Cyclin A1 Is Predominantly Expressed in Hematological Malignancies with Myeloid Differentiation

Home , Cyclin A1

Leukemia (1998) 12, 893–898  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Cyclin A1 is predominantly expressed in hematological malignancies with myeloid differentiation ¨ A Kramer, A Hochhaus, S Saußele, A Reichert, A Willer and R Hehlmann ¨ III. Medizinische Klinik, Klinikum Mannheim der Universitat Heidelberg, Wiesbadener Str. 7–11, 68305 Mannheim, Germany

Cyclin A is a cell cycle regulatory protein that functions in by complexes of CDK4/6 and cyclin D as well as CDK2 and mitotic and S phase control in mammalian cells. However, in cyclin E.12 As a result, transcription factors E2F and CBP/cycA contrast to other G1 phase regulatory proteins, such as cyclin INK4A are released from Rb binding, and transcription of multiple D, retinoblastoma protein and p16 , cyclin A seems not to 5,8,9,13 be commonly involved in tumorigenesis. Recently, a second genes that regulate cell proliferation is induced. Both human cyclin A – cyclin A1 – has been identified. In contrast CBP/cycA as well as E2F have been reported to transactivate to cyclin A which is expressed throughout embryonic develop- cyclin A transcription8,9,14 and CDK2–cyclin A activity is ment and in adult tissue, the expression of cyclin A1 has been 10,11 necessary for the transition from late G1 to S phase. At this reported to be restricted to embryonic and germ line cells. We point the cell enters an irreversible state of the cell cycle. The have confirmed the absence of cyclin A1 mRNA from normal kinase activity of the CDK4/6–cyclin D complex is inhibited peripheral blood leukocytes of seven healthy donors by single INK4A INK4B step reverse transcriptase-polymerase chain reaction (RT- by p16 and p15 , two members of a recently cloned PCR). Furthermore, we have examined the expression of cyclin family of CDK inhibitors.15,16 A1 mRNA in 173 peripheral blood samples of 162 patients with Deregulation of the cell cycle machinery, especially G1/S various hematological malignancies. Cyclin A1 mRNA was phase deregulation, is known to contribute to the develop- detectable in 11 of 11 patients with acute myeloid leukemia, ment of solid tumors as well as hematological malignancies. three of three patients with acute biphenotypic leukemia, eight While cyclins have been implicated as oncogenes when their of eight patients with myelodysplastic syndrome, 59 of 69 17–19 patients with chronic myelogenous leukemia (CML) at diag- expression is deregulated, some CDK inhibitors are nosis, 13 of 15 patients with CML in blastic transformation, 10 known to act as tumor suppressors in a wide range of malig- of 18 patients with chronic lymphocytic leukemia, two of nine nancies.16,18,20 Especially for lymphoid neoplasias several patients with essential thrombocythemia, and only two of 10 alterations of G1/S phase-regulating genes have been reported. patients with acute lymphoblastic leukemia (ALL) with both Overexpression of cyclin D1 which acts as a positive regulator cyclin A1 RT-PCR positive ALL leukemias being undifferentiated relapses. In addition, cyclin A1 mRNA was found in one of G1 phase progression by rearrangement of the bcl-1/PRAD1 of six leukapheresis products, harvested from individuals with- locus on chromosome 11q13 with the immunoglobulin heavy out hematological disorders. Taken together, cyclin A1 is chain gene on chromosome 14q23,19 is the hallmark of expressed in the majority of myeloid and undifferentiated hem- mantle cell lymphoma21 and has also been described in mul- atological malignancies as well as in normal hematopoietic pro- tiple myeloma22 and atypical chronic lymphocytic leuke- genitor cells. We conclude that cyclin A1, a protein potentially mia.23 In addition, homozygous deletions of the p16INK4A and involved in G1/S phase progression of immature cells, might INK4B be necessary for proliferation of early hematopoietic progenitor p16 CDK-inhibitor genes and loss