Hepatocyte growth factor receptor (HGFR) gene is overexpressed in some cases of human leukemia and lymphoma

Leukemia Research Vol. 18, No. 1, pp. 7-16, 1994. Printed in Great Britain.

0145-2126/94 $6.00 + .00 (~) 1993 Pergamon Press Ltd

THE Met/HEPATOCYTE GROWTH FACTOR RECEPTOR (HGFR) GENE IS OVEREXPRESSED IN SOME CASES OF HUMAN LEUKEMIA AND LYMPHOMA MANFRED J(}CKER,*t ANDREASGONTHER,~: GEORG GRADL,§ CHRISTAFONATSCH,§ GERHARD KRUEGER,~ VOLKERDIEHLt and HANS TESCHt tMedizinische Klinik I, Universit~t K61n, F.R.G.; ~:Immunpathologisches labor, Institut ffir Pathologie, Universit~it K61n, F.R.G.; and §Institut ftir Humangenetik, Medizinische Universit~it zu Liibeck, F.R.G. (Received 13 October 1992. Revision accepted 7 August 1993) Abstract--The proto-oncogene c-met encodes a heterodimeric (~, fl) tyrosine kinase receptor which binds the hepatocyte growth factor (HGF). Recently, overexpression of the Met/HGF receptor gene has been detected in fresh samples of carcinomas and in epithelial tumor cell lines but not in cell lines derived from human leukemia and lymphoma. Our analysis of 50 primary samples of human leukemia and lymphoma and 23 hematopoietic cell lines revealed expression of mRNA and protein of the met/HGF receptor in 6 out of the 73 hematopoietic tumor samples analyzed. Four of the six samples positive for expression of the Met/HGF receptor gene were derived from patients with Hodgkin's disease. In addition, in one Burkitt's lymphoma cell line and in one acute myeloid leukemia (AML), expression of the Met/HGF receptor gene was detected. In normal unstimulated lymphocytes, granulocytes or monocytes we did not find expression of the Met/HGF receptor gene. Upon stimulation with the phorbol ester TPA we detected a weak expression of Met/HGF receptor specific transcripts of 9.0 kb in peripheral blood mononuclear cells of a healthy donor. Cytogenetic analyses of three of the four cell lines which express the Met/HGF receptor gene revealed structural or numerical abnormalities of the long arm of chromosome 7, where the Met/HGFR gene is located, in each of the three cell lines analyzed. In one of these cell lines (L540) the Met/HGFR gene is translocated to a marker chromosome. Southern blot and pulsed field gel electrophoresis experiments did not show any rearrangement in a region of 600 kb around the Met/HGF receptor gene excluding an activation of Met/HGFR by a TPR/Met oncogenic rearrangement as described for MNNG-HOS cells and for some gastric tumors. Our data indicate that the Met/HGFR gene is deregulated in a few cases of human leukemia, Burkitt's lymphoma and Hodgkin's disease possibly by chromosomal rearrangements resulting in an overexpression of the normal Met/HGF receptor mRNA and protein without formation of a hybrid gene. Key words: Oncogene expression, c-met, hepatocyte growth factor receptor, human leukemia, non-Hodgkin's lymphoma, Hodgkin's disease.

Introduction THE PROTO-ONCOGENEc-met encodes a tyrosine kin-

ase receptor with an apparent molecular weight of Abbreviations: TPA, 12-O-tetradecanoylphorbol 13acetate; kb, kiiobases, kDa, kiloDalton; PBMC, peripheral blood mononuclear cells; PFGE, pulsed field gel electrophoresis; HGFR, hepatocyte growth factor receptor; IL, Interleukin; HD, Hodgkin's disease; Ig, immunoglobulin; TCR, T-cell receptor. Correspondence to: Dr. H. Tesch, Medizinische Klinik I, Universit~it K61n, Joseph-Stelzmannstr. 9, 5000 K61n 41, F.R.G. Tel.: 49-221-478-4456; Fax: 49-221-478-6383. *Present address: Department of Microbiology and Immunology, BRB, Room 13-043, University of Maryland, School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, U.S.A.

190kDa (p190 met) [1, 2]. The glycosylated mature protein p190 met is a heterodimer of two disulphide linked chains of 50 kDa (tr-chain) and 145 kDa (#chain) which arise from a single polypeptide precursor by endoproteolytic processing [2]. Recently, the hepatocyte growth factor receptor (HGFR) has been identified as the product of the c-met protooncogene [3, 4]. Due to its heterodimeric (o~ #) subunit structure the M e t / H G F R protein is the prototype of a new class of tyrosine kinase receptors [5]. The Met oncogene has been originally identified in a human osteosarcoma cell line by transfection analysis in NIH 3T3 ceils [6]. The Met oncogene was activated by a DNA rearrangement that fused sequences of the TPR locus (translocated promotor

M, JOCKERet al.

