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Mutations spanning P53 exons 5–9 detected by non-isotopic RNAse cleavage assay and protein expression in human colon cancer
Cancer Genetics and Cytogenetics 129 (2001) 40–42
Mutations spanning P53 exons 5–9 detected by non-isotopic RNAse cleavage assay and protein expression in human colon cancer Stefania Tommasia, Marta Abatangeloa, Rosanna Lacalamitaa, Severino Montemurroa, Francesco Marzulloa, Angelo Paradisoa,* a
National Cancer Institute, Experimental Clinical Oncology Laboratory, Via Amendola, 209-70126 Bari, Italy Received 18 September 2000; received in revised form 22 January 2001; accepted 25 January 2001
Abstract
The non-isotopic assay (NIRCA), based on the observation that RNAse is able to specifically cleave a single mismatch in RNA/RNA duplexes, has been recently proposed to detect p53 mutations. To verify the use of this method as a valid screening for P53 mutations in a routinely collected cancer series, we used this assay on 3 cases with normal and 5 cases with abnormal P53 expression detected by Western blots. In all cases, P53 exons 5–6, 7 and 8–9 regions were analyzed. There were mutations only in the five overexpressed cases: two cases showed mutations in exon 5, one between intron 6 and exon 6 and two in the region spanning exons 8 and 9. Our experience showed NIRCA to be fast, reliable and providing the ability to study long target regions in a single step, thus making this assay useful for genetic screenings. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction The P53 is a multifunctional protein involved in both tumorigenesis and progression of many human tumors, especially colon carcinoma. In particular, it plays a leading role in tumorigenicity modulating gene transcription, policing cell cycle checkpoints, activating apoptosis, controlling DNA replication and repair, maintaining genomic stability and responding to genetic insults [1,2]. During tumor progression, it modulates the response to physical and chemical agents, including the response to chemotherapy or radiation, and modulates the biological aggressiveness by cell cycle arrest and programmed cell death. All these functions are essentially related to P53 gene mutation and/or high protein expression level, which confer a selective advantage to the cells favoring cancer formation. The p53 protein levels are regulated by post-translational mechanisms and most P53 mutations disrupt the degradation pathway, resulting in a higher level of mutant protein. Mutations of P53 gene occur in approximately 50% of colon carcinomas of which nearly 87% could be detected in the region spanning exons 5–8 [3,4]. Clinical relevance of P53 detection was essentially based on studies utilizing immunohistochemistry expression assay [5,6], even though immunohistochemical staining does not
perfectly correspond to P53 gene mutations [7]. Furthermore, the clinical relevance of P53 alterations seems to be related to the type of mutation as the most important mutations are in the most preserved region of the gene. However, the techniques traditionally used (SSCP and direct sequencing) for molecular analysis of P53 are time consuming, labor intensive, and need a large quantity of source material. Recently, a non-isotopic assay has been proposed as a quick, reliable and sensitive test to detect P53 mutations [8,9]. Previous studies have utilized the non-isotopic RNAse cleavage assay (NIRCA) to evaluate proteins like Factor IX system, parvovirus B19, and AML1 protein, demonstrating that the major advantages of this method are: (1) the ability to screen longer target regions in each experiment; (2) the lack of radioisotope utilization; (3) the rapidity of execution; and the (4) relatively lower laboratory/material costs for genetic screening than for the classical methods. To determine if this method is a valid screening for P53 mutations in a routinely collected cancer series, we used this assay (Ambion kit, Austin, TX, USA) in cases with normal and abnormal P53 expression. 2. Material and methods 2.1. Western blot
* Corresponding author. Tel.: 39-080-555556; fax: 39-080-555556. E-mail address: [email protected] (A. Paradiso).
