Stem Cells, Vol. 14, No. 6, 725-729,
November 1996
© 1996 AlphaMed Press
Mutations and Expression of the ras Family Genes in Leukemias
Dora M. Gougopouloua,b,
Hippokratis Kiarisa,b,
Mary Ergazakia,b,
Nikolaos I. Anagnostopoulosc,
Vasiliki Grigorakic,
Demetrios A. Spandidosa,b
a Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece;
b Medical School, University of Crete, Heraklion, Greece;
c Department of Haematology, General Hospital of Athens, Athens, Greece.
Key Words. H-ras • K-ras • N-ras • Mutation • Expression • Leukemia
Professor Dr. D.A. Spandidos, University of Crete, Faculty of Medicine, PO Box 1393, Heraklion 71409, Crete, Greece.
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Abstract
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The levels of expression and the incidence of codon 12 point mutation of the ras family genes were studied in 18 cases of leukemia, seven with acute myeloblastic leukemia (AML), three with acute lymphoblastic leukemia (ALL), four cases with chronic myelogenic leukemia (CML) and four cases with chronic lymphocytic leukemia (CLL). Elevated expression of the ras genes was found for 39%, 61% and 67% of the specimens for the H-ras, K-ras and N-ras, respectively. A trend was found between the overexpression of the N-ras gene and the acute leukemias: all 10 acute leukemias exhibited overexpression of the N-ras gene, while only two of the CML cases, both in blastic crisis, showed elevated levels of the N-ras gene. Codon 12 point mutations at the N-ras gene were found in two of seven cases (28%) with AML and one of the four cases (25%) with CML. The only K-ras codon 12 point mutation was found in a patient with CLL. No mutations were found in the codon 12 of H-ras. Our data suggest that apart from the point mutations, overexpression of the ras family genes is important in the development of the disease.
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Introduction
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Molecular alterations targeting to the activation of proto-oncogenes and the inactivation of tumor suppressor genes (TSGs) play a key role in the development of multistage carcinogenesis [1]. The members of the ras family of genes (H-, K- and N-ras) are involved in a wide range of human tumors, including pancreatic, lung and colorectal cancer [2]. Point mutations, usually at codons 12, 13 and 61, alter the GTPase activity of the Ras protein and provide the cells bearing this alteration, a tumorigenic potential [3]. Although the majority of human solid tumors harbor a K-ras point mutation, in leukemias approximately 30% of the patients harbor an activated N-ras oncogene at codon 12. In several cases the presence of N-ras point mutations has been associated with particular clinicopathological parameters [4, 5].
Although point mutations represent an important mechanism of activation for the ras genes, they are not the only implication of these genes in carcinogenesis. In vitro experiments showed that apart from the mutant form, the overproduction of the normal Ras protein is sufficient to confer a transforming potential in cultured cells [6]. Furthermore, experiments in human tumor specimens revealed that elevated levels of ras encoded RNA and protein is a consistent feature in a wide range of human cancers [7]. In addition, elevated levels of ras mRNA have been associated with particular clinicopathological parameters such as favorable prognosis in head and neck cancer [8], suggesting an important role for the regulation of the ras family genes in human tumors. Interestingly, in several cases overexpression was not accompanied by point mutations, pointing to an alternative implication of the ras genes in carcinogenesis apart from structural alterations [9].
Although several studies have recognized the important role of the ras family genes and N-ras in particular, in leukemias on the frequency of point mutations. [10-13], the data on the level of expression are limited. In the present study we investigated the implication of the ras family genes in a set of myeloblastic and lymphocytic leukemias, of both the acute and the chronic phase (acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenic leukemia (CML) and chronic lymphocytic leukemia (CLL), respectively). The analysis involved the assessment of both codon 12 point mutations and the levels of expression at the H-, K- and N-ras genes. Our results suggest that apart from the mutations, overexpression of the ras family genes is a frequent event, and regardless of the presence of mutations, plays an important role in the development of the disease.