8

region) on chromosome 1 with the c-met proto-oncogene locus on chromosome 7 [7]. Using a very sensitive PCR procedure this T P R / M e t oncogenic rearrangement and the fused T P R / M e t - R N A has been detected in four out of four cell lines derived from gastric tumors and in 12 out of 22 biopsy samples from human gastric tumors [8]. In addition to the activation by chromosomal rearrangements the Met/ H G F R gene can be activated by overexpression of the normal M e t / H G F R gene [9, 10]. The Met/ H G F R gene is consistently overexpressed in human tumors of the gastrointestinal tract [11, 12] as well as in thyroid papillary carcinoma [12] indicating that overexpression of the M e t / H G F R gene is involved in the pathogenesis of certain human carcinomas. To investigate whether the M e t / H G F R gene is also overexpressed in human hematopoietic tumors we analyzed the expression of the M e t / H G F R gene in normal and transformed hematopoietic cells at the m R N A and protein level.

Materials and Methods

Cell lines and primary tissues The cell lines analyzed are derived from patients with Burkitt's lymphoma (BL2, BL17, BL33, BL36, BL41, BL60, BL67, BL74, LY67, RAJI) [13, 14], Hodgkin's disease (I_A28, L540, CO, DEV, L591, HDLM2, KMH2) [15], B-cell leukemia (L660) [16], T-cell leukemia (CEM, JM) [17, 18], acute myelocytic leukemia (HL60) [19], chronic myeloid leukemia (K562) [20] and histiocytic lymphoma (U937) [21]. All cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS) at 37°C in a humidified, 5% CO2 atmosphere. Primary biopsies of lymph nodes were obtained from patients with Hodgkin's disease and non-Hodgkin's lymphoma. Primary specimens were frozen in liquid nitrogen. Frozen sections were used for immunophenotypic analysis. Peripheral blood mononuclear cells (PBMC) were obtained from a healthy donor by Ficoll gradient sedimentation. PBMC were stimulated with 12-O-tetra-decanoylphorbol-13-acetate (TPA) at a concentration of 10 ng ml-1. Pulsed field gel electrophoresis (PFGE) Cell concentration was determined in a counting chamber and agarose plugs (100 ~tl, containing about 10 ~tg of DNA) were prepared with 1 x 1 0 7 cellsm1-1 in 0.5% low melting point agarose (BRL). Plugs were digested in 0.5M EDTA pH9.5, 1% N-lauroylsarcosine, lmgm1-1 proteinase K, (1 rag/plug) at 50°C for 48 h. After washing the plugs three times for 2h in 1 × TE (5rag/plug), including 0.1 mM phenylmethansulfonylfluoride (PMSF), the plugs were incubated in the recommended restriction enzyme buffers for 1 h (5 ml/block) and subsequently digested in 200 Ixl of fresh buffer, with 40 U of restriction enzyme per plug. DNA was separated by rotaphor II chamber (Biometra, GOttingen, F.R.G.) using yeast chromosomes (Saccharomyces cerevisiae, strain WAY4A),

lambda multimers and HindlII digested lambda DNA as size markers. Gels were stained with ethidium bromide, blotted on nylon membranes (Zetaprobe, Biorad Inc.) and hybridized as described [22].

DNA and RNA hybridizations Southern and Northern blot analyses were performed as described previously [23]. DNA probes were labeled by random hexanucleotide priming [24]. With specific activities of 1-2 x 1 0 9 cpm/~tg. Hybridization was performed as described previously [23]. Probe pmetD is a 1.1 kb EcoRI fragment [6], probe pmetG is a 2.3 kb PstI fragment [7], and probe pmetH is a 1.6 kb EcoRI/SalI fragment [25] in pBR322. Plasmid pLcn2 contains a 2.4 kb EcoRI/HindlII fragment in pUC9 [26]. Western blotting Western blot analyses were performed as described previously [27]. Immunophenotypic analysis Immunophenotyping was done on frozen sections using the alkaline phosphatase-antialkaline phosphatase method (APAAP) [28] as described previously [29]. Monoclonal anti-Met antibody 19S is directed against a bacterially expressed Met protein (p50 met) containing the Met kinase domain [30, 31]. For control staining two monoclonal antibodies with an IgG~ and IgG2b isotype were used which are directed against bacterial peroxidase and an idiotope on mouse immunoglobulin, respectively. Results

Expression of the Met/HGFR gene in human hematopoietic cell lines Expression of the M e t / H G F R gene was analyzed in 23 human hematopoietic cell lines by Northern blot analyses. The results are summarized in Table 1. In three out of seven Hodgkin's disease derived cell lines (L428, L540 and HDLM2) we detected M e t / H G F R specific transcripts of 9 and 7 kb as described previously [23]. Western blot analyses revealed expression of p145 me~in the three Hodgkin's disease derived cell lines L428, L540 and H D L M 2 which express M e t / H G F R m R N A (Fig. 1). We also analyzed 10 Burkitt's lymphoma cell lines (BL2, BL17, BL33, BL36, BL41, BL60, BL67, BL74, LY67 and RAJI) for expression of the M e t / H G F R gene. M e t / H G F R specific transcripts of 9 kb and p145 met were detected in RAJI cells (Fig. 1) but not in nine other Burkitt's lymphoma cell lines. In six leukemia cell lines of B-cell (L660), T-cell (CEM, JM) or myeloid origin (HL60, K562, U937) expression of the M e t / H G F R gene was not detected (data not shown). Expression of the Met/HGFR gene in human leukemia and lymphoma cells We further analyzed expression of the M e t / H G F R

