p53 expression, p53 and Ha-ras mutation and telomerase activation during nitrosamine-mediated hamster pouch carcinogenesis

Kuo-Wei Chang, Sara Sarraj1, Shu-Chun Lin, Pei-I Tsai and Dennis Solt1,2

Faculty of Dentistry and Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan, ROC and
1 Department of Pathology, Northwestern University, Chicago, IL 60611, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Squamous cell carcinomas (SCC) induced in hamster buccal pouch (HBP) by 22 weeks of topical N-methyl-N-benzylnitrosamine (MBN) treatment (twice-weekly, 10 mg MBN/ml propylene glycol) were evaluated for: (i) altered expression of p53 using immunohistochemistry (IHC); (ii) mutations in Ha-ras and p53 using PCR/single strand conformation polymorphism (SSCP); (iii) telomerase activity using the telomerase repeat amplification protocol (TRAP). Precancerous lesions were also evaluated using p53 IHC. Hamsters were killed for lesion analysis at either 3 days (group A, eight hamsters, 89 carcinomas) or 7 weeks (group B, six hamsters, 105 carcinomas) following the final MBN application. Between 3 days and 7 weeks post-treatment the proportion of tumors exhibiting p53 IHC activity (at least 10% of nuclei stained using D07 antibodies for detection of both mutant and wild-type p53) fell from 91 to 50%. However, during this same post-treatment period the frequency of tumors analyzed exhibiting confirmed sequence alterations in the conserved exons (E5–E8) of p53 remained constant (5/15 = 33% in group A versus 14/45 = 31% in group B). Heightened expression of wild-type p53 resulting from DNA damage in the immediate post-treatment period is likely to have contributed to the high proportion of group A tumors exhibiting p53 IHC activity. Nearly 80% of the identified p53 mutations were G->A and C->T transitions. The identified p53 point mutations occurred at or near (within three codons) of the corresponding hot-spot codons (175, 245, 248 and 273) of human oral SCC. The proportion of group A and group B tumors analyzed exhibiting Ha-ras mutations was 1/15 (7%) and 7/45 (16%), respectively. Only four of the observed eight Ha-ras mutations occurred in codons known to result in activation of this gene. Telomerase activation was demonstrated in 11 of 13 group A tumors (85%) and in 23 of 24 (96%) group B tumors analyzed. The alterations in p53, Ha-ras and telomerase activity observed in this HBP–MBN model are similar in many respects to those observed in the analogous human lesions of the head and neck. This model may be particularly useful for development of cancer chemoprevention regimens and multimodality cancer therapies.

Abbreviations: HBP, hamster buccal pouch; HBPC, hamster buccal pouch carcinoma; IHC, immunohistochemical; MBN, N-methyl-N-benzylnitrosamine; PBS, phosphate-buffered saline; PG, propylene glycol; SCC, squamous cell carcinoma; SSCP, single strand conformation polymorphism; TRAP, telomerase repeat amplification protocol.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In many respects the oral mucosa and the contiguous mucosa of the upper aerodigestive tract constitute a functional unit that is uniquely accessible and amenable to development of more effective diagnostic, preventive and therapeutic strategies for cancer control (112). The rapid development of novel and improved approaches for cancer control in this anatomical region requires the utilization of appropriate experimental models (821). In this regard, chemical carcinogenesis in the hamster buccal pouch (HBP) appears to be a particularly relevant in vivo model (8,9,1121). The development of squamous cell carcinoma (SCC) in HBP during exposure to specific chemical carcinogens recapitulates all of the structural (1921) and many of the biological (8,2429) and molecular alterations (8,23,24,27,3034) which occur during head and neck carcinogenesis in humans voluntarily exposed to the constituents of tobacco products and alcoholic beverages.