of Rb expression cells and their leukemic counterparts being blocked at that are among the most frequently observed abnormalities in stage of differentiation. lymphoid tumors.24–31 Keywords: cyclin A; hematological malignancies; AML; ALL; cell The human cyclin A gene was originally identified as the cycle; G1 phase site of hepatitis B virus integration in a hepatocellular carcinoma, and this integration and subsequent cyclin A overexpression may contribute to tumorigenesis.32 In fibroblasts Introduction overexpression of cyclin A appears to advance S phase entry and confers anchorage-independent growth.33,34 In hematol- Progression through the cell cycle is controlled by the sequen- ogical malignancies, cyclin A expression correlates with the 1 tial activation of a set of cyclin-dependent kinases (CDK). The percentage of cells in S plus G2-M phase and thus with the activities of the CDK are regulated by association with positive proliferation rate of these disorders.35 However, except for the regulators – cyclins – as well as negative regulators referred single case of hepatocellular carcinoma mentioned above, to as CDK inhibitors. Cyclin gene expression is tightly regu- cyclin A seems, per se, not to be implicated in tumorigen- lated at the transcriptional level in a phase-specific manner. esis.36 Instead, its expression in tumors seems to be a conse- 2 Expression of cyclin D1 is induced by serum growth factors quence of alterations earlier in G1 phase. 3 and is required for progression through early G1 phase. Recently, a second human cyclin A – cyclin A1 – has been 37 Cyclin E is synthesized in late G1 phase and activates CDK2 identified. Cyclin A1 has 48% amino acid sequence identity by complex formation.4 Cyclin E–CDK2 activity is regulated with human cyclin A and 84% identity to the previously by the adhesion status of the cell5,6 and is rate-limiting for the cloned murine cyclin A1. In mice and Xenopus, the 7 transition through late G1. Cyclin A gene transcription, which expression of cyclin A1 is limited to embryonic and germ line 8,9 38,39 is cell cycle- and adhesion-dependent, starts at the G1/S tissue. In analogy, the expression of human cyclin A1 has boundary and is required for S phase entry, DNA replication been reported to be restricted to testis and, to a lesser extent, 10,11 37 and passage through G2. Phosphorylation and concomi- the brain. In contrast, cyclin A is expressed throughout tant inactivation of the retinoblastoma protein (Rb) is induced embryonic development and in adult tissues in Xenopus, mouse and man. The only available information on cyclin A1 expression in neoplasias has been provided by in vitro studies ¨ 37 Correspondence: A Kramer; Fax: 49 621 383 4201 of tumor cell lines. The aim of the present report was there- Received 14 January 1998; accepted 9 March 1998 fore to test for expression of cyclin A1 in tumor cells obtained Cyclin A1 expression in hematological malignancies ¨ A Kramer et al 894 from patients with various hematological malignancies as well Processing of samples as in normal hematopoietic progenitor cells in leukapheresis products (LP) from individuals without hematological malig- Leukocytes from patients and normal individuals were isolated nancies. Since cyclin A1 expression is known to be restricted from 10–20 ml of peripheral blood by red-cell lysis of centri- to immature cells and expression of cyclin A1 mRNA has been fuged buffy-coat preparations. For analysis of LP, 1 ml aliquots demonstrated in acute leukemia cell lines,37 we speculated were used. Total RNA was extracted from the leukocyte prep- that cyclin A1 might be expressed in early hematopoietic pro- arations, 108 cells per cell line, and LP aliquots by ultracentri- genitor cells as well as in hematological malignancies fugation (CsCl gradient) or using the RNeasy kit (Qiagen, derived thereof. Hilden, Germany) according to the manufacturer’s recommendations. The RNA was reverse transcribed into cDNA with MMLV reverse transcriptase using random hexamers as Materials and methods primers.46 Samples

