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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. RAPID COMMUNICATION Homozygous Loss of the Cyclin-Dependent Kinase 4-Inhibitor (p16) Gene in Human Leukemias By Seishi Ogawa, Naoto Hirano, Naomi Sato, Tokiharu Takahashi, Akira Hangaishi, Kozo Tanaka, Mineo Kurokawa, Tomoyuki Tanaka, Kinuko Mitani, Yoshio Yazaki, and Hisamaru Hirai Recently, it has been shownthat the homozygous deletion of the cyclin-dependent kinase-4inhibitor (CDK41; p161 gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4 gene is thoughtto be a putative tumor-suppressor gene. We report here that both alleles of the CDKU gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4l gene locus by Southern blot analysis. We found that a part or the whole of the CDK41 gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia(ALL), and 13with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4l gene was detected in a variety of cell lineages, whereas all 4 cases showingthe homozygous deletion were confined to ALL. It should be noted that 2 of them of chromosome 9. Our rehad no cytogenetic abnormalities sults suggestthat loss of the CDKU function may contribute to immortalizationof human leukemiac e l l s and play a causative role at least in development of human lymphocytic leukemias. 0 1994 by The American Societyof Hematology. 10% fetal calf serum. TFl, W, CMK, and F36E are factor-depenANY LINES OF evidences have established the sigdent cell lines and are cultured in the presence of 5 ng/mL of recomnificance of inactivation of tumor-suppressor gene(s) binant human granulocyte-monocyte colony-stimulating factor in development of human malignancies, including leuke(rhGM-CSF). mias.I4 Many investigators have reported mutations andor Patients and preparation of sample cells. Bone marrow or pedeletion of p53, RB, WT1, and NF1 genes in a wide variety ripheral blood samples from 72 patients, including 45 patients with of both familial and sporadic cases of human cancer~.~*~-’lacute myelocytic leukemia (AML), 14 patients with acute lymphoThese genetic abnormalities are considered to deteriorate cytic leukemia (ALL), and 13 patients with chronic myelogenous normal functions of the tumor-suppressor gene products leukemia (CML) in blastic crisis, were collected after informed conquantitatively or qualitatively and to make a contribution to sent was obtained. In all samples examined, the proportion of tumor carcinogenesis.’” The cell cycle in eukaryotes is regulated cells exceeded 70%. From the samples, mononuclear cells were separated on Ficoll-Hypaque density gradients. by the cyclin-dependent kinases (CDKS).”*’~ The sequential Synthetic primers. The reverse transcription polymerase chain activation of CDKs and their cosequent phosphorylation of reaction (RT-PCR) primers were designed to amplify almost the critical substrates stimulates progressionofthe cell entire coding sequence (nucleotide [nt] 32 to 485) ofthe CDK4I The complexes formed by CDK4 and the D-type cyclins cDNA previously reported by Serrano et ai?’ Their sequences are control passage through the G1 phase of the cell as follows: the antisense primer for RT (nt 526 to 507), 5‘-AGGThe CDK4-inhibitor (CDK4I; p16) is a protein of 16 kD ACCITCGGTGACTGAT-3’; the primers for the first PCR, 5’that binds to and inhibits the catalytic activity of the CDK4/ CAGCATGGAGCCITCGG-3’ (sense) (nt 15 tont 31) and 5’-TCTcyclin D c ~ m p l e x e s . ~Because ’ - ~ ~ CDK4I is a negative reguAAGTITCCCGA-GGTITC-3’ (antisense) (nt 505 to nt 486); and lator of cell-cycle progression, it has been thought to be the primers for the second PCR, 5”CTGACTGGCTGGCCAinactivated during cancer d e v e l ~ p m e n t Recently, .