), 0.2 ␮m each primer (P16AS206, P16S57, P16BS40, BA299S) except for BA538ASP16AS which were added at 0.5 nm,2␮Ci of ␣-32P dCTP, 0.5 U recombinant Taq DNA polymerase (Takara), and 1 ␮l dimethyl sulfoxide (Wako Pure Chemical Industries, Osaka, Japan). Reaction mixtures were overlaid with mineral oil (Sigma, St Louis, MO, USA) and PCR amplification was started by placing the capped tubes on the block in the DNA thermal cycler (Perkin Elmer, Norwalk, CT, USA) which had already been heated to over 90°C. Each cycle constituted denaturation (1 min at 94°C, first cycle 5 min), annealing (2 min at 64°C), and extension (3 min at 72°C, last cycle 10 min). PCR was run for 21 cycles unless otherwise stated. Five microliters of each PCR reaction were separated on a 4.5% polyacrylamide gel followed by autoradiography. An optical scanner was used and densitometrical analysis was made using NIH image 1.59 software (NIH, Bethesda, MD, USA). As negative controls, distilled water instead of cDNA or RT reactions without reverse transcriptase was subjected to Figure 1 Quantitative RT-PCR for relative expression levels of PCR and we confirmed no false positive reaction. p16INK4A and p14ARF with endogenously expressed ␤-actin used as an PCR primers were synthesized by Greiner Japan (Tokyo, internal control. (a) Schematic presentation of primer setting on Japan) or GIBCO BRL (Tokyo, Japan) and are listed in Table p16INK4A, p14ARF and ␤-actin sequences. Thick lines indicate coding 3. Schematic presentation of design of the PCR primers is regions and thin lines represent truncated non-coding regions. Thick arrows indicate primers used in the PCR. The common primer, depicted in Figure 1a. Expected sizes of the PCR products are INK4A INK4A ARF ␤ P16AS206, is derived from the identical region between the p16 as follows: p16 , 150 bp; p14 , 197 bp; -actin, 260 bp. and p14ARF sequence. The composite primer, BA538ASP16AS, con- Three specific upstream primers, a shared downstream primer, sisted of P16AS206 in the 5Ј part and ␤-actin sequence in the 3Ј part. and a composite downstream primer were used. The shared The common primer, P16AS206, is shared in amplification of the downstream primer, P16AS206, corresponded to 187–206 three PCR products, while P16S57, P16BS40, and BA299S are specific nucleotide (nt) in the sequence of p16INK4A (␣ form)3 and 217– to p16INK4A, p14ARF, and ␤-actin sequences, respectively. (b) PCR of INK4A ARF ␤ ARF ␤ 13 p16 , p14 and -actin cDNA templates. The indicated tem- 236 nt in the sequence of p14 ( form). The upstream ␤ ARF INK4A INK4A plate, -actin, p14 or p16 cDNA plasmid, was added at the primer specific to p16 , P16S57, corresponded to 57– indicated concentration while the other two were kept at 2 × 104 3 74 nt in the sequence. The upstream primer specific to molecules/␮l for p14ARF and p16INK4A, and at 2 × 105 molecules/␮l for p14ARF, P16BS40, corresponded to 40–60 nt in the sequence ␤-actin and PCR was done, as described in ‘Materials and methods’. and was the same as P1 primer described by Duro et al.13 The After electrophoresis, the gels were dried and exposed to X-ray films upstream primer specific to ␤-actin, BA299S, corresponded to at −80°C with intensifying screens. Arrows indicate PCR products cor- INK4A ARF ␤ 299–318 nt in the sequence of ␤-actin cDNA.23 The 3Ј portion responding to p16 , p14 and -actin. INK4A/ARF in hematological malignancies T Taniguchi et al 1764 of the composite downstream primer, BA538ASP16AS, corre- p16INK4A and p14ARF expression in hematopoietic cell sponded to 519–538 nt in the ␤-actin sequence23 and the 5Ј lines portion was the same as P16AS206. The level of p16INK4A or p14ARF expression was determined We studied p16INK4A and p14ARF mRNA expression in hemato- as the ratio of the signal of the p16INK4A or p14ARF PCR product poietic cell lines, by RT-PCR described above and Northern to those of the internal control (␤-actin). RT-PCR for cyclin D1 blot analysis. As shown in Figures 2a and b, the RT-PCR and overexpression was done, as described.21 Northern blot analysis gave similar results and thus, the quan- titative nature of the PCR was re-confirmed. The RT-PCR had a more sensitive detectability. Western blot analysis for pRb In 22 (54%) of 41 cell lines, neither p16INK4A nor p14ARF mRNA was detected by the RT-PCR, which is consistent with Cells were lyzed with 1 × sample buffer (Tris-HCl 60 mm, SDS frequent deletions of the INK4A/ARF locus in cell lines 12,27 2%, dithiothreitol 0.1 m, pH 6.8) and boiled for 5 min. Protein reported previously. In seven (17%) of 41 cell lines, only ARF INK4A concentration was