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Materials and Methods
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Specimens
The diagnosis of leukemia was made with standard bone marrow and blood smears and immunophenotyping data. The 10 patients of this study were diagnosed as AML or ALL according to the FAB classification. None of the patients had received therapy. Four patients fulfilled the criteria for the diagnosis of CML, two of them in chronic phase (CP) and two in blastic crisis (BC) of the disease. Four patients with CLL were included. All had >20 x 10–9/L monoclonal CD19+CD5+ cells in peripheral blood and were classified according to the International Recommendation. Peripheral blood from 18 patients and two healthy donors was collected in tubes with EDTA and mononuclear cells were separated after Ficoll-Hypaque density gradient centrifugation and stored immediately at -70°C. Total DNA and RNA was extracted using TRIzol Reagent (GIBCO BRL; Grand Island, NY) following the manufacturer's instructions.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RNA Quantitation
Two hundred ng of total RNA were reverse transcribed in a 50 µL reaction (10 mM Tris HCl, Ph 8.3; 50 mM KCl; l mM MnCl2; 200 µM dNTPs; 200 ng antisense primer; and 2.5 U Tth polymerase) for 15 min at 70° C. PCR amplification of cDNA was performed by adding 50 µL of buffer containing 75 mM Tris-HCl, pH 9.0; 20 mM (NH4)2SO4; 1.5 mM MgCl2; 0.01% Tween-20; 0.75 mM EDTA; and 200 ng sense primer. The oligonucleotide primers used and the subsequent PCR products have been previously described [14].
The PCR programs consisted of 1 min steps at 95°C, 60°C and 72°C for 28 cycles. Preliminary experiments had revealed the conditions in which the amplification reaction remained in the exponential phase (data not shown) and thus the results could be used for RNA quantitation. Ten µL of the PCR product were electrophoresed through a 2% agarose gel, and the intensity of the bands was analyzed by a UVP image analysis system
Detection of ras Mutations at Codon 12 by Restriction Fragment Length Polymorphism (RFLP)
PCR amplification of the ras genes surrounding codon 12 was performed as described above. Thirty µL of the PCR product were digested overnight with 20 U of the restriction endonucleases MspI (H-ras) and BstNI (K-ras and N-ras) in conditions recommended by the suppliers, electrophoresed in a 2% agarose gel and stained with ethidium bromide.
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Results
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Codon 12 Point Mutations of the ras Family Genes
The incidence of codon 12 point mutations of the H-, K- and N-ras genes was assessed in 18 cases of leukemia including AML, CML, CLL and ALL. N-ras mutations were found in two of seven (28%) cases with AML and one of four (25%) cases with CML, both in blastic crisis (Fig. 1
), while no mutations were found in the four CLL and the three ALL cases tested. The only K-ras codon 12 point mutation was found in a patient with CLL. No mutations were found in the codon 12 of the H-ras gene.
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Figure 1. Detection of N-ras codon 12 point mutations by a combined PCR-RFLP assay. Lanes 1, 5 and 6 correspond to mutant specimens and lanes 2, 3, 4 and 7 to normal specimens. Lane 8 corresponds to undigested PCR product.
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Differential Expression of the ras Family Genes
The levels of expression of the H-ras, K-ras and N-ras proto-oncogenes were assessed at the level of RNA in 18 patients with AML, CML, CLL and ALL. RNA preparations from two normal individuals were used as controls for the evaluation of the background levels of ras expression. The levels of differential expression for the ras family genes were expressed as the ratio of the expression in each specimen versus the average expressional levels of the normal specimens. Amplification of the ß-actin transcript was performed in each RNA preparation in order to assess the quality and the quantity of the RNA template (Fig. 2). Cancer cells have a cell-kinetic system in which the p21ras signal transduction pathway is induced. Thus, we arbitrarily considered as overexpression levels of expression higher than 1.5-fold versus the RNA levels of the normal specimens [8,14]. The aforementioned manipulation revealed that H-ras was overexpressed in seven (39%), K-ras in 11 (61%) and N-ras in 12 (67%) specimens (Table 1). Association was found between overexpression of the N-ras proto-oncogene and the type of the disease: all 10 specimens classified as acute leukemias (either myeloblastic or lymphoblastic) exhibited overexpression of the N-ras gene, while two cases of CML in BC showed higher than 1.5-fold levels of N-ras expression, none of two cases of CML in CP and none of the three ALL cases exhibited overexpression of the N-ras. However, the precise figures of these results should be considered with caution since the absence of "homogeneous" cell population makes the comparison difficult.
