Mutations of the p53 Gene in Lymphoid Leukemia

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Mutations of the p53 Gene in Lymphoid Leukemia By Koichi Sugimoto, Hideo Toyoshima, Ryuichi Sakai, Kiyoshi Miyagawa, Koichi Hagiwara, Hisamaru Hirai,

Fuyuki Ishikawa, and Fumimaro Takaku p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase-polymerase chain reaction, single strand conformationpolymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemiaswere examined and two with acute lymphoblastic leukemia and one with Waldenstrem’s macroglobulinemia were identified to have mutations in the coding region of the

p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore,expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias. 6 1991 by The American Society of Hematology.

T

coding region of the p53 gene in two patients with Philadelphia chromosome (Ph’) negative acute lymphoblastic leukemia (ALL) and one patient with Waldenstrom’s macroglobulinemia. Wild-type p53 mRNA has not been detected in leukemia cells from these three patients.

HE NUCLEAR PHOSPHOPROTEIN p53 was first

detected because it formed a tight complex with SV40 large-T antigen and was coimmunoprecipitated by anti-T antibodies from extracts of SV40-transformed cells.’ Originally considered to be an oncogene, several lines of research indicated that the wild-type p53 gene actually functions as a tumor suppressor gene? For example, transfection of the wild-type p53 cDNA inhibits the ability of adenovirus E1A or mutant p53 gene plus activated ras oncogene to transform primary rat embryo fibroblast^.^ The human p53 gene is located on chromosome 17p, the allelic loss of which is a frequent feature of various human malignancies, including colorectal cancer, small cell lung cancer, breast cancer, and astrocytoma.4-’The allelic loss of chromosome 17p is considered to occur not as a sole genetic alteration but as one of multiple changes involved in the For example, progression of some human malignan~ies?~.~ in case of colorectal tumors, some genetic alterations, such as activation of ras oncogenes, were often shown in both benign adenomas and carcinomas, whereas allelic loss of 17p was usually detected only in carcinomas? Interestingly, in tumors with such allelic deletion of 17p, the p53 gene on the remaining (nondeleted) allele was often shown to contain mutations?-” Therefore, these observations suggest that the loss of normal p53 function is involved in the progression to the malignant state in various human tumors. As for hematologic malignancies, several groups showed alterations of the p53 gene in blast crisis of chronic myelogenous leukemia (CML).12-14 The observation suggests that alteration of the p53 may be involved in the progression of CML from chronic phase to blast crisis. These findings led us to the idea that alterations of p53 may be a frequent feature in acute leukemias, and urged us to study mutations of the p53 gene in various types of human leukemias. In the present report, we have analyzed 14 cases of human leukemias for mutations of the p53 gene using reverse transcriptase-polymerase chain reaction (RT-PCR) method and single strand conformation polymorphism (SSCP) analysis.*5The RT-PCWSSCP analysis can detect even a point mutation in the coding region of the p53 gene, and, moreover, can make it clear whether the wild-type p53 gene is expressed or not. We have detected mutations in the Blood, Vo177, No6 (March 15),1991: pp 1153-1156

MATERIALS AND METHODS Patients. Bone marrow samples from 14 patients, including six patients with acute myelogenous leukemia (AML), four with Ph’-negative ALL, three with Ph’-positive ALL, and one with Waldenstrom’s macroglobulinemia, were collected after informed consent. The percentages of leukemic cells in the bone marrow samples were more than 80% in seven cases, 50% to 80% in two cases, and 20% to 50% in five cases. RT-PCR method. Primers used in this study were prepared by the 381A DNA synthesizer (Applied Biosystems, Foster City, CA). Using the nucleotide numbers of the sequence published by Zakut-Houri et the sense primners were: ST1, nucleotide (nt) 361 to 380; SN2, nt 373 to 392; and SC3, nt 603 to 622. The antisense primers were: AST1, nt 1,OOO to 981; ASN2, nt 777 to 758; and ASC3, nt 980 to 961. The RT-PCR was performed as follows. Complementary DNA was synthesized from 1 kg of total cellular RNA from bone marrow mononuclear cells using 100 ng of 3’-primer ASTl and 200 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD) in a 25 kL solution containing 200 kmol/L each of all four dNTPs, 80 U of RNase inhibitor, 50 mmol/L Tris-HC1 (pH 8.3), 75 mmol/L KCI, 10 mmol/L dithiothreitol (D’IT), 3 mmol/L MgCI,. The reaction was allowed to proceed for 60 minutes at 37°C and used as substrate for the PCR. To the RT reaction solution, 25 kL of a solution containing 250 FmoVL each