Mutations in the p53 (35,36) and Ha-ras (37) genes and activation of the ribonucleoprotein enzyme telomerase (38,39) are among the specific molecular alterations which have been observed in human cancers of the head and neck. The purpose of this study was to characterize carcinomas of the HBP induced by the potent mucosal carcinogen N-methyl-N-benzylnitrosamine (MBN) with regard to telomerase activation, p53 and Ha-ras mutation and immunohistochemical (IHC) expression of p53 protein. The occurrence of these molecular alterations and the IHC expression of p53 in both the carcinomas and precancerous lesions observed in this animal model resemble those reported for the analogous head and neck lesions of man. The results of this study further advance this variant of the HBP model as a potential tool for the development of novel strategies for chemoprevention, diagnosis and treatment of head and neck cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal treatment and tissue sampling
Sixteen male Syrian golden hamsters, 3–5 weeks of age, were treated by painting both buccal pouches, each Monday and Friday for 22 weeks, with a 1% solution (10 mg/ml) of MBN dissolved in propylene glycol (PG) (22). Two control hamsters were similarly treated topically with PG alone. Induction of wild-type p53 (40,41) and a myriad of other metabolic effects (e.g. induction of phase I and phase II enzymes and induction of DNA repair processes) occur within hours or days following exposure of target tissues to chemical carcinogens. Accordingly, groups of hamsters were killed at 3 days and 7 weeks following the final MBN application, in order to compare the IHC expression of p53 in tumors and surrounding tissues, when DNA damage and metabolic effects resulting from carcinogen exposure are likely to be either maximal (i.e. at 3 days) (42) or largely diminished (i.e. after several weeks). Three days after the final MBN treatment, eight hamsters were killed and tumor tissues were harvested for IHC analysis and extraction of DNA and protein as described below. Two of the remaining MBN-treated hamsters died unexpectedly in week 6 following carcinogen treatment and could not be included in the study. The remaining six MBN-treated hamsters were killed during the following week (i.e. week 7) for tumor analysis. At the time of death the tumor-bearing pouches were immediately excised, cleansed by thoroughly rinsing twice in cold phosphate-buffered saline (PBS), blotted dry and spread out with connective tissue side down on pieces of cardboard. The tumors were then enumerated, diagrammed and their maximum dimensions were recorded in 0.5 mm increments. Representative exophytic tumor masses, comprised predominantly of grayish glistening neoplastic tissue, were identified and individually bisected using a clean blade to avoid cross-contamination of tumor macromolecules. One half of each tumor thus obtained was frozen in liquid nitrogen for DNA and protein isolation. The remaining tumor tissues were processed for routine histological evaluation and p53 immunohistochemical evaluation. Individual PG-treated control hamsters were similarly killed and their normal pouch tissues were sampled for histological, immunohistochemical and mutation analysis, at 3 days and 7 weeks, respectively, following completion of treatment.

Histology, p53 immunohistochemistry and analysis
After excision of portions of several individual tumors for DNA and protein extraction, the remaining pouch tissues were fixed in 10% neutral buffered formalin and embedded in paraffin within 48 h. Sections of 5 µm were prepared and stained with hematoxylin and eosin for routine histological evaluation. Contiguous sections were processed for immunohistochemical identification of p53 protein using an indirect immunoperoxidase technique employing the primary monoclonal antibody NCL-p53-D07 (mAb D07; Novocastra, Newcastle, UK) and the avidin–biotin complex method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) with 3,3-diaminobenzidine substrate. The mAb D07 antibodies detect both wild-type and mutant forms of p53 (43). Tissue sections mounted on gelatin–chrome alum-coated slides were deparaffinized in xylene (twice) and rehydrated with a descending series of absolute, 95%, 70% and 30% ethanol, followed by water. After blocking endogenous peroxidase activity with 1% H2O2, antigenicity was unmasked by microwave heating for 3 min in a 10 mM solution of sodium citrate. A blocking solution of 2% dry milk in PBS (with 0.02% sodium azide) was applied to reduce non-specific staining. The sections were incubated with mAb D07, at a dilution of 1:200, for 2 h at 25°C in a humidification chamber. After rinsing with PBS, the sections were incubated with biotinylated anti-mouse IgG antibody for 30 min, washed again with PBS and then incubated with avidin–biotin complex conjugated to horseradish peroxidase for another 30 min. After a PBS rinse, the sections were stained with 1 mg/ml 3,3-diaminobenzidine tetrahydrochloride (Novocastra) and 0.30% H2O2 for 1 min. After rinsing with PBS, the slides were washed in tap water for at least 5 min before counterstaining with hematoxylin. A p53-staining human breast carcinoma sample served as a positive control for p53 nuclear staining. This same tumor without primary antibody was used as a negative control.