determined by spectrophotometry using p14 was detected, which might be related to p16 pro- ARF BCA Protein Assay Reagent (Pierce, Rockford, IL, USA).24 A moter hypermethylation (Table 1). p14 expression without INK4A total of 30 ␮g protein per lane was loaded on an 8% SDS- p16 expression was frequently (4/11) observed in B cell polyacrylamide gel and subjected to Western blot analysis, as lines, while it was infrequent (1/12) in non-HTLV-1-infected T INK4A described.25 Anti-pRb monoclonal (MAb1; TRITON cell lines (Figure 2c). In contrast, p16 expression without ARF Diagnostics, Alameda, CA, USA), at 0.4 ␮g/ml, was used. p14 expression was observed only in two non-HTLV-1- infected T cell lines, KOPT-K1 and PEER (Figure 2b). Li et al7 reported that p16INK4A mRNA accumulates to a high Statistical analyses level in cells lacking Rb function and that transcription of p16INK4A is repressed by Rb. Therefore, we studied pRb expression in these cell lines by Western blot analysis (Table Univariate (Kaplan–Meier) analysis was used to assess survival 1). However, reciprocal expression of p16INK4A and pRb is not differences. Survivals between different subgroups were necessarily expected. ARF is reported to be expressed highly compared using the Wilcoxon test. in cell lines lacking p53.16 p53 mutation, deletion or rearrangement was noted in at least 18 of the cell lines listed in Table 1.28–37 However, mutation of p53 and lack of p14ARF Results expression were not mutually exclusive. Seven cell lines showed smears in Northern blot analysis of Quantitative RT-PCR to compare expression levels of p14ARF mRNA and JK-1 showed a smear of p16INK4A mRNAs, p16INK4A, p14ARF and ␤-actin although the RT-PCR revealed no corresponding signals (Table 1 and Figure 2b). Among them, two cell lines (KOPT-K1 and Co-amplification of targets (p16INK4A and p14ARF) and control SKW-3) also showed p14ARF bands of aberrant sizes. (␤-actin) in a tube was designed to circumvent difficulties in conventional competitive RT-PCR assays (Figure 1a). Simple co-amplification of control and targets reveals the ratio of p16INK4A and p14ARF expression in patients with target to control templates by comparing target and control hematological malignancies products generated during the exponential phase of the PCR reaction.26 Since ␤-actin is expressed at a level much higher As shown in Figure 3a, in normal PB and BM MNCs and than those of the targets, comparable co-amplification can be lymph nodes of reactive lymphadenitis, p14ARF mRNAs were difficult. Therefore, we used a composite primer at a low at low levels and p16INK4A mRNAs were barely detectable. In concentration to reduce ␤-actin signals at a constant ratio to clinical specimens of hematological malignancies, abnor- the level comparable to the target signals. mally high expression levels of p16INK4A and/or p14ARF com- As shown in Figure 1a, we designed a common primer, pared to normal tissues were frequently observed (Figure 3). P16AS206, derived from 16 bases in 5Ј end of exon 2 and On the other hand, p16INK4A mRNA expression was lacking four identical bases in 3Ј ends of exons 1␣ and 1␤, and a in 60 of 137 patients and p14ARF mRNAs were undetectable in composite primer, BA538ASP16AS, the 3Ј part of which is 19 of 137. Almost all patients without p14ARF mRNAs lacked derived from the sequence of ␤-actin and the 5Ј part of which p16INK4A expression (Table 2) except for one patient with ALL is the same as the common primer, P16AS206. The three spe- who had B cell phenotype and TCR rearrangement. cific primers for p16INK4A, p14ARF and ␤-actin are P16S57, p16INK4A and p14ARF expression differed among types of dis- P16BS40 and BA299S, respectively. Amplification of ␤-actin eases. All patients with FL expressed p14ARF while nine (39%) cDNA must be initiated with BA299S and BA538ASP16AS, of 23 patients with diffuse large B cell lymphoma (DLBCL) then, P16AS206 takes the place of BA538ASP16AS because lacked p14ARF expression, thus corresponding to the relatively we use BA538ASP16AS at a lower concentration. Final pro- high incidence of p16INK4A deletion in diffuse lymphoma ducts for ␤-actin were made by virtue of P16AS206 and (13%).12 On the other hand, p16INK4A expression was fre- BA299S. We confirmed identity of the PCR products by direct quently undetectable in both DLBCL (15/23, 65%) and FL sequencing (data not shown). We found exponential amplifi- (13/30, 43%). MM was similar in expression to FL. These find- cation phases of three genes overlapped up to 21 cycles with ings are consistent with the previous reports that homozygous a cDNA from MEG-01s cell line used as a template (data not deletion of the INK4A/ARF locus in FL and MM is rare and shown). Therefore, PCR was next run for 21 cycles. In that p16INK4A is hypermethylated in 40% of FL and in 75% of addition, using cDNA plasmids for the genes as templates, we MM.12,38,39 In a study of p16INK4A protein expression in NHLs confirmed the ratio of PCR products reflecting that of added by Western blot analysis, loss of expression was rare (6%) in templates (Figure 1b). typical FL but frequent (28%) in large cell lymphoma.40 Two INK4A/ARF in hematological malignancies T Taniguchi et al 1765

