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Molecular Analysis of Cyclin-Dependent Kinase Inhibitors

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Molecular Analysis of Cyclin-Dependent Kinase Inhibitors Leukemia (1997) 11, 1696–1699 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Molecular analysis of cyclin-dependent kinase inhibitors in human leukemias S Hayette1, X Thomas2, Y Bertrand3, I Tigaud1,4, M Callanan1, A Thiebaut1, C Charrin1, E Archimbaud1,4, J-P Magaud1,4 and R Rimokh4 1Laboratoire d’He´matologie et de Cytoge´ne´tique, Hoˆ pital Edouard Herriot; 2Service d’He´matologie, Hoˆpital Edouard Herriot; 3Service d’He´matologie, Hoˆpital Debrousse; and 4INSERM Unite´ 453, Centre Le´on Be´rard, Lyon, France Recurrent anomalies of the short arm of chromosome 9, includ- in 6–40% of cases (0–83% in T cell ALL and 6–22% in B ing interstitial deletions and translocations, have often been cell ALL).17 In other hematologic malignancies: acute myeloid described. Recently two cyclin-dependent kinase inhibitors, known as P16 (INK4A/MTS1) and P15 (INK4B/MTS2), which map leukemia (AML), chronic lymphocytic leukemia (CLL), the to 9p21, have been found deleted in a wide range of tumors incidence of P16/P15 inactivation is low, probably less than and particularly in leukemic cells. We report here Southern blot 1–2%. Concerning others CDIs, P27 and P21 have been rarely analyses of cyclin-dependent kinase inhibitors (P16, P15, P21, implicated in human malignancies.18–20 and P27) status in primary tumoral cells of 121 patients with We report here Southern blot analyses of P16, P15, P21 and acute lymphoblastic leukemias, 85 patients with acute myeloid P27 status in primary tumoral cells of 121 patients with ALL, leukemias and 42 patients with B-chronic lymphocytic leuke- mias. P16 inactivation was found in 25 of 38 T-ALLs and in 28 85 patients with AML and 42 patients with B-CLL. We show of 83 B-lineage ALLs. In eight cases (three T-ALLs and five B- that P16 was inactivated in 25 of 38 T-ALLS and in 28 of 83 lineage ALLs), one or both alleles of P16 locus were B-lineage ALLs. With a few exceptions (seven cases) both P16 rearranged. In these cases, breakpoints occurred within the and P15 were deleted in the leukemias analyzed. P21 was two major breakpoints cluster regions previously described in never found deleted and monoallelic deletion of P27 was T-ALLs. Homozygous P16 deletions were observed in two of 85 present in four of 85 AML tested. These data confirm that AMLs but in none of the 42 B-CLL cases tested. Our results suggest that P16 inactivation are the most frequent event deletional inactivation of P16/P15 locus is the most frequent observed in ALL (44%), are quite rare in AML (,2%) and seem event in ALLs and especially in T-ALLs. to be absent in CLL. Search for P27 and P21 deletion was nega- tive in B/T-lineage ALLs and monoallelic deletions of P27 were found in four AML cases (5%). Keywords: INK4A; CDK inhibitors; leukemia; tumor suppressor Materials and methods gene Cells samples Introduction Bone marrow (BM) or peripheral blood (PB) samples were obtained from newly diagnosed ALL (121 cases), AML (85 Cyclin-dependent-kinases (CDKs) play a key role in the con- cases) and B-CLL (42 cases). Diagnosis was established 1,2 trol of the cell cycle in eukaryotes. Their sequential acti- according to the standard French–American–British (FAB) cri- vation and the consequent phosphorylation of critical sub- teria. Cell suspension immunophenotypic studies were perfor- strates stimulate progression through the cell cycle. In med using a panel of lineage-specific monoclonal antibodies. particular, G1 phase is positively regulated by CDK4/6-cyclin In all samples, tumor cells represented more than 80% of the D and CDK2-cyclin E complexes. Based upon sequence hom- nucleated cells. ology, two classes of CDK activity inhibitors (CDIs) have been identified these last years: one class includes P15, P16, P18 and P19 genes, P21 and P27 form another class. P16 (INK4A/MTS1) and P15 (INK4B/MTS2) inhibit CDK4/CDK6 Cytogenetic studies activity,3,4 P21 and P27 inhibit CDK2/CDK4 activity.5–7 With regard to their function, CDIs can be considered as tumor sup- Tumoral cells isolated from BM or PB were analyzed using R pressor genes.8 The organization of the INK4A and INKB locus banding after 24- or 48-h unstimulated cultures. The karyo- is represented in Figure 1. The INK4A locus contains four types were established according to the International System exons that could give rise to two transcripts and two unrelated for Human Cytogenetic Nomenclature.21 proteins.9–11 P16 is encoded by exons E1a, E2 and E3,12 and P19ARF by a different 5′ exon (E1b) plus the same exons E2 and E3.13 P16 and P15 locus which lie closely on chromo- some 9p21, are inactivated by deletion or less frequently by Table 1 Incidence of P16 inactivation in acute lymphoblastic