The proportion (percentage) of tumor cells exhibiting p53 nuclear staining was determined by microscopic examination of randomly selected high power fields in each tumor. The average proportion of p53-stained cells was then calculated for each tumor and recorded in 10% gradations, as follows: 0–<10% = 0%, >=10–<20% = 10%, >=20–<30% = 20%, etc. These determinations were made independently by two pathologists, who typically agreed within one gradation. When initial determinations were discordant, a consensus figure was achieved by joint re-examination and evaluation of the tumors in question. A tumor was recorded as p53-positive when the proportion of tumor cells exhibiting nuclear staining was determined to be >=10%.

DNA isolation and PCR
DNA was isolated from tumor samples by conventional proteinase K/phenol/chloroform extraction (44). Segments of the hamster p53 gene were amplified in vitro by PCR, as previously described (45), using the oligonucleotide primers shown in Table IGo.


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Table I. The oligonucleotide primers used
 
A hamster Ha-ras sequence of 161 bp, including exon 1, was amplified using the synthetic oligonucleotide hamster-specific primers Ha-ras E1S (5'-TGGCAGCCTCTATAGAAGC-3') and Ha-ras E1A (5'-GCCAGAGCCCAGCAGGTAG-3'). A hamster Ha-ras sequence of 179 bp, including exon 2, was amplified using the hamster-specific primers Ha-ras E2S (5'-GACT- CCTACCGGAAACAGGT-3') and Ha-ras E2A (5'-CTGTACTGATGGA- TGTCTTC-3'). Parameters for amplification were 1 min at 94°C, 1 min at 55–57°C and 1 min at 72°C for 35 cycles. All amplifications were performed using a TouchDownTM thermal cycling system (Hybaid, Teddington, UK).

Single strand conformation polymorphism (SSCP)
A primary amplification was executed in duplicate as described above with unlabeled primers. Each sense strand was analyzed in a second amplification in which only the sense oligonucleotide was end-labeled with [{gamma}-32P]ATP (DuPont/New England Nuclear, Wilmington, DE) using T4 polynucleotide kinase (Promega, Madison, WI). The amplified product with only one strand radioactively tagged was added to a formamide-based loading buffer, denatured and loaded onto a 1x MDE gel matrix (AT Biochem, Malvern, PA) with 1x Tris–borate/EDTA buffer (45). Electrophoreses were done at a constant 20 W at room temperature for 12–16 h. The gels were transferred to Whatman 3MM paper and vacuum dried at 70°C. Autoradiography was done at –70°C for 2–5 h with Hyperfilm-MP (Amersham, Arlington Heights, IL) (45).

Alternatively, a silver staining detection system was used in addition to isotopic labeling. For this purpose the amplicons were denatured and electrophoresed using the GeneGel Excel 12.5/24 (12.5% T, 2% C) kit (Pharmacia Biotech, Uppsala, Sweden) executed at a constant 15 W for 2 h at 5°C (GenePhor Electrophoresis Unit; Pharmacia Biotech). Gels were stained using the Plus One Silver Staining Kit® (Pharmacia) according to the manufacturer's specifications.

DNA sequencing
In cases where SSCP demonstrated band shifts of >=20%, as determined by image densitometry (Viber Lourmat image system and Bio-ProfilTM analysis program; Marne La Vallee, France) direct sequencing of gel-purified amplicons (Qiaex II Gel Extraction Kit; Qiagen, Hilden, Germany) was performed with the use of a Tag/DyeDeoxy terminator sequencing kit and 377 automatic DNA sequencer (ABI, Foster City, CA) as specified by the manufacturer.

In cases where band shifts of <20% were observed, the bands were eluted from the gel and PCR was repeated prior to direct sequencing. In order to obtain unequivocal results, sequencing was routinely performed in both the sense and antisense directions.