Expression of Met/HGFR in human leukemia and lymphoma TABLE 1. EXPRESSION OF THE M e t / H G F R GENE IN HUMANHEMATOPOIET1CCELL LINES

Cell line RAJI BL2 BL17 BL33 BL36 BL41 BL60 BL67 BL74 LY67 L428 L540 CO L591 KMH2 HDLM2 DEV L660 JM CEM HL60 K562 U937

Origin Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Burkitt's lymphoma Hodgkin's disease Hodgkin's disease Hodgkin's disease Hodgkin's disease Hodgkin's disease Hodgkin's disease Hodgkin's disease B-cell leukemia T-cell leukemia T-cell leukemia Promyelocytic leukemia Chronic myeloid leukemia Histiocytic lymphoma

Met/HGFR mRNA

Met/HGFR protein

+ + ++ + -

+ nd nd nd nd nd nd nd nd nd ++ +++ + nd nd nd nd nd nd nd

+ to + + + indicate different amounts of M e t / H G F R m R N A or protein detected by Northern blot or Western blot analyses, respectively. nd: not done.

FIO. 1. Detection of M e t / H G F R expression in human lymphoma cell lines by Western blot analysis. Protein extracts of 1 x 106 cells of the indicated cell lines derived from patients with Hodgkin's disease (L428, L540, CO, L591, KMH2 and HDLM2) and Burkitt's lymphoma (RAJI) were separated by SDS--PAGE and electroblotted onto nitrocellulose. Monoclonal anti-Met antibody 19S [29] was used to detect p145 met. Numbers on the left indicate the positions of molecular weight protein markers, kDa, kilo Dalton.

10

M. JIJCKER et al. TABLE

2.

EXPRESSION OF THE M e t / H G F R GENE IN PRIMARY HUMAN LEUKEMIA AND LYMPHOMA CELLS

Cell type

No. positive/ no. tested

Met/HGFR expression*

Chronic lymphoid leukemia Acute lymphoid leukemia Chronic myeloid leukemia Acute myeloid leukemia

0/3 0/5 0/7 1/14

+

Leukemia

Non-Hodgkin's lymphoma Immunocytoma Lymphoblastic lymphoma Centroblastic lymphoma Centrocytic lymphoma

Hodgkin's disease Nodular sclerosis Mixed cellularity Lymphocyte predominant

0/2 0/1 0/1 0/1

1/10 0/5 0/1

+

* Expression of the Met/HGFR gene was analyzed by Northern blot experiments in leukemia and non-Hodgkin's lymphoma and by immunohistochemistry in Hodgkin's disease.

gene in human leukemia and lymphoma cells. The results are summarized in Table 2. The leukemia cells analyzed were derived from the peripheral blood of patients with acute lymphoid leukemia (ALL, n = 5), chronic lymphoid leukemia (CLL, n = 3), acute myeloid leukemia (AML, n = 14) and chronic myeloid leukemia (CML, n = 7). In one case of an acute myeloid leukemia we detected strong expression of M e t / H G F R specific transcripts of 9.0 kb which is shown in Fig. 2. In the remaining 28 leukemias, M e t / H G F R specific transcripts were not detected (data not shown). In peripheral blood cells from five patients with non-Hodgkin's lymphoma (two immunocytoma, one cenroblastic lymphoma, one centrocytic lymphoma and one lymphoblastic lymphoma) with infiltration of lymphoma cells in the peripheral blood (cell counts between 150,000 and 500,000 leukocytes per ~tl) we did not detect Met/HGFR-specific mRNA by Northern blot analysis (data not shown). In addition, expression of p145 met was analyzed by immunohistochemical staining on frozen tissues from lymph nodes from patients with Hodgkin's disease. Expression of p145 met was detected in Hodgkin and Reed-Sternberg cells in one out of 16 lymph nodes from patients with Hodgkin's disease. The subtype of this particular case of Hodgkin's disease was nodular sclerosis (data not shown).

Expression of the Met/HGFR gene in normal hematopoietic cells Expression of the M e t / H G F R gene was also analyzed in hematopoietic cells from a healthy donor

by Northern blot experiments. In peripheral blood mononuclear cells (PBMC), granulocytes or tonsil cells we did not detect M e t / H G F R mRNA (data not shown). However, upon stimulation of PBMC with the phorbol ester TPA we detected a weak expression of the 9.0 kb M e t / H G F R mRNA in a time-dependent manner. Most M e t / H G F R mRNA was observed at 15h upon stimulation (Fig. 3). In addition very weak signals were observed at 8 and 48 h (Fig. 3).