Proteins were extracted from 100 g of tumor tissue and 100 g of adjacent normal mucosa by homogenization in
0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 4 2 2 - 8
S. Tommasi et al. / Cancer Genetics and Cytogenetics 129 (2001) 40–42
41
RIPA buffer (NaCl 0.5M, Triton X100 1%, NP40 0.5%, Deoxycolic acid 1%, SDS 3.5 mM, Tris HCl 8.3 mM pH 7.4, Tris base 1.6 mM) and treated with 20% protease inhibitor cocktail (SIGMA). Total proteins were measured by the Bradford method and 50 g were electrophoretically separated on 12.5% acrilamide gel. Pab1801 (Santa Cruz, CA, USA) monoclonal antibody was used to detect P53 protein and a mouse-HRP was used as a secondary antibody. Signal was detected by a chemoluminescence assay (ECL-Plus, Amersham Life Science, UK). Expression level was evaluated by MultiAnalyst software (Biorad, Hercules, CA) using colored filter photographs for normalization. Protein extract from A431 human epidermoid carcinoma cell line and from human fibroblasts was used as positive- and negativecontrol, respectively. 2.2. NIRCA We analyzed three normally expressed (GS, GC, RR) and five overexpressed (DR, FDP, GG, AM, VM) cases. Tumor tissue and respective normal mucosa of all cases were analyzed to evaluate P53 exons 5–6, 7 and 8–9 regions. DNA was extracted from 80 mg of tumor tissue and from an equal amount of normal adjacent mucosa modifying a previously reported method [10]. Pulverized samples were incubated in lysis buffer (Sucrose 0.25 M, KCl 0.15 M, Tris Hcl 10 mM pH 7.5, EDTA 1 mM) at 4C, centrifuged and incubated with a solution containing proteinase K 100 g/l at 37C overnight. Exogenous RNAse was added to avoid RNA contamination and DNA was purified by phenol/chloroform extraction and ethanol precipitation. One microgram of DNA was used to amplify each region using specific primers. Nested PCR was conducted using specific internal primers containing the T7 and SP6 promoters. We performed all reactions separately presuming homozygous samples. Sense and antisense RNA were transcribed in separate reactions with T7 and SP6 polymerase. An equal volume of T7 transcripts reacted to SP6 transcripts to hybridize in double strand RNA as follows: normal T7 transcript to normal SP6 transcript, tumor T7 transcript to tumor Sp6 transcript, as internal controls, and normal T7 to tumor Sp6 transcripts and normal SP6 to tumor T7 transcripts to evidence eventual mutations in one or both strands. The double strands RNA were cleaved by a mixture of three different RNAses (a bacterial RNAse 1, RNAse T1 from Aspergillus and RNAse A), which cleave distinct but overlapping subsets mismatches, as suggested by the kit information. We previously used the three RNAse separately, but the mixture was more efficient. The cleavage pattern was electrophoretically visualized on 2% agarose gel in 1 TBE.
3. Results Fig. 1 shows P53 expression of 6 of the 8 analyzed cases. The three cases with normal P53 expression did not show any P53 gene mutation, whereas the five cases with P53
Fig. 1. The P53 expression pattern of six of eight analyzed cases. T: tumor tissue; N: normal mucosa; A431: human epidermoid carcinoma cell line; HF: human fibroblast.
overexpression presented mutations at least in one of the three regions analyzed: cases DR and FDP showed a mutation in the antisense strand within exon 5; case GG had a mutation in the sense strand in the sequence between intron 6 and exon 6 and cases AM and VM presented a mutation in the antisense strand in the region spanning exons 8 and 9. Fig. 2 shows the pattern relative to case DR. To confirm what was found in cases AM and VM, we performed amplification using the outer primer (sense) of region 2 and the inner primer (antisense) of region 3. The cleavage pattern of this overlapping 5 and 3 fragments, confirmed the mutation localized at about 120 bp in exon 8.
Fig. 2. Example of P53 mutation in exon 5 case DR. MW: molecular weight marker; WT: normal control (kit provided); M: positive control, with point mutation in exon 5 (kit provided); N: RNA duplex from normal tissue of patient DR, the smaller band of about 200 bp represent a polymorphism specific cleavage; T: RNA duplex showing the two RNAse cleavage products derived from a mismatched base pairing (arrows) in the double stranded RNA from DR tumor patient; (about 300–350 bp); Ta: RNA heteroduplex showing the mutation in the tumor antisense strand hybridizing with the normal tissue RNA sense strand. Ts: double strand RNA formed by tumor sense strand and normal tissue antisense strand.
