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Mutations of the RAS genes in childhood acute myeloid leukemia, myelodysplastic syndrome and juvenile chronic myelocytic leukemia
Leukemia Rerearch Vol 21, No. 8, pp. 697-701, 1997. D 1997 Elsewer Science Lrd All rights reserved Printed in Grear Britain 0145-2126197 $17.00 + 0.00
Pergamon PII: SO145-2126(97)00036-Z
MUTATIONS OF THE RAS GENES IN CHILDHOOD ACUTE MYELOID LEUKEMIA, MYELODYSPLASTIC SYNDROME .AND JUVENILE CHRONIC MYELOCYTIC LEUKEMIA Xiao Ming Sheng”, Machiko Kawamura”, Hiroaki Ohnishi”, Kohmei Ida*, Ryoji Hanadat, Seiji Kojimal, Miyuki Kobayashi *, Fumio Bessho*, Masayoshi Yanagisawa* and Yasuhide Hayashi* *Department of Pediatrics, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113 Japan, TDivision of Hematology/Oncology, Saitama Children’s Medical Center, Saitama, Japan and fDivision
of Hematology/Oncology,
Nagoya First Red Cross Hospital, Nagoya, Japan
(Received 17 February 1997. Accepted 26 February 1997) Abstract-Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108 fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examined for mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 1 lq23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. All mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies. c 1997 Elsevier Science Ltd Key words: leukemia.
RAS gene, leukemia,
myelodysplastic
Introduction
syndrome,
juvenile
chronic
myelocytic
by a point mutation at one of the three codons 12, 13, or 61 [I]. Early studies of mutations of RAS genes have suggested that important differences in the frequencies of mutations of RAS genes may exist among tumors derived from different tissues and even among tumors from the same tissues if they are histopathologically different [ 11.For instance, studies in adults have showed that the incidence of the K-f?AS gene mutation was as high as 90% in pancreatic tumors and the incidence of the N-RAS gene mutation was about 30% in acute myeloid leukemia (AML), whereas RAS mutations are found only rarely in breast carcinoma [ 1,6]. Previous studies have shown that RAS mutations appear to be an early event in the development of some tumors, such as colon cancers and acute leukemias [6,7], but they seemed to be associated with later stages of carcinogenesis in other tumors, such as melanoma and multiple myeloma [ 1,8,9]. It has been reported that patients with the RAS gene mutations had a poorer
Alterations in the cellular genome affecting the expression or the function of genes controlling cell growth and differentiation are considered to be the main cause of cancer. RAS genes consist of a family of genes which are frequently found to be mutated in human tumors [I]. This family consists of three functional genes, H-, K-, and N-RAS genes [l-4], which encode proteins with molecular weights of 21,000 and have mutually high homology [5]. These proteins function as guanosine nucleotide triphosphate (GTP)-binding proteins (~21) with intrinsic GTPase activity. It is noted that certain structural alterations in RAS proteins lead to the loss of this GTPase activity. Thus, the proteins lose their ability to become inactivated and continue to stimulate growth or differentiation. These alterations can be brought about Correspondence to: Y. Hayashi, Department of Pediatrics, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan (Tel.: 81 3 3815 541 I ext. 3452; Fax: 81 3 3816 4108). 697
698
Xiao Ming Sheng et al.
prognosis than those without them [lo, 111, and that myelodysplastic syndrome (MDS) with Z&G gene mutation tends to transform into leukemia [l, 10-161. Studies concerning RAS gene mutations in leukemia have been concentrated on adult leukemia, and studies on childhood leukemia are few. Because childhood leukemia differs in many aspects from adult leukemia, it is important to know the exact frequency of RAS gene mutations to understand if RAS genes play any role in childhood leukemia. In this study, we investigated mutations of exons 1 and 2 of N-, K- and H-RAS genes in 12 AML cell lines and 67 fresh AML samples, 29 MDS, and 12 juvenile chronic myelogenous leukemias (JCML) in children, using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method and direct sequencing.
Table 1. Mutation of RAS genesin cell lines and fresh tumors, of childhood AML, MDS and JCML
Disease AML MDS JCML Total
Cell lines
Fresh tumors
Total
mutations/ samples(%)
mutations/
mutations/
samples(%)
samples(%)
2167 (3.0%) l/29 (3.4%) l/l2 (8.3%) 41108 (3.7%)
6/79 (7.6%) l/29 (3.4%) l/12 (8.3%) 81120 (6.7%)
4112 (33.3%) 4112 (33.3%)
PCR method Most RAS gene mutations have been demonstrated to be localized in exons 1 and 2. Therefore, fragments from regions carrying exon 1 or 2 of the K-RAS gene (Kl or K2), the H-RAS gene (Hl or H2) and the N-RAS gene (Nl or N2) were amplified by PCR. The sequences of primers used for PCR were the same as reported previously [ 18, 191. DNA samples (50 ng) in the mixture (5 ~1) with appropriate unlabelled primers and [a 32P] dCTP (20 p Ci per tube, 3000 Ci/mmol, Amersham), were subjected to 30 cycles of the reaction.
