Int. J. Cancer: 50,500-504 (1992) 0 1992 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union InternationaleContre le Cancer

ENHANCED INSULIN-RECEPTOR TYROSINE KINASE ACTIVITY ASSOCIATED WITH CHROMOSOMAL TRANSLOCATION (1;19) IN A PRE-B-CELL LEUKEMIA LINE Julie D. NEWMA".3,Leonard C. HARRISON',Glenn S. ECKARDT' and Ian JACK' 'Bumet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050; and 'Virus Laboratories, Royal Children's Hospital, Parkville, Kctoria 3052, Australia. The gene for the insulin receptor has been assigned to chromosome I 9 near the breakpoint of the translocation t( I ;19) which occurs in 25% of pre-B-cell leukemias. Insulin receptors in a pre-B-cell leukemia cell line (ACV) with t( I ;19) were found to have 2-fold higher affinity for insulin, 5-fold higher basal and insulin-stimulated fl sub-unit autophosphorylation, and 2-fold higher basal and 4-fold higher insulin-stimulated fl sub-unit kinase activity on the synthetic peptide poly(Glu,Tyr), compared to receptors in a B-cell line (ADD) with normal karyotype from the same patient. ACV cells had a novel 13-kb receptor mRNA species and expressed a DNA polymorphism localized to the tyrosine kinase domain of the receptor gene. These findings suggest that t(1;19) in the ACV cell may result in rearrangement of the insulin receptor gene and translation of a receptor with enhanced tyrosine kinase activity.

Pre-B-cell leukemia is an acute lymphoblastic leukemia involving cells of the B-cell lineage which produce cytoplasmic but not cell-surface immunoglobulin heavy chains (Vogler et al., 1978). A specific chromosome rearrangement, the translocation t(1;19)(q23;p13.3), occurs in 25% of pre-B-cell leukemias (Carroll et al., 1984). Translocations may result in activation of proto-oncogenes situated near the breakpoints on either chromosome (reviewed by Cory, 1986); specific examples include activation of c-myc with t(8;14) in Burkitt lymphoma (Nishikura et al., 1983), bcl with t(14;18) in follicular lymphoma (Tsujimoto et al., 1984) and c-abl with t(9;22) in chronic myelogenous leukemia (Shtivelman et al., 1985). The human insulin receptor, whose tyrosine kinase domain has sequence homology with other tyrosine-specific protein kinases encoded by the src family of oncogenes (Ullrich et al., 1985; Ebina et al., 1985; Neckameyer and Wang, 1985), is encoded by a gene assigned near the breakpoint of t(l;19) in pre-B-cell leukemia (Yang-Feng et al., 1985). To obtain evidence that the insulin receptor might be a candidate proto-oncogene activated by t( l;l9), we have examined the structure, function and genetic expression of the receptor in a cell line (ACV) with t( 1;19) established from the bone marrow of a child with pre-B-cell leukemia (Jack et al., 1986). A cell line (ADD) of normal karyotype established by transformation of B cells from the same patient served as the control.

MATERIAL AND METHODS

Serum containing antibodies to the insulin receptor (B-2) was obtained from a patient with the type-B syndrome of severe insulin resistance and acanthosis nigricans (Kahn and Harrison, 1981). Na['251], [a-32P]adenosine 5'-triphosphate (ATP) and [Y-~'P]ATP were supplied by Amersham (Ailesbury UK). The synthetic co-polymer poly(Glu,Tyr 4:l) was obtained from Sigma (St. Louis, MO). [lzI]-labelled insulin (12G-140 pCi/pg) was prepared by a modification of the chloramine-T method (Roth, 1975). Plasmids containing cDNAs spanning the entire reading frame of the human insulin receptor (phIR 18-2 and phIR 13-1) were kindly provided by Dr. G.I. Bell, Howard Hughes Medical Institute, Chicago, IL. The cDNA

