Human Molecular Genetics, Vol. 1, No. 3
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A novel mutation in the p53 gene in a Burkitt's lymphoma cell line Kishor Bhatia, Marina I.Gutierrez and Ian T.Magrath Lymphoma Biology Section, Pediatric Branch, NCI, NIH, Bethesda, MD 20892, USA Submitted March 10, 1992 Wild type p53 has been shown to possess antiproliferative properties (1). Thus, as one would expect, loss of p53 protein is associated with an oncogenic phenotype. p53, however, does not always act as a true tumor supressor gene; indeed, earlier studies with p53 showed that it has a dominant transforming activity (2). Subsequently, it was realized that this transforming property was a result of missense mutations within the p53 coding region (3). Mutations in the p53 gene have now been described in human colorectal (4), lung (5), breast (6), esophageal (7), lymphoid (8, 9) and brain tumors (10). Some of these mutations may be oncogenic because of loss of function of the p53 gene, especially those that are associated with transforming potential only in the absence of a normal allele, e.g. those associated with the Li-Fraumeni Syndrome (11). However other mutations will confer their transforming activity even in the presence of a normal allele. This dominant negative action of p53 results either from sequestration of the normal p53 (12) by mutated p53, or because the mutated p53 protein had acquired the ability to promote proliferation. For this reason, dominant negative mutations have been also referred to as gain of function mutations (13). Most of the mutations described within the p53 gene occur in a stretch of about 200 amino acids and are largely confined to exons 5 - 8 (10). A common theme of all p53 gain of function mutations is that they are misense mutations in conserved domains of the protein and they confer increased stability upon mutated p53, thus leading to accumulation of p53 protein (14). This common property (stabilization) of pathogenetcally relevant p53 mutations has been used to detect mutant p53 in various tumors by immunohistochemical (15) and Western blot analysis (Bhatia et al., unpublished data). A compendium of published data on mutations within the p53 gene reveals that almost all mutations are single base changes (16). A few deletions and insertions have been reported but all those described to date cause a change in the reading frame, leading eventually to a premature termination of the translated product. We and others have reported that p53 mutations occur frequently (37%) in primary Burkitt's lymphomas (8, 9). The frequency of these mutations is even greater (70%) in cell lines. In vitro establishment of cell lines is successful only in a fraction (30%) of lymphomas but is more successful (80%) in recurrent tumors. These high mutation rates make it probable that these mutations play a role in the pathogenesis of BL. We report here an unusual mutation in a Burkitt's lymphoma cell line, SG588A. This cell line was derived from a sporadic Burkitt's tumor biopsy obtained from a patient at the Pediatric branch, NCI. The mutation involves duplication of two codons, 175 and 176, in exon 5 of p53. This duplication retains the open reading frame of p53. Like other dominant negative mutations the resultant p53 protein has gained the ability to accumulate within cells, suggesting that it is pathogenotically relevant. It is of interest that
although codon 175 is frequently mutated in colorectal tumors, it is rarely involved in Burkitt's lymphomas (8). The mutation was identified through screening a number of Burkitt lymphoma cell lines by SSGE analysis (17). Individual PCR amplifications were carried out using primers flanking exons 5 through 8 (9). Radiolabelled amplified products were run on a 6% polyacrylamide gel and migration patterns of the denatured bands were determined by autoradiography. Abnormally migrating amplified products were subject to direct sequencing using internal primers. Figure 1 is an autoradiograph of the sequencing reaction using the amplified product from exon 5 of SG588A p53. As a control, wild type sequence obtained from a similar reaction using DNA from a non-mutated cell line is also shown. Since the mutation was evident as a SSCP, and since the sequence was obtained directly from the PCR amplified product without subcloning, the mutation cannot be ascribed to errors in the polymerization by Taq polymerase. However to definitely exclude the possibility that a Taq error occurred at a very early round of the PCR, multiple sequencing reactions were carried out using as a template, amplified products from independent PCRS. All of these sequencing reactions demonstrated the same mutation. Sequencing of exon 5 of the p53 gene obtained from the DNA of the original tumor biopsy also revealed the same mutation. Furthermore, protein analysis showed stabilization of p53 in this cell line confirming a mutation (Figure 2). SSGE analysis extended to other exons fail to reveal other mutations.
SG588A
WILD TYPE
A G C T A G C T
Figure 1. Sequence of p53 exon 5 mutation in SG588A. PCR amplified DNA was sequenced by the double stranded method. Sequence obtained from a normal exon 5 is also shown for comparison. Mutation in SG588A was confirmed in six independent reactions. DNA obtained from the biopsy also demonstrated the same mutation.