Materials and Methods Cell lines Twelve AML cell lines were analyzed in this study. They were obtained from the Japanese Cancer Research Resources Bank and cultured in RPM1 1640 medium suppletiented with 10% fetal bovine serum.
SSCP analysis After adding 45 pl of formamide denaturing dye mixture (95% formamide: 20 mM EDTA: 0.05% xylene cyanol: 0.05% bromophenol blue), the PCR mixture was heated at 80°C for 3 min, and then 1 ~1 of the diluted mixture was loaded on to one lane of a 5% polyacrylamide gel containing 45 mM Tris-borate (pH 8.3) and 4 mM EDTA. The gel contained 10% glycerol when it was specified. Electrophoresis was performed at 40 W for l-3 h with cooling. The gel was dried on filter paper and exposed to X-ray film.
Patient samples Bone marrow or peripheral blood samples were collected from 67 children with AML, 29 with MDS and 12 with JCML from various institutes after obtaining informed consent. Diagnosis of AML, JCML and MDS were based on the criteria of the FrenchAmerican-British (FAB) classification. In AML samples, the proportion of leukemic cells exceeded 80%. Mononuclear cells were separated on a Ficoll-Hypaque (Lymphoprep) density gradient, suspended in RPM1 medium containing 10% of dimethylsulfoxide, and kept at -80°C until use.
Direct sequencing of PCR-amplifiedfiagments Direct sequencing was performed as previously described [20] with some modifications [ 17, 191. A small piece of the gel corresponding to the abnormal band detected by SSCP analysis was removed, immersed in 20 ~1 of water, heated at 80°C for 15 min, and centrifuged. DNA in the extract (1 ~1) was subjected to
Preparation of DNA High-molecular weight DNA was prepared by the proteinase K-phenol-chlorofolm extraction method as previously reported [ 171.
Table 2. RAS gene mutations and nucleotide changes in AML cell lines Cell lines P39/TSU KG-l P3 l/FUF THP- 1 CTY-1
Type of AML
Type of RAS
No. of codon
AML AML AMOL AMOL AMOL
N N N N K
61 12 12 12 62
Gln: glutamine; Gly: glycine; Glu: glutamic acid; Leu: leucine; Asp: asparagine; Ser: serine.
Nucleotide and amino acid changes CAA(Gln)+CTA(Leu) GGT(Gly)-+GAT(Asp) GGT(Gly)+GAT(Asp) GGT(Gly)+AGT(Ser) GAG(Glu) +GAA(Glu)
699
Mutations of RAS genes in childhood malignancies
Table 3. RAS gene mutations and nucleotide changes in childhood fresh tumors of AML, MDS and JCML Diseases AMOL AMOL MDS JCML
Ages*/sext
Type of RAS
No. of codon
5 months/male 10 years/male 5 years/female 1 year/male
K K N N
61 12 13 12
Nucleotide and amino acid changes
Survival time
CAA(Gln)-+CAC(His) GGT(Gly)-+GAT(Asp) GGT(Gly)+GTT(Val) GGT(Gly)+GTT(Val)
16 day 5 months 27 months + 84 months +
*m: month, y: year. **M: male, F: female. +: Alive
30 cycles of PCR, and the products were purified with Microcon 100 (Amicon). The DNA fragments thus obtained were sequenced by the dideoxy chain termination method using 5’-32P labelled primers and Taq DNA polymerase (dsDNA Cycle Sequencing System, Gibco BRL, Gaithersburg, MD, U.S.A.). Primers for sequencing were the same as those used for PCR-SSCP. Results Of the 12 AML cell lines, four were found to contain mutation in N-RAS genes and one in the K-RAS gene (Tables 1 and 2). Nucleotide sequence analysis revealed that mutations of the N-RAS gene in four cell lines were missense mutations and mutation of the K-RAS gene in one cell line was a silent mutation (Tables 1 and 2). The cell line P-39/TSU contained a CAA to CTA transversion in the second base of codon 61 of the N-RAS gene, resulting in the substitution of leucine for glutamine. The cell lines KG- 1 and P3 1-FUF contained the GGT to GAT transition in the second base of codon 12 of the NRAS gene, causing the substitution of asparagine for
a. SSCP
glycine. The cell line THP-1 contained a GGT to AGT transition in the first base of codon 12 of the N-RAS gene, causing the substitution of serine for glycine. The cell line CTY-1 had a GAG to GAA silent mutation in the 3rd base of codon 62 of the K-RAS gene. DNA samples from 67 children with AML were screened for point mutations at exons 1 and 2 of N-, Kand H&IS genes (Tables 1 and 3). Two of them were found to contain a K-RAS gene mutation. One had mutation of a GGT to GAT transition in the second base of codon 12 of the K-RAS gene, causing the substitution of asparagine for glycine, whereas another had a CAA to CAC transversion in the 3rd base of codon 61 of the KRAS gene, causing the substitution of histamine for glutamate. These two patients were diagnosed as having AMOL-MS and had 1lq23 translocations. DNA sample from 12 JCML patients and 29 MDS patients were screened for point mutations in exons 1 and 2 of the N-, K- and H-RAS genes (Tables 1 and 3). One of the 12 JCML samples and 1 of the 29 MDS samples contained a GGT to GTT transversion at codon 12 and 13 of the N-RAS gene, respectively (Fig. 1).
b. I
sequence WT patient r-in TCGA TCGA
Fig. 1. (a) SSCP analysis of N-RAS gene in MDS patient. The arrow points to an abnormal band shift of a MDS patient. (b) Mutation of the N-RAS gene identified in a MDS sample. Direct sequencing of the PCR products amplified from the abnormally shifted band demonstrating a mutation of GGT(Gly)+GTT(Val) at codon 13 in N-RAS gene. WT, wild type; T, thymine; C, cytosine; G, guanine; A. adenine.