~~ Nobori CGGCC-3’ (sense) (nt 32 to nt 51) and5’-TCAGAGCCTCTCTGGTT%TT-3’ (antisense) (nt 485 to nt 466). et alZ4and Kamb et al” have proposed that the CDK4I gene RT-PCR analysis. Total RNA was isolated from normal bone is a novel candidate of a tumor-suppressor gene. They have marrow mononuclear cells with the single step method described by reported that the CDK4I gene is deleted or mutated with Chomczynski and Sacchi.26 Five hundred nanograms of total RNA surprisingly high frequencies in human cancer cell lines, was mixed in a reaction mixture containing 2 pmol of RT primers including melanoma, glioma, lung cancers, and leukemias. for the CDK4I gene, 3.8 pL of 5X RT buffer (250 mmoVL TrisThese findings prompted us to investigate the existence of HCI [pH 8.31, 375 mmol/L KCI, 15 mmoVL MgCI,) in a volume of homozygous loss of this gene in samples from patients with 12.5 pL. The mixture was heated at 95°C for 3 minutes, chilled on leukemias as well as in human hematopoietic cell lines by Southern blot analysis. M MATERIALSANDMETHODS Cell lines. Thirty-seven human leukemia cell lines were included in this study and consist of 11 myelocytidmonocytic cell lines (KG1, HL60, HEF-2, KU8 12, SKHI, JOSK-I, JOSK-K, JOSK-S, P31 FUJ, P39 TSU, and THP-l), 4 erythroid cell lines (TF-I, F36E, HEL, and K562). 4 megakaryocytic cell lines (W,CMK, MegJ, and MOLMI), 1 1 B-lymphocytic cell lines (SCMCL-Ll, SCMCLL3, SCMCL-LA, BALLl, P32 ISH, P30 OHK, I“9, Daudi, HA, M5, and Raji), and 7 T-lymphocytic cell lines (MOLT16, Jurkat, MT2, CEM, MOLT4, SKW3, and A3KWA). The cells were grown in suspension culture inRPM1 medium 1640 supplemented with Blood, Vol 84, No 8 (October 15), 1994 pp 2431-2435 FromThe Third Department of Inteml Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Submitted June 23, 1994; accepted July 22, 1994. Address reprint requests to Hisamaru Hirai, MD, The Third Department of Inteml Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advettisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8408-06$3.00/0 2431 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. OGAWA ET AL 2432 with Hindnl, separated through 0.8% agarose gels, and blottedonto the nylon filters. The RT-PCR products of CDK41 cDNA were excised from low melting temperature agarose gels, extracted from the gels with phenolkhloroform. and used as a probe for detecting the a kindgift CDK4IDNA. A humanAMLlprobe,C6E3SS2,was A kb 23 9.4 6.6 - Table 1. Homozygous Loss or Rearrangement of the CDK4l Gene in Hematodetic Cell tines B kb 23 9.4 6.6 1 - " " " - 2 3 4 5 6 7 8 9 1 0 1 1 1 2 ~ - ""~r""" I * Fig 1. Southern blot analysis of human hematopoietic c e l lines. The CDK4 gene was detected as the threediscrete bands of approximately 20, 12, and 6 kb in size by Hindlll digestion of genomic DNA. The AMLl gene fragment of 7 kb in size was hybridized as an internal control and indicated by an arrow. (A) Lane 1, Jurkat; lane 2, MOLT4; lane 3, SCML-L4; lane 4,MOLT%; lane 5, SCML-U; lane 6, SKW3; lane 7, JOSK-S; lane 8, MT2; lane 9, P39-TSU; lane 10, P3O-OHK; lane 11, TF1; lane 12, normal bone marrow.(B) Lane 1, KG1; lane 2, MP1; lane 3, SKH1; lane 4,HEL; lane 5, CMK; lane 6, MegJ; lane 7, KU812; lane 8, UT7; lane 9, F36E; lane 10, K562; lane 11, M5; lane 12, JOSK1. Myelocytidmonocytic cell lines HL60 SKHl KG1 KU812 HEF2 JOSK-I JOSK-K JOSK-S P39 TSU P31 FUJ THPl Erythroid cell lines F36E K562 HEL TF1 Megakaryocytic cell lines UT7 CMK MegJ MOLMl B-lymphocytic cell line BALL1 Daudi Raji IM9 M5 HA P32 ISH SCMCL-L1 SCMCL-L3 SCMCL-L4 P30 OHK ice, and incubated at 37°Cfor 15 minutes for annealing. The mixture was then supplemented with 0.2 pL of 5 X RT buffer, 2 pL of 0.1 moVL D'IT, 4.0 pL of 2.5 mmol/L each of dNTPs, 20 U of RNase inhibitor(Takara,Kyoto, Japan), and 1 0 0 U ofMoloneymurine BRL, Gaithleukemia virus (M-MLV) reverse transcriptase (GIBCO ersburg, MD) to a volume of 20 pL and incubated at 37°C for 60 minutes. The RT reaction products were again heated at 96°C for 2 minutes and chilled on ice. The 2 pL of the RT reaction products was subjected to the first PCR amplification in 50 pL of reaction mixture containing IO mmol/L Tris-HCI (pH 8.8). 