Figure 2 Analysis of hematopoietic cell lines. (a) Comparison between the RT-PCR and Northern blot analysis. RNAs extracted from the indicated cells on each lane were subjected to the RT-PCR (upper part) and to Northern blot analysis (middle part) as described in ‘Materials and methods’. RNA loading in each lane is shown in the photograph of the ethidium bromide-stained gel at the bottom. (b) Comparison between the RT-PCR and Northern blot analysis of a part of T cell lines. Note smears shown in lanes for CEM, KOPT-K1, MOLT-4, PEER and SKW-3, and bands of aberrant sizes in lanes for SKW-3 and KOPT-K1 in the lower panel. (c) The RT-PCR analysis of remaining hematopoietic cell lines for expression of p16INK4A and p14ARF. Arrows indicate PCR products corresponding to p16INK4A, p14ARF and ␤-actin or each transcript signal of Northern blot analysis. other immunohistochemical studies revealed that the inci- or FL. We found no significant difference in OS between the dence of p16INK4A protein loss in FL is zero whereas in diffuse presence and absence of p16INK4A expression for patients with large lymphoma it is as high as 44–65%.41,42 Taken together, DLBCL or FL. Regarding p14ARF expression, when survival in a portion of FL, p16INK4A may be hypermethylated and its time for patients with DLBCL was measured from the time of mRNA levels may be low, but not low enough to silence pro- sampling for molecular analysis, patients with increased tein expression. In almost all patients with acute myeloid leu- p14ARF expression (p14ARF/␤-actin Ͼ0.19) compared to nor- kemia (AML), both expressions were detectable, such being mal controls tended to have a longer OS than did other consistent with the rarity of homozygous deletion of patients (P = 0.096, Figure 4b). Since all patients with FL INK4A/ARF locus in this disease.12 High levels of p16INK4A expressed p14ARF, we determined if the high levels of p14ARF expression (defined as INK4A/␤-actin Ͼ0.2 arbitrarily) were expression affected survival time. When high levels of p14ARF significantly more frequent in those with acute leukemia, expression were defined arbitrarily as p14ARF/␤-actin Ͼ0.6, including ALL, AML and blast crisis of chronic myelogenous patients with high p14ARF expression had a significantly leukemia (CML-BC) than in other hematological malignancies shorter OS from the time of diagnosis than did other patients (12/33 vs 6/104, P Ͻ 0.01, chi square test). (P = 0.030, Figure 5a). OS measured from the time of molecu- We also examined cyclin D1 mRNA expression in these lar analysis showed no significant difference (P = 0.21) (Figure patients using the RT-PCR assay that we had devised;20,21 we 5b). We found no significant correlations of p14ARF over- identified 15 patients (six NHLs, one WM, five MMs, one CLL expression and other clinical parameters including age, LDH and two PLLs) with cyclin D1 overexpression. There was no levels and clinical stage in FL. apparent relationship between p16INK4A and p14ARF expression and cyclin D1 overexpression (Table 2). Discussion

Clinical correlation We used a quantitative RT-PCR assay to examine relative expression levels of p16INK4A and p14ARF in primary tumors We then asked whether p16INK4A or p14ARF expression status of various hematological malignancies and hematopoietic cell affected overall survival time (OS) for patients with DLBCL lines. Major mechanisms of gene inactivation of this locus are INK4A/ARF in hematological malignancies T Taniguchi et al 1766

Figure 4 Overall survival time for diffuse large B cell lymphoma patients, with or without increased p14ARF mRNA expression. (a) There is no significant difference in the overall survival time from the time of diagnosis between patients with increased p14ARF expression (solid line, n = 11) and those without (dotted line, n = 12). (b) Patients without increased p14ARF expression (dotted line) tended to have a shorter overall survival time from the time of molecular analysis than did other patients (solid line).