700
Xiao Ming Sheng ef al.
Discussion The technique to examine RAS gene point mutations has been mainly performed by the synthetic dot blot hybridization method. Using this method, frequency of the i?AS gene mutation of adult leukemia was high (i.e. 27% in AML, 30% in MDS) [I, lO-13,21-231. In childhood leukemia only few studies concerning RAS gene mutations have been reported [ 14-161. In these studies, the synthetic dot blot hybridization method was also used, and the frequency was also relatively high [ 1, 151. The technique of the synthetic dot blot hybridization method is known to give false positive results at high frequencies and can examine only the confined codons. In contrast, the SSCP method give less false positives and can simultaneously cover mutations of all codons [20]. This method is also faster, simpler and more accurate. In the previous studies on RAS mutations in other tumors, we obtained good results with this method combined with direct sequencing [ 181. Results of direct sequencing for normal bands in SSCP showed the wild type of the RAS gene, and all abnormal bands in SSCP showed changes of bases. Therefore, the findings of our study could be considered to be true incidences of RAS gene mutations, although frequencies of RAS gene mutations in AML, JCML and MDS in children was very low in this study (3.0%, 8.3%, 3.4%, respectively). Relatively high incidence of N-RAS gene mutation has been reported in JCML [24], which was not compatible with our results. The difference between them is unknown. The frequency of the mutation in our study was also lower than that of mutation for adults which was obtained with the same PCR-SSCP technique [I, 131. Thus, the frequency of RAS mutations in AML and MDS for children may be lower than that for adults. One possible reason for the difference in frequency of RAS gene mutations between children and adults may be due to the difference in leukemogenesis. That is, children are less exposed to poisonous environments than adults so they have less chance for mutation than adults. Notably, a recent study revealed that mutation of the RAS genes was rare in follicular lymphoma, using the PCR-SSCP method [25]. Frequency of RAS gene mutations may depend on the technique used. In this study, observed mutations were limited to Nand K-RAS genes, and positions of mutation were at codons, 12, 13 and 61, as reported in adults. This means that the role of mutation in leukemogenesis is not different between children and adults. These three positions are related to the domain of GTPase activity. If any of these three positions suffer from mutations, activity of GTPase will be affected, and its activity will decrease. As a result, activated p21 loses its ability to become inactivated and continue to stimulate growth or
differentiation. K-RAS gene mutations were found in two of 67 fresh AML samples, although N-RAS gene mutations were usually found in hematological malignancies. Interestingly, these two patients had AMOLM5 and llq23 translocations. Frequency of the mutation in 12 cell lines was apparently higher than that of fresh AML, being compatible with the previous studies in other tumors [ 1, 17,261. There have been different studies concerning the timing of RAS gene mutations in the course of the development of cancer. This timing seems to depend on the type of tumors. In leukemia, it was demonstrated to occur in the early stage of leukemogenesis, whereas in melanoma and myeloma, it appears to occur in a later stage. Our findings supported the view that in AML, mutation of the RAS gene occurs in the later stage of the disease instead of the early stage. Further larger scale investigations on AML at diagnosis, at relapse, and cell lines is necessary to clarify this problem. In conclusion, low frequency of RAS gene mutations in this study suggested that the mutation of the RAS gene does not play an important role in the development of childhood myeloid malignancies. Acknowledgements-The authors wish to thank clinicians for providing the samples and clinical data on patients. They would also like to thank Mrs Shyoko Soma for technical assistance. Supported in part by a Grant-in Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and a Grant-in Aid for scientific Research from The Ministry of Education, Science of Culture (044545668, 04454276) Japan.
