ARTICLE

Splicing Mutations in TP53 in Human Squamous Cell Carcinoma Lines Influence Immunohistochemical Detection

Correspondence to: Wolfgang Eicheler, Uniklinikum, Klinik für Strahlentherapie und Radioonkologie, Fetscherstr. 74, 01307 Dresden, Germany. E-mail: wolfgang.eicheler@mailbox.tu-dresden.de

	Summary

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The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.

(J Histochem Cytochem 50:197–204, 2002)

Key Words: p53, squamous cell carcinoma, mutation, immunohistochemistry, splicing, exon skipping

	Introduction

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EXAMINATION of the mutational status of the p53 gene (TP53) by immunohistochemistry (IHC) or sequencing is widely used in cancer research. Immunohistochemical detection is based on the fact that the half-life of the mutated p53 protein is often increased, leading to its accumulation in the nucleus. In contrast, the wild-type protein has a shorter half-life and steady-state levels are not detectable by conventional IHC. When compared systematically, the correlation of IHC staining of p53 protein and the mutational status of TP53 was only weak in some studies (Kropveld et al. 1999 ; Saunders et al. 1999 ; Nieder et al. 2000 ; Petersen et al. 2001 ) but strong in others (Calzolari et al. 1997 ).

In human head and neck squamous cell carcinoma (hnSCC), p53 overexpression, as assessed by IHC, varied between 34 and 79% in different studies (Raybaud-Diogene et al. 1996 ). Immunostaining of p53, but not the mutational status of exons 5–9, was a prognostic factor for the outcome of therapy in hnSCC (Sauter et al. 1995 ), suggesting that p53 IHC might be useful for selection of patients for individualized treatment. On the other hand, reviews of the available clinical data revealed only a minor role or no role whatsoever of the TP53 status in anti-cancer therapy (Bosari and Viale 1995 ). In hnSCC, a prognostic role for the TP53 status was seen only when the analysis was restricted to mutations that cause an obvious change in p53 protein function (Nylander et al. 2000 ).

Conflicting reports were also given with regard to the effect of TP53 mutations in the response of hnSCC cell lines to radiation therapy (Brachman et al. 1993 ; Servomaa et al. 1996 ; Pulkkinen et al. 2000 ). Despite the importance of TP53 in tumor biology, there are only a few studies concerning the effect of a specific mutation in TP53 on IHC (Nylander et al. 2000 ). Mutation screening in hnSCC has been performed in most studies to date by sequencing all or only a selected part of the exons of the TP53 gene using chromosomal DNA, at times preceded by single-strand conformational polymorphism analysis (SSCP) (Greenblatt et al. 1994 ; Ahomadegbe et al. 1995 ; Fracchiolla et al. 1995 ; Nylander et al. 1995 ; Sauter et al. 1995 ; Ganly et al. 2000 ; Pulkkinen et al. 2000 ). In most cases no systematic search for alternative splicing products, which may occur in TP53 because of intronic mutations (Harris 1996 ) or to alternative splicing (Han and Kulesz-Martin 1992 ; Flamman et al. 1996 ), has been done. An analysis based on RT-PCR found a 91% incidence of TP53 mutations, with 33% of them located outside the conserved region, and a 36% incidence of truncated p53 protein by sequencing the complete reading frame of TP53 in hnSCC biopsies of 25 patients (Kropveld et al. 1999 ). A recent review by van Houten et al. 2000 reported a mutation frequency of 60% in hnSCC primary tumors by sequencing exons 2–11 at the genomic level. These authors found only an additional 6% of the mutations outside the conserved region of the gene.

In the present study we examined 12 different hnSCC lines, which are currently used in our laboratory for radiobiological experiments. We sequenced the complete reading frame of TP53 using cDNA derived from the mRNA to account for splicing artifacts and alternative splicing and, if necessary, the corresponding intron sequences at the DNA level to identify the underlying intronic mutation. The mutational status was compared to the detection of the p53 protein in IHC on paraffin sections and in Western blotting.

	Materials and Methods

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hSCC Cell Lines
Twelve different squamous cell carcinoma (SCC) lines from the head and neck were grown SC in male and female NMRI (nu/nu) nude mice from the specific pathogen-free animal breeding facility of the Experimental Center of the Medical Faculty of the Technical University of Dresden. The animal facilities and experiments were approved according to the German animal welfare regulations. To further immunosuppress the nude mice, they were whole body-irradiated 1–2 days before transplantation with 4 Gy using 200 kV X-rays (0.5 mm Cu) at a dose of 1.1 Gy/min (Suit et al. 1988 ; Baumann et al. 1992 ).