inserts were purified by standard methods (Maniatis et al., 1982). Cell culture The ACV cell line was established from a bone-marrow aspirate from a child with acute B-lymphoblastic leukemia (Jack et al., 1986). It lacks Epstein-Barr virus (EBV) nuclear antigen and exhibits the chromosome translocation t( 1;19)(q23; p13.3) and trisorny 8. The ADD cell line of normal karyotype was established by EBV-transformation of peripheral B cells from the same patient. Cells were grown in RPMI-1640 medium supplemented with heat-inactivated FCS. Insulin binding studies Cells at a late log-stationary stage of growth were washed twice with phosphate-buffered saline (PBS) and resuspended in lymphocyte binding buffer (50 mM HEPES, 120 mM NaCI, 1 mM MgSO,, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, 1% BSA, 45 U/ml bacitracin, PH 7.6). Insulin binding to 2 X lo6cells was measured at steady-state (90 min, 1 5 T ) in a standard competition assay in which the binding of a fixed tracer amount of 1Z51-labelled insulin (20,000 cpm; 0.04 nM) was measured in the presence of increasing amounts of unlabelled insulin. Following incubation, cells were washed once with ice-cold lymphocyte binding buffer and cell pellets were counted in a 1260 LKB Multigamma counter (Bromma, Sweden). Cell-suface (I2'I) labelling and receptor immunoprecipitation Cells were washed 3 times in PBS containing 20 mM glucose, resuspended in PBS/glucose (5 x lo7 cells/ml) and labelled with Na['Z'I] (1 mCi) by lactoperoxidase (150 pg/ml) and glucose oxidase (1 U/ml) (Markwell and Fox, 1978) for 10 min at room temperature. Cells were then washed 3 times with PBS/l mM KI, pelleted and solubilized in 0.1 M sodium phosphate, PH 7.4, 1% Triton X-100,2 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM EDTA, 1,000 U/ml aprotinin, 100 U/ml bacitracin for 60 min at 4°C. After centrifugation at 100,000 g at 4°C for 1 hr to remove insoluble material, glycoproteins from solubilized cells were purified by wheatgerm-lectin/agarose chromatography (Hedo et al., 1981). Solubilized receptors were precipitated from glycoprotein fractions with either control serum (1:250 final dilution) or anti-receptor serum B-2 (1500 final dilution). Samples were mixed with a 50-fold excess of Staphylococcus protein A for 1 hr at 4"C, washed and solubilized at 60°C for 15 min in 2% SDS sample buffer/100 mM dithiothreitol (DTT), and then analyzed on 7.5% acrylamide SDS-PAGE gels under reducing conditions and autoradiographed. ~~

~

'Present address, to which correspondence and reprint requests should be sent: Department of Biochemistry, Monash Medical Centre, Clayton Campus, Clayton, Victoria, 3168, Australia. Received: July 29, 1991.

ENHANCED INSULIN-RECEPTOR TYROSINE KINASE ACTIVITY

Autophosphorylationof the 6-sub-unitof the insulin receptor Cells were solubilized for 60 rnin at 4°C in 50 mM HEPES, PH 7.4, 1% Triton X-100, 10 pg/ml leupeptin, 1,000 U/ml aprotinin, 4 mM PMSF and 2 mM EDTA. Insoluble material was removed by centrifugation at 100,000 g for 60 rnin at 4°C and glycoproteins were prepared by wheat-germ-lectinl agarose chromatography. Equal amounts of receptors (13 pmol, determined by Scatchard analysis) of ACV and ADD glycoproteins were incubated in a final volume of 40 p,l with varying concentrations of insulin (0-100 nM) for 1 h at 4°C. Phosphorylation was then initiated by the addition of MgCl,, MnCI, and [Y-~~PIATP (final concentrations 12 mM, 2 mM, 50 p~ respectively). After 10 min at 4”C, the reaction was stopped by the addition of 10% SDS sample buffer containing ATP and D n in final concentrations of 500 pM and 100 mM, respectively. Samples were immediately boiled for 3 min and labelled proteins were identified by autoradiography of SDS-PAGE acrylamide gels. The extent of phosphorylation of the p subunit was quantified by excising the M, 95,000 p sub-unit phosphoproteins from the gels and measuring the radioactivity by scintillation counting. In addition, after phosphorylation had been performed, reactions were stopped by adding 10 vol of ice-cold 50 mM HEPES PH 7.4 containing 0.1% Triton X-100,lO mM sodium fluoride, 4 mM EDTA and 1mM sodium vanadate. Receptors were then precipitated with anti-receptor serum B-2 (1500 final dilution) and analysed by SDS-PAGE and autoradiography. In some experiments, wheat-germ-lectin-purified glycoproteins were incubated for 30 rnin at 22°C in the presence or absence of 1 p~ insulin before carrying out phosphorylation of the synthetic co-polymer poly(Glu,Tyr 4:l) as described by Zick et a1 (1985).

