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E-mail: [email protected]. *These authors contributed equally ... expression levels of the gravin gene in the CD34+/blast cells of a range of.
short report

Low expression of the putative tumour suppressor gene gravin in chronic myeloid leukaemia, myelodysplastic syndromes and acute myeloid leukaemia

Jacqueline Boultwood,1,* Andrea Pellagatti,1,* Fiona Watkins,1 Lisa J. Campbell,1 Noor Esoof,1 Nicholas C. P. Cross,2 Helen Eagleton,1 Tim J. Littlewood,1 Carrie Fidler1 and James S. Wainscoat1 1

Leukaemia Research Fund Molecular

Haematology Unit, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford, UK, and 2Human Genetics Division, University of Southampton School of Medicine, Southampton, UK

Received 18 March 2004; accepted for publication 18 May 2004

Summary The putative tumour suppressor gene gravin is down-regulated in several solid tumours and is implicated in tumorigenesis. We have evaluated the expression levels of the gravin gene in the CD34+/blast cells of a range of myeloid malignancies as compared with controls using real-time quantitative polymerase chain reaction (PCR). Gravin was markedly down-regulated in 41 of 41 patients with acute myeloid leukaemia (AML), nine of 10 patients with myelodysplastic syndromes (MDS) and 33 of 33 patients with chronic myeloid leukaemia (CML), of whom 24 were in blast crisis (BC). We have shown that gravin is consistently down-regulated in the CD34+/blast cells of myeloid malignancies and may play a role in the molecular pathogenesis of these disorders. Keywords: tumour suppressor gene, acute myeloid leukaemia, myelodysplastic syndromes, chronic myeloid leukaemia, real-time quantitative polymerase chain reaction.

Correspondence: Jacqueline Boultwood, Leukaemia Research Fund Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. E-mail: [email protected] *These authors contributed equally to this work.

The gravin/AKAP12 gene encodes a scaffolding protein and is a member of the cyclic AMP-dependent kinase-anchoring proteins (AKAPs) (Nauert et al, 1997). Gravin and its rodent ortholog SSeCKS regulate cell signalling, cell adhesion, mitogenesis and development, and differentiation through selective binding of signalling proteins such as protein kinase A, protein kinase C, cyclins and the beta(2)-adrenergic receptor (Lin et al, 1995; Gelman, 2002). There is evidence that gravin/SSeCKS may function as a tumour suppressor (Lin & Gelman, 1997; Perou et al, 2000; Gelman, 2002). The SSeCKS gene is downregulated in src- and ras-transformed fibroblasts and re-expression of SSeCKS has been shown to suppress srcinduced oncogenesis (Lin et al, 1995; Lin & Gelman, 1997). The expression of gravin/SSeCKS is strongly suppressed in several solid tumours, including prostate, ovary, lung, thyroid doi:10.1111/j.1365-2141.2004.05067.x

and breast (Perou et al, 2000; Welsh et al, 2001; Xia et al, 2001; Wikman et al, 2002; Wasenius et al, 2003). It has also been shown that gravin/SSeCKS is a potential metastasis inhibitor in prostate cancer (Xia et al, 2001). Given the widespread down-regulation of gravin/SSeCKS in solid tumours and the increasing evidence that it may play an important role in tumorigenesis we sought to investigate the expression levels of this gene in myeloid malignancy.

Materials and methods Patients and samples Ten patients with a myelodysplastic syndrome (MDS) (five refractory anaemia, two refractory anaemia with ringed

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 508–511

Short Report sideroblasts and three refractory anaemia with excess blasts), 41 patients with acute myeloid leukaemia (AML) (entered into the UK MRC adult AML trials) and 33 patients with chronic myeloid leukaemia (CML) were included in the study. Seven patients with CML were in chronic phase (CP), two were in accelerated phase (AP) and 24 in blast crisis (BC), comprising 20 in myeloid blast crisis and four in lymphoid blast crisis. Three additional CP patients were in cytogenetic remission (Goldman & Melo, 2003) following successful treatment with imatinib mesylate (Gleevec; STI-571). This study was approved by the ethics committee (C00.198) and informed consent was obtained. Bone marrow samples were obtained and CD34+ cells isolated from all MDS patients, CML patients in CP and AP, and 10 healthy controls (patients undergoing hip replacement). Mononuclear cells were purified by Histopaque (Sigma-Aldrich, Dorset, UK) density gradient centrifugation, labelled with CD34 MicroBeads and CD34+ cells were isolated using magnetic cell separation columns (MACS) (Miltenyi Biotec Inc., Auburn, CA, USA) according to the manufacturer’s recommendations. CD34+ cell purity was evaluated with FACS and was greater than 80% (with the majority greater than 90%). Peripheral blood or bone marrow samples were obtained from patients with AML and BC CML and the leukaemic blast cells (mononuclear fraction) isolated by Histopaque density gradient centrifugation.