References 1. Bos, J. L., Ras oncogenes in human cancer. A review. Cancer Research, 1989, 49, 4682. 2. Harvey, J. J., An unidentified virus which causes the rapid production of tumours in mice. Nature, 1964, 204, 1104. 3. Kirsten, W. H. and Mayer, L. A., Morphologic responses to a murine erythroblastosis virus. Journal of the National Cancer Institute, 1967, 39, 3 11. 4. Shimizu, K., Goldfarb, M., Perucho, M. and Wigler, M., Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line. Proceedings of the National Academy of Science USA, 1983, 80, 383. 5. Taparowsky, E., Shimizu, K., Goldfarb, M. and Wigler, M., Structure and activation of the human N-ras gene. Cell, 1983, 34, 581. 6. Almoguera, C., Shibata, D., For-rester, K., Martin, J., Amheim, N. and Perucho, M., Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell, 1988, 53, 549. 7. BOS, J. L., Fearon, E. F., Hamilton, S. R., Verlaan-de Vriws, M., van Boom, L. K., van der Eb, A. J. and Vogelstein, B., Prevalence of RAS gene mutations in human colorectal cancers. Nature, 1987, 327, 293. 8. Portier, M., Moles, J. P., Mazards, G. R., Jeanteur, P.,
Mutations of RAS genes in childhood malignancies Bataille, R., Klein, B. and Theillet, C., p.53 and RAS gene mutations in multiple myeloma. Oncogene, 1992, 7, 2539. 9. Bell, N. J., Yohn, J. J., Morelli, J. G., Norris, D. A., Golitz, L. E. and Hoeffler, J. P., RAS mutations in human melanoma: a marker of malignant progression. Journal of Investiaative Dermatolonv, 1994. 102, 285. IO. Hirai, H., Kobayashi, Y, Mano, H., Hagiwara, K., Maru, Y., Omone, M., Mizuguchi, H., Nishida, J. and Takaku, F., A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome. Nature, 1987, 327, 430. 11. Hirai, H., Okada, M., Mizoguchi, H., Mano, H., Kobayashi, Y., Nishida, J. and Takaku, F., Relationship between an activated N-ras oncogene and chromosomal abnormality during leukemia progression from myelodysplastic syndrome. Blood, 1988, 71, 256. 12. Paquette, R. L., Landaw, E. M., Pierre, R. V., Kahan, .I., Lubbert, M., Lazcano, O., Lsaac, G., McCormick, F. and Koeffler, H. P., N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome. Blood, 1993, 82, 590. 13. Nakagawa, T., Saitoh, S., Imoto, S., Itoh, M., Tsutsumi, M., Hikiji, K., Nakamura, H., Matozaki, S., Ogawa, K. and Nakao, Y., Multiple point mutation of N-ras and K-ras oncogenes in myelodysplastic syndrome and acute myelogenous leukemia. Oncogene, 1992, 49, 114. 14. Neubauer, A., Shannon, K. and Liu, E., Mutations of the ras proto-oncogenes in childhood monosomy 7. Blood, 1991, 77, 594. 15. Vogelstein, B., Civin, C. L., Preisinger, A. C., Krisccher, J. P., Steuber. P., Ravindranath, Y., Weinstein, H., Elfferich, P. and Bos, J., RAS gene mutation in childhood acute myeloid leukemia: A Pediatric Oncology Group Study. Genes Chromosomes Cancer, 1990, 2, 159. 16. Lubbert, M., Mirro, J. J., Kitchingman, G., McCormick, F., Mertelsmann, R., Herrmann, F. and Koeffler, H. P., Prevalence of N-ras mutation in children with myelodysplastic syndromes and acute myeloid leukemia. Oncogene, 1992, 7, 263. 17. Kawamura, M., Kikuchi, A., Kobayashi, K., Hanada, R.,
Yamamoto. K., Horibe, K., Shikano, T., Ueda, K., Hayashi, K., Sekiya, T. and Hayashi, Y., Mutations of
701
the ~53 and ras genes in childhood t( 1;19)-acute lymphoblastic leukemia. Blood, 1995, 85, 2546. 18. Suzuki, Y., Orita, M., Shiraishi, M., Hayashi, K. and Sekiya, T., Detection of ras gene mutations in human lung cancers by single strand conformation polymorphism analysis of polymerase chain reaction product. Oncogene, 1990, 5, 1037. 19. Ohnishi, H., Kawamura, M., Ida, K., Sheng, X. M., Hanada, R., Nobori, T., Yamamori, S. and Hayashi, Y., Homozygous deletion of p 16/MTS 1 gene are frequent but mutation are infrequent in childhood T-cell acute lymphoblastic leukemias. Blood, 1995, 86, 1269. 20. Suzuki, Y., Sekiya, T. and Hayashi, K., Allele-specific polymerase chain reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants. Analytical Biochemistv, 1991, 192, 82. 21. Needleman, S. W., Kraus, M. H., Srivastava, S. K., Levine,
P. H. and Aaronson, S. A., High frequency of N-RAS activation in acute myelogenous leukemia. Blood, 1986, 67, 753. 22. Bos, J. L., Verlaan-de Vries, M., van der Eb, A. J., Janssen,
J. W., Delwel, R., Lowenberg, B. and Golly, L. P., Mutations in N-RAS predominate in acute myeloid leukemia. Blood, 1987, 69, 1237. 23. Browertt, P. J. and Norton, J. D., Analysis of RAS gene mutations and methylation state in human leukemias. Oncogene, 1989, 4, 1029. 24. Miyauchi, J., Asada, M., Sasaki, M., Tsunematsu, Y., Kojima, S. and Shuki Mizutani, S., Mutation of the N-ras gene in juvenile chronic myelogenous leukemia. Blood, 1994,83, 2248. 25. Clark, H. M., Yano, T., Sander, C., Jaffe, E. S. and Raffeld,
M., Mutation of the ras genes is a rare genetic event in the transformation histologic of follicular lymphoma. Leukemia, 1996, 10, 844. 26. Souyri, M. and Fleissner, E., Identification by transfection of transforming sequences in DNA of human T-cell leukemias. Proceedings of the National Academy of Science USA, 1983, 80. 6676.