FaDu (ATCC HTB-43), an undifferentiated human hypopharygeal SCC cell line (Rangan 1972 ) was obtained in 1999 from the American Type Culture Collection (ATCC; Rockville, MD).

FaDu_DD is a subline of ATCC HTB-43, which has been used in different laboratories for radiobiological experiments in nude mice and in vitro since the 1980s (Suit et al. 1990 ; Baumann 1994 ; Petersen et al. 1998 ).

SKX was established as a xenograft line in nude mice from a biopsy of a moderately differentiated SCC of the floor of the mouth in 1991. In 1999, a cell line was established in vitro from the xenograft line (Dorfler et al. 2000 ).

GL is a moderately differentiated keratinizing SCC line, which was established as a permanent xenograft line in nude mice from a surgical specimen of an advanced human laryngeal carcinoma (Petersen et al. 1998 ).

The MKG7 xenograft tumor line was derived from a gingival tumor (courtesy of Dr. R. Schimming; Department of Oral and Maxillofacial Surgery, University of Dresden, Germany).

The UT-SCC-5, UT-SCC-10, UT-SCC-14, UT-SCC-15, UT-SCC-16a, and UT-SCC-24b tumor cell lines were established from tumors of the mobile tongue. The UT-SCC-33 tumor line came from the gingiva of the maxilla. The tumor lines designated "UT-SCC" were established at the Department of Otorhinolaryngology–Head and Neck Surgery (Turku University; Turku, Finland) as described earlier (Grenman et al. 1992 ; Lansford et al. 1999 ).

As wild-type controls, human lymphocytes were isolated from the peripheral blood of voluntary donors by centrifugation (1200 x g, 20 min) through a Ficoll 400 cushion (D = 1.077 at 20C; Biochrom, Berlin, Germany).

Immunohistochemistry
Dissected xenograft tumors were fixed overnight in neutral buffered 4% formalin and embedded in paraffin. Specimens were cut at 3-µm thickness, mounted on poly-L-lysine-coated slides, dried overnight at 37C, deparaffinized in xylene, and rehydrated. Endogenous peroxidase was blocked with 0.3% H₂O₂ in H₂O for 10 min. After blocking with horse serum (Vector; Burlingame, CA), sections were incubated for 1 hr at 37C with a primary monoclonal antibody (Mab) with target epitopes either at the N-terminus (amino acids 19–26, clone DO-7; DAKO, Hamburg, Germany) or within the C-terminus (amino acids 296–389, clone G59-12; Becton Dickinson/PharMingen, San Diego, CA). The primary antibodies were diluted 1:100 (clone DO-7) or 1:20 (clone G59-12) in PBS. Sections were washed three times for 5 min with PBS, covered with diluted (1:200) biotinylated anti-mouse IgG (Vector), and incubated for 45 min at RT. After thorough rinsing in PBS, a peroxidase–streptavidin complex (1:100 in PBS) was applied for 30 min at RT. The sections were rinsed in PBS and then incubated in 3'-diaminobenzidine (10 mg dissolved in 100 ml PBS containing 0.03% H₂O₂) for 10 min at RT. Cells were counterstained with hematoxylin. Sections were dehydrated and mounted in Entellan (Merck; Karlsruhe, Germany). As a negative control, the primary antibody was replaced by PBS. Results were negative if not otherwise stated.