From The Third Department of Intemal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Submitted September 5,1990; accepted December 31,1990. Supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the M i n k e of Education, Science and Culture in Japan. Address reprint requests to Koichi Sugimoto, MD, The Third Department of Intemal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section I734 sole& to indicate thD fact. 6 1991 by The American Society of Hematology. 0006-4971I91/7706-O038$3.0010 1153


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of all four dNTFk, 100 ng of 5’-primer ST1, 10 mmol/L Tris-HCI (pH 8.3), 50 mmol/L KCI, and 3 U of Tag polymerase (Perkin Elmer-Cetus, Norwalk, CT)was added. PCR was performed for 25 cycles of 94°C (1 minute), 50°C (1 minute) and 72°C (2 minutes). SSCP analysk. For the SSCP analysis, 5’- and 3’-side p53 cDNA fragments were generated using 1 pL (one-fiftieth) of RT-PCR reaction solution by second PCR of 30 cycles (94°C 1 minute, 50°C 1 minute, and 72°C 2 minutes). The 5’-ends of the primers used in the second PCR were labeled with [y-”P] adenosine triphosphate (ATP) and T4 polynucleotide kinase (Takara, Kyoto, Japan). Primers SN2 (100 ng) and ASN2 (100 ng) were used to amplify the 5’-side fragment, and primers SC3 (100 ng) and ASC3 (100 ng) were for the 3’-side fragment. The reaction solution (25 pL) was mixed with 450 KL of 95% formamide, 20 mmoVL EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol, and heated at 94°C for 3 minutes. The mixture was quickly chilled on ice and 2 WLwas loaded onto 5% polyacrylamidegels containing 90 mmol/L Tris-borate pH 8.3, and 4 mmol/L EDTA with or without 10% gylcerol. Electrophoresiswas performed at room temperature and at 4°C. Sequencing. For direct sequencing, 1 KL of RT-PCR reaction solution was used in 50-cycle second PCR with a 10- to 20-fold reduction of one of the primers. The resulting single-stranded DNAwas purified and sequenced by the dideoxy chain termination method. Sequencing primers were SS4 for nt 622 to 641 (sense) and ASS4 for nt 962 to 943 (antisense). For conventional sequence analysis, the 5’- and 3‘-side p53 cDNA fragments were cloned and sequenced by the dideoxy chain termination method. RESULTS

RT-PCRISSCP analysk. The positions of p53 gene mutations previously reported are clustered in four regions spanning about 650 bp of the p53 coding sequence that are highly conserved among wide species (regions A, B, C, and D in Fig l).” Therefore, the RT-PCR method was used to amplify the 650-bp coding sequence. Because SSCP analysis can detect mutations effectively in DNA fragments less than 400 bp, the 650-bp RT-PCR product was analyzed in two fragments (5’- and 3‘-side fragments generated by the second PCR). The DNA fragment was denatured into separate single strands and each strand folded back on itself in a unique conformation during electrophoresis under nondenaturing conditions. Even a single nucleotide substitution can usually be detected, because it alters the three-dimensionalconformation of the single-strandedDNA fragment and, therefore, its electrophoretic mobility.” Figure 2 shows the SSCP analysis of 3’- and 5’-side p53 cDNA fragments performed at room temperature in the A

CODON

I

1

B

I .

four highly conserved regions

I

100

C

D

I

I

I

I

200

300

I

393

Y-side SSCP fragment 3’-side SSCP fragment

Fig 1. Strategiesfor amplificationof p53 gene sequences. 5‘- and 3’-side p53 cDNA fragments amplified by second PCR using the 850-bp RT-PCR product as a template are indicatedby the black bars. Positions of the primersfor PCR were designed to include four highly conserved regions in the p53 coding sequence indicated by the black rectangles marked A through D.

B

A

t? PN

t

Pt. 1

Kt t Pt. 3

Pt. 2

Fig 2. SSCP analysis of the 5’- and 3‘-side p53 cDNAfragments.(A) 3’-side SSCP. The lane at the left end (P) shows positive control SW480 ( C G F + CAT and C C P + TCC). The next lane IN) shows negativecontrol (normal mononuclearcells of a volunteer). The arrow points to the aberrantly migrating fragments of patient 1 (Pt 1). (B) 5’-side SSCP. The lane at the left end (P) shows positive control T47D (ClT’= + IT). The arrows point to the aberrantly migrating fragments of patient 3 (Pt 3) and patient 2 (Pt 2).

