Karyotypic Analysis of Gastric Carcinoma Cell Lines Carrying an Amplified c-met Oncogene Giovanna Rege-Cambrin, Patrizia Scaravaglio, Franca Carozzi, Silvia Giordano, Carola Ponzetto, Paolo M. Comoglio, and Giuseppe Saglio

ABSTRACT: MKN 45 is a poorly differentiated gastric carcinoma cell line from which the subclone GTL 16 was obtained. Both lines carry an amplification unit derived from chromosome 7 sequences and containing an activated c-met oncogene. Karyotypic analysis showed that GTL 16 derived from a subclone of MKN 45 after endoreduplicotion. Several clonal abnormalities are evident in both lines; some are frequently observed in gastrointestinal tumors (loss of 17p and m o n o s o m y 18). Other consistent anomalies include 6 q - , t(8;10) and t(5;8), and inv(16). A m a r k e r chromosome ((M1), which was previously shown to contain the c-met amplification unit, is constantly duplicated in all GTL 16 metaphases; in contrast, most unidentified markers are retained in only a single copy in GTL 16 cells. These data are in agreement with the hypothesis that the c-met oncogene activation in these gastric cancer cell lines might be related to a gene dosage effect.

INTRODUCTION MKN 45 is a h u m a n cell line derived from a poorly differentiated gastric adenocarcinoma of the m e d u l l a r y type [1]. The cell line GTL 16, w h i c h has been shown to carry an activated c-met protooncogene [2], was obtained by subcloning MKN 45. Both cell lines contain an amplification unit w h i c h is at least 3,000 kilobases (kb) long and includes c-met, w h i c h is activated and overexpressed, as well as the CF and irp genes, w h i c h in contrast are not expressed [3]. To gain further insight into the chromosome abnormalities associated with this amplification unit and to define how the two cell lines have diverged, we performed cytogenetic analysis of both MKN 45 and GTL 16 ceils. MATERIALS AND METHODS GTL 16 was obtained from MKN 45 by limit-dilution subcloning.

Chromosome Studies Chromosomes were analyzed by a direct technique; Colcemid (GIBCO) 0.1 ~tg/ml was a d d e d to the cell line cultures for I

From the Department of Biomedical Sciences and Oncology, University of Turin (G. R. C., P. S., S. G., C. P., P. M. C.), lst Medical Clinic, University of Pemgia, (G. S.), Center of lmmunogenetics and Histocompatibility of CNR (E C.), Turin, Italy. Address reprint requests to: Giovanna Rege-Cambrin, M.D., Dipartimento di Scienze Biomediche e Oncologia Umana, Via Geneva 3, 10126 Torino, Italy. Received October 25, 1991; accepted July 30, 1992. 170 Cancer Genet Cytogenet 64:170-173 [1992) 0165-4608/92/$05.00

hour. Cells were harvested by standard procedures, and metaphases were G-banded w i t h Wright stain. Twenty mitoses were fully karyotyped from either MKN 45 and GTL 16 cell lines;.chromosome abnormalities were classified on the basis of the International System for H u m a n Cytogenetic Nomenclature [4]. RESULTS Both cell lines show a complex karyotype with several clonal chromosomal abnormalities and a high degree of c e l l - c e l l variation. MKN 45, w h i c h derived from a female patient [I], is a h y p o d y p l o i d cell line w i t h a m o d a l chromosome number of 42. The major population has the following karyotype: 4 2 , X , - X,der(1)t(1;2) (q32 ;q13), - 2,t(2 ;15)(p16;q14),del(3)(p21), - 4,der(4)t(4;?)(q33;?),der(5)t(5;?8)(q31;?q21),del(6)(q21),der(7) del(7)(p14)del(7)(q32), - 8, - 8, - 9,der(10)t(8;10)(q13;p13), der(11)inv(11)(q13q24)t(11;?)(p15;?), - 12, - 13,der(14)t(14;?) (p11;?),inv(16) (p13q22),der(17)t(17;?) (p13 ;?), - 18, - 20, - 22, + M1, + M2, + M3, + M4, + M5, + M6, + M7 (Fig. 1). In a few cells, two normal chromosomes 3 are present, whereas the m o n o s o m i c chromosome 12 has extra material derived from chromosome 1 on its short arm: der(12)t(1;12) (q32;p11). A l t h o u g h r a n d o m chromosome loss is observed in each metaphase, some derivative chromosomes are always present, including der(4), t(5;?8), del(6q), der(7), t(8;10), inv(16), and markers M1-M3. GTL 16 is a hypotetraploid cell line w i t h a m o d a l chromosome n u m b e r of 78. The karyotype of the major GTL 16 cell p o p u l a t i o n is 78,XX, + der(1)t(1;2)(q32;q13),t(2;15)(p16; q14) x 2, + 3, + 3,der(4)t(4;?)(q33;?) x 2, + der(5)t(5;?8)(q31;?q21)