50 mmoVL KCI, 3.5 mmol/L MgC12,0.01% bovine serum albumin (BSA), 200 pmoV L dNTPs, 5% dimethylsulfoxide, 15% glycerol, I pmoVLeachof T-lymphocytic cell lines thefirst PCRprimers,and2.5 U TaqDNA polymeme (PerkinJurkat Elmer Cetus, Norwalk, CT). The PCR were repeated for 40 cycles of96°C (1 minute), 50°C (1 minute),and72°C (2 minutes). Two A W W MOLT4 microliters of the first reaction products was subjected to the nested MOLT16 second PCR reaction, essentially under the same conditions as the SKW3 first reaction except that the pH of the buffer was 9.3, the concentraCEM tion of MgC12 was 1.5 mmol/L, and the primer set for the second MT2 PCR was used. Sequencing of RT-PCR products. For subsequent use as a probe for Southern blot analysis, the RT-PCR product of the CDK4l cDNA Total from the normal bone marrowsample was subcloned into pBluscript Abbreviations: DD, neither of both alleles of the CDK41 gene were SK(-) (Stratagene, La Jolla, CA) andsequencedby the dideoxy detected; Dm, solely a rearranged allele was detected, indicating loss chain termination method. of the CDK41 genein one allele and a rearrangement in another allele; Southern hlor analysis. Ten micrograms of DNAs extracted from cultured leukemia cell lines and from patients' samples was digested -, negative for homozygous loss of the CDK41 locus. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CDK41 ( ~ 1 6 GENE ) IN HUMAN LEUKEMIAS 2433 1 2 3 4 " " 5 6 7 8 9 1 0 1 1 1 2 - kb 23 9.4 6.6 4.4 2.3 2.0 " 9 -- " " " " " " I C 4- Fig 2. Southem blot analysis of samples from patients with leukemias. Lanes l and 2, normal bone marrow; lanes 3 through 6, patients with ALL showing homozygous loss or rearrangement of the CDK491 gene; lanes 7 through 9, patients with AML; and lanes l 0 through 12, patients with CML in blastic crisis. The AMLl gene as an internal control was indicated by an arrow. from Dr H. Miyoshi (Radiobiology Division, National Cancer ResearchInstitute.Tukiji.Tokyo,Japan)and was usedtoconfirm integrity of the sample DNA and completeness of Hind111 digestion. The 25 pg of both probes was labeled to high specific activities with [(r--'*P]dCTF'by the random priming method and used as probes for detection of the Hindlll-digested fragments of the CDMI and AMLlgenomic DNA. Hybridimtionwasperformed in a solution with 50% formamide, 5 X SSC, 5 X Denhart's solution, and 0.5% sodium dodecyl sulfate (SDS)at 42°C overnight. After highly stringent wash with 0.1X SSC and 0.1% SDS at 65°C. filters were exposed to x-ray films with an intensifying screen at -70°C. RESULTS Sequencing of the RT-PCR product. A 463-bp cDNA fragment was amplified using RNA from normalhuman bone marrow cells with the RT-FCR method and confirmed to be the authentic CDK4I cDNA fragment by nucleotide sequencing?' Southern blot analysis of DNAs of hematopoietic cell lines and samples from leukemia patients. We first analyzed 37 leukemia cell lines for the configuration of the CDK4I gene locus (Fig 1A and B). The normal CDK4I genomic DNA Table 2. Homozygous Loss of the CDK41 Gene in Blood Samples of Leukemia Patients Homozygous Loss of the CDKIl Gene Disease 0145 AML ALL CML-BC Total 4/14 0113 (5.6%) Abbreviation: CML-BC, CML in blastic crisis. 