Analysis of Met/HGFR gene structure To analyze whether the M e t / H G F R gene is rearranged in Hodgkin's disease or Burkitt's lymphoma cell lines which express the M e t / H G F R gene, Southern blot experiments were performed using four restriction enzymes (EcoRI, HindlII, KpnI and BgllI). After hybridization with probe pmetG we did not detect a rearrangement or amplification in the DNA of L428 and L540 cells (data not shown). As in L540 cells a translocation of M e t / H G F R gene to a marker chromosome was confirmed by in situ hybridization [32], the M e t / H G F R locus was analyzed by pulsed field gel electrophoresis. The restriction map obtained for the restriction enzymes Sill and NotI around the M e t / H G F R gene is shown in Fig. 4(A). Complete digestion of DNA from Hodgkin's disease derived cell line L540 and normal human fibroblasts cultures (Fil; Fi2) with Sill resulted in a fragment of 170 kb after hybridization with probe metH (Fig. 4(B)). Partial digestion with Sill resulted in two fragments of 170 and 600kb (Fig. 4(B)). There was no difference in fragment size

Expression of Met/HGFR in human leukemia and lymphoma

FIG. 2. Northern blot analysis of Met/HGFR m R N A expression in human acute myeloid leukemia. A 20~tg sample of total RNA from peripheral blood cells of 14 patients with acute myeloid leukemia was size fractionated in denaturing formaldehyde gels, transferred to nylon membranes and hybridized with a 'multiprime' [32p]dCTPlabeled M e t / H G F R probe (pmetG). The same blot was hybridized with fl-actin to control the integrity of RNA in all lanes (data not shown). RNA of different sizes (RNA ladder, BRL) and the position of 28S and 18S were used as size markers, kb, kilobase.

FIG. 3. Expression of M e t / H G F R m R N A in TPA stimulated normal hematopoietic cells. Peripheral blood mononuclear cells from a healthy donor were stimulated with 10 ng/ml TPA for varying lengths of time. Total RNA was extracted and Northern blot analysis w/is performed as described in Fig. 2. Lane 1: unstimulated PBMC; lane 2: 30 min.; lane 3: 2h; lane 4: 8h; lane 5: 15h: lane 6: 48h. kb, kilobase.

11

A

600 35O 170

s J |,

.

lOOkb

/

N

SI

I,

J

L c n 2 ~

N

S n

NI I

I

N2 I

metH

H I

Lcn2 i

~'~

t

lkb B

FI6. 4. Analysis of the Met/HGFR locus in human lymphoma cells by pulsed field gel electrophoresis (PFGE). (A) Map of the Met/HGFR gene locus. The localization of the Met/HGFR gene and the upstream located Lcn gene is indicated by the probes metH and Lcn2, respectively, used in hybridization experiments for detection of large restriction fragments after PFGE and Southern transfer. Sizes of restriction fragments obtained after hybridization with probe metH are indicated. S, SfiI; S*, partial digested SfiI site; N, non-polymorphic NotI site; N1 and N2; polymorphic NotI sites; E, EcoRI; H, HindIIl. (B) Southern blot analysis of the Met/HGFR gene locus. High molecular weight DNA of Hodgkin's disease derived cell line L540 (lane 2) and normal human fibroblasts (lanes 1 and 3) were digested with the indicated restriction enzymes, separated by PFGE and transferred to nylon membrane. The sizes of restriction fragments obtained after hybridization with probe metH are indicated in kilobases. Restriction enzymes used are NotI (N) and Sill (S). Sp is a partial digest with SfiI. N + Sp is a digest with Nod after a partial digest with S¢/1.

E I

Expression of Met/HGFR in human leukemia and lymphoma between L540 cells and normal fibroblasts. The same results in SfiI digests were obtained for L428 cells and RAJI cells (data not shown). The published Notl polymorphism of 880 and 550 kb was observed in the DNA of two fibroblast cultures (Fig. 4(B)) [33]. Double digestion of partially SfiI digested DNA with NotI revealed two fragments of 170 and 350 kb (Fig. 4(B)), independent of the NotI polymorphism, indicating that the non-polymorphic NotI site is located on the 600kb Sill fragment. This 600kb Sill fragment, however, was not cleavable with Nod in cell lines L540 (Fig. 4(B)) and RAJI (data not shown). Since digestion of DNA with NotI is methylation-dependent, the lack of cleavage of this NotI site is most likely due to DNA methylation of cytosine residues in the NotI recognition sequence. To rule out a deletion in the region of this NotI site, hybridization with probe pLcn2.1 which is located on a 6.6kb EcoRI fragment near to the NotI site was performed. In cell lines L428, L540 and RAJI we detected the normal 6.6 kb EcoRI fragment in all cell lines (data not shown). In addition, digestion with Sill and partial digestion with Sill of DNA from L428, L540 and RAJI cells revealed the normal fragments of 430 and 600 kb, respectively (data not shown). Discussion We have analyzed the expression of the protooncogene c-met which encodes the hepatocyte growth factor receptor in human leukemia and lymphoma cells in order to obtain information about the putative involvement of this proto-oncogene in the pathogenesis of human hematopoietic tumors. Our analyses show that the M e t / H G F R gene is expressed in a few cases of human leukemia and lymphoma, but not in normal unstimulated hematopoietic cells. In total, expression of the M e t / H G F R gene was detected in 6 out of 73 cases (8.2%) of all hematopoietic tumors analyzed. Four of the six samples positive for expression of the M e t / H G F R gene were derived from patients with Hodgkin's disease. In addition, in one Burkitt's lymphoma cell line and in one acute myeloid leukemia (AML) expression of the Met/ H G F R gene was detected. In normal unstimulated lymphocytes, granulocytes or monocytes we did not reveal expression of the M e t / H G F R gene. The expression of M e t / H G F R detected in normal peripheral blood mononuclear cells upon stimulation with the phorbol ester TPA indicates that expression of the M e t / H G F R gene can be regulated via the protein kinase C signal transduction pathway. The M e t / H G F R gene is normally expressed as a transcript of 9.0 kb detected in normal human fibroblasts [7]. In all cases analyzed, the size of Met/