Pergamon PII: SO145-2126(97)00036-Z
MUTATIONS OF THE RAS GENES IN CHILDHOOD ACUTE MYELOID LEUKEMIA, MYELODYSPLASTIC SYNDROME .AND JUVENILE CHRONIC MYELOCYTIC LEUKEMIA Xiao Ming Sheng”, Machiko Kawamura”, Hiroaki Ohnishi”, Kohmei Ida*, Ryoji Hanadat, Seiji Kojimal, Miyuki Kobayashi *, Fumio Bessho*, Masayoshi Yanagisawa* and Yasuhide Hayashi* *Department of Pediatrics, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113 Japan, TDivision of Hematology/Oncology, Saitama Children’s Medical Center, Saitama, Japan and fDivision
of Hematology/Oncology,
Nagoya First Red Cross Hospital, Nagoya, Japan
(Received 17 February 1997. Accepted 26 February 1997) Abstract-Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108 fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examined for mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 1 lq23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. All mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies. c 1997 Elsevier Science Ltd Key words: leukemia.
RAS gene, leukemia,
myelodysplastic
Introduction
syndrome,
juvenile
chronic
myelocytic
by a point mutation at one of the three codons 12, 13, or 61 [I]. Early studies of mutations of RAS genes have suggested that important differences in the frequencies of mutations of RAS genes may exist among tumors derived from different tissues and even among tumors from the same tissues if they are histopathologically different [ 11.For instance, studies in adults have showed that the incidence of the K-f?AS gene mutation was as high as 90% in pancreatic tumors and the incidence of the N-RAS gene mutation was about 30% in acute myeloid leukemia (AML), whereas RAS mutations are found only rarely in breast carcinoma [ 1,6]. Previous studies have shown that RAS mutations appear to be an early event in the development of some tumors, such as colon cancers and acute leukemias [6,7], but they seemed to be associated with later stages of carcinogenesis in other tumors, such as melanoma and multiple myeloma [ 1,8,9]. It has been reported that patients with the RAS gene mutations had a poorer
Alterations in the cellular genome affecting the expression or the function of genes controlling cell growth and differentiation are considered to be the main cause of cancer. RAS genes consist of a family of genes which are frequently found to be mutated in human tumors [I]. This family consists of three functional genes, H-, K-, and N-RAS genes [l-4], which encode proteins with molecular weights of 21,000 and have mutually high homology [5]. These proteins function as guanosine nucleotide triphosphate (GTP)-binding proteins (~21) with intrinsic GTPase activity. It is noted that certain structural alterations in RAS proteins lead to the loss of this GTPase activity. Thus, the proteins lose their ability to become inactivated and continue to stimulate growth or differentiation. These alterations can be brought about Correspondence to: Y. Hayashi, Department of Pediatrics, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan (Tel.: 81 3 3815 541 I ext. 3452; Fax: 81 3 3816 4108). 697
698
Xiao Ming Sheng et al.
prognosis than those without them [lo, 111, and that myelodysplastic syndrome (MDS) with Z&G gene mutation tends to transform into leukemia [l, 10-161. Studies concerning RAS gene mutations in leukemia have been concentrated on adult leukemia, and studies on childhood leukemia are few. Because childhood leukemia differs in many aspects from adult leukemia, it is important to know the exact frequency of RAS gene mutations to understand if RAS genes play any role in childhood leukemia. In this study, we investigated mutations of exons 1 and 2 of N-, K- and H-RAS genes in 12 AML cell lines and 67 fresh AML samples, 29 MDS, and 12 juvenile chronic myelogenous leukemias (JCML) in children, using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method and direct sequencing.
Table 1. Mutation of RAS genesin cell lines and fresh tumors, of childhood AML, MDS and JCML
Disease AML MDS JCML Total
Cell lines
Fresh tumors
Total
mutations/ samples(%)
mutations/
mutations/
samples(%)
samples(%)
2167 (3.0%) l/29 (3.4%) l/l2 (8.3%) 41108 (3.7%)
6/79 (7.6%) l/29 (3.4%) l/12 (8.3%) 81120 (6.7%)
4112 (33.3%) 4112 (33.3%)
PCR method Most RAS gene mutations have been demonstrated to be localized in exons 1 and 2. Therefore, fragments from regions carrying exon 1 or 2 of the K-RAS gene (Kl or K2), the H-RAS gene (Hl or H2) and the N-RAS gene (Nl or N2) were amplified by PCR. The sequences of primers used for PCR were the same as reported previously [ 18, 191. DNA samples (50 ng) in the mixture (5 ~1) with appropriate unlabelled primers and [a 32P] dCTP (20 p Ci per tube, 3000 Ci/mmol, Amersham), were subjected to 30 cycles of the reaction.
Materials and Methods Cell lines Twelve AML cell lines were analyzed in this study. They were obtained from the Japanese Cancer Research Resources Bank and cultured in RPM1 1640 medium suppletiented with 10% fetal bovine serum.