Real-time quantitative PCR analysis Total RNA from each patient and control CD34+/blast cell sample was extracted using TRIZOL (Invitrogen-Life Technologies, Paisley, UK). Two micrograms of total RNA from each sample was reverse-transcribed using RETROscript kit (Ambion Inc., Austin, TX, USA). The generated first-strand cDNA was diluted and used as template for real-time quantitative polymerase chain reaction (PCR) analysis (Heid et al, 1996). The expression level of the ABL gene was used to normalize for differences in input cDNA, with the exception of CML where the b2-microglobulin gene was used (as this disorder has an ABL translocation). Predeveloped TaqMan Assays were used (Assays-on-Demand, Applied Biosystems, Foster City, CA, USA). PCR amplifications occurred in a volume of 25 ll and fluorescence detected using an ABI Prism 5700 Sequence Detection System. Each sample was performed in triplicate. The expression ratio was calculated as 2n, where n is the CT value difference (gravin minus ABL or b2microglobulin) normalized by the average CT difference of the samples from healthy volunteers. Statistically significant differences between disease and control groups were investigated using the Mann–Whitney U-test. Statistical analysis was performed using the StatView 5.0 software package (SAS, Institute, Cary, NC, USA).

Mutation analysis Genomic DNA was extracted from the neutrophil fraction obtained from five patients with MDS, from the blasts of 16

patients with AML and from the peripheral blood of 50 normal healthy individuals. The coding sequence and intron/exon boundaries corresponding to exons 1–5 of the gravin gene (Nauert et al, 1997) (http://www.ensembl.org) and the promoter regions (identified using ‘Promoter Scan’ and ‘Promoter Inspector’ promoter prediction programs) located within exon 1 ()79 to 188 bp) and intron 1 (524–775 bp), respectively, were amplified using PCR and screened for mutations using cycle sequencing.

Results and discussion The AML, CML and MDS are clonal disorders of the haematopoietic stem cell. We have evaluated the expression levels of the putative tumour suppressor gene gravin in the CD34+/blast cell populations of a range of myeloid malignancies including AML, CML and MDS as compared with the CD34+ cell population of a group of healthy individuals using real-time quantitative PCR (Heid et al, 1996). We have shown that the gravin gene was down-regulated in the leukaemic blast cells of 41 of 41 patients with de novo AML, a proportion of which showed absent expression (Table I; Fig 1). Gravin was found to be down-regulated in the CD34+/blast cell populations of 33 of 33 CML patients and at all phases of the disease (Table I; Fig 1). Interestingly, the expression levels of gravin were reduced in AP and BC as compared with CP and thus we suggest that a progressive reduction in the expression levels of gravin may be associated with disease evolution in CML. In the CD34+ cell populations of the three additional CP CML patients in remission following successful treatment with imatinib the gravin expression levels were slightly higher than those observed in normal healthy individuals (Table I; Fig 1), reflecting a restoration of haematopoiesis towards normal. We have also shown that gravin was down-regulated in the CD34+ cell populations of nine of 10 patients with MDS (Table I; Fig 1). These data demonstrate that the gravin gene is consistently down-regulated in myeloid malignancies and in several cases shows absent expression. Table I. Descriptive statistics of disease and control groups. Median value and range of expression of gravin in each disease group and in the control group.

Controls (n ¼ 10) MDS (n ¼ 10) AML (n ¼ 41) CML–CP (n ¼ 7) CML–AP (n ¼ 2) CML–BC (n ¼ 24) CML–CP STI (n ¼ 3)

Median

Range

P-value

0Æ895 0Æ18 0Æ02 0Æ19 0Æ05 0Æ025 3Æ3

0Æ61–2Æ29 0–0Æ74 0–0Æ42 0Æ09–0Æ22 0Æ02–0Æ08 0–0Æ52 1Æ98–4Æ06

– 0Æ0004