13

H G F R specific transcripts of M e t / H G F R encoded proteins was not altered in the leukemia or lymphoma cells with the exception of two Hodgkin's disease derived cell lines (L428 and L540) where two transcripts of 9.0 and 7.0 kb were detected. An additional Met/HGFR-specific 7.0 kb transcript has also been detected in an epithelial lung carcinoma cell line (CALU-1) and in a human osteogenic sarcoma cell line (HOS). The HOS cell line expresses in addition to the 9.0 kb and 7.0 kb M e t / H G F R transcripts, a 6kb M e t / H G F R specific transcript. It has been shown that the three HOS M e t / H G F R RNAs (9.0, 7.0 and 6.0 kb) are 3' coterminal indicating that they have unique 5' ends or share a common 5' end and are differentially spliced onto a common set of 3' exons [7]. We do not know whether the 7.0 kb Met/ H G F R transcript in the Hodgkin's disease derived cell lines is aberrant and contributes to an altered phenotype. Since in the Hodgkin's disease derived cell lines only the normal p145 met protein was detected there is no indication that an aberrant protein is expressed from the 7.0 kb transcript. Activation of the M e t / H G F R oncogene was originally identified in a chemically treated human osteosarcoma cell line by transfection analysis in NIH3T3 cells [6]. It has been shown that a chromosomal DNA rearrangement created a hybrid TPR/Met gene with upstream sequences from a locus on chromosome 1 (designated TPR for translocated promotor region) fused to downstream sequences of the M e t / H G F R gene locus located on chromosome 7q21-31 [25]. The activated TPR/Met oncogene is expressed as a fusion transcript of 5 kb that encodes a 65 kDa protein [31]. This 65 kDa TPR/Met protein contains the kinase domain, but not the transmembrane domain from the M e t / H G F R proto-oncogene and p65 tpr-mc~ is autophosphorylated in vitro on tyrosine residues [1, 31]. The TPR/Met oncogenic rearrangement is present and expressed in several human gastric carcinoma and precursor lesions [8]. In our analysis we did not detect the aberrant Met/TPR hybrid transcript of 5.0 kb in the hematopoietic tumor cells or a rearrangement within the M e t / H G F R locus in cells expressing the M e t / H G F R gene. This is in agreement with a previous analysis of TPR/Met oncogenic rearrangement by polymerase chain reaction, where a TPR/Met oncogenic rearrangement could not be detected in two leukemia and lymphoma cell lines [34]. Thus our data indicate that the M e t / H G F R gene is not activated by a TPR/Met rearrangement in hematopoietic tumors. Overexpression of a normal full length mouse Met// H G F R cDNA in NIH3T3 cells resulted in morphological transformation and these cells exhibit properties of malignant cells including growth in soft

14

M. JOCKERet al.

TABLE 3. CYTOGENETIC ANALYSIS OF HEMATOPOIETIC CELL LINES EXPRESSING THE M e t / H G F R GENE*

TABLE 4. CHARACTERIZATION OF CELL LINES EXPRESSING

Cell line

Chromosome 7 configuration

Cell line

Origin

L428 L540 RAJI

7, inv dup(7)(qll.2q31.2) 3 x 7, t(7;21)(q22/31;p12) No normal chromosome 7 der(7)del(7)(q31.2) der(7)t(7;11 ?)(q31.2;q13?)

L428" L540t HDLM2~ RAJI

HD HD HD BL

* The Met/HGFR gene is located on chromosome 7@131 [251.

agar and induction of tumors in nude mice [10]. Elevated levels of M e t / H G F R mRNA of normal size were also detected in these transformants [10]. These data show that the M e t / H G F R proto-oncogene can also be activated by an overexpression of the normal M e t / H G F R gene. We detected expression of the normal M e t / H G F R transcripts and protein in a few cases of human leukemia and lymphoma, but not in normal hematopoietic cells. Our failure to detect Met/HGFR expression in normal hematopoietic tissues is in agreement with a previous investigation in which expression of the M e t / H G F R gene was not detected in normal mouse hematopoietic tissues like spleen, thymus, bone marrow or activated macrophages [10]. However, in a recent report Kmiecik and coworkers describe the expression of the Met/ HGFR gene in unfractionated and progenitor enriched murine bone marrow cell populations [35]. We could not detect M e t / H G F R expression in peripheral blood cells which are predominantly mature cells. These data suggest that the M e t / H G F R gene is differentially expressed during hematopoiesis and that some immature, but not mature hematopoietic cells express the M e t / H G F R gene. The M e t / H G F R gene has been mapped to human chromosome 7q21-31 [25]. Our cytogenetic analyses of hematopoietic cells which express the M e t / H G F R gene have revealed structural or numerical alterations of the long arm of chromosome 7 (summarized in Table 3): (i) the M e t / H G F R expressing cell line L540 bears three normal chromosome 7 as well as a translocation t(7,21)(q22/31;p12) [32]; (ii) cell line L428 has a normal chromosome 7 and a chromosome 7 with a duplicated long arm--dup (7)(qll.2q31.2) [36]; (iii) the Burkitt's lymphoma cell line RAJI bears two rearranged chromosomes 7, one with an elongated long arm due to a translocation t(7;ll?)(q31.2;q13?) and one with a deletion of a part of chromosome 7--del(7)(q31.2) (C. Fonatsch, unpublished data). Analysis of the organization of the M e t / H G F R gene in M e t / H G F R expressing cells, however, did not reveal any rearrangement within