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S. Tommasi et al. / Cancer Genetics and Cytogenetics 129 (2001) 40–42
4. Discussion
Acknowledgments
Evaluation of P53 mutations by NIRCA have been previously conducted only in a series of patients affected by prostate carcinoma [11] and in 6 cases of colon cancer [9]. This latter article considered, the P53 region spanning exons 7 and 8 in colon cancer and showed the ability of this mutation detection strategy to identify a single-base mutation when the amount of DNA is very low or when the mutant allele is present in 4% of the total population of alleles of the total genomic DNA. The authors showed strict correspondence between NIRCA (Ambion, Austin, TX, USA) P53 mutations detection results and direct sequencing data. Of course, NIRCA cannot distinguish results if the sequence variation is due to a polymorphism rather than to a mutation. However, the usage of the normal tissue of the same patient as a normal control prevents evaluating as mutation a potential polymorphism only if the polymorphism is homozygous. Our study approached the P53 alteration problem from a pragmatic point of view: we analyzed the entire hot spot region (exon 5–9) for mutations; we verified the relationship between P53 NIRCA detection status and protein expression. Our results indicate a strict correlation between P53 mutations and overexpression in human colon carcinoma in respect to presence of mutations when p53 protein is expressed at a higher level. Our data are the first showing the correlation between Western blot and NIRCA analyses. Previous studies [7] on P53 expression evaluated by immunohistochemical antibody staining sometimes fail to show a concordance between expression level and gene mutations. In our hands, Western blot and NIRCA can be considered both useful and complementary to detect P53 alterations. In conclusion, the NIRCA is less time consuming than other molecular techniques used for gene mutation detection, allows the study of long target regions in a single step and uses no radioisotopes. Therefore, this assay is a potential candidate for genetic screenings in those cancers with higher rates of P53 mutations.
This work was supported by Italian Ministry of Health project PF96/296 and partially supported by CHR and AIRC.
References [1] Albrechtsen N, Dornreiter I, Grosse F, Kim E, Wiesmuller L, Deppert W. Maintenance of genomic integrity by p53: complementary roles for the activated and non-activated p53. Oncogene 1999;18:7706–17. [2] Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855–78. [3] Sheikh MS, Fornace AJ Jr. Role of p53 family in apoptosis. J Cell Physiol 2000;182:171–81. [4] Goh H-S, Yao J, Smith DR. p53 point mutation and survival in colorectal cancer patients. Cancer Res. 1995;55:5217–21. [5] Paradiso A, Tommasi S, Barletta A, Leone B, Lacava J, Vellejo C, Labriola A, Marzullo F, Altieri R, Schittulli F, De Lena M. Metastatic site and p53 primary tumor expression in previously untreated stage IV breast cancer patients. Anticancer Res 1999;19:4523–8. [6] Paradiso A, Simone G, Petroni S, Leone B, Vellejo C, Lacava J, Romero A, Machiavelli M, De Lena M, Allegra CJ, Johnston PG. Thymidylate synthase and p53 primary tumor expression as predictive factors for advanced colorectal cancer patients. Br J Cancer 2000;82:560–7. [7] Sjögren S, Inganäs M, Torbjörn N, Lindgren A, Nordgren H, Holmberg L, Bergh J. The p53 gene in breast cancer: Prognostic value of complementary DNA sequencing versus immunohistochemistry. J.N.C.I. 1996;88:173–81. [8] Goldrick MM, Kimball GR, Liu Q, Martin LA, Sommer SS, Tseng JY. NIRCA: a rapid robust method for screening for unknown point mutations. Biotechniques 1996;21:106–12. [9] Prescott J, Patel H, Tillman S, McHugh T, Ralph D. Cleavage of double-stranded copy RNA by Rnase 1 and Rnase T1 provides a robust means to detect p53 gene mutations in clinical specimens. Electrophoresis 1999;20:1149–61. [10] Macera MJ, Godec CJ, Sharma N Verma RS. Loss of heterozygosity of the p53 tumor suppressor gene and detection of point mutations by the non-isotopic RNAse cleavage assay in prostate cancer. Cancer Genet Cytogenet 1999;108:42–7. [11] Cotter FE, Hall PA, Young BD. Extraction of DNA from small sections of frozen tissue with simultaneous histological examination. J Clin Pathol 1998;41:1125–5.