point mutations in a large number of malignant tumors.14–16 In leukemias hematologic malignancies, somewhat controversial data have been recently published. In primary acute lymphoblastic leu- ALL p16-R No. kemia (ALL), biallelic deletions of P16/P15 have been reported Children T 78% 47% 18 B 33% 40 Adults T 55% 41% 20 B 35% 43 Total 121 Correspondence: R Rimokh, INSERM Unite´ 453, Centre Le´on Be´rard, 28 rue Lae¨nnec, 69373 Lyon Cedex 08, France P16-R, P16 inactivation (deletions or rearrangements); No., No. Received 17 February 1997; accepted 25 June 1997 reported cases. Status of CDK inhibitors in leukemias S Hayette et al 1697 Figure 1 Restriction map of the INK4A/INK4B locus. Transcriptional sense from centromere to telomere is indicated by arrows. Exons are indicated by black boxes. Probes used in Southern blot experiments are shown. H, HindIII. Southern blot analysis High molecular weight DNA was extracted from mononuclear cells obtained from PB or BM samples after isolation by Ficoll– Hypaque centrifugation. After digestion with XbaI, HindIII and SacI, DNA fragments were separated by electrophoresis in 0.8% agarose gel according to standard procedure. DNA loading was verified by visual inspection of ethidium-stained gels and by hybridization of stripped blots to chromosome 19p13 (R13/19) and 3q27 (LAZ3/BCL6) probes (gift from C Bastard, Rouen, France). These probes were chosen because chromosome 3 and 19 are exceptionally involved in numeri- cal anomalies frequently encountered in leukemias and especially in ALL. P15, P16 and P27 probes used in this study consist of poly- merase chain reaction (PCR)-amplified human DNA segments encompassing nucleotides 6 to 142 on the P16 exon 1a sequence (5′ P16 probe, Figure 1), nucleotides 702 to 898 on the P16 exon 3 sequence (3′ P16 probe),3 nucleotides 35 to 218 on the P15 sequence (P15 probe)4 and nucleotides 216 to 396 on the P27 sequence.7 There is no sequence homology Figure 2 Representative Southern blot experiment showing some between the amplified P15 and P16 DNA segments used as ALL cases with P16/P15 deletions. The filter, containing HindIII- probes in this study. All PCR products were cloned in the digested tumor DNAs was hybridized with a mixture of 5′ P16,3′ Bluescript II SK (+) vector and sequenced before use. The P21 P16, R13/19 and P15 probes. The samples 2 to 5 and 7 to 9 showed cDNA probe was a gift from P Savatier (ENS, Lyon, France), homozygous P16 deletion with (or without) bi- or monoallelic P15 the TAL-1 gene probe (pG26.5) was kindly provided by O deletion. C, control. Bernard (IGM, Paris, France) and consists of a 5 kb DNA frag- ment. Probes a-32P-labelling, prehybridization, hybridization and washing were carried out as previously described.22 Monoallelic P16 deletion has only been observed in three cases. In some monoallelic cases, a frameshift mutation was found on the remaining allele.27,31 This kind of P16 alteration Results and discussion is likely to be very rare in ALL28 and has not been studied in this report. The 121 ALLs studied by Southern blot included 58 children In AML, homozygous P16 deletions were observed in only aged <15 years and 63 adults aged .15 years, 38 T-ALLs and two of 85 AML cases (,2%), one AML-M5 and one AML-M6. 83 B-lineage ALLs. Examples of homozygous P16 deletions Similar results have already been reported by other authors.32 and P16 rearranged cases analyzed by Southern blot are None of the 42 B-CLL tested demonstrated anomaly of the shown in Figures 2 and 3, respectively. Data are summarized P16/P15 locus (data not shown) confirming the rarity of this in Table 1. P16 inactivation by deletion and/or rearrange- genetic alteration in this pathology.33 Finally, search for P27 ments were found in 25 of 38 T-ALLs (66%) and in 28 of 83 B- and P21 deletion was negative in B/T-ALL cases and mono- lineage ALLs (34%). There is no significant difference between allelic deletions of P27 were found in four (5%) AML cases adults and children in this study. Homozygous P16 deletions (one AML-M4, one AML-M6, one AML-M7 and one AML- are significantly more frequent in T-ALLs than in B-lineage M8) (data not shown). In three of these cases, anomalies of ALLs (P , 0.001). This distribution may reflect a different chromosome 12p were observed: del(12p) in two cases and tumor suppressor activity of the P16 gene with respect to the monosomy 12 in one case. So, P16 inactivation is the most lymphoid lineages. The frequency of P16 inactivation pre- prevalent genetic defect found in ALLs. viously reported in T-ALLs varies from 0%23 to 83–95%.24,25 Out of 40 ALLs with P16 defect, P15 biallelic deletions A similar incidence to the one we report here was observed were observed in 22 cases (55%) and monoallelic deletions in 22 German childhood T-ALLs (77%)26 and in 59 French T- in 11 cases (28%).
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