THE Met/HGFR GENE Differentiation antigens CD30, CD19, CD25 CD30, CD2, CD4 CD30, CD2 IgM, ;.

EBV +

* L428 cells have Ig and TCR gene rearrangements and express cy and TCR-o: mRNAs [42]. t L540 cells have rearranged TCR-o:, TCR-/3 and TCRy genes and express TCR-tr mRNA [42]. HDLM2 cells have rearranged TCR-/3 and TCR-?' genes [43]. HD, Hodgkin's disease; BL, Burkitt's lymphoma; CD, cluster of differentiation; IgM, immunoglobulin M; EBV, Epstein-Barr virus.

the Met/HGFR gene or in a region of about 600 kb around the M e t / H G F R gene by pulsed field gel electrophoresis experiments. This situation might be comparable to the variant translocations in Burkitt's lymphoma where the breakpoints on chromosome 8 occur at distances of more than 140 kb downstream of c-myc [37]. Interestingly, four out of six samples positive for expression of the M e t / H G F R gene were derived from patients with Hodgkin's disease including three Hodgkin's disease derived cell lines and one primary biopsy. The characterization of cell lines expressing the M e t / H G F R gene is shown in Table 4. The Hodgkin's disease derived cell lines L428, L540 and HDLM2 express the CD30 antigen which is characteristic but not specific for Hodgkin's and ReedSternberg cells and express B- or T-cell differentiation antigens. In addition, these Hodgkin's disease derived cell lines have rearranged Ig and TCR genes and express Ig and TCR specific mRNAs. Epstein-Barr virus has not been detected in the Hodgkin's disease derived cell lines expressing the M e t / H G F R gene. The analysis of other cytokine receptors in Hodgkin's disease revealed expression of IL-6 receptors in Hodgkin and Reed-Sternberg cells in 8 out of 16 affected lymph nodes from patients with Hodgkin's disease and in five out of six Hodgkin's disease derived cell lines [29]. Expression of IL-2 receptor o: and/3 chains have been detected in Hodgkin's disease derived cell lines and in some primary cases of Hodgkin's disease suggesting a functional high affinity IL-2 receptor at least in some cases of Hodgkin's disease [38]. In addition, expression of the M-CSF receptor gene (c-fms) has been detected in Hodgkin's disease derived cell lines L428 and its variants L428KS and L428KSA [39]. Comparing these previous studies for receptor expression in HD

Expression of Met/HGFR in human leukemia and lymphoma with our data on M e t / H G F R expression it becomes clear that the cell lines expressing the M e t / H G F R gene (L428, L540 and H D L M 2 ) also express the IL-6 receptor and IL-2 receptor genes. Analysis of primary tissues revealed expression of the IL-6 receptor gene in Hodgkin and Reed-Sternberg cells in sections from the same lymph node where Met/ H G F R gene expression was detected [29]. Therefore, cells expressing the M e t / H G F R gene also express other cytokine receptor genes suggesting that these receptors have a synergistic effect on the proliferation a n d / o r differentiation of Hodgkin and Reed-Sternberg cells. Indeed, it has been shown that the hepatocyte growth factor ( H G F ) which is the ligand of the M e t / H G F R synergizes with IL-3 and GM-CSF in stimulating the proliferation of myeloid progenitor bone marrow cells [35]. Whether H G F can also synergize with IL-6 or IL-2 has to be determined in further experiments. In summary, our analyses showed that the M e t / H G F R gene was expressed in a few cases of hematopoietic tumors but not in normal unstimulated hematopoietic cells. Despite the failure to detect a rearrangement in the M e t / H G F R locus in Met/ H G F R expressing cells, cytogenetic data suggest that expression of the M e t / H G F R gene in these hematopoietic tumors might be due to chromosomal rearrangements. However, we cannot exclude the possibility that expression of the M e t / H G F R gene is activated during in vitro culturing of the cell lines or during processing of the primary tumor samples. It might also be possible that expression of the Met/ H G F R gene in some tumor samples reflects simply a distinct immature differentiation stage of these cells, because expression of the M e t / H G F R gene has been detected in bone marrow cells [35]. Since the M e t / H G F R has a cytoplasmic protein kinase it is possible that M e t / H G F R expressing cells stimulate their own proliferation by a deregulated kinase activity. Alternatively the growth of H G F R expressing cells may be triggered in a paracrine manner by H G F produced by other ceils. Indeed expression of H G F has been detected in several organs and in the human plasma [40, 41]. Further experiments are necessary to prove whether hematopoietic tumor cells expressing the H G F R stimulate their own growth in an autocrine or paracrine manner via a deregulated kinase activity.