501

Richmond, CA) by blotting in 1.5 M NaCI, 5 mM NaOH. The filter was probed using a mixture of nick-translated, ”P-labelled cDNA fragments of 1 kb and 4 kb spanning the entire reading frame of the human insulin receptor. To control for RNA loading, each lane of the ethidium-bromide-stained gels was scanned using densitometry.

Southem-blotanalysis Genomic DNA was extracted from ACV and ADD cells by digestion overnight at 37°C with proteinase K (200 pg/ml) in 10 mM Tris PH 7.6, 10 mM EDTA, 50 mM NaCI, 0.2% SDS, followed by extraction with phenol/chloroform/isoamyl alcohol. The DNA was precipitated with 2 vol of isopropanol, dissolved in 10 mM Tris PH 7.6,l mM EDTA and stored at 4°C. DNA samples (7 pg) were digested for 2 hr with HincII and then overnight with StuI or XbaI, fractionated in 0.8% agarose gels, soaked in 0.15 M HCl for 10 rnin and then transferred on to a nylon membrane (Biotrace, Gelman Sciences Inc., Ann Arbor, MI) in 0.4 M NaOH. Filters were probed by means of a HincII fragment of insulin receptor cDNA (bases 3187-3871). RESULTS

The number of insulin receptors on ACV cells (17,000 c 2,00O/cell; mean -r- SEM)determined by Scatchard analysis of competitive ‘zI-labelled insulin-binding data was half that on ADD cells (38,000 c 3,00O/cell), but the affinity of receptors

RNA isolation and Northem-blot analysis Poly(A)’ RNA was prepared from proteinase-K-digested cells using oligo (dT)-cellulose chromatography. RNA was fractionated on a denaturing formaldehyde, 0.8% agarose gel and transferred to a nylon membrane (Zetaprobe, Biorad,

Scatchard Plot

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Insulin bound (nMf FIGURE1- Insulin-binding studies. Binding of ‘251-labelledto intact ACV and ADD cells ( 2 X lo6cells/tube) in the presence of increasing concentrations of unlabelled insulin was measured at steady state (90 min, 15T) in a standard competition assay and expressed in the form of Scatchard plots. This Figure is representative of 4 separate experiments.

FIGURE 2 - Identification of insulin-receptor sub-units by cellsurface labelling and immunoprecipitation. ACV and ADD cells (lo8)were surface-labelled with Na’”I using lactoperoxidase and glucose oxidase and solubilized in 1% Triton X-100 phosphate buffer. Receptors eluted from a wheat-germ-lectin column were precipitated with anti-receptor serum B-2 (lanes 2 and 4) or control serum (lanes 1 and 3) and analyzed by SDS-PAGE and autoradiography,as described in “Material and Methods”.

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NEWMAN ET AL.

on ACV cells (K, = 0.6 nM, where K, = concentration of unlabelled insulin required for half-maximal displacement of ‘=I-insulin)was twice that on ADD cells (K,, = 1.2 nM) (Fig. 1). Cell-surface labelling with ‘*’I and precipitation with antireceptor serum B-2 revealed that insulin receptors on ACV