Mutations spanning P53 exons 5–9 detected by non-isotopic RNAse cleavage assay and protein expression in human colon cancer Stefania Tommasia, Marta Abatangeloa, Rosanna Lacalamitaa, Severino Montemurroa, Francesco Marzulloa, Angelo Paradisoa,* a
National Cancer Institute, Experimental Clinical Oncology Laboratory, Via Amendola, 209-70126 Bari, Italy Received 18 September 2000; received in revised form 22 January 2001; accepted 25 January 2001
Abstract
The non-isotopic assay (NIRCA), based on the observation that RNAse is able to specifically cleave a single mismatch in RNA/RNA duplexes, has been recently proposed to detect p53 mutations. To verify the use of this method as a valid screening for P53 mutations in a routinely collected cancer series, we used this assay on 3 cases with normal and 5 cases with abnormal P53 expression detected by Western blots. In all cases, P53 exons 5–6, 7 and 8–9 regions were analyzed. There were mutations only in the five overexpressed cases: two cases showed mutations in exon 5, one between intron 6 and exon 6 and two in the region spanning exons 8 and 9. Our experience showed NIRCA to be fast, reliable and providing the ability to study long target regions in a single step, thus making this assay useful for genetic screenings. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction The P53 is a multifunctional protein involved in both tumorigenesis and progression of many human tumors, especially colon carcinoma. In particular, it plays a leading role in tumorigenicity modulating gene transcription, policing cell cycle checkpoints, activating apoptosis, controlling DNA replication and repair, maintaining genomic stability and responding to genetic insults [1,2]. During tumor progression, it modulates the response to physical and chemical agents, including the response to chemotherapy or radiation, and modulates the biological aggressiveness by cell cycle arrest and programmed cell death. All these functions are essentially related to P53 gene mutation and/or high protein expression level, which confer a selective advantage to the cells favoring cancer formation. The p53 protein levels are regulated by post-translational mechanisms and most P53 mutations disrupt the degradation pathway, resulting in a higher level of mutant protein. Mutations of P53 gene occur in approximately 50% of colon carcinomas of which nearly 87% could be detected in the region spanning exons 5–8 [3,4]. Clinical relevance of P53 detection was essentially based on studies utilizing immunohistochemistry expression assay [5,6], even though immunohistochemical staining does not
perfectly correspond to P53 gene mutations [7]. Furthermore, the clinical relevance of P53 alterations seems to be related to the type of mutation as the most important mutations are in the most preserved region of the gene. However, the techniques traditionally used (SSCP and direct sequencing) for molecular analysis of P53 are time consuming, labor intensive, and need a large quantity of source material. Recently, a non-isotopic assay has been proposed as a quick, reliable and sensitive test to detect P53 mutations [8,9]. Previous studies have utilized the non-isotopic RNAse cleavage assay (NIRCA) to evaluate proteins like Factor IX system, parvovirus B19, and AML1 protein, demonstrating that the major advantages of this method are: (1) the ability to screen longer target regions in each experiment; (2) the lack of radioisotope utilization; (3) the rapidity of execution; and the (4) relatively lower laboratory/material costs for genetic screening than for the classical methods. To determine if this method is a valid screening for P53 mutations in a routinely collected cancer series, we used this assay (Ambion kit, Austin, TX, USA) in cases with normal and abnormal P53 expression. 2. Material and methods 2.1. Western blot
* Corresponding author. Tel.: 39-080-555556; fax: 39-080-555556. E-mail address: [email protected] (A. Paradiso).