Acknowledgements--The authors would like to thank Drs G. F. Vande Woude and D. L. Faletto for providing anti-Met antibodies and c-met DNA clones, Dr D. Eick for providing Burkitt's lymphoma cell lines, Dr H. Lehrach for providing plasmid pLcn2, Harry Abts for helpful advice and Ingrid Pahl for technical assistance. This work was

15

supported by the Deutsche Krebshilfe, Mildred Scheel Stiftung e.V. and the Deutsche Forschungsgemeinschaft.

References 1. Park M., Dean M., Kaul K., Braun M. J., Gonda M. A. & Vande Woude G. (1987) Sequence of Met protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors. Proc. natn. Acad. Sci. 84, 6379. 2. Giordano S., Ponzetto C., Di Renzo M. F., Cooper C. S. & Comoglio P. M. (1989) Tyrosine kinase receptor indistinguishable from the c-met protein. Nature 339, 155. 3. Bottaro D. P., Rubin J. S., Faletto D. L., Chan A. M. L., Kmiecik T. E., Vande Woude G. F. & Aaronson S. A. (1991) Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251,802. 4. Naldini L., Vigna E., Narsimhan R., Gaudino G., Zarnegar R., Michalopoulos G. K. & Comoglio P. M. (1991) Oncogene 6, 501. 5. Giordano S., Di Renzo M. F., Narsimhan R. P., Cooper C. S., Rosa C. & Comoglio P. M. (1989) Biosynthesis of the protein encoded by the c-met protooncogene. Oncogene 4, 1383. 6. Cooper C. S., Park M., Blair D. G., Tainsky M. A., Huebner K., Croce C. M. & Vande Woude G. F. (1984) Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 311, 29. 7. Park M., Dean M., Cooper C. S., Schmidt M., O'Brien S. J., Blair D. G. & Vande Woude G. F. (1986) Mechanism of met oncogene activation. Cell 45, 895. 8. Soman N. R., Correa P., Ruiz B. A. & Wogan G. N. (1991) The TPR-Met oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions. Proc. hath. Acad. Sci. 88, 4892. 9. Cooper C. S., Tempest P. R., Beckman M. P., Heldin C. H. & Brookes P. (1986) Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts. E M B O J. 5, 2623. 10. Iyer A.. Kmiecik T. E., Park M., Daar I., Blair D., Dunn K. J., Sutrave P., Ihle J. N., Bodescot M. & Vande Woude G. F. (1989) Structure, tissue-specific expression, and transforming activity of the mouse met protooncogene. Cell Growth & Differentiation 1, 87. 11. Liu C., Park M. & Tsa M.-S. (1992) Overexpression of c-met proto-oncogene but not epidermal growth factor receptor or c-erB-2 in primary human colorectal carcinomas. Oncogene 7, 181. 12. Di Renzo M. F., Narsimhan R. P., Olivero M., Bretti S., Giordano S., Medico E., Gaglia P., Zara P. & Comoglio P. M. (1991). Expression of the Met/HGF receptor in normal and neoplastic human tissues. Oncogene 6, 1907. 13. Lenoir G. M., Preud'homme J. L., Bernheim A. & Berger R. (1982) Correlation between immunoglobulin light chain expression and variant translocation in Burkitt's lymphoma. Nature 298, 474. 14. Lenoir G. M., Vuillaume M. & Bonnardel C. (1985) The use of lymphomatous and lymphoblastoid cell lines in the study of Burkitt's lymphoma. In Burkitt's Lymphoma. A Human Cancer Model (Lenoir G. M.,