presence of 10% glycerol. Positive and negative control fragments are also included (P and N, respectively). One patient for the 3’-side (patient 1in Fig 2A) and two patients for the 5‘-side SSCP analysis (patients 2 and 3 in Fig 2B) can be identified to have aberrantly migrating fragments. All three samples lacked normally migrating fragments, suggesting that the leukemic cells of these patients did not express wild-type p53 mRNA. The SSCP analysis under all of the four conditions (at room temperature or at 4°C in the presence or absence of 10% glycerol) detected aberrantly migrating fragments in the three patients. We also performed the 5’- and 3’-side SSCP analysis on 32 healthy volunteers at room temperature in the presence of 10% glycerol and found no aberrantly migrating fragments (data not shown). Sequencing analysk. The 3’-side p53 cDNA fragment of patient 1 was directly sequenced to determine the nucleotide change responsible for the mobility shift in SSCP analysis. If both wild-type and mutant p53 mRNAs are expressed, we can obtain sequence ladders of both wildtype and mutant p53 mRNA with different intensities depending on their relative amount. The analyzed sequence exactly matched the published except for a G to C transition at nt 826 (Fig 3, panel 2), which results in a change of the encoded amino acid from alanine to proline at codon 276. A faint band at nt 826 of the G lane in this panel may reflect the wild-type p53 mRNA from the residual normal cells, which accounted for 20% of the marrow nuclear cells. For patients 2 and 3, we cloned the 5’-side p53 cDNA fragment into a Mlfderived vectors and sequenced four independent clones for each fragment to avoid random errors generated by the PCR. Patient 2 was shown to have a triplet deletion (CCX), which removes


1155

~ 5 GENE 3 IN LYMPHOID LEUKEMIA

ACGT

AGCT

AGCT

M

Fig 3. Examples of sequencing reactions demonstrating p53 gene mutations. Direct sequencing showed that cDNA of the patient 1 contained a point mutation at codon 276 (CCC, panel 2). instead of the wild-type sequence (GCC) found in the cDNA from normal mononuclear cells of a volunteer (panel 1). Nucleotide sequencing of the p53 cDNA from patient 3 showed that a T nucleotide was inserted between nucleotides 714 and 715 (panel 4) compared with the correspondingregionof the normal control p53 cDNA (panel 3).

v

I’

2

proline at condon 190 (data not shown). In patient 3, a single nucleotide (T) insertion was identified between nucleotides 714 and 715, and the inserted T with the following AA nucleotides generated a termination signal at codon 239 (Fig 3, panel 4). Ten normally migrating 5’-or 3’-side p53 cDNA fragments from other patients were sequenced and confirmed to contain no mutations (data not shown). DISCUSSION

We have identified three mutations of the p53 gene in two cases of Ph’-negative ALL (pre-B cell and null cell types) and one case of Waldenstrom’s macroglobulinemia by RT-PCWSSCP analysis and sequencing (Table 1). Furthermore, our results suggested the possibility of the absence of wild-type p53 mRNA expression in the leukemia cells of the three patients. Because the p53 gene is now considered to be a tumor suppressor gene, it is important to show inactivation of the gene in both alleles to demonstrate that the p53 gene has a role in leukemogenesis. RT-PCWSSCP analysis is useful for this purpose because it can separate mRNAs from both alleles unless they are identical. However, if there is far less mutant p53 mRNA than that of wild type in the bone marrow specimen, the SSCP analysis cannot detect the aberrantly migrating fragments. We chose 14 cases containing leukemic cells more than 20% of bone marrow cells, because PCR-SSCP method was reported to detect point mutations of about 10% of total DNA.18 To optimize the detection of the p53 gene mutation, we performed electrophoresis at room temperature or at 4°C in the presence or absence of 10% glycerol. The SSCP analysis detected aberrantly migrating fragments in the three patients under all four conditions. Mobility shifts of mutant p53 cDNA fragments from the patients and positive controls were best resolved when electrophoresis was performed at room temperature in the presence of 10% glycerol. Because the effect of sequence alterations on electro-