Determination of telomerase activity
Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) as described by Kim et al. (46), with minor modifications (47). Protein extract for TRAP was obtained by the following procedures. Approximately 10 mg of frozen tissue sample was homogenized, mixed with 100 µl lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM ß-mercaptoethanol, 0.5% CHAPS and 10% glycerol), incubated on ice for 30 min, then the lysate was centrifuged at 12 000 g for 30 min at 4°C. The supernatant was repeatedly centrifuged under similar conditions. One microliter of supernatant was used to measure the protein concentration with a Bradford protein assay kit (Pierce, Rockford, IL). Then the protein concentration was adjusted to 3 µg/µl. The adjusted aliquots were stored at –70°C until use. TRAP analysis was performed as follows. An aliquot of 2 µl of adjusted extract (6 µg, equal to 2x104 cultured KB cells) was assessed in a 50 µl reaction mixture containing 2 µl (10 pmol) of TS primer (5'-AATCCGTCGAGCAGAGTT-3'), 2 µl (10 pmol) of CX primer (5'-CCCTTACCCTTACCCTTACCCTAA-3'), 5 µl of 10x TS buffer (0.2 M Tris–HCl, pH 8.3, 15 mM MgCl2, 630 mM KCl, 0.05% Tween 20, 10 mM EGTA and 1 mg/ml BSA), 2 µl (10 mM) dNTP, 0.8 µl (5 U/µl) Taq polymerase (Promega) and 36.2 µl of ddH2O overlayed with mineral oil in a 0.5 ml tube. After 30 min incubation at 25°C to allow formation of TS primer-dependent telomerase products, the reaction tube was subjected to 90°C for 3 min to inactivate the telomerase enzyme activity. It was followed by 31 cycles of PCR reaction consisting of 94°C for 30 s, 57°C for 30 s and 72°C for 30 s. Then, 25 µl of PCR products were analyzed by electrophoresing in a 12.5% non-denaturing polyacrylamide gel and 1x TAE buffer until the bromophenol blue dye had reached the lower border of the gel. The gel was then stained with a PlusOne DNA silver staining kit (Pharmacia) in a Hoefer automated gel stainer (Pharmacia). Photographic films were obtained by an image analyzer (Vilber Lourmat) under predetermined conditions (45). Telomerase activity was demonstrated by prominent successive bands (typically 10 or more).

Reproducibility of TRAP data was confirmed by two or more repeat experiments. Optimization testing demonstrated a proportional relationship between band number and band intensity. To check whether there were telomerase (or PCR) inhibitors preventing adequate amplification, samples negative in the TRAP reaction were diluted 10-fold in an attempt to recover telomerase activity. As an additional check for the presence of TRAP inhibitory factors, telomerase-negative extracts were also run in combination with positive extracts (1:1 ratio). To exclude the possibility of DNA contamination in positive samples, 2 µl samples of extract were incubated with 1 µl RNase A (1 mg/ml) for 20 min at 25°C, then subjected to TRAP assay. A negative control (sample-free, lysis buffer only) and a positive control (KB cell extract) were included with each round of TRAP assay.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A summary of the results, including tumor frequency and the proportion of carcinomas exhibiting p53 IHC activity, frequency of mutations in the p53 and Ha-ras genes and telomerase activity, is shown in Table IIGo.


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Table II. Frequency of carcinomas, p53 IHC and telomerase activity and p53 and Ha-ras mutations at 3 days and 7 weeks post-MBN treatment
 
All MBN-treated hamsters exhibited exophytic tumors of the buccal pouch, innumerable surface irregularities and regions of hyperkeratosis. Examples of the range of histopathological and p53 IHC results observed in MBN-induced precancerous lesions and SCC are shown in Figure 1Go. The tumors included well-differentiated and moderately differentiated SCC and occasional hyperplastic inflammatory lesions. The latter were not included in the tumor analysis. In the group A hamsters, evaluated 3 days after the last MBN application, 81 of the 89 carcinomas (91%) exhibited nuclear p53 IHC activity in 10% or more of the tumor cells. In both group A and B hamsters, patchy p53 IHC activity was also observed throughout the grossly tumor-free MBN-exposed epithelium. In these areas, p53 activity was observed primarily in sites of microscopic hyperplasia, structural precancerous lesions (i.e. dysplasia and carcinoma in situ) and in microinvasive SCC (Figure 1A–DGo). In non-dysplastic areas, p53 IHC activity was usually limited to the basal aspect of the epithelium (Figure 1AGo).