Proteins were extracted from 100 g of tumor tissue and 100 g of adjacent normal mucosa by homogenization in
0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 4 2 2 - 8
S. Tommasi et al. / Cancer Genetics and Cytogenetics 129 (2001) 40–42
41
RIPA buffer (NaCl 0.5M, Triton X100 1%, NP40 0.5%, Deoxycolic acid 1%, SDS 3.5 mM, Tris HCl 8.3 mM pH 7.4, Tris base 1.6 mM) and treated with 20% protease inhibitor cocktail (SIGMA). Total proteins were measured by the Bradford method and 50 g were electrophoretically separated on 12.5% acrilamide gel. Pab1801 (Santa Cruz, CA, USA) monoclonal antibody was used to detect P53 protein and a mouse-HRP was used as a secondary antibody. Signal was detected by a chemoluminescence assay (ECL-Plus, Amersham Life Science, UK). Expression level was evaluated by MultiAnalyst software (Biorad, Hercules, CA) using colored filter photographs for normalization. Protein extract from A431 human epidermoid carcinoma cell line and from human fibroblasts was used as positive- and negativecontrol, respectively. 2.2. NIRCA We analyzed three normally expressed (GS, GC, RR) and five overexpressed (DR, FDP, GG, AM, VM) cases. Tumor tissue and respective normal mucosa of all cases were analyzed to evaluate P53 exons 5–6, 7 and 8–9 regions. DNA was extracted from 80 mg of tumor tissue and from an equal amount of normal adjacent mucosa modifying a previously reported method [10]. Pulverized samples were incubated in lysis buffer (Sucrose 0.25 M, KCl 0.15 M, Tris Hcl 10 mM pH 7.5, EDTA 1 mM) at 4C, centrifuged and incubated with a solution containing proteinase K 100 g/l at 37C overnight. Exogenous RNAse was added to avoid RNA contamination and DNA was purified by phenol/chloroform extraction and ethanol precipitation. One microgram of DNA was used to amplify each region using specific primers. Nested PCR was conducted using specific internal primers containing the T7 and SP6 promoters. We performed all reactions separately presuming homozygous samples. Sense and antisense RNA were transcribed in separate reactions with T7 and SP6 polymerase. An equal volume of T7 transcripts reacted to SP6 transcripts to hybridize in double strand RNA as follows: normal T7 transcript to normal SP6 transcript, tumor T7 transcript to tumor Sp6 transcript, as internal controls, and normal T7 to tumor Sp6 transcripts and normal SP6 to tumor T7 transcripts to evidence eventual mutations in one or both strands. The double strands RNA were cleaved by a mixture of three different RNAses (a bacterial RNAse 1, RNAse T1 from Aspergillus and RNAse A), which cleave distinct but overlapping subsets mismatches, as suggested by the kit information. We previously used the three RNAse separately, but the mixture was more efficient. The cleavage pattern was electrophoretically visualized on 2% agarose gel in 1 TBE.
3. Results Fig. 1 shows P53 expression of 6 of the 8 analyzed cases. The three cases with normal P53 expression did not show any P53 gene mutation, whereas the five cases with P53
Fig. 1. The P53 expression pattern of six of eight analyzed cases. T: tumor tissue; N: normal mucosa; A431: human epidermoid carcinoma cell line; HF: human fibroblast.
overexpression presented mutations at least in one of the three regions analyzed: cases DR and FDP showed a mutation in the antisense strand within exon 5; case GG had a mutation in the sense strand in the sequence between intron 6 and exon 6 and cases AM and VM presented a mutation in the antisense strand in the region spanning exons 8 and 9. Fig. 2 shows the pattern relative to case DR. To confirm what was found in cases AM and VM, we performed amplification using the outer primer (sense) of region 2 and the inner primer (antisense) of region 3. The cleavage pattern of this overlapping 5 and 3 fragments, confirmed the mutation localized at about 120 bp in exon 8.
Fig. 2. Example of P53 mutation in exon 5 case DR. MW: molecular weight marker; WT: normal control (kit provided); M: positive control, with point mutation in exon 5 (kit provided); N: RNA duplex from normal tissue of patient DR, the smaller band of about 200 bp represent a polymorphism specific cleavage; T: RNA duplex showing the two RNAse cleavage products derived from a mismatched base pairing (arrows) in the double stranded RNA from DR tumor patient; (about 300–350 bp); Ta: RNA heteroduplex showing the mutation in the tumor antisense strand hybridizing with the normal tissue RNA sense strand. Ts: double strand RNA formed by tumor sense strand and normal tissue antisense strand.
42
S. Tommasi et al. / Cancer Genetics and Cytogenetics 129 (2001) 40–42
4. Discussion
Acknowledgments
Evaluation of P53 mutations by NIRCA have been previously conducted only in a series of patients affected by prostate carcinoma [11] and in 6 cases of colon cancer [9]. This latter article considered, the P53 region spanning exons 7 and 8 in colon cancer and showed the ability of this mutation detection strategy to identify a single-base mutation when the amount of DNA is very low or when the mutant allele is present in 4% of the total population of alleles of the total genomic DNA. The authors showed strict correspondence between NIRCA (Ambion, Austin, TX, USA) P53 mutations detection results and direct sequencing data. Of course, NIRCA cannot distinguish results if the sequence variation is due to a polymorphism rather than to a mutation. However, the usage of the normal tissue of the same patient as a normal control prevents evaluating as mutation a potential polymorphism only if the polymorphism is homozygous. Our study approached the P53 alteration problem from a pragmatic point of view: we analyzed the entire hot spot region (exon 5–9) for mutations; we verified the relationship between P53 NIRCA detection status and protein expression. Our results indicate a strict correlation between P53 mutations and overexpression in human colon carcinoma in respect to presence of mutations when p53 protein is expressed at a higher level. Our data are the first showing the correlation between Western blot and NIRCA analyses. Previous studies [7] on P53 expression evaluated by immunohistochemical antibody staining sometimes fail to show a concordance between expression level and gene mutations. In our hands, Western blot and NIRCA can be considered both useful and complementary to detect P53 alterations. In conclusion, the NIRCA is less time consuming than other molecular techniques used for gene mutation detection, allows the study of long target regions in a single step and uses no radioisotopes. Therefore, this assay is a potential candidate for genetic screenings in those cancers with higher rates of P53 mutations.
This work was supported by Italian Ministry of Health project PF96/296 and partially supported by CHR and AIRC.
References [1] Albrechtsen N, Dornreiter I, Grosse F, Kim E, Wiesmuller L, Deppert W. Maintenance of genomic integrity by p53: complementary roles for the activated and non-activated p53. Oncogene 1999;18:7706–17. [2] Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855–78. [3] Sheikh MS, Fornace AJ Jr. Role of p53 family in apoptosis. J Cell Physiol 2000;182:171–81. [4] Goh H-S, Yao J, Smith DR. p53 point mutation and survival in colorectal cancer patients. Cancer Res. 1995;55:5217–21. [5] Paradiso A, Tommasi S, Barletta A, Leone B, Lacava J, Vellejo C, Labriola A, Marzullo F, Altieri R, Schittulli F, De Lena M. Metastatic site and p53 primary tumor expression in previously untreated stage IV breast cancer patients. Anticancer Res 1999;19:4523–8. [6] Paradiso A, Simone G, Petroni S, Leone B, Vellejo C, Lacava J, Romero A, Machiavelli M, De Lena M, Allegra CJ, Johnston PG. Thymidylate synthase and p53 primary tumor expression as predictive factors for advanced colorectal cancer patients. Br J Cancer 2000;82:560–7. [7] Sjögren S, Inganäs M, Torbjörn N, Lindgren A, Nordgren H, Holmberg L, Bergh J. The p53 gene in breast cancer: Prognostic value of complementary DNA sequencing versus immunohistochemistry. J.N.C.I. 1996;88:173–81. [8] Goldrick MM, Kimball GR, Liu Q, Martin LA, Sommer SS, Tseng JY. NIRCA: a rapid robust method for screening for unknown point mutations. Biotechniques 1996;21:106–12. [9] Prescott J, Patel H, Tillman S, McHugh T, Ralph D. Cleavage of double-stranded copy RNA by Rnase 1 and Rnase T1 provides a robust means to detect p53 gene mutations in clinical specimens. Electrophoresis 1999;20:1149–61. [10] Macera MJ, Godec CJ, Sharma N Verma RS. Loss of heterozygosity of the p53 tumor suppressor gene and detection of point mutations by the non-isotopic RNAse cleavage assay in prostate cancer. Cancer Genet Cytogenet 1999;108:42–7. [11] Cotter FE, Hall PA, Young BD. Extraction of DNA from small sections of frozen tissue with simultaneous histological examination. J Clin Pathol 1998;41:1125–5.