One hundred and eighty peripheral blood samples were RT-PCR analysis obtained from 162 patients with various hematological malignancies and from seven healthy individuals. Eleven patients had acute myeloid leukemia (AML), three patients had acute PCR amplification of cyclin A1 cDNA was done with primers biphenotypic leukemia (BPL) according to the EGIL score sys- CycA1+ (5′-GCCTGGCAAACTATACTGTG-3′) and CycA1− tem,40 eight patients had myelodysplastic syndromes (MDS), (5′-CTCCATGAGGGACACACACA-3′). The primers were 113 patients had chronic myelogenous leukemia (CML), 18 designed to span a region of 197 base pairs (bp) of the cyclin patients had chronic lymphocytic leukemia (CLL), nine A1 cDNA,37 as published. The PCR was set up in 22 ␮l vol- patients had essential thrombocythemia (ET), and 10 patients ume containing 10 mM Tris, pH 8.3, 50 mM KCl, 2.0 mM ␮ had acute lymphoblastic leukemia (ALL). Of the 11 patients MgCl2, 200 M each dATP, dCTP, dGTP and dTTP, 0.5 mM with AML, three had AML/FAB-M1, three had AML/FAB-M2, each 5′ and 3′ primer, 0.5 U Taq polymerase and 2 ␮l cDNA. and one had AML/FAB-M5. For the remaining four patients All PCR experiments included a negative control reaction with AML, no FAB classification was available. Six patients without DNA template. Rigorous precautions were taken to had primary AML, two and three patients had AML secondary avoid the possibility of false positive results, as described else- to MDS and myeloproliferative disease, respectively. Of the where.47 Thermocycling conditions were: one cycle of denat- eight patients with MDS, two had MDS/FAB-RAEB, one had uring at 100°C for 10 s and 96°C for 1 min, annealing at 60°C MDS/FAB-RAEB-T, and four had MDS/FAB-CMML. For the for 3 min, and extension at 72°C for 2 min; 32 cycles of denat- remaining MDS patient no FAB classification was available. uring at 100°C for 10 s and 97°C for 20 s, annealing at 60°C From the 113 patients with CML, 69 peripheral blood samples for 50 s, extension at 78°C for 10 s, and 73°C for 50 s, fol- were obtained at diagnosis, 29 in chronic phase with hydroxy- lowed by a final extension at 73°C for 10 min. Ten microliters urea- and/or interferon ␣-therapy, and 15 in blastic transform- of reaction products were electrophoresed through ethidium ation. Four CML blast crises had lymphoblastic, four myelo- bromide-stained 1.8% agarose gel, visualized and photo- blastic, one monoblastic, and one megakaryoblastic graphed under UV light. cDNA quality was ensured by a sin- differentiation. For five patients with CML in blastic transform- gle step multiplex PCR for BCR-ABL and BCR transcripts46 ation, no lineage determination was available. Of the 10 and/or single step PCR for ABL transcripts.48 patients with ALL, nine had common-ALL and one had T-ALL. From seven ALL patients cDNA was obtained at diagnosis. In the remaining three cases, the cDNAs stemmed from the first, second and third relapse, respectively. Furthermore, six LP Southern blot analysis were examined. One LP was harvested from a patient with stage I breast cancer. Peripheral blood stem cells were col- Ten microliters of PCR product were electrophoresed through lected during recombinant human granulocyte colony-stimul- a 1.8% agarose gel, transferred by Southern blotting to a nylon ating factor (rhG-CSF; 300 ␮g/day, s.c.)-enhanced bone mar- membrane, pre-hybridized at 43°C for 2 h and hybridized at row recovery following a mobilization chemotherapy with 43°C overnight to a 25-mer synthetic oligonucleotide cyclin ifosfamide (10 g/m2) and epirubicin (100 mg/m2). Five LP were A1 probe (5′-TAATTGCTTGCTGAGGTCGATGGGG-3′). The collected from four healthy individuals during stimulation of membrane was washed in 2 × SSC plus 0.1% SDS at room hematopoiesis with rhG-CSF (11–13 ␮g/kg body weight/day). temperature and exposed to radiographic film at −70°C for Informed consent was obtained from all subjects in the study. 6h.