homozygous deletion and hypermethylation, both leading to lack of expression. This assay cannot detect some modes of gene inactivation such as point mutation. However, p16INK4A inactivation by point mutation is rare in hematological malig- nancies12 and mutations in p14ARF exon 1␤ are not found in tumor-derived lung, bladder, glioma or melanoma cell lines or in primary T-ALL cells.18 To maintain the quantitative nature of our RT-PCR assay, we limited the PCR cycles to 21 and the sensitivity of PCR is somewhat compromised. In reactive lymphoid tissues used as controls, p16INK4A mRNAs were barely detectable with this INK4A INK4A ARF assay, although p16 nuclear immunoreactivity has been Figure 3 p16 and p14 mRNA expression in normal tissues reported.41 p16INK4A protein production from levels of and clinical specimens of various hematological malignancies. (a) INK4A Representative results of the RT-PCR analysis are shown. Arrows indi- p16 mRNA undetectable in our RT-PCR assay is possible INK4A cate PCR products corresponding to p16INK4A, p14ARF and ␤-actin. (b) and it should be noted that the ‘lack of p16 expression’ Histogram of relative p16INK4A and p14ARF mRNA expression levels does not necessarily lead to complete silencing of the p16INK4A determined by RT-PCR. Expression levels were standardized using the gene. Therefore, our assay may overestimate the inactivation ␤ -actin signal of each sample as an internal control and are plotted of p16INK4A. However, it has as good a quantitative nature as INK4A ␤ over a ratio of p16 or p14ARF to -actin signal. LN, lymph node Northern blot analysis and is useful when comparing the of reactive lymphadenitis; PB MNC, mononuclear cells from normal INK4A ARF peripheral blood; BM MNC, mononuclear cells from normal bone expression levels of p16 and p14 mRNAs among a marrow; NHL, non-Hodgkin’s lymphoma; DLBCL, diffuse large B cell number of samples. To the best of our knowledge, this is the lymphoma; FL, follicular lymphoma; WM, Waldenstro¨m’s macroglob- first report that characterizes simultaneously mRNA ulinemia; MM, multiple myeloma; CLL, chronic lymphocytic leuke- expression levels in various hematological malignancies. mia; PLL, B cell prolymphocytic leukemia; ALL, acute lymphoblastic Our results were fairly consistent with the genomic status of leukemia; AML, acute myeloid leukemia; CML-BC, blast crisis of the p16INK4A gene reported in hematopoietic cell lines (Table chronic myelogenous leukemia; ATL, adult T cell 12,27,37,43,44 INK4A leukemia/lymphoma. Dotted lines indicate the maximum levels of 1). In the cell lines in which p16 genes were expression observed in controls. *, Specimens were obtained during relapse or progression of diseases. INK4A/ARF in hematological malignancies T Taniguchi et al 1767 negative for p16INK4A by the RT-PCR. This smear could be transcripts derived from exon 1␣ without an efficient termin- ation signal, because in these cells, exon 2 is deleted.44 These transcripts would not produce functional p16INK4A protein, because exon 2 is essential for p16INK4A function.1 In primary tumors, p16INK4A expression more frequently than p14ARF expression was lacking. In only one ALL patient was p16INK4A expressed without p14ARF. Therefore, in many patients with hematological malignancies, the target of inacti- vation is p16INK4A alone, or both p16INK4A and p14ARF, but not p14ARF alone, although there remains the possibility that p16INK4A was expressed below detectable levels. However, in ALL, p14ARF may be a primary target of inactivation as sug- gested by findings in one ALL patient with TCR rearrangement and two T cell lines, KOPT-K1 and PEER, which expressed p16INK4A without p14ARF, probably through an illegitimate recombination mechanism reported by other investi- gators.18,46 Garcia-Sanz et al47 reported that deletions and rearrange- ments of p16INK4A are associated with a poor prognosis in B cell NHLs. Loss of p16INK4A expression and deletions of p16INK4A gene are associated with aggressive variants of mantle cell lymphomas48 and homozygous deletions of p16INK4A are associated with histologic progression in FL.49 Interestingly, when