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cells had a gross sub-unit composition which was similar, overall, to that of insulin receptors on ADD cells and other lymphoblastoid cell lines, with an (Y sub-unit of molecular weight (MW) 130 kDa and a p sub-unit of MW 95 kDa (Fig. 2). The insulin receptor p sub-unit contains an insulin-sensitive tyrosine kinase domain which is auto-phosphorylated, can phosphorylate other substrates and is postulated to play a key role in signal transduction (Gammeltoft and Van Obberghen, 1986). Basal and insulin-stimulated phosphorylation of the p sub-unit in ACV cells were each 5 times higher than in ADD cells (p < 0.05) (Fig. 3a, 6). Other phosphoproteins of approx. 180 and 64 kDa were also detected in ACV cells but their abundance was not influenced by insulin. Immunoprecipitation using anti-receptor serum confirmed the identity of the phosphorylated p sub-unit in ACV cells and revealed the 210-kDa pro-receptor (Hedo et al., 1983) which was also phosphorylated in a dose-dependent manner by insulin (results not shown). In the presence of equal amounts of receptor binding sites (13 pmol, estimated by Scatchard analysis) partially purified from ACV and ADD cells by wheat-germ-lectin chromatography, insulin stimulated the phosphorylation of the synthetic peptide poly(Glu,Tyr) by 6-fold and 3-fold, respectively (Fig. 4). Basal phosphorylation was 2-fold higher and insulinstimulated phosphorylation 4-fold higher in ACV cells than in ADD cells. Lymphoblastoid cells express up to 6 insulin receptor mRNA transcripts varying in size between 2 and 11 kb (Goldstein et aZ., 1987). Northern-blot hybridization revealed that the ACV cell expressed an additional, larger mRNA species of 13 kb (Fig. 5). This larger species was not detected in ADD cells even when Northern blots were carried out using twice the amount of ADD mRNA compared to ACV mRNA (results not shown). In addition, the relative amounts of the mRNA species varied between the cell lines, the amount of 11-kb species being greater in ACV cells and the amount of 7.5-kb species being greater in ADD cells (Fig. 5). Southern blots probed with 2 insulin-receptor cDNAs spanning the full coding sequence revealed no differences in band patterns between ACV and ADD cells or amplification of the receptor gene when DNA was digested with AvaI, BamHI, BglII, BstNI, EcoRI, HaeIII, HindIII, KpnI, MspI, PstI, PVuII, TaqI or XbaI. However, there appeared to be a slight differ-

n L

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600.

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P ACV (+insulin)

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b FIGURE 3 - Autophosphorylation of the p sub-unit of the insulin receptor. Glycoproteins were purified by wheat-germ-lectinagarose chromatography of solubilized cells, and equal amounts of ACV and ADD receptor-bindingsites (13 pmoles, determined by Scatchard analysis) were incubated with increasing concentrations of insulin (0-100 nM) in a final volume of 40 pl for 1 hr at 4°C. Phosphorylation and SDS-PAGE were carried out as described in the “Material and Methods” ( a ) . The insulin-receptor p sub-unit phosphoprotein 95-kDa bands were excised from the gels and quantitated by scintillation counting (b).

2

/ ADD (+insulin)

20,000

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3

I

6 Time (minutes)

ACV (-insulin) ADD (-insulin)

I

9

FIGURE 4 - Phosphorylation of synthetic poly(Glu,Tyr) by partially purified insulin receptor preparations from ACV and ADD cells. Wheat-germ-lectin-purified glycoproteins from ACV and ADD cells, containing equal amounts (13 pmoles) of receptorbinding sites as determined by Scatchard analysis, were incubated in a volume of 40 pl for 30 min at 22°C in the presence or absence of 1 p~ insulin. Phosphorylation of poly(Glu,Tyr) was carried out as described by Zick et al. (1985).

503

ENHANCED INSULIN-RECEPTOR TYROSINE KINASE ACTIVITY

FIGURE 5 - Northern analysis of RNA transcripts in ACV and ADD cells. Poly(A)'RNA (10 pg) isolated from ACV and ADD cells by proteinase-K digestion and oligo (dT)-cellulose chromatography was fractionated on a denaturing formaldehyde 0.8% agarose gel and transferred to a nylon membrane (Zetaprobe) by blotting in 1.5 M NaC1, 5 mM NaOH. The filter was hybridized using a mixture of 32Pnick-translated cDNA fragments of 1 kb and 4 kb spanning the entire reading frame of the human insulin receptor. The pattern shown was reproduced in another 2 experiments. The ACV and ADD lanes contained equivalent amounts of RNA as measured by densitometry of ethidium-bromide-stained gels.

ence in size of the largest fragment generated by HincII digestion of ACV and ADD DNA. D N A was therefore digested with combinations of HincII and other restriction enzymes. A HincII fragment of receptor cDNA (bases 31873871) (Ullrich et a i , 1985), which spans most of the genomic region between exons 17 and 22 encoding the tyrosine kinase domain (Seino et al., 1989), hybridized to extra bands of 2.5 kb and 1.5 kb in a HincII/StuI, but not HincII digest of ACV cell D N A (Fig. 6a). This HincII probe contains 2 StuI sites at positions 3233 and 3726 in exons 17 and 21 (Fig. 6b). However, as the HincII and StuI restriction sites in the intervening (intron) sequences have not been reported, exact mapping of the StuI polymorphism in the ACV D N A is not possible at this time. DISCUSSION