Cell lines cDNA sequencing cDNA from cell lines K562, BV173, KCL22, KYO1, KU812, LAMA, SD-1 and HL6041–43 was kindly provided by Dr Junia V Melo (Imperial College School of Medicine, London, UK). Cyclin A1 RT-PCR products were electrophoresed through a ¨ KG-1a44 cells were donated by Dr Stefan Fruhauf 1.8% agarose gel, the 197 bp band was extracted by electroe- (Medizinische Klinik und Poliklinik V, University of Heidel- lution and sequenced using the Pharmacia T7 sequencing kit berg, Germany). Cell lines K562 and NB445 were purchased (Pharmacia, Erlangen, Germany) according to the manufac- from Deutsche Sammlung von Mikroorganismen und Zellkul- turer’s recommendations. The sequencing products were run turen (Braunschweig, Germany). on a 6.0% polyacrylamide gel and exposed for 24 h. Cyclin A1 expression in hematological malignancies ¨ A Kramer et al 895 Results

Cyclin A1 expression in hematological malignancies

A total of 173 peripheral blood samples from 162 patients with various hematological malignancies were analyzed by single step RT-PCR. Cyclin A1 mRNA could be detected in peripheral blood samples from 11 of 11 (100%) patients with AML, three of three (100%) patients with BPL, eight of eight (100%) patients with MDS, 59 of 69 (86%) patients with BCR- ABL positive CML at diagnosis, 12 of 29 (41%) patients with chronic phase CML and hydroxyurea- and/or interferon ␣- therapy, 13 of 15 (87%) patients with BCR-ABL positive CML in blast crisis, and 10 of 18 (56%) patients with CLL (Table 1). In contrast, only two of nine (22%) peripheral blood samples from patients with ET and two of 10 (20%) samples from patients with ALL contained detectable cyclin A1 mRNA. From the patients with lymphoblastic differentiation of CML blast crisis, three out of four were cyclin A1 RT-PCR positive. Whereas the patient with monoblastic differentiated CML blast crisis was cyclin A1 RT-PCR negative, all patients with mye- Figure 1 (a) Representative examples showing results of reverse loblastic or megakaryoblastic differentiated blast crises gave transcriptase-polymerase chain reactions (RT-PCR) or PCR for cyclin cyclin A1 RT-PCR positive results. The two cyclin A1 RT-PCR A1. Lanes 1–10, cDNA of peripheral blood (PB) from patients with positive ALL peripheral blood samples were obtained from the chronic myelogenous leukemia (CML) at diagnosis, CML in chronic ␣ two common-ALL patients at second and third relapse. Mor- phase during therapy with interferon , CML blast crisis with lymphoblastic differentiation, CML blast crisis with monoblastic differen- phologically, these relapses were judged to be undifferen- + tiation, chronic lymphocytic leukemia, essential thrombocythemia, tiated. In addition, both relapses were CD34 . One had lost myelodysplastic syndrome (CMML), acute lymphoblastic leukemia, CD10 expression. No correlation between cyclin A1 acute biphenotypic leukemia, acute myeloid leukemia; lane 11, gen- expression and the presence of the Philadelphia translocation omic DNA of PB from a healthy donor; lane 12, cDNA of PB from a in the ALL samples was found. healthy donor; lane 13, cDNA of leukapheresis product from a patient Of the 162 patients, one individual with BPL was RT-PCR with stage I breast cancer; lane 14, blank; lane M, 100 bp marker. (b) Southern blot analysis of the PCR products shown in (a). The PCR positive for cyclin A1 at diagnosis but became RT-PCR nega- products were transferred onto a nylon membrane and probed for tive after induction of complete remission by chemotherapy. cyclin A1 cDNA and/or genomic DNA using 5′- Four CML patients achieved complete cytogenetic remission TAATTGCTTGCTGAGGTCGATGGGG-3′ as a probe. after treatment with interferon ␣ and/or hydroxyurea but sub- sequently relapsed. Three of four were cyclin A1 RT-PCR 197 bp cDNA product fragment and the 1300 bp genomic negative during remission but all four were cyclin A1 RT-PCR DNA product (Figure 1b). To further confirm the identity of positive at relapse. In contrast, one CML patient who did not the cDNA-specific PCR product, this band was sequenced. As respond to interferon ␣-therapy was persistently RT-PCR expected, the sequence was identical to that of the published positive for cyclin A1. cyclin A1 cDNA sequence.37 No statistical significant differ- The cyclin A1 RT-PCR product is 197 bp in size (Figure 1a). ence between the white blood cell count of cyclin A1 RT-PCR Amplification of genomic DNA yielded two bands of approxi- positive and negative peripheral blood samples was found. mately 900 bp and 1300 bp; a 197 bp fragment was not amplified from genomic DNA. Southern blot analysis with a specific probe for cyclin A1 demonstrated hybridization to the Cyclin A expression in leukemic cell lines Four of six CML blast crisis cell lines examined expressed Table 1 Results of cyclin A1 RT-PCR in peripheral blood samples cyclin A1 mRNA in a RT-PCR assay (BV173, KCL22, KU812, of patients with various hematological malignancies LAMA; Table 2). The CML blast crisis cell lines K562 (two dif-