208 Human Molecular Genetics, Vol. 1, No. 3 1
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3
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mutant p53
Figure 2. Western blot analysis of total cellular extracts of Burkitt's lymphoma cell lines with and without p53 mutation. Cells were grown in RPMI 1640 with 10% fetal bovine serum. For Western analysis cell pellets were lysed in 150 mM NaCl, 50 mM Tris pH 8.0, O.5 mM EDTA, 1% NP40 and 1 mMPMSF. Following sonication on ice, the lysates were subject to denaturing PAGE and the proteins transferred to an Immobilion membrane. The membranes were blocked with milk and probed with the antibody pAb240 and detected using alkaline phosphatase Lane 1 is an extract from a Burkitt's lymphoma cell line with wild type p53. Lanes 2 and 3 contain lysates from the two independent cell lines (JD38PB and JD38 ASQ derived from a sporadic Burkitt's lymphoma with a mutation in exon 6, codon 234, lane 4 contains lysate from the Burkitt's lymphoma cell line (ST486) with a mutation in exon 5, codon 158. The lysate from SG588A is in lane 5. Protein standards were loaded in lane 6.
We conclude that the BL cell line SG588A carries a novel mutation which duplicates codons 175 and 176. The mutation does not alter the reading frame of the p53 protein and only adds two extra amino, acids. This mutation is hemizygous (or homozygous) and in SG588A causes p53 protein to accumulate abnormally. Since heterozygous mutations of this kind have not been observed, it is not yet possible to determine whether it is dominant negative or recessive.
REFERENCES 1. Baker.S.J., Markowitiz.S., Fearon.E.R., WilsonJ.K.V. and Vogelstein.B. (1990) Science 249, 912-915. 2. Eliyahu.D., Raz.A., Gruss.P., Givol.D. and Oren.M. (1984) Nature 312. 646-649. 3. Hinds.P., Finlay.C. and Levine.A. (1989) /. Virol 63. 739-746. 4. Baker.S.J., Fearon.E.R., NigroJ.M., Harrulton.S.R.. Preisinger.A.C, JessupJ.M , vanTuinen.P., LedbeOer.D.H., Barker.D.F. and Nakamura.Y. (1989) Science 244, 217-221. 5. Chiba.L, Takahashi.T.. Nau.M.M., D'Amico.D., Curiel.D.T., Mitsudomi.T., Buchhagen.D.L., Carbone.D., Piantadosi.S., Koga.H., Reissman.P.T., Slamon.DJ., Holmes.E.C.and MinnaJ.D. (1990) Oncogene 5. 1603-1610. 6. BartekJ., Iggo.R., GannonJ. and Lane.D.P. (1990) Oncogene 5, 893-899. 7. Holistein.M., Metcalf.R.A., WebhJ., Montesano.R. and Harris.C.C. (1990) Proc. Nail Acad. Sci. USA 87, 9958-9961. 8. Gutierrez.M.L, Magrath.I.T., Huppi.K., Siwarski.D. and Bhatia.K. (1991) Blood 78 (Suppl. 1), 324. 9. Gaidano.G., Ballerina,P.. Gong.J.Z., Inghirami.G.. Neri.A.. Newcomb.E.W., Magrath.I.T.. Knowles.D.M., Dalla-Favera.R. (1991) Proc. Natl. Acad. Sci. USA 88. 5413-5417. 10. NigroJ.M.. Baker.S.J.. Preisinger.A.C.. JessupJ.M.. Hosterer.R.. Cleary.K., Bigner.S.H., Davidson.N., Baylin.S., Devilee.P., Glover.T.. Collins.F.S., Weston.A., Modali.R . Harris.C.C. and Vogelstein.B. (1989) Nature 342. 705-708.
11. Srivastava.S., Zou.Z.Q., Pirollo.K., Blattner.W. and Chang.E.H. (1990) Nature 348, 747-749. 12. MilnerJ., Medcalf.E.A. and Cook.A.C. (1991) Moi. Cell Bid. 11. 12-19. 13. Michalovitz,D., Hatevy.O. and Oren.M. (1991)/ Ceil. Biochem. 45, 22-29. 14. Lane.D P. and Benchimol.S. (1990) Genes Dn: 4. 1 - 8 . 15. Iggo,R.,Gaiter,K., BartekJ., Lane.D. and Harris.A L. (1990) Lancet 335, 675-679. 16. Holistein.M., Sidransky.D., Vogelstein.B. and Harris.C.C. (1991) Science 253, 49-53. 17. Orita.M , Suzuki.Y., Sekiya.T. and Hayashi.K. (1989) Genomics 5. 874-879.