16

M. JUCKERet al.

O'Connor & Olmeny C. L. M., eds), Vol. 60, p. 309. IARC, Lyons. 15. Schaadt M., Kalle C. von, Tesch H., Burrichter H. & Diehl V. (1988). Immunologic, functional and molecular-genetic properties of Hodgkin-derived cell lines. Cancer Rev. 10, 108. 16. Fonatsch C., Burrichter H., Schaadt M., Kirchner H. H., & Diehl V. (1982) Translocation t(8;22) in peripheral lymphocytes and established lymphoid cell lines from a patient with Hodgkin's disease followed by acute lymphatic leukemia. Int. J. Cancer 30, 321. 17. Foley G. E., Lazarus H., Farber S., Uzmann B. G., Boone B. A. & McCarthy R. E. (1965) Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18, 522. 18. Schwenk H. U. & Schneider U. (1975) Cell cycle dependency of a T-cell marker on lymphoblasts. Blut 31, 299. 19. Collins S. J., Galo R. C. & Gallagher R. E. (1977) Continuous growth and differentiation of human myeloid leukemia cells in suspension culture. Nature 270, 347. 20. Lozzio C. B. & Lozzio B. B. (1975) Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45, 321. 21. Sundstr6m C. & Nilsson K. (1976) Establishment and characterization of a human histiocytic lymphoma cell line (13-937). Int. J. Cancer 17, 565. 22. Reed K. C. & Mann D, A. (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucl. Acids. Res. 13, 7207. 23. Jiicker M., Schaadt M., Diehl V., Poppema S., Jones D. & Tesch H. (1990) Heterogeneous expression of proto-oncogenes in Hodgkin's disease derived cell lines. Hemat. Oncol. 8, 191. 24. Feinberg A. P. & Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analyt. Biochem. 132, 6. 25. Dean M., Park M., Le Beau M. M., Robins T. S., Diaz M. O., Rowley J. D., Blair D. G. & Vande Woude G. F. (1985) The human met oncogene is related to the tyrosine kinase oncogenes. Nature 318, 385. 26. Michiels F., Burmeister M. & Lehrach H. (1987) Derivation of clones close to met by preparative field inversion gel electrophoresis. Science 236, 1305. 27. Jiicker M., Roebroek A. J. M., Mautner J., Koch K., Eick D., Diehl V., Van de Ven W. J. M. & Tesch H. (1992) Expression of truncated transcripts of the protooncogene c-fpsffes in human lymphoma and lymphoid leukemia cell lines. Oncogene 7, 943. 28. Cordell J. L., Falini B., Erber W. N, Ghosh A. K., Abdulaziz Z., Macdonald S., Pulford K. A. F., Stein H. & Mason D. Y. (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J. Histochem. Cytochem. 32, 219. 29. Jiicker M., Abts H., Li W., Schindler R., Merz H., Giinther A., Kalle C. von, Schaadt M., Diamantstein T., Feller A. C., Krueger G. R. F., Diehl V., Blankenstein T. & Tesch H. (1991). Expression of interleukin-6 and interleukin-6 receptor in Hodgkin's disease. Blood 77, 2413.

30. Faletto D., Tsarfaty I., Kmiecik T. E., Gonzatti M., Suzuki T. & Vande Woude G. F. (1992) Evidence for non-covalent clusters of the c-met proto-oncogene product. Oncogene 7, 1149. 31. Gonzatti-Haces M., Seth A., Park M., Copeland T., Oroszlan S. & Vande Woude G. F. (1988). Characterization of the TPR-Met oncogene p65 and the Met protooncogene p140 protein-tyrosine kinases. Proc. hath. Acad. Sci. 85, 21. 32. Fonatsch C., Gradl G., Kolbus U., Rieder H. & Tesch FI. (1990) Chromosomal in situ hybridization of a Hodgkin's disease-derived cell line (L540) using DNA probes for TCRA, TCRB, MET, and rRNA. Hum. Genet. 84, 427. 33. Julier C. & White R. (1988) Detection of a Notl polymorphism with the pmetH probe by pulsed-field gel electrophoresis. Am. J. Hum. Genet. 42, 45. 34. Soman N. R., Wogan G. N. & Rhim J. S. (1990) TPRMet oncogenic rearrangement: detection by polymerase chain reaction amplification of the transcript and expression in human tumor cell lines. Proc. natn. Acad. Sci. 87, 738. 35. Kmiecik T. E., Keller J. R., Rosen E. & Vande Woude G. F. (1992) Hepatocyte growth factor is a synergistic factor for the growth of hematopoietic progenitor cells. Blood 80, 2454. 36. Fonatsch C., Diehl V., Schaadt M., Burrichter H. & Kirchner H. (1986) Cytogenetic investigations in Hodgkin's disease: 1. Involvement of specific chromosomes in marker formation. Cancer Genet. Cytogenet. 20, 39. 37. Henglein B., Synovzik H., Groitl P., Bornkamm G. W., Hartl P. & Lipp M. (1989) Three breakpoints of variant t (2;8) translocations in Burkitt's lymphoma cells fall within a region 140 kilobases distal from cmyc. Mol. Cell. Biol. 9, 2105. 38. Tesch H., Giinther A., Abts H., Jiicker M., Klein S., Krueger G. R. F. & Diehl. V. (1993) Expression of IL-2Ra~and IL-2Rfl in Hodgkin's disease. Am. J. Path. 142, 1714. 39. Paietta E., Racevskis J., Stanley E. R., Andreeff M., Papenhausen P. & Wiernik P. H. (1990) Expression of the macrophage growth factor, CSF-1 and its receptor c-fms by a Hodgkin's disease derived cell line and its variants. Cancer Res. 50, 2049. 40. Tashiro K., Hagiya M., Nishizawa T., Seki T., Shimonishi M., Shimizu S. & Nakamura T. (1990) Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues. Proc. natn. Acad. Sci. 87, 3200. 41. Zarnegar R., Muga S., Rahija R. & Michalopoulos G. (1990) Tissue distribution of hepatopoietin-A: a heparin-binding polypeptide growth factor for hepatocytes. Proc. natn. Acad. Sci. 87, 1252. 42. Falk M. H., Tesch H., Stein H., Diehl V., Jones D. B., Fonatsch C. & Bornkamm G. W. (1987) Phenotype versus immunoglobulin and T-cell receptor genotype of Hodgkin-derived cell lines: activation of immature lymphoid cells in Hodgkin's disease. Int. J. Cancer 40, 262. 43. Drexler H. G., Leber B. F., Norton J., Yaxley J., Tatsumi E., Hoffbrand A. V. & Minowada J. (1988) Genotypes and immunophenotypes of Hodgkin's disease derived cell lines. Leukemia 2, 371.