Entity, stage No. studied No. (%) Cyclin A1 RT- Table 2 Results of cyclin A1 RT-PCR in leukemic cell lines PCR positive Name Origin Cyclin A1 RT-PCR AML 11 11 (100) BPL 3 3 (100) K562 (41) CML-BC − MDS 8 8 (100) BV173 (41) CML-BC + CML, diagnosis 69 59 (86) KCL22 (41) CML-BC + CML, chronic phase 29 12 (41) KYO1 (41) CML-BC − CML, blast crisis 15 13 (87) KU812 (41) CML-BC + CLL 18 10 (56) LAMA (41) CML-BC + ET 9 2 (22) SD-1 (42) ALL + ALL 10 2 (20) HL60 (43) AML + KG-1a (44) AML + + AML, acute myeloid leukemia; BPL, acute biphenotypic leukemia; NB4 (45) AML MDS, myelodysplastic syndrome; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; ET, essential throm- CML-BC, chronic myelogenous leukemia in blast crisis; AML, acute bocythemia; ALL, acute lymphoblastic leukemia. myeloid leukemia; ALL, acute lymphoblastic leukemia. Cyclin A1 expression in hematological malignancies ¨ A Kramer et al 896 ferent sources) and KYO1 gave negative results. The ALL cell the myeloid cell lines KCL22, KU812, HL60 and NB4. KG- line SD-1 as well as the AML cell lines HL60, KG-1a, and 1a, a undifferentiated, promyeloblastic variant derived from NB4 were cyclin A1 RT-PCR positive. the AML cell line KG-1, was cyclin A1 RT-PCR positive, as has been reported for the parental cell line KG-1.37 In addition, we found cyclin A1 mRNA expression in the CML blast crisis cell Cyclin A1 expression in individuals without lines BV173 and LAMA as well as in the lymphoblastic cell hematological malignancies line SD-1, whereas the CML blast crisis cell lines KYO1 and K562 gave negative results. Since Yang et al.37 reported cyclin Using single step RT-PCR, cyclin A1 could not be detected in A1 mRNA expression in K562 cells detected with the same peripheral blood samples from seven healthy individuals. In method, we repeated the cyclin A1 RT-PCR with K562 cells addition, six LP were examined for cyclin A1 expression from a different source, the result being negative again. (Table 3). Whereas the LP harvested from a patient with stage All of the genes involved in Rb regulation have been shown I breast cancer was cyclin A1 RT-PCR negative, one out of to be frequently altered in malignant disorders.17–31 In con- ﬁve LP collected from healthy individuals gave a positive trast, alterations of cyclin A are rare.36 Instead, in tumors + result. With respect to the CD34 cell count in the LP, the expression of cyclin A in late G1 phase seems to be a conse- cyclin A1 RT-PCR positive LP had the highest CD34+ cell quence of upstream alterations of the Rb/cyclin D/p16 path- count among the LP harvested from healthy individuals. way which results in release of the transcription factors E2F and CBP/cycA from Rb binding and subsequent transcriptional activation of the cyclin A gene. Although the function of Discussion cyclin A1 is still unclear, its expression in embryonic cells in Xenopus39 and in germ line cells in mice and humans37,38 as

In this study, we examined the expression of cyclin A1 in nor- well as the association with CDK2 may point to a G1/S phase mal and malignant hematopoietic cells. First, we have shown regulatory role in germ line cells and early embryos. This con- for the first time that cyclin A1 mRNA is expressed in the tention is supported by the finding that disruption of the majority of myeloid and undifferentiated hematological malig- murine cyclin A gene leads to delayed early embryonic lethal- nancies. While cyclin A1 was expressed in all samples of ity around day 5.5 p.c. which might be explained by comp- patients with AML, BPL and MDS as well as in the majority lementation of cyclin A function by cyclin A1 during early of samples from patients with CML, both at diagnosis and in embryonic development.49 Since cyclin A1 mRNA is absent blastic transformation examined, only two of 10 samples from from mature peripheral blood leukocytes but expressed in a patients with ALL were cyclin A1 RT-PCR positive. Moreover, LP with a high CD34+ cell count from a healthy donor, we both cyclin A1 RT-PCR positive ALL were undifferentiated suggest that cyclin A1 might be necessary for proliferation and relapses. Furthermore, we were able to demonstrate that treat- differentiation of early hematopoietic progenitor cells. Conse- ment of chronic phase CML with hydroxyurea and/or inter- quently, the leukemic counterparts of