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Fig. 1. Range of MBN-induced HBP precancerous changes and carcinomas exhibiting nuclear p53 immunostaining. (A) Hyperkeratotic epithelium with mild basal hyperplasia and patchy intense p53 staining of basal cell nuclei (hematoxylin and p53, x500). (B) Dysplastic epithelium showing p53 nuclear staining of variable intensity (hematoxylin and p53, x330). (C and D) Microinvasive carcinomas (center) with p53 immunostained nuclei (hematoxylin and p53, x330). (E) Periphery of SCC exhibiting intense p53 immunostaining and scattered grossly enlarged nuclei (hematoxylin and p53, x330). (F) Interior of carcinoma showing generalized intense p53 nuclear staining (hematoxylin and p53, x500). (G) Moderately differentiated MBN-induced SCC invading muscle (lower left) (hematoxylin and eosin, x500). (H) SCC infiltrating desmoplastic stroma. The nuclear p53 immunostaining is of variable intensity. The focus of carcinoma shown in the center of the field has notable nuclear enlargement, nuclear pleomorphism and intense p53 staining, suggestive of tumor progression (hematoxylin and p53, x330).

 
The proportion of carcinomas exhibiting p53 IHC activity in group B hamsters was 53 of 105 (50%). Thus, the proportion of carcinomas exhibiting p53 IHC activity dropped significantly (P < 0.001, two-tailed Student's t-test) between 3 days and 7 weeks after MBN treatment. In general, there was no apparent relationship between the degree of differentiation of the carcinomas and the proportion or intensity of neoplastic cells exhibiting p53 IHC activity. Nor was there a relationship between the size of the tumors and the proportion of tumor cells exhibiting p53 IHC activity (Table IIIGo and Figure 2Go). However, an occasional carcinoma exhibited cellular regions or foci with both a greater degree of cytological atypia and more intense expression of p53 IHC activity than the tumor overall (see Figure 1HGo). Lesions with this pattern suggest a mechanistic link between tumor progression and expression of p53 IHC. The buccal pouch mucosa of individual control hamsters, evaluated at 3 days and 7 weeks following the last application of the vehicle PG, exhibited neither morphological change nor p53 IHC activity.


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Table III. Summary of group B carcinomas exhibiting p53 IHC activity and p53 and Ha-ras mutations
 


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Fig. 2. Lack of correlation between tumor size and percentage of tumor cell nuclei exhibiting p53 IHC activity. •, values of individual tumors; {blacktriangleup}, values of two or more tumors. r2 = 0.72.

 
A combined total of 60 buccal pouch carcinomas from group A (15 carcinomas) and B (45 carcinomas) hamsters were analyzed by SSCP and DNA sequencing in order to identify specific p53 and Ha-ras mutations (Tables II–VGoGo). Approximately 32% of these carcinomas exhibited p53 mutations (Table IIGo). Representative results for group B carcinomas, analyzed by SSCP and DNA sequencing of p53 exons 5 and 7, are shown in Figures 3 and 4GoGo, respectively. Eleven of the 14 group B carcinomas (i.e. 79%) exhibiting p53 mutations, as determined by either SSCP or a combination of SSCP and DNA sequencing, also exhibited p53 IHC activity in 10% or more of their constituent cells (see Table IIIGo). The specific mutations identified in exons 5–8 of the p53 gene are presented in Table IVGo. Of the 18 carcinomas wherein specific p53 mutations were identified by SSCP and DNA sequencing, G->A and C->T transitions accounted for 44 and 33%, respectively.