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Cytogenetics of Gastric Cancer Cell Lines

173

× 2, + del(6)(q21), + del(6), + der(7)del(7)(p14)del(7)(q32), + der(7) del(7)(p12)del(7)(q32), - 8, - 8, + der(10)t(8;10)(q13;p13) × 2, + der(11)inv(11)(q13q25)t(11;?)(p15;?) × 2,der(12)t(1;12)(q32;p13) × 2, + 13, + 13,der(14)t(14;?)(p11;?), + der(14), + 15, + 15, + inv(16) (p13q22) × 2, + der(17)t(17;?)(p!3;?) × 2, + 19, + 19, + 21, + 21, + M1, + M1, + M2, + M3, + M4, + M5, + M6, + M7 (Fig. 2). Most derivative chromosomes observed in MKN 45 are generally present in two copies in GTL 16, except der(1) (always in a single copy) and del(3p), w h i c h is not observed. Because of the der(12)t(1;12), the karyotype of GTL 16 appears to derive from the subclone of MKN 45 containing two normal chromosomes 3 a n d a dar(12) anomaly. M1 is a large submetacentric chromosome; the long arm, which contains the c-met amplification unit, has a long darkb a n d e d region that shows no similarity with other chromosomes. Duplication of the M1 marker chromosome is observed in all GTL16 mitoses.

Our data demonstrate that the M1 derivative chromosome is present in a single copy in all analyzed MKN 45 cells, whereas it is always observed in two copies in every GTL 16 metaphase. This behavior has not been observed for the other unidentified marker chromosomes, w h i c h are usually present in a single copy in the hypotetraploid GTL-16 mitoses and are r a n d o m l y lost in part of the metaphases. Therefore, cytogenetic analysis showed that the gene dosage of the amplified c-met is maintained during cell line karyotypic evolution.

DISCUSSION

REFERENCES

Few studies have been performed to analyze the cytogenetic pattern of h u m a n gastric cancer [5-8]; the presence of specific chromosome abnormalities, involving particularly chromosomes 8 (trisomy or isochromosome for the long arm), has been suggested [5-9]. The chromosomal pattern of MKN 45 is characteristic of a monosomic type-near-diploid tumor, as defined in colorectal adenocarcinomas [10]. Cytogenetic analysis clearly shows that GTL 16 is derived from a subclone of MKN 45 that underwent e n d o r e d u p l i c a t i o n . This mechanism of karyotypic evolution is frequently reported to occur in vivo in intestinal tumors [10]. Multiple chromosome abnormalities are observed in both cell lines. A m o n g these, the deletion 6 q - is a frequent finding both in hematopoietic and solid tumors [9]; m o n o s o m y 18 and the presence of an abnormal 17p with loss of the short arm are also frequently observed in h u m a n malignancies, particularly in colorectal carcinomas, in w h i c h the latter often is associated w i t h loss of the p53 gene and mutation of the remaining allele [11, 12]. This is in agreement with previous data suggesting that some c o m m o n features might be shared by colorectal and gastric tumors [7]. On the other hand, inv(16) is u s u a l l y found in acute n o n l y m p h o b l a s t i c leukemia with eosinophilia (ANLL-M4Eo) [13], and the significance of such a finding in this gastric cancer cell line is unknown. No normal chromosome 8 is observed in both lines; the 8q is involved in two translocations (with chromosomes 5 and 10), confirming a possible role of chromosome 8 abnormalities in stomach tumors. MKN 45 and GTL 16 also contain one and two copies, respectively, of a derivative chromosome 7 (with loss of material from both the short and long arm) in a d d i t i o n to the normal homolog. However, the c-met amplicon, w h i c h contains about 3,000 kilobases of genetic material originated from chromosome 7, has not been located on either the normal or the rearranged c h r o m o s o m e Z but on the long arm of a novel marker c h r o m o s o m e indicated as M1 (3). This indicates that sequences from 7q, i n c l u d i n g c-met, underwent both amplification and relocation. The c-met oncogene has been shown to be activated and overexpressed in GTL-16 cells [3]; the activation is not related to mutations in c-met's coding sequence, w h i c h is conserved, and the role of a genedosage effect has been suggested [3].