SSCP analysis After adding 45 pl of formamide denaturing dye mixture (95% formamide: 20 mM EDTA: 0.05% xylene cyanol: 0.05% bromophenol blue), the PCR mixture was heated at 80°C for 3 min, and then 1 ~1 of the diluted mixture was loaded on to one lane of a 5% polyacrylamide gel containing 45 mM Tris-borate (pH 8.3) and 4 mM EDTA. The gel contained 10% glycerol when it was specified. Electrophoresis was performed at 40 W for l-3 h with cooling. The gel was dried on filter paper and exposed to X-ray film.
Patient samples Bone marrow or peripheral blood samples were collected from 67 children with AML, 29 with MDS and 12 with JCML from various institutes after obtaining informed consent. Diagnosis of AML, JCML and MDS were based on the criteria of the FrenchAmerican-British (FAB) classification. In AML samples, the proportion of leukemic cells exceeded 80%. Mononuclear cells were separated on a Ficoll-Hypaque (Lymphoprep) density gradient, suspended in RPM1 medium containing 10% of dimethylsulfoxide, and kept at -80°C until use.
Direct sequencing of PCR-amplifiedfiagments Direct sequencing was performed as previously described [20] with some modifications [ 17, 191. A small piece of the gel corresponding to the abnormal band detected by SSCP analysis was removed, immersed in 20 ~1 of water, heated at 80°C for 15 min, and centrifuged. DNA in the extract (1 ~1) was subjected to
Preparation of DNA High-molecular weight DNA was prepared by the proteinase K-phenol-chlorofolm extraction method as previously reported [ 171.
Table 2. RAS gene mutations and nucleotide changes in AML cell lines Cell lines P39/TSU KG-l P3 l/FUF THP- 1 CTY-1
Type of AML
Type of RAS
No. of codon
AML AML AMOL AMOL AMOL
N N N N K
61 12 12 12 62
Gln: glutamine; Gly: glycine; Glu: glutamic acid; Leu: leucine; Asp: asparagine; Ser: serine.
Nucleotide and amino acid changes CAA(Gln)+CTA(Leu) GGT(Gly)-+GAT(Asp) GGT(Gly)+GAT(Asp) GGT(Gly)+AGT(Ser) GAG(Glu) +GAA(Glu)
699
Mutations of RAS genes in childhood malignancies
Table 3. RAS gene mutations and nucleotide changes in childhood fresh tumors of AML, MDS and JCML Diseases AMOL AMOL MDS JCML
Ages*/sext
Type of RAS
No. of codon
5 months/male 10 years/male 5 years/female 1 year/male
K K N N
61 12 13 12
Nucleotide and amino acid changes
Survival time
CAA(Gln)-+CAC(His) GGT(Gly)-+GAT(Asp) GGT(Gly)+GTT(Val) GGT(Gly)+GTT(Val)
16 day 5 months 27 months + 84 months +
*m: month, y: year. **M: male, F: female. +: Alive
30 cycles of PCR, and the products were purified with Microcon 100 (Amicon). The DNA fragments thus obtained were sequenced by the dideoxy chain termination method using 5’-32P labelled primers and Taq DNA polymerase (dsDNA Cycle Sequencing System, Gibco BRL, Gaithersburg, MD, U.S.A.). Primers for sequencing were the same as those used for PCR-SSCP. Results Of the 12 AML cell lines, four were found to contain mutation in N-RAS genes and one in the K-RAS gene (Tables 1 and 2). Nucleotide sequence analysis revealed that mutations of the N-RAS gene in four cell lines were missense mutations and mutation of the K-RAS gene in one cell line was a silent mutation (Tables 1 and 2). The cell line P-39/TSU contained a CAA to CTA transversion in the second base of codon 61 of the N-RAS gene, resulting in the substitution of leucine for glutamine. The cell lines KG- 1 and P3 1-FUF contained the GGT to GAT transition in the second base of codon 12 of the NRAS gene, causing the substitution of asparagine for
a. SSCP
glycine. The cell line THP-1 contained a GGT to AGT transition in the first base of codon 12 of the N-RAS gene, causing the substitution of serine for glycine. The cell line CTY-1 had a GAG to GAA silent mutation in the 3rd base of codon 62 of the K-RAS gene. DNA samples from 67 children with AML were screened for point mutations at exons 1 and 2 of N-, Kand H&IS genes (Tables 1 and 3). Two of them were found to contain a K-RAS gene mutation. One had mutation of a GGT to GAT transition in the second base of codon 12 of the K-RAS gene, causing the substitution of asparagine for glycine, whereas another had a CAA to CAC transversion in the 3rd base of codon 61 of the KRAS gene, causing the substitution of histamine for glutamate. These two patients were diagnosed as having AMOL-MS and had 1lq23 translocations. DNA sample from 12 JCML patients and 29 MDS patients were screened for point mutations in exons 1 and 2 of the N-, K- and H-RAS genes (Tables 1 and 3). One of the 12 JCML samples and 1 of the 29 MDS samples contained a GGT to GTT transversion at codon 12 and 13 of the N-RAS gene, respectively (Fig. 1).
b. I
sequence WT patient r-in TCGA TCGA
Fig. 1. (a) SSCP analysis of N-RAS gene in MDS patient. The arrow points to an abnormal band shift of a MDS patient. (b) Mutation of the N-RAS gene identified in a MDS sample. Direct sequencing of the PCR products amplified from the abnormally shifted band demonstrating a mutation of GGT(Gly)+GTT(Val) at codon 13 in N-RAS gene. WT, wild type; T, thymine; C, cytosine; G, guanine; A. adenine.