The insulin receptor in pre-B-cell leukemic ACV cells with t(1;19) is abnormal in several respects compared to ADD cells from the same patient with normal karyotype, o r indeed unrelated lymphoblastoid cells. It has a higher affinity and higher basal and insulin-stimulated kinase activity. This increased activity of the receptor kinase in the ACV cell in

I I 3726 3871 ~ -1 1 , - - ~1 -1 ~ I - l u t 51 3187 3233

3025

3270

3381

3541

3611

3806

b FIGURE6-Southern analysis of ACV- and ADD-cell DNA. Genomic DNA (7 pg) from ACV and ADD cells was digested for 2 hr with HincII and then overnight with either StuI or XbaI, fractionated on 0.8% agarose gels, soaked in 0.15 M HC1 for 10 min and then transferred on to a nylon membrane (Biotrace) in 0.4 M NaOH. (a) Hybridization using a HincII fragment of receptor cDNA (bases 3187-3871). (b) Region of the human insulin receptor gene spanned by the HincII probe used in (a) Exons (numbered) are indicated as solid boxes and introns (not to scale) as dashed lines. response to insulin is not accounted for solely by the increase in receptor affinity and appears to be due to an increase in intrinsic activity. It could be similar to the case of the c-abl protein in the K562 leukemia cell line with t(9;22), in which a change in conformation is postulated to unmask the tyrosine kinase (Konopka et al., 1984). ACV cells also have a novel high-MW insulin-receptor mRNA species. Novel transcripts have been detected in other leukemias and lymphomas with chromosomal translocations. For example, the c-abl proto-oncogene from chromosome 9 is linked to the bcr gene on chromosome 22 in chronic myelogenous leukemia (CML) (Groffen et al., 1984; Heisterkamp et at., 1985) and CML cells have a predominant, novel, larger abl-related transcript that represents a fused c-abllbcr transcript (Shtivelman et al., 1985). In follicular B-cell lymphomas

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with t(14;18) the bcl-2 gene is linked to the immunoglobulin heavy-chain (IgH) locus, resulting in the formation of chimeric bcl-IgH transcripts (Tsujimoto et al., 1984; Hua et al., 1988). The larger 13-kb receptor mRNA species in the ACV cell might, therefore, arise from fusion of the receptor gene with an unknown gene at the breakpoint on chromosome 1. Alternatively, it may represent unspliced nuclear RNA, as a 12-kb receptor mRNA species has been demonstrated in nuclear RNA preparations from rat hepatoma Fao cells (Goldstein et al., 1987). We have also shown that ACV cells expressed a DNA polymorphism localized to the tyrosine kinase domain of the insulin receptor. Southern blot analyses using other restriction enzymes did not however reveal any further polymorphisms indicating that the ACV cell may have a relatively small translocated insertion. This rearrangement of the ACV receptor gene may be linked to the enhanced tyrosine kinase activity in the ACV cell. Unfortunately, further mapping of this polymorphism will not be possible until the genomic map of the insulin receptor is available. Kaplan rt al. (1989) have reported over-expression of the insulin receptor in another pre-B-cell leukemia line with t(1;19), but the comparison was made with an unrelated

pre-B-cell line without t( 1;19) which had a low concentration of receptors. These workers did not report evidence of rearrangement of the receptor gene or an increase in receptor tyrosine kinase activity. Mellentin et al. (1989) have demonstrated that the gene E2A coding for the immunoglobulin enhancer binding proteins E12/E47 maps to the breakpoint site of the t(1;19) in acute lymphoblastic leukemias. Their results suggest that alteration of this transcription factor gene has a direct oncogenic role. However, this does not exclude a role of this kind for other genes close to the breakpoint. Our findings suggest that t(1;19) in the ACV cell may have resulted in a rearrangement of the insulin-receptor gene and translation of a unique receptor whose enhanced tyrosine kinase activity could contribute to the neoplastic state of the ACV cell. ACKNOWLEDGEMENTS

The authors were supported by the National Health and Medical Research Foundation of Australia. The authors gratefully acknowledge the secretarial assistance of Mrs. M. Thompson and thank Drs. L. Ellis, J. Adams and D. Cram for helpful discussions regarding this report.

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