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A novel mutation in the p53 gene in a Burkitt's lymphoma cell line Kishor Bhatia, Marina I.Gutierrez and Ian T.Magrath Lymphoma Biology Section, Pediatric Branch, NCI, NIH, Bethesda, MD 20892, USA Submitted March 10, 1992 Wild type p53 has been shown to possess antiproliferative properties (1). Thus, as one would expect, loss of p53 protein is associated with an oncogenic phenotype. p53, however, does not always act as a true tumor supressor gene; indeed, earlier studies with p53 showed that it has a dominant transforming activity (2). Subsequently, it was realized that this transforming property was a result of missense mutations within the p53 coding region (3). Mutations in the p53 gene have now been described in human colorectal (4), lung (5), breast (6), esophageal (7), lymphoid (8, 9) and brain tumors (10). Some of these mutations may be oncogenic because of loss of function of the p53 gene, especially those that are associated with transforming potential only in the absence of a normal allele, e.g. those associated with the Li-Fraumeni Syndrome (11). However other mutations will confer their transforming activity even in the presence of a normal allele. This dominant negative action of p53 results either from sequestration of the normal p53 (12) by mutated p53, or because the mutated p53 protein had acquired the ability to promote proliferation. For this reason, dominant negative mutations have been also referred to as gain of function mutations (13). Most of the mutations described within the p53 gene occur in a stretch of about 200 amino acids and are largely confined to exons 5 - 8 (10). A common theme of all p53 gain of function mutations is that they are misense mutations in conserved domains of the protein and they confer increased stability upon mutated p53, thus leading to accumulation of p53 protein (14). This common property (stabilization) of pathogenetcally relevant p53 mutations has been used to detect mutant p53 in various tumors by immunohistochemical (15) and Western blot analysis (Bhatia et al., unpublished data). A compendium of published data on mutations within the p53 gene reveals that almost all mutations are single base changes (16). A few deletions and insertions have been reported but all those described to date cause a change in the reading frame, leading eventually to a premature termination of the translated product. We and others have reported that p53 mutations occur frequently (37%) in primary Burkitt's lymphomas (8, 9). The frequency of these mutations is even greater (70%) in cell lines. In vitro establishment of cell lines is successful only in a fraction (30%) of lymphomas but is more successful (80%) in recurrent tumors. These high mutation rates make it probable that these mutations play a role in the pathogenesis of BL. We report here an unusual mutation in a Burkitt's lymphoma cell line, SG588A. This cell line was derived from a sporadic Burkitt's tumor biopsy obtained from a patient at the Pediatric branch, NCI. The mutation involves duplication of two codons, 175 and 176, in exon 5 of p53. This duplication retains the open reading frame of p53. Like other dominant negative mutations the resultant p53 protein has gained the ability to accumulate within cells, suggesting that it is pathogenotically relevant. It is of interest that
although codon 175 is frequently mutated in colorectal tumors, it is rarely involved in Burkitt's lymphomas (8). The mutation was identified through screening a number of Burkitt lymphoma cell lines by SSGE analysis (17). Individual PCR amplifications were carried out using primers flanking exons 5 through 8 (9). Radiolabelled amplified products were run on a 6% polyacrylamide gel and migration patterns of the denatured bands were determined by autoradiography. Abnormally migrating amplified products were subject to direct sequencing using internal primers. Figure 1 is an autoradiograph of the sequencing reaction using the amplified product from exon 5 of SG588A p53. As a control, wild type sequence obtained from a similar reaction using DNA from a non-mutated cell line is also shown. Since the mutation was evident as a SSCP, and since the sequence was obtained directly from the PCR amplified product without subcloning, the mutation cannot be ascribed to errors in the polymerization by Taq polymerase. However to definitely exclude the possibility that a Taq error occurred at a very early round of the PCR, multiple sequencing reactions were carried out using as a template, amplified products from independent PCRS. All of these sequencing reactions demonstrated the same mutation. Sequencing of exon 5 of the p53 gene obtained from the DNA of the original tumor biopsy also revealed the same mutation. Furthermore, protein analysis showed stabilization of p53 in this cell line confirming a mutation (Figure 2). SSGE analysis extended to other exons fail to reveal other mutations.
SG588A
WILD TYPE
A G C T A G C T
Figure 1. Sequence of p53 exon 5 mutation in SG588A. PCR amplified DNA was sequenced by the double stranded method. Sequence obtained from a normal exon 5 is also shown for comparison. Mutation in SG588A was confirmed in six independent reactions. DNA obtained from the biopsy also demonstrated the same mutation.