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Discussion The technique to examine RAS gene point mutations has been mainly performed by the synthetic dot blot hybridization method. Using this method, frequency of the i?AS gene mutation of adult leukemia was high (i.e. 27% in AML, 30% in MDS) [I, lO-13,21-231. In childhood leukemia only few studies concerning RAS gene mutations have been reported [ 14-161. In these studies, the synthetic dot blot hybridization method was also used, and the frequency was also relatively high [ 1, 151. The technique of the synthetic dot blot hybridization method is known to give false positive results at high frequencies and can examine only the confined codons. In contrast, the SSCP method give less false positives and can simultaneously cover mutations of all codons [20]. This method is also faster, simpler and more accurate. In the previous studies on RAS mutations in other tumors, we obtained good results with this method combined with direct sequencing [ 181. Results of direct sequencing for normal bands in SSCP showed the wild type of the RAS gene, and all abnormal bands in SSCP showed changes of bases. Therefore, the findings of our study could be considered to be true incidences of RAS gene mutations, although frequencies of RAS gene mutations in AML, JCML and MDS in children was very low in this study (3.0%, 8.3%, 3.4%, respectively). Relatively high incidence of N-RAS gene mutation has been reported in JCML [24], which was not compatible with our results. The difference between them is unknown. The frequency of the mutation in our study was also lower than that of mutation for adults which was obtained with the same PCR-SSCP technique [I, 131. Thus, the frequency of RAS mutations in AML and MDS for children may be lower than that for adults. One possible reason for the difference in frequency of RAS gene mutations between children and adults may be due to the difference in leukemogenesis. That is, children are less exposed to poisonous environments than adults so they have less chance for mutation than adults. Notably, a recent study revealed that mutation of the RAS genes was rare in follicular lymphoma, using the PCR-SSCP method [25]. Frequency of RAS gene mutations may depend on the technique used. In this study, observed mutations were limited to Nand K-RAS genes, and positions of mutation were at codons, 12, 13 and 61, as reported in adults. This means that the role of mutation in leukemogenesis is not different between children and adults. These three positions are related to the domain of GTPase activity. If any of these three positions suffer from mutations, activity of GTPase will be affected, and its activity will decrease. As a result, activated p21 loses its ability to become inactivated and continue to stimulate growth or
differentiation. K-RAS gene mutations were found in two of 67 fresh AML samples, although N-RAS gene mutations were usually found in hematological malignancies. Interestingly, these two patients had AMOLM5 and llq23 translocations. Frequency of the mutation in 12 cell lines was apparently higher than that of fresh AML, being compatible with the previous studies in other tumors [ 1, 17,261. There have been different studies concerning the timing of RAS gene mutations in the course of the development of cancer. This timing seems to depend on the type of tumors. In leukemia, it was demonstrated to occur in the early stage of leukemogenesis, whereas in melanoma and myeloma, it appears to occur in a later stage. Our findings supported the view that in AML, mutation of the RAS gene occurs in the later stage of the disease instead of the early stage. Further larger scale investigations on AML at diagnosis, at relapse, and cell lines is necessary to clarify this problem. In conclusion, low frequency of RAS gene mutations in this study suggested that the mutation of the RAS gene does not play an important role in the development of childhood myeloid malignancies. Acknowledgements-The authors wish to thank clinicians for providing the samples and clinical data on patients. They would also like to thank Mrs Shyoko Soma for technical assistance. Supported in part by a Grant-in Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and a Grant-in Aid for scientific Research from The Ministry of Education, Science of Culture (044545668, 04454276) Japan.