4/72 was detected as three distinct bands of approximately 20, 12, and 6 kb in size, whereas in 14 of 37 cell lines (38%). one or more of the three bands for the CDK4I gene failed to be detected. Because the AMLl probe could successfully detect a 7.0-kb HindIII genomic fragment in every lane, it was confirmed that all sample DNAs examined were highly intact and completely digested with HindIII. Therefore, it is concluded that all or a part of the CDK4I gene locus was homozygously deleted in these cell lines. In lanes 1, 4, and IO ofFig 1A and in lanes 2, 4, 6, 8, 9, and 10 of Fig IB, we could not detect any of the three HindIII-digested fragments of the CDK4I gene with this probe (a 17-kb band seen in most lanes of Fig 1A is a nonspecific band), whereas in lanes 2 and 6 of Fig I A, the partially deleted CDK4 gene fragments with or without rearrangement were observed. These observations were confirmed by digestion with another restriction enzyme, EcoRI, suggesting that the deleted region of the CDK4I gene locus is variable among cell lines. The results were summarized in Table 1, which suggests that, in the cell lines analyzed, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages. To test the possibility that the homozygous deletion could also occur in leukemia cells from patients, we examined the blood samples from 72 leukemia patients. Because, in contrast to cell lines, patients' blood samples inevitably contain residual normal bloodcells and thus introduced normal DNA into the prepared sample DNA,whichmight disturb the accurate evaluation of the results obtained by Southern blot analysis, we have included only the samples that were assessed to contain more than 70% leukemic cells so that the DNA of normal cells could be neglected or properly evaluated by using the internal control, ie, the AMLl gene. Taking this into consideration, it was concluded that, in 4 of 14 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 2434 OGAWA ET AL Table 3. Clinical and Cytogenetic Characteristics of 4 Patients With Loss of the CDK41 Gene Patient No. Age (yr)/Sex Diagnosis Blast (%l 1 2 3 4 2/F 6OlF 30lM 52/F Null ALL Pre-B ALL Null ALL Pre-B ALL 90.0 88.8 93.6 98.6 Karyotype +8,51, +7,XX, del(l)(q32), f21, 46, XX, -1. -4, 2q-, 5q+, 6p-, 41, XY, -6, -6, -9, -13, -17, -22, 46,XX. -18, l q - , 13q-, 4p+, samples from ALL patients, homozygous loss of a part or the whole of the CDK4I gene was detected, compared with the AMLl band being clearly detected (Fig 2). However, in the other samples from patients, including 45 cases with AML and 13 cases with CML in blastic crisis, we could not detect the homozygous deletion or rearrangements of the gene (Table 2). As for the cell lineages of the 4 ALL cases with the homozygous deletion of the CDK4I gene, 2 are preB ALL and the other 2 are null ALL. Despite the CDK4I gene locus on the short arm of human chromosome 9, 2 of the 4 cases did not show any cytogenetical abnormalities involving chromosome 9. DISCUSSION The cell division cycle is regulated by a number of protein kinases known as CDK4s,””‘ among which the CDK4 is considered to associate with the D-type cyclins and to control cell proliferation through the G1 phase.”-” The CDK4I was originally identified as a protein of relative molecular mass of 16 kD (p16), which associates with CDK4 and inhibits catalytic activity of the CDK4kyclin D complexes and cell proliferati~n?~.~~ In this context, the recent reports that the CDK4I gene is homozygously lost in a wide variety of human tumor cell linesz3-z5seem to strongly support the idea that it is a tumor-suppressor gene that had long been quested for in the 9p21 locus, a frequently deleted chromosomal region in ALL and other various t ~ m o r s . ~For ~ ” ~further confirmation of the idea, therefore, we analyzed 72 leukemia patients as well as 37 leukemia cell lines for loss of the CDK4I gene by Southern blot analysis. As a result, it is shown that at least a part of the CDK4I gene was homozygously lost, witha surprisingly high frequency (38%) in hematopoietic cell lines. Considering that the incidence of mutations of both p53 alleles is estimated around 75% in human leukemia cell lines (Cheng and H a a ~ Sugimoto ,~~ et al,” and our unpublished observations), this suggests that the inactivation of the CDK4I gene may similarly be important for cell immortalization as that of the p53 