208 Human Molecular Genetics, Vol. 1, No. 3 1
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mutant p53
Figure 2. Western blot analysis of total cellular extracts of Burkitt's lymphoma cell lines with and without p53 mutation. Cells were grown in RPMI 1640 with 10% fetal bovine serum. For Western analysis cell pellets were lysed in 150 mM NaCl, 50 mM Tris pH 8.0, O.5 mM EDTA, 1% NP40 and 1 mMPMSF. Following sonication on ice, the lysates were subject to denaturing PAGE and the proteins transferred to an Immobilion membrane. The membranes were blocked with milk and probed with the antibody pAb240 and detected using alkaline phosphatase Lane 1 is an extract from a Burkitt's lymphoma cell line with wild type p53. Lanes 2 and 3 contain lysates from the two independent cell lines (JD38PB and JD38 ASQ derived from a sporadic Burkitt's lymphoma with a mutation in exon 6, codon 234, lane 4 contains lysate from the Burkitt's lymphoma cell line (ST486) with a mutation in exon 5, codon 158. The lysate from SG588A is in lane 5. Protein standards were loaded in lane 6.
We conclude that the BL cell line SG588A carries a novel mutation which duplicates codons 175 and 176. The mutation does not alter the reading frame of the p53 protein and only adds two extra amino, acids. This mutation is hemizygous (or homozygous) and in SG588A causes p53 protein to accumulate abnormally. Since heterozygous mutations of this kind have not been observed, it is not yet possible to determine whether it is dominant negative or recessive.
REFERENCES 1. Baker.S.J., Markowitiz.S., Fearon.E.R., WilsonJ.K.V. and Vogelstein.B. (1990) Science 249, 912-915. 2. Eliyahu.D., Raz.A., Gruss.P., Givol.D. and Oren.M. (1984) Nature 312. 646-649. 3. Hinds.P., Finlay.C. and Levine.A. (1989) /. Virol 63. 739-746. 4. Baker.S.J., Fearon.E.R., NigroJ.M., Harrulton.S.R.. Preisinger.A.C, JessupJ.M , vanTuinen.P., LedbeOer.D.H., Barker.D.F. and Nakamura.Y. (1989) Science 244, 217-221. 5. Chiba.L, Takahashi.T.. Nau.M.M., D'Amico.D., Curiel.D.T., Mitsudomi.T., Buchhagen.D.L., Carbone.D., Piantadosi.S., Koga.H., Reissman.P.T., Slamon.DJ., Holmes.E.C.and MinnaJ.D. (1990) Oncogene 5. 1603-1610. 6. BartekJ., Iggo.R., GannonJ. and Lane.D.P. (1990) Oncogene 5, 893-899. 7. Holistein.M., Metcalf.R.A., WebhJ., Montesano.R. and Harris.C.C. (1990) Proc. Nail Acad. Sci. USA 87, 9958-9961. 8. Gutierrez.M.L, Magrath.I.T., Huppi.K., Siwarski.D. and Bhatia.K. (1991) Blood 78 (Suppl. 1), 324. 9. Gaidano.G., Ballerina,P.. Gong.J.Z., Inghirami.G.. Neri.A.. Newcomb.E.W., Magrath.I.T.. Knowles.D.M., Dalla-Favera.R. (1991) Proc. Natl. Acad. Sci. USA 88. 5413-5417. 10. NigroJ.M.. Baker.S.J.. Preisinger.A.C.. JessupJ.M.. Hosterer.R.. Cleary.K., Bigner.S.H., Davidson.N., Baylin.S., Devilee.P., Glover.T.. Collins.F.S., Weston.A., Modali.R . Harris.C.C. and Vogelstein.B. (1989) Nature 342. 705-708.
11. Srivastava.S., Zou.Z.Q., Pirollo.K., Blattner.W. and Chang.E.H. (1990) Nature 348, 747-749. 12. MilnerJ., Medcalf.E.A. and Cook.A.C. (1991) Moi. Cell Bid. 11. 12-19. 13. Michalovitz,D., Hatevy.O. and Oren.M. (1991)/ Ceil. Biochem. 45, 22-29. 14. Lane.D P. and Benchimol.S. (1990) Genes Dn: 4. 1 - 8 . 15. Iggo,R.,Gaiter,K., BartekJ., Lane.D. and Harris.A L. (1990) Lancet 335, 675-679. 16. Holistein.M., Sidransky.D., Vogelstein.B. and Harris.C.C. (1991) Science 253, 49-53. 17. Orita.M , Suzuki.Y., Sekiya.T. and Hayashi.K. (1989) Genomics 5. 874-879.