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Table V. Ha-ras mutations in hamster pouch carcinomas
 


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Fig. 3. SSCP/sequencing of p53 exon 5. (A) SSCP. Lane 1, B1-L6; lane 2, B3-R6; lanes 3 and 4, normal control; lane 5, B3-L7; lane 6, B3- L2; lane 7, B3-L5. Arrows designate mobility shifts. (B) Sequencing. (Upper) Normal sequences; (lower left) a CGT->CAT mutation at codon 158 indicated by sequencing the PCR products of the shifted band in lane 2; (lower right) a CCC->TCC mutation at codon 177 indicated by sequencing the PCR product of the shifted band in lane 6.

 


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Fig. 4. SSCP/sequencing of p53 exon 7. (A) SSCP. Lane 1, B4-R8; lane 2, B5-R2; lane 3, B1-L6; lane 4, B3-R6; lane 5, B4-L3; lane 6, normal control. Arrows designate mobility shifts at identical positions in lanes 2 and 5. (B) Sequencing. (Upper) Normal sequence; (lower) a CGG->TGG mutation at codon 251 indicated by sequencing the PCR product of B5-R2.

 

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Table IV. p53 mutations in group A and B carcinomas
 
Of the 60 carcinomas analyzed by SSCP and DNA sequencing to identify mutations in the Ha-ras gene, only eight (13%) exhibited a point mutation in this gene. Figures 5 and 6GoGo show representative SSCP/sequencing analyses of Ha-ras gene exons 1 and 2, respectively, in group B buccal pouch carcinomas. A summary of the Ha-ras mutation analysis is shown in Table VGo. Activation of the ras gene family is known to occur primarily through mutation at codons 12, 13 and 61 (48,49). In three of the eight buccal pouch carcinomas harboring Ha-ras mutations, the mutations were detected in either codon 12 (two carcinomas) or 13 (one carcinoma). However, mutations were also identified in codons 7 (three carcinomas), 60 (one carcinoma) and 63 (one carcinoma).



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Fig. 5. SSCP/sequencing of H-ras exon 1. (A) SSCP. Lane 1, normal control; lane 2, B5-R3; lane 3, B2-R4; lane 4, B4-L1; lane 5, B5-L4. Arrows designate mobility shifts. (B) Sequencing. (Upper) Normal sequences; (lower left) a GGC->GAC mutation at codon 13 by sequencing the PCR product of B5-R3; (lower right) a GTG->ATG mutation at codon 7 by sequencing the PCR product of B5-L4.

 


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Fig. 6. SSCP/sequencing of H-ras exon 2. (A) SSCP. Lane 1, B3-L4; lanes 2 and 3, normal control; lane 4, B2-R4. Arrows indicate mobility shifts. (B) (Upper) Normal sequences; (lower left) a GGC->GAC mutation at codon 60 by sequencing the PCR product of B3-L4; (lower right) a GAG->AAG mutation at codon 63 by sequencing the PCR product of SB2-R4.

 
A total of 37 MBN-induced hamster buccal pouch carcinomas (HBPCs) were examined for telomerase activity using TRAP analysis. Telomerase activity was observed in 34 (92%) of these tumors (Table IIGo). Representative TRAP analyses of group A and B carcinomas are shown in Figure 7Go. Telomerase activity was not detected in PG-treated control HBP epithelium.



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Fig. 7. TRAP analysis. Lane M, 25 bp ladder molecular weight marker; lanes 1–3, representative carcinomas of group A; lanes 4–7, representative carcinomas of group B; lane 8, normal control; lane 9, a positive control of human oral SCC. Sample 5 is the only sample exhibiting negativity of telomerase activation in group B.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study examined the frequency of telomerase activation, distribution of p53 IHC activity and the character and genomic distribution of p53 and Ha-ras mutations in HBPCs induced by topical application of MBN. The pattern of p53 IHC activity and its persistence were examined in both precancerous and malignant MBN-induced lesions.