gene. Moreover, inactivation of both CDK4I and p53 occurred in THP1, F36E, K562, UT7, Molt 16, Jurkat, Daudi, UT7, CEM, Molt4, and CMK (Cheng and H a a ~ , 3Sugimoto ~ et al,” and our unpublished observations), implicating that inactivation of multiple tumor-suppressor genes may occur during development of human leukemias. The frequency of the homozygous deletion in cell lines were about 40% in our study and may comparable to 9 of 14 cell lines (64.3%) in the report by Nobori et aLZ4However, in patients’ samples, the frequency is relatively low, as is the case with that of the p53 gene alteration^.^.^^.'^ There could be two possible explanations for the difference between cell lines and pa- (t4;11)(q21;q23). 6q-, 9p+, 9p-, 14q-, 14q+, lp-, 3p-, 7pf. lop+, 15p+, 15q- 19p+, +mar. +mar. -2Op+, +mar. tients’ samples. One explanation is that leukemic cells with CDK4I inactivation, more probably by homozygous loss, take advantage of acquiring immortality (a cause for cell immortalization), and the other is that, after immortalization, cell lines may become more prone to deletion of this locus (a result of cell imm~rtaIization).~~~’ We have shown loss of both alleles of this gene in 4 of 72 (5.6%) samples from patients with acute phase of leukemias and 4 of 14 (28%) of patients with ALL. Our findings suggest that CDK4I gene inactivation was not merely the result of establishment of a cell line, but possibly related to de novo development of human leukemia. The clinical and cytogenetical findings in these 4 cases were summarized in Table 3. Although the deletion seems to occur in every cell lineage in cell lines, all 4 patients with the homozygous loss were confined to ALL, which may reflect the findings that loss of 9p21 locus is much more frequently found in ALL?’” In this context, it is worth noting that inactivation of the CDK4I gene was detected in 2 cases without cytogenetic abnormalities in 9p2 l locus, suggesting the actual frequency of loss of function of this gene may be higher than predicted by cytogenetic analysis in ALL, which may in turn implicate the importance of inactivation of this gene in ALL leukemogenesis. In contrast, the homozygous loss of the CDK4I gene was not detected in 14 cases with blastic crisis of CML and 45 cases with AML. We may safely conclude that inactivation of the CDK4I through deletion of both alleles is, if it exists, rare in AML. However, in this study, we did not examine the other mechanisms through which the CDK4I become inactivated, eg, point mutations of this gene. It is well known that tumor-suppressor genes, including p53 and RB genes, undergo inactivation by point mutation^."^ The existence of point mutations of the CDK4I gene were shown at a high frequency in melanoma cell linesz4and, therefore, this possibility should also be extensively addressed in leukemia patients in further studies. ACKNOWLEDGMENT We thank Dr Yasuhide Hayashi (Department of Pediatrics, University of Tokyo, Tokyo, Japan) and Dr T. Shikano (Department of Pediatrics, University of Hokkaido, Hokkaido, Japan) for providing samples from patients. We also give thanks to Dr Miyoshi for his generous gift of an AML 1 probe, C6E3SS2. REFERENCES 1. Weinberg RA: Tumorsuppressor gene. Science 2541 138, 1991 2. 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Yin Y, Tainsky MA, Bischoff F Z , Strong LC, Wahl GM: Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 70:937, 1992 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1994 84: 2431-2435 Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16) gene in human leukemias S Ogawa, N Hirano, N Sato, T Takahashi, A Hangaishi, K Tanaka, M Kurokawa, T Tanaka, K Mitani and Y Yazaki Updated information and services can be found at: http://www.bloodjournal.org/content/84/8/2431.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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