Sequencing of TP53
Total RNA and genomic DNA were isolated with the TRIZOL reagent (Life Technologies; Karlsruhe, Germany). Total RNA was reverse-transcribed with the Reverse-iT first strand synthesis kit and anchored oligo-dT primers from ABgene (Surrey, UK). Exons 2–11 were amplified from the cDNA with p53RNAsense3 (5'-GTG ACA CGC TTC CCT GGA T-3') and p53RNAantisense3 (5'-TTC TGA CGC ACA CCT ATT GC-3') primers. They were deduced from GenBank entry U94788, using the Primer3 software from the Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). The cDNA was isolated using QiaQuick columns (Qiagen; Hilden, Germany), and both strands were sequenced by dye-deoxy cycle-sequencing with the p53RNAsense3 and p53RNAantisense3 primers using AbiPrism sequencers. The sequencing was performed by a commercial subcontractor (SeqLab; Göttingen, Germany). For detection of splicing artifacts, total RNA was amplified by using RT-PCR with the p53-RNAsense2 (5'-CCC AGA AAA CCT ACC AGG GC-3') p53-RNAantisense2 (5'-CGA AGC GCT CAC GCC CAC GG-3') primers spanning exons 4–10 and analyzed in agarose gels. In tumor cell lines showing amplicons with aberrant size, the corresponding intron sequences were amplified from the chromosomal DNA and sequenced to identify putative intronic or splice site mutations. The following primers were used for PCR and sequencing: for intron 6 in FaDu and FaDu_DD, p53-6sense (5'-TCC TCA CTG ATT GCT CTT-3') and p53-6antisense (5'-CAC ATC TCA TGG GGT TAT-3'); intron 4 in UT-SCC-15, p53-4sense (5'-TTT TCA CCC ATC TAC AGT CC-3') and p53-4antisense (5'-CGG CCA GGC ATT GAA GTC TC-3'); intron 7 to exon 8 in UT-SCC-14, p53-7sense (5'-TCA TCT TGG GCC TGT GTT AT-3') and p53-8antisense (5'-TTG TCC TGC TTG CTT ACC TC-3'); exon 7 to intron 9, p53-8sense (5'-TGC TTC TCT TTT CCT ATC CT-3') and p53-8antisense (5'-TTG TCC TGC TTG CTT ACC TC-3'); intron 5 in UT-SCC-10, p53-5neu-sense (5'-CAC TTG TGC CCT GAC TTT CA-3') and p53-5neu-antisense (5'-CAA ATT TCC TTC CAC TCG GA-3'). The sequences for the genomic primers were taken from the literature (Servomaa et al. 1996 ) or deduced from the genomic sequence as described above.

LOH Analysis for TP53 Locus in FaDu and FaDu_dd
To investigate the allelic status of TP53 in the two FaDu sublines, the repeat polymorphism in intron 3 of TP53 (1952–11967)_1–2 (Lazar et al. 1993 ) was amplified by PCR with the primers p53-2-3sense (5'-CAG CCA TTC TTT TCC TGC TC-3') and p53-2-3antisense (5'-GGG GAC TGT AGA TGG GTG AA), resolved on 10% thin-layer polyacrylamide gels (ETC; Kirchentellinsfurt, Germany), and silver stained. In all other tumors, the allelic status of a given mutation was judged by the phenotype of the corresponding peaks in the electropherograms from the sequencer. The absence of double peaks in the electropherogram was interpreted as homozygosity or, more likely, hemizygosity of the respective base.

Sequence Data Analysis
The sequences of both strands of each amplicon were compared using the BLAST software (http://www.ncbi.nlm.nih gov/BLAST). The consensus sequences were compared with the GenBank entry U94788 using BLAST. To identify frameshifts, the DNA sequences were translated into protein sequence using the Translate^L tool from the ExPASy software package (Swiss Institute of Bioinformatics; http://www.expasy.ch/). Molecular weights of the truncated proteins were calculated using the Compute pI/Mw tool from the same website.

Western Blotting
The protein fraction was extracted from the supernatants after DNA/RNA isolation using TRIZOL, and 20 µg of total protein was separated in 7.5–15% polyacrylamide gradient gels and semi-dry blotted onto nitrocellulose membranes. Unspecific binding sites were blocked in blocking buffer (5% low-fat milk powder, 0.05% Tween-20 in PBS) and incubated with the DO-7 primary antibody (1:300 in PBS) overnight at 4C. After incubation with anti-mouse–horseradish peroxidase conjugates (1:500 in PBS; Santa Cruz Biotechnology, Santa Cruz, CA), the blots were incubated for 1 min in luminol reagent (Santa Cruz Biotechnology), exposed to ECL films for 30 sec to 3 min (Amersham; Aylesbury, UK), and automatically processed in a medical film processor (Konica Europe; Hohenbrunn, Germany).

	Results

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All 12 SCC lines showed mutations in TP53 (Table 1). SKX, GL, FaDu, UT-SCC-5, UT-SCC-16a, UT-SCC-33, and MKG7 had missense mutations resulting in amino acid changes. FaDu had an additional heterozygous splicing site mutation causing a 49-bp insertion of intron 7 sequence with a frameshift and a premature stop codon. The FaDu_DD subline showed only the splicing site mutation, indicating the loss of one allele. This loss of heterozygosity (LOH) was confirmed by the analysis of a repeat polymorphism in TP53 (Fig 1). UT-SCC-10 showed two heterozygous nonsense mutations causing premature stops in codon 144 and codon 306. The mutation in codon 306 was not seen in the RNA sequence, indicating that these mutations were located on different alleles, one of them being not transcribed. All other base changes found were classified as homo- or hemizygous according to the absence of double peaks in the electropherograms of the sequencing reaction. UT-SCC-15 showed erroneous splicing with the insertion of 17 bp of intron 10 due to a splicing site mutation. In UT-SCC-24b, a splicing site mutation caused an in-frame insertion of a 69-bp intronic sequence with the creation of additional 23 codons after codon 224. UT-SCC-14 exhibited a complete skipping of exon 8 in the mRNA. However, we could not identify any underlying genomic mutation. The flanking sequences from intron 7 to 9 as well as the affected exon 8 were wild-type in three individually sequenced specimens (Table 1). The sequence of intron 7 in UT-SCC-14 differed significantly from the published GenBank sequence (Genbank entries U94788 and X54156) but was identical to the sequence found in the control lymphocytes and the published sequence of the A549 (ATCC CCL-185) human lung carcinoma cell line and the human malignant glioma-derived cell line M059J (Anderson and Allalunis-Turner 2000 ; GenBank entries AF136270 and AF135120).