References 1. Bos, J. L., Ras oncogenes in human cancer. A review. Cancer Research, 1989, 49, 4682. 2. Harvey, J. J., An unidentified virus which causes the rapid production of tumours in mice. Nature, 1964, 204, 1104. 3. Kirsten, W. H. and Mayer, L. A., Morphologic responses to a murine erythroblastosis virus. Journal of the National Cancer Institute, 1967, 39, 3 11. 4. Shimizu, K., Goldfarb, M., Perucho, M. and Wigler, M., Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line. Proceedings of the National Academy of Science USA, 1983, 80, 383. 5. Taparowsky, E., Shimizu, K., Goldfarb, M. and Wigler, M., Structure and activation of the human N-ras gene. Cell, 1983, 34, 581. 6. Almoguera, C., Shibata, D., For-rester, K., Martin, J., Amheim, N. and Perucho, M., Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell, 1988, 53, 549. 7. BOS, J. L., Fearon, E. F., Hamilton, S. R., Verlaan-de Vriws, M., van Boom, L. K., van der Eb, A. J. and Vogelstein, B., Prevalence of RAS gene mutations in human colorectal cancers. Nature, 1987, 327, 293. 8. Portier, M., Moles, J. P., Mazards, G. R., Jeanteur, P.,
Mutations of RAS genes in childhood malignancies Bataille, R., Klein, B. and Theillet, C., p.53 and RAS gene mutations in multiple myeloma. Oncogene, 1992, 7, 2539. 9. Bell, N. J., Yohn, J. J., Morelli, J. G., Norris, D. A., Golitz, L. E. and Hoeffler, J. P., RAS mutations in human melanoma: a marker of malignant progression. Journal of Investiaative Dermatolonv, 1994. 102, 285. IO. Hirai, H., Kobayashi, Y, Mano, H., Hagiwara, K., Maru, Y., Omone, M., Mizuguchi, H., Nishida, J. and Takaku, F., A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome. Nature, 1987, 327, 430. 11. Hirai, H., Okada, M., Mizoguchi, H., Mano, H., Kobayashi, Y., Nishida, J. and Takaku, F., Relationship between an activated N-ras oncogene and chromosomal abnormality during leukemia progression from myelodysplastic syndrome. Blood, 1988, 71, 256. 12. Paquette, R. L., Landaw, E. M., Pierre, R. V., Kahan, .I., Lubbert, M., Lazcano, O., Lsaac, G., McCormick, F. and Koeffler, H. P., N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome. Blood, 1993, 82, 590. 13. Nakagawa, T., Saitoh, S., Imoto, S., Itoh, M., Tsutsumi, M., Hikiji, K., Nakamura, H., Matozaki, S., Ogawa, K. and Nakao, Y., Multiple point mutation of N-ras and K-ras oncogenes in myelodysplastic syndrome and acute myelogenous leukemia. Oncogene, 1992, 49, 114. 14. Neubauer, A., Shannon, K. and Liu, E., Mutations of the ras proto-oncogenes in childhood monosomy 7. Blood, 1991, 77, 594. 15. Vogelstein, B., Civin, C. L., Preisinger, A. C., Krisccher, J. P., Steuber. P., Ravindranath, Y., Weinstein, H., Elfferich, P. and Bos, J., RAS gene mutation in childhood acute myeloid leukemia: A Pediatric Oncology Group Study. Genes Chromosomes Cancer, 1990, 2, 159. 16. Lubbert, M., Mirro, J. J., Kitchingman, G., McCormick, F., Mertelsmann, R., Herrmann, F. and Koeffler, H. P., Prevalence of N-ras mutation in children with myelodysplastic syndromes and acute myeloid leukemia. Oncogene, 1992, 7, 263. 17. Kawamura, M., Kikuchi, A., Kobayashi, K., Hanada, R.,
Yamamoto. K., Horibe, K., Shikano, T., Ueda, K., Hayashi, K., Sekiya, T. and Hayashi, Y., Mutations of
701
the ~53 and ras genes in childhood t( 1;19)-acute lymphoblastic leukemia. Blood, 1995, 85, 2546. 18. Suzuki, Y., Orita, M., Shiraishi, M., Hayashi, K. and Sekiya, T., Detection of ras gene mutations in human lung cancers by single strand conformation polymorphism analysis of polymerase chain reaction product. Oncogene, 1990, 5, 1037. 19. Ohnishi, H., Kawamura, M., Ida, K., Sheng, X. M., Hanada, R., Nobori, T., Yamamori, S. and Hayashi, Y., Homozygous deletion of p 16/MTS 1 gene are frequent but mutation are infrequent in childhood T-cell acute lymphoblastic leukemias. Blood, 1995, 86, 1269. 20. Suzuki, Y., Sekiya, T. and Hayashi, K., Allele-specific polymerase chain reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants. Analytical Biochemistv, 1991, 192, 82. 21. Needleman, S. W., Kraus, M. H., Srivastava, S. K., Levine,
P. H. and Aaronson, S. A., High frequency of N-RAS activation in acute myelogenous leukemia. Blood, 1986, 67, 753. 22. Bos, J. L., Verlaan-de Vries, M., van der Eb, A. J., Janssen,
J. W., Delwel, R., Lowenberg, B. and Golly, L. P., Mutations in N-RAS predominate in acute myeloid leukemia. Blood, 1987, 69, 1237. 23. Browertt, P. J. and Norton, J. D., Analysis of RAS gene mutations and methylation state in human leukemias. Oncogene, 1989, 4, 1029. 24. Miyauchi, J., Asada, M., Sasaki, M., Tsunematsu, Y., Kojima, S. and Shuki Mizutani, S., Mutation of the N-ras gene in juvenile chronic myelogenous leukemia. Blood, 1994,83, 2248. 25. Clark, H. M., Yano, T., Sander, C., Jaffe, E. S. and Raffeld,
M., Mutation of the ras genes is a rare genetic event in the transformation histologic of follicular lymphoma. Leukemia, 1996, 10, 844. 26. Souyri, M. and Fleissner, E., Identification by transfection of transforming sequences in DNA of human T-cell leukemias. Proceedings of the National Academy of Science USA, 1983, 80. 6676.