normal hematopoietic feron ␣ leads to a 50% reduction of the rate of cyclin A1 progenitors may express cyclin A1 because they are arrested RT-PCR positivity. The induction of a complete cytogenetic at that early stage of differentiation. remission of CML yielded in cyclin A1 RT-PCR negativity in Recently, the cyclin A1 promoter was cloned.50 It contains three of four patients. multiple binding sites for the transcription factors c-myb, Sp1 Southern blotting of RT-PCR amplification products with a and E2F. Preliminary results suggest that cyclin A1 transcrip- 32P end-labeled probe internal to the cyclin A1 sequence cor- tion is driven by c-myb.50 While v-myb, the viral counterpart roborated our results. All PCR-positive lanes hybridized to this of c-myb, is a potent inducer of myeloid leukemias,51 c-myb probe, whereas none of the PCR-negative lanes yielded a posi- is known to be involved in the regulation of hematopoiesis. tive hybridization signal. DNA sequencing of the cyclin A1 C-myb expression is mostly restricted to the hematopoietic RT-PCR amplification product from one peripheral blood sam- system and correlates with both the immaturity and the pro- ple of a CML patient at diagnosis yielded a sequence identical liferative state of these cells.52 It cooperatively regulates tran- to the one originally reported.37 scription of myeloid-specific genes and blocks terminal differ- Second, we confirmed the absence of cyclin A1 mRNA entiation of myeloid cells.53,54 Targeted mutagenesis of the c- from mature peripheral blood leukocytes of normal individ- myb gene in mice leads to a reduced number of erythroid and uals. However, since we found cyclin A1 mRNA expression myeloid progenitor cells.55 In view of this, our finding of in a LP with a high CD34+ cell count from a healthy donor, cyclin A1 expression in the majority of myeloid and undiffer- cyclin A1 seems to be expressed in at least a subset of normal entiated hematological malignancies and even in two of nine hematopoietic progenitor cells. peripheral blood samples from patients with ET might be a Third, we confirmed the expression of cyclin A1 mRNA in consequence of c-myb expression in these cells. Furthermore, expression of A-myb, another member of the myb transcription factor family that recognizes the same DNA sequence as Table 3 Results of cyclin A1 RT-PCR in leukapheresis products c-myb, has been shown to be expressed in most B-ALL and in a subset of CLL but not in common-ALL or null-ALL.56 This Diagnosis CD34 positive Cyclin A1 RT-PCR finding might explain the lack of cyclin A1 expression in the cells/␮l majority of common-ALL cases as well as cyclin A1 RT-PCR positivity of 56% of CLL cases investigated by us. Breast cancer, stage I 6372 − Normal individual 886 − Interestingly, the rates of cyclin A1 RT-PCR positivity of Normal individual 992 − CML in blastic transformation and at diagnosis were similar Normal individual 1084 − (87 and 86%, Table 1). Since cyclin A1 seems to be expressed Normal individual 1139 − early in hematopoiesis, continued expression of cyclin A1 Normal individual 2875 + might contribute to a proliferative advantage of a multipotent, BCR-ABL-transformed hematopoietic cell at an early stage of Cyclin A1 expression in hematological malignancies ¨ A Kramer et al ¨ 897 CML. The acquisition of genetic alterations during the course K, Jansen-Durr P, Henglein B. Cell cycle regulation of the cyclin of the disease might confer additional growth advantage to A gene promoter is mediated by a variant E2F site. Proc Natl Acad these cells, associated with disease progression to blast crisis. Sci USA 1995; 92: 11264–11268. 15 Hannon GJ, Beach D. p15INK4B is a potential effector of TGF-␤ Taken together, in this work we describe expression of induced cell cycle arrest. Nature 1994; 371: 257–261. cyclin A1 in the majority of myeloid and undifferentiated 16 Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tav- hematological malignancies as well as in normal hematopo- tigian SV, Stockert E, Day RS III, Johnson BE, Skolnick MH. A cell ietic progenitor cells. Thus, expression of cyclin A1 in these cycle regulator potentially involved in genesis of many tumor malignancies might be a consequence of a differentiation types. Science 1994; 264: 436–440. arrest of the tumor cells at a stage where they normally express 17 Hartwell LH, Kastan MB. Cell cycle control and cancer. Science this protein. As an effector of c-myb, cyclin A1, a protein 1994; 266: 1821–1828. 18 Hunter T, Pines J. Cyclins and cancer II: cyclin D and CDK inhibi- potentially involved in G1/S phase progression of immature tors come of age. 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