4

3

phoretic mobility is unpredictable, it is true that some of the sequence mutations may not appreciably affect the mobility. However, Orita et a1 reported that single base changes can be detected as mobility shift with SSCP analysis in all 12 arbitrary chosen tumor cell lines that are known to contain mutated H-rus, K-rus, or N-rus.” We observed normal migration of the p53 gene in the SSCP analysis on the 32 normal samples. Moreover, we sequenced 10 normally migrating fragments and confirmed that they contain no mutations. These findings suggest that the SSCP analysis has relatively high sensitivity and, therefore, usefulness for detecting mutations. The mutations of patients 1and 3 are mapped in the four regions highly conserved among five species from Xenopus Zuevis to human (regions D and C, respectively, in Fig 1). Although the triplet deletion at codon 190 of patient 2 is out of these regions, the amino acid sequences between condons 189and 200 are also highly conserved.” Therefore, the mutations of all the three cases may affect the normal function of the p53 protein. Previous cytogenetic and molecular genetic studies show that various types of human tumors have a 17p allelic deletion with a mutation in the remaining p53 gene, and that p53 gene mutations can be detected in some human tumors without 17p allelic deletions?-” Loss of 17p coupled with the mutation of the p53 gene in patient 2 coincides with the idea that the wild-type p53 gene functions as a tumor suppressor gene (Table 1). In the cases of patients 1 and 3, mutations of the p53 gene occur without loss of chromosome 17p. However, the SSCP analysis (patients 1 and 3) and the direct sequencing (patient 1) showed that normally migrating p53 cDNA fragments were lacking in both cases, suggestingthe absence of wild-type p53 mRNA expression from the nonmutated allele. At least two possible explanationscan account for this result. First, there may exist inner or whole deletion of another p53 gene, which can not be detected by conventional cytogenetic analysis. Second, mutations may exist in some regions that regulate

Table 1. PatientslWith p53 Gene Mutations Mutation Patient

Diaanosis

Blabt (Oh)

1 2 3

Pre-B cell ALL Null-cell ALL Waldenstrh‘s macroglobulinemia

80

99 90

Ka~otv~e

Normal WW.6q-, 17P-I [46xy, -1, -6, -16,3p-]

Codon

Nucleotide

Amino Acid

276

GCCjCCC Deleted CCT Inserted a T

Ala+Pro Deleted Pro Asn + Stop

190 239


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SUGIMOTO ET AL

the p53 gene expression. In any case, our results suggest that leukemic cells of the three patients lacked the wildtype p53 mRNA expression. Both activation of oncogene(s) and inactivation of tumor suppressor gene(s) are recently considered to be necessary for tumorigenesis in some human malignancies! Alterations of the p53 gene seem to act in concert with the activated ab1 protein, a bcr-abl fusion product, in blast crisis of CML. In the case of patient 2, we detected K-ras gene activation at codon 12 (unpublished result), which may suggest that the inactivated p53 gene may cooperate with the activated K-ras gene in the leukemogenesis of this patient. If both of the p53 alleles are transcriptionally inactivated in leukemic cells, then the RT-PCR reaction may amplify wild-type p53 mRNA from the residual normal cells in the

bone marrow samples. Some of the cases showing the normally migrating fragments in the SSCP analysis may, therefore, have had leukemic cells that entirely lacked the p53 gene expression. Our results suggest that loss of the normal p53 function may be important in the genesis of some human leukemias. More extended study of the p53 gene mutation will give an important insight into the mechanism of human leukemogenesis. ACKNOWLEDGMENT

We thank Dr A. Aoyagi of Yokosuka Mutual Aid Hospital, Dr Y. Miura of the Department of Hematology, Jichi Medical School, and Dr A. Fujita of Showa Hospital for providing samples from patients, and Dr K. Hayashi of National Cancer Center Research Institute for his kind advice.

REFERENCES

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11. Nigro JM, 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: Mutations in the p53 gene occur in diverse human tumour types. Nature 342705,1989 12. Ahuja H, Bar-Eli M, Advani SH, Benchimol S, Cline MJ: Alterations in the p53 gene and the clonal evolution of the blast crisis of chronic myelocytic leukemia. Proc Natl Acad Sci USA 86:6783,1989 13. Kelman Z, Prokocimer M, Peller S, Kahn Y, Rechavi G, Manor Y, Cohen A, Rotter V: Rearrangements in the p53 gene in Philadelphia chromosome positive chronic myelogenous leukemia. Blood 742318,1989 14. Mashal R, Shtalrid M, Talpaz M, Kantarjian H, Smith L, Beran M, Cork A, Trujillo J, Gutterman J, Deisseroth A Rearrangement and expression of p53 in the chronic phase and blast crisis of chronic myelogenous leukemia. Blood 75:180,1990 15. Orita M, Suzuki Y, Sekiya T, Hayashi K: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874,1989 16. Zakut-Houri R, Bientz-Tadmor B, Givol D, Oren M: Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. EMBO J 4:1251,1985 17. Jenkins JR, Sturzbecher H - W The p53 oncogene, in Reddy EP, Skalka AM, Curran T (eds): The Oncogene Handbook. New York, NY,Elsevier Science, 1988, p 403 18. Suzuki Y, Orita M, Shiraishi M, Hayashi K, Sekiya T: Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 5:1037,1990


1991 77: 1153-1156

Mutations of the p53 gene in lymphoid leukemia K Sugimoto, H Toyoshima, R Sakai, K Miyagawa, K Hagiwara, H Hirai, F Ishikawa and F Takaku

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