1. Motoyama T, Hojo H, Watanabe H (1986): Comparison of seven cell lines derived from human gastric carcinomas. Acta Pathol Jpn 36:65-83. 2. Giordano S, Ponzetto C, Di Renzo MF, Cooper CS, Comoglio PM (1989): Tyrosine kinase receptor indistinguishable from the c-met protein. Nature 339:155-156. 3. Ponzetto C, Giordano S, Peverali F, Della Valle G, Abate ML, Vaula G, Comoglio PM (1991): C-met is amplified but not mutated in a cell line with an activated met tyrosine kinase. Oncogene 6:553-559. 4. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP (eds.); published in collaboration with Cytogenetic Cell Genet (Karger, Basel, 1985); also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 5. Saal K, Vollmers HP, Hohn H, Muller-Hermelink HK (1989); Cytogenetic studies on human gastric cancers. Cancer Genet Cytogenet 38:196. 6. Ochi H, Douglass HO Jr, Sandberg AA (1986): Cytogenetic studies in primary gastric cancer. Cancer Genet Cytogenet 22:295-30. 7. Ferti-Passantonopoulou AD, Panani AD, Vlachos JD, Raptis SA (1987): Common cytogenetic findings in gastric cancer. Cancer Genet Cytogenet 24:63-73. 8. Castedo S, Correira C, David L, Sobrinho-Simoes M (1991): Isochromosome 8q: A recurrent change in gastric carcinoma. Cancer Genet Cytogenet 54:137-138. 9. Mitelman F (1988): Catalog of Chromosome Aberrations in Cancer, 3rd Ed, Alan R. Liss, New York. 10. Muleris M, Delattre O, Olschwang S, DutrillauxAM, Remvikos Y, Salmon RJ, Thomas G, Dutrillaux B (1990): Cytogenetic and molecular approaches of polyploidizationin colorectal adenocarcinomas. Cancer Genet Cytogenet 44:107-118. 11. Vogelstein B, Fearon BA, Hamilton SR, Scott EK, Preisinger BA, Leppert M, Nakamura Y, White R, Smits AMM, Bos JL (1988): Genetic alteration during colorectal-tumor development. N Engl J Med 319:525-532. 12. Nigro JlVl,Baker SJ, Preisinger AC, Jessup JM, Hostetter R, Cleary K, Bigner SH, Davidson N, Baylin S, Devilee P, Glover T, Collins FS, Weston A, Modali R, Harris CC, Vogelstein B (1989): Mutations in the p53 gene occur in diverse human tumor types. Nature 342:705-707. 13. Le Beau/viM, Larson ILK,Bitter MA, Vardiman JW, Golomb HM, Rowley JD (1983): Association of an inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia: A unique cytogenetic-clinic-pathologicalassociation. N Engl J Med 309:630-636.

This work was supported by grants from the Associazione Italiana Ricerca sul Cancro and CNR, PF-ARCO grant no. 92-02263.PF39 to G.S.P.S. is a fellow of Comitato Gigi Ghirotti. The authors thank Dr. Paola Dal Cin, University of Leuven, Belgium, for helpful advice.

Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene.

MKN 45 is a poorly differentiated gastric carcinoma cell line from which the subclone GTL 16 was obtained. Both lines carry an amplification unit deri...
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