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Figure 2. Expression of the ras family genes in leukemias. K-ras was stained with silver while N-ras, H-ras and ß-actin were stained by ethidium bromide. N indicates a pool of two normal specimens, used in order to assess the background levels of ras expression.
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Table 1. Mutations and expression of the ras family genes in 18 specimens from patients with AML, ALL, CML and CLL
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Discussion
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In the present study 18 patients with AML, ALL, CML or CLL were investigated for aberrant expression and codon 12 point mutations of the H-, K- and N-ras genes. For the detection of codon 12 point mutations a sensitive PCR-RFLP assay was employed. N-ras mutations were found in two patients with AML and one patient with CML. The only K-ras mutation was found in a CLL patient. These results confirm previous investigations that recognized a higher prevalence of N-ras mutations in patients with AML and also a detectable incidence of K-ras mutations [2].
Furthermore, in the present study we assessed the levels of expression of the ras family genes at the RNA level, in the same panel of specimens by quantitative RT-PCR. Fifteen out of 18 specimens tested exhibited overexpression of at least one member of the ras family. Similar data were obtained in our previous investigation in laryngeal cancer [14]. The pattern of expression of the ras genes was diverse. Overexpression of the N-ras gene was found in 12, K-ras in 11 and the H-ras gene in eight cases. Interestingly, all 10 cases with acute myeloblastic or lymphocytic leukemia exhibited overexpression of the N-ras gene, while only two of the nine chronic leukemia cases exhibited elevated levels of the N-ras RNA. These results suggest that the transcriptional activation of the N-ras gene may play an important role during the transition of the acute phase of the disease. It is also interesting that the same member of the ras family is frequently activated by point mutations in AML cases. Apart from the mutational activation, the present study suggests that N-ras is also implicated in the development of the disease in a quantitative manner, by deregulation of its expression at the RNA level. We may postulate that among the ras family genes, N-ras is overexpressed in a particular subset of the cases and when a mutation occurs, it provides a proliferative advantage to the cells with the aforementioned aberration. This creates a predictable hypothesis: if indeed overexpression of the N-ras gene is important for the acquisition of the malignant phenotype in association with an N-ras mutation, we should expect in this case to detect elevated levels of the mutant N-ras transcript as compared to its normal counterparts, in the heterozygous patients. A similar phenomenon has been described in colon cancer for the K-ras gene which plays an important role in the development of the disease [15]. Unfortunately in the present study, due to the presence of a high percentage of normal cells in the peripheral blood, this experiment could not be performed with accuracy. A subset of the cases did not show overexpression of any of the ras genes. It might be postulated that an alternative pathway is activated which does not require ras activation.
It could be argued that the importance of ras overexpression is decreased by the fact that tumor cells may exhibit an induction of the genes that participate in a signal transduction pathway. However, ras genes did not exhibit a uniform pattern of overexpression, suggesting that the transcriptional activation of specific ras genes in particular specimens may have importance in the development of the disease. Furthermore, it suggests that although ras genes are considered housekeeping genes, factors that may result in their transcriptional activation should be considered as a possibility. The latter, apart from the transactivation of ras genes by member-specific factors, may also include mutations within the regulatory regions of these genes or gene amplification.
This is the first report to our knowledge on the differential expression of the ras family genes in leukemias. Although the number of patients tested in the present study is limited to support a strong association with particular clinicopathological parameters, it provides strong evidence on the implication of the N-ras gene in leukemias. We postulate that N-ras is not only involved in the development of the disease at the level of point mutations, but also at the level of overexpression. Furthermore, the present study points to the existence of factors that may differentially activate ras family genes with important consequences in carcinogenesis. More in-depth studies are required in order to establish the precise role of the ras family genes in the development of the disease.
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Footnotes
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Provisionally accepted July 5, 1996.
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References
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Received May 10, 1996;
accepted for publication August 12, 1996.
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Abstract
Introduction