survival time for patients with DLBCL was measured from the time of molecular analysis (not from the time of diagnosis), the difference in OS between the absence and presence of increased p14ARF expression became bigger, but not significant (Figure 4a and b). Inactivation of the INK4A/ARF locus may occur by chance and change the Figure 5 Overall survival time for follicular lymphoma patients, subdivided according to the expression levels of the p14ARF mRNAs. course of the disease. (a) p14ARF highly-expressing patients (solid line, n = 6) had a signifi- On the other hand, in FL without homozygous deletion of cantly shorter overall survival time from the time of diagnosis than INK4A/ARF locus, p14ARF overexpression seemed to be a poor other patients (dotted line, n = 24). (b) There is no significant differ- prognostic factor. Several mitogenic stimuli such as E1A, , ence in the overall survival time from the time of molecular analysis oncogenic ras, V-abl and -1 upregulate ARF leading to p53 ARF between patients with high p14 expression (solid line) and those stabilization (reviewed in Ref. 2). p14ARF is negatively regu- with low p14ARF expression (dotted line). lated by wild-type p53 expression.16,19 Therefore, the high expression of p14ARF observed in this study may reflect onco- deleted, neither p16INK4A nor p14ARF mRNAs were detected, genic stimuli in tumor cells and/or inactivation of other tumor except for MEG-01 cells, contradictory results were suppressors such as p53 and Rb. Since we analyzed only a reported.44,45 In cell lines reported to have methylated small number of patients, the relation to other prognostic p16INK4A gene, p16INK4A mRNAs were either not detected or factors remains to be clarified. were at low to intermediate levels, and p14ARF mRNA levels p16INK4A mRNA accumulates to a high level in cells lacking were almost always elevated. This means that methylation of Rb function7 and in senescent cells caused by an increase in the p16INK4A gene does not necessarily lead to complete gene the number of population doublings.9 High expression levels silencing and that even if the p16INK4A gene is silenced by of p16INK4A mRNAs especially in acute leukemias might be methylation, p14ARF mRNA expression can be elevated. This is related to a rapid increase in population doublings of leu- consistent with reported data that human ARF promoter region kemic cells. Recently, Mekki et al50 reported that childhood hypermethylation is a low-frequency event in cell lines.19 ALL patients with high p16INK4A expression have shorter Smears and bands of aberrant sizes observed in seven cell disease-free survival than other such patients. lines by Northern blot analysis of p14ARF could be transcripts In conclusion, p16INK4A and p14ARF expression is differen- derived from exon 1␤ without exon 2. Since we used primers tially affected and is more frequently deregulated in hemato- for RT-PCR on exon 1␤ and exon 2, breakpoints of rearrange- logical malignancies than speculated from documented gen- ment may be located between exon 1␤ and exon 2 in these etic data. In addition to inactivation, overexpression may have cell lines. In fact, a breakpoint cluster region exists within 3- clinical significance and further study on the relation between kb 3Ј to exon 1␤ and MOLT-4 has a breakpoint in this expression and prognosis is warranted. region.46 We confirmed the expression of exon 1␤ in these cell lines (except for KOPT-K1) using an RT-PCR assay and primer pairs within exon 1␤ (data not shown). These aberrant transcripts derived from exon 1␤ may produce functional Acknowledgements p14ARF protein, because the aminoterminal domain encoded by exon 1␤ is both necessary and sufficient for inducing G1 This work was supported in part by grants from the Ministry arrest.1 Whether functional are actually made of Education, Science, Sports and Culture of Japan. We thank remains to be elucidated. JK-1 cells also showed a smear on M Yoshikawa for technical assistance and M Ohara for langu- Northern blot analysis for exon 1␣ (data not shown) and are age assistance. 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