Telomerase activation was demonstrated in 34 of 37 (92%) of the HBPC examined. This frequency of carcinomas exhibiting telomerase activation is comparable with that observed in carcinomas of the head and neck (38,39,47,50). p53 mutations and persistent p53 IHC activity were observed in 32 and 50% of the carcinomas examined, respectively. Ha-ras mutations were observed in <15% of the buccal pouch carcinomas examined. Whereas Ha-ras mutational activation is reportedly common in Asian populations wherein chewing tobacco is a major risk factor (37), Ha-ras mutations are much less common in Western populations whose head and neck carcinomas are most frequently attributable to the use of tobacco products and alcoholic beverages (5154). Similarly, the frequency of p53 mutations in head and neck cancers is high in populations using tobacco products and alcoholic beverages (35,36,55,56), but comparatively low in populations where these specific risk factors are not strongly implicated (55,5761). Thus the frequency of occurrence of these molecular alterations in carcinomas induced in the MBN–HPB model is comparable with that observed in Western cultures where tobacco smoking and excessive use of alcoholic beverages are the major risk factors for head and neck cancer.

p53 mutation was demonstrated in 32% of the buccal pouch tumors examined using SSCP and sequencing (Table IIGo). The frequency of p53 mutations observed is in agreement with the frequency of 35% observed in an earlier study undertaken with this model (33) and approximates that of 40–50% reported for human head and neck cancers (35,36,55,56,62). Additional p53 mutations may have gone undetected in this study owing to limitations of the SSCP technique (63,64) or as a consequence of restricting the analysis to exons 5–8. However, the human p53 data indicate that mutations outside these conserved exons are uncommon (36,62), accounting for only ~2% of all p53 mutations in carcinomas of the head and neck (62). Many of the p53 mutations identified in the MBN-induced carcinomas occurred at or near codons corresponding (65) to the `hot-spot' codons 175, 245, 248 and 273, identified in human oral cancers and cancers of other head and neck locations (36,62). In this study, G->A transition mutations predominated (15/19 = 79%), as is characteristic of mutations induced by MBN (66,67). G->A transitions are also the most common form of p53 mutations occurring in human head and neck cancer, including those of oral mucosa (35,36,62). G->A transitions are also the predominant type of p53 mutation in esophageal carcinomas occurring in high risk areas of China (64,68), where MBN has been implicated as an environmentally derived agent contributing to this disease (69,70).

p53 IHC activity was observed in the majority (81/89 = 91%) of carcinomas harvested at day 3 post-MBN treatment and in 53 of the 105 (50%) carcinomas of the group B hamsters examined at 7 weeks following MBN exposure. The differences in frequency of p53 IHC expression between these groups is striking. Whereas wild-type p53 protein is rapidly degraded (72) and not usually detectable by IHC techniques, mutant forms are more stable (72,73), thus enabling detection by IHC (7477). Stabilization of p53 protein secondary to mutation accounts for the frequent IHC detection of this protein in human neoplasms (64,7682), including carcinomas of the head and neck (35,36,62,82,83), and is the most likely determinant of the persistent p53 IHC activity observed in the present study at 7 weeks post-MBN treatment. Of the 45 group B tumors analyzed by both IHC and SSCP/DNA analysis, concordant results (i.e. both analyses either positive or negative) were observed in 29 (64%) of the carcinomas, according to the criteria selected for IHC positivity used in this study (i.e. 10% or more of the tumor cell nuclei exhibiting p53 activity). This result is comparable with data reported for cancers of the head and neck, for which concordance between overexpression of p53 and molecular confirmation of mutation reportedly ranges from ~50 to 90% (36,62,82,83). It is also noteworthy that in this experimental group 11 of the 13 carcinomas (i.e. 85%) with confirmed mutations in exons 5–8 also exhibited p53 IHC activity. These results indicate that, at most, only a distinct minority of carcinomas harboring missense mutations in this experimental model will fail to exhibit persistent p53 IHC activity.

p53 overexpression has been demonstrated in squamous cell carcinomas of the head and neck (36,61,82,8487) and other sites (82,88), without apparent p53 mutations. The high proportion of HBP carcinomas exhibiting p53 IHC activity at day 3 post-MBN treatment cannot be attributed to p53 mutation alone. It is well established that biological perturbations other than mutations (41,76,84,8994