View larger version (59K): [in this window] [in a new window]   Figure 1. PCR analysis of the repeat polymorphism in TP53 (11952–11967)1–2, showing a loss of the upper allele of TP53 in the FaDuDD subline compared to FaDu. This LOH is in line with the different staining patterns in immunohistochemistry and Western blotting. PAGE 10%, silver staining.

Owing to the aberrant size, the mRNA in FaDu_DD, UT-SCC-14, and UT-SCC-24b was classified as mutant after agarose gel electrophoresis (Fig 2), but the 17-bp insertion in UT-SCC-15 was detected only in high-resolution polyacrylamide gel electrophoresis (not shown).

View larger version (109K): [in this window] [in a new window]   Figure 2. Differing mRNA sizes in a 2% agarose gel indicate splicing mutations. RT-PCR with primers spanning exons 4–9 reveals a PCR product of 720 bp in most of the tumors. UT-SCC-14, UT-SCC-24b, and FaDuDD show aberrant amplicon sizes of 583 bp, 789 bp, and 769 bp, respectively. The 17-bp deletion in UT-SCC-15 is not resolved in this gel. Marker lane, 100-bp ladder.

The IHC staining did not reflect well the mutation status. Nine of 12 tumor cell lines showed nuclear staining with the DO-7 antibody, and three tumor cell lines with premature stops at codons 144, 202, and 244, respectively, were negative (Fig 3). UT-SCC-14, which had a premature stop at codon 290, showed immunoreaction with the DO-7 antibody. The G59-12 antibody, which detects an epitope at the C-terminus of p53, positively stained only seven of the tumor cell lines. No immunoreaction with G59-12 was seen in all tumor lines with premature stop codons, regardless of the size of the truncated protein. In addition, the UT-SCC-24b, which showed 23 additional codons between codon 224 and codon 225, was negative with G59-12. As seen with DO-7, all tumor cell lines harboring a missense mutation (including FaDu with the additional heterozygous splicing mutation) showed nuclear accumulation of p53 with the G59-12 antibody.

View larger version (193K): [in this window] [in a new window]   Figure 3. p53 immunostaining of paraffin-embedded tumor xenografts with the antibody DO-7. Nuclear staining is seen in FaDu, SKX, GL, MKG7, UT-SCC-5, UT-SCC-14, UT-SCC-16a, UT-SCC-24b, and UT-SCC-33. No specific staining is present in FaDuDD and UT-SCC15. UT-SCC-10 shows faint to moderate cytoplasmic immunoreaction.

Both the full-length and the truncated p53 were detected in Western blotting in six tumor cell lines, with the exeption of the truncated protein in FaDu_DD, UT-SCC-10, UT-SCC-15, and the extended protein in UT-SCC-24b, which were negative (Fig 4).

View larger version (29K): [in this window] [in a new window]   Figure 4. Western blotting analysis with the antibody DO-7, showing the predicted aberrant size of 46 kD of the p53 protein in UT-SCC-14 and the full-length protein of 53 kD in FaDu, SXK, GL, UT-SCC-5, UT-SCC-16a, UT-SCC-33, and MKG7. Corresponding to the immunohistochemical staining pattern, the truncated p53 proteins with expected sizes of 26 kD and 24 kD are not detected in FaDuDD, UT-SCC-10, and UT-SCC-15 tumors. In contrast to the immunohistochemical staining, the p53 with the expected size of 55 kD is not detected in Western blotting.

	Discussion

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The IHC staining did not reflect the mutational status of human SCC lines in this study. Despite the fact that only a limited set of tumor models was examined, some correlations between IHC staining and mutation type were obvious. Truncated p53 protein, as it appears after erroneous splicing, appears to have a shorter half-life and escapes IHC detection. In the hnSCC lines described here, we found missplicing in 5/12 tumor lines due to intronic point mutations or to a still unknown mechanism in one case. These alterations resulted in frameshifts and eventually premature stop codons, and gave rise to a high incidence (25–40%) of false-negative p53 immunostaining. FaDu was heterozygous for a splicing mutation and on the other allele an additional missense mutation in codon 248. Therefore, the nuclear immunostaining and the 53-kD band in Western blots were derived from the correctly spliced allele, whereas the other allele was transcribed only at very low levels (Reiss et al. 1993 ). In contrast, the FaDu_DD subline, which has lost the allele with the missense mutation and therefore was hemizygous for the splicing site mutation, was negative in IHC and in Western blotting. Whether or not truncated p53 protein was visible in immunohistochemistry and Western blotting was dependent on the antibody used and on the location of the premature stop. In UT-SCC-14, the p53 truncated at codon 290 was detected IHC at least with the N-terminal antibody DO-7, whereas the shorter p53 proteins in UT-SCC-15 and FaDu_DD with sizes of 202 and 244 amino acids, respectively, appear to be more vulnerable to proteolytic degradation. Loss of the carboxy terminus normally abolishes p53 protein function in human lymphocytes (Flamman et al. 1996 ). In contrast to this, the p53 protein in UT-SCC 14 still includes the nuclear localization signal (NLS) and the oligomerization domains and may therefore have preserved partial or complete p53 function.

In UT-SCC-14, we found incorrect splicing even with correct splicing sites and an intact putative branch point 40 bp upstream of the 3' splicing site of intron 7, compared to the corresponding sequence in normal human lymphocytes and the published sequences for the cell lines A549 (ATCC CCL-185) and M059J. The exact homologies between our human lymphocytes, UT-SCC-14, M059J, and A549 suggest that they represent the wild-type sequence for intron 7 rather than the sequences in the GenBank entries U94788 and X54156 (Anderson and Allalunis-Turner 2000 ; Eicheler and Baumann 2001 ). If this holds true, the splicing out of exon 8 in UT-SCC 14 is not caused by alterations in the flanking intron sequences, indicating the presence of not yet defined other sequences for this process, e.g., exonic splicing enhancers (Lorson et al. 1999 ). Skipping of exons of TP53 without mutations in the flanking introns, i.e., due to a yet unknown mechanism, was reported for exon 8 in a rat hepatoma cell line (Fukuda and Ogawa 1992 ) and for multiple exons in human chronic myelogenous leukemia cells (Nakai et al. 1994 ).

Most of the truncating mutations described in head and neck SCC thus far were attributed to intra-exon insertions rather than to erroneous splicing (Ahomadegbe et al. 1995 ).

Sequencing of TP53 either by manual, automated, or a new gene chip assay was shown to yield substantial false-negative rates in primary lung cancer tissue due to the mixture of mutant and wild-type cells in that material (Ahrendt et al. 1999 ). This could explain in part differences in the reported mutation rates between primary tumors and deduced tumor cell lines, which might be much more homogeneous due to selection during cultivation. On the other hand, the systematic analysis of all 11 exons and the complete mRNA of TP53 revealed a high mutation rate of 91% in primary hnSCC biopsies (Kropveld et al. 1999 ). Thirty-three percent of these mutations were located outside the core domain; 36% of the mutations resulted in a truncated p53 protein due to splice site mutations, insertions, or deletions. The data from hnSCC lines reported here are in line with these observations on clinical material. Compared to the 60% mutation frequencies in exons 2–11 in hnSCC primary tumors recently published by van Houten et al. 2000 , the numbers reported here and by Kropveld et al. 1999 are unusually high. These higher numbers are in part attributed to the fact that we and Kropveld et al. screened for splice mutations using RT-PCR, and indeed these mutations were found.

In conclusion, the high incidence of TP53 mutations recently reported for clinical tumors is in agreement with our findings in a set of 12 hnSCC lines and supports the value of these model systems for oncological research. The potential pitfall of false-negative results in IHC due to a high incidence of truncating mutations in TP53 suggests that more attention needs to be focused on this type of p53 inactivation, presumably by sequencing the RNA messenger rather than the exons at the DNA level.

	Acknowledgments

Supported by the Deutsche Forschungsgemeinschaft (Grant Ba 1433/2).

We thank Ms M. Oelsner and Ms S. Balschukat for skillful technical assistance and Dipl Ing V. Lieder (Computing Center of the Medical Faculty Carl Gustav Carus, Technical University Dresden) for help with the artwork.

Received for publication March 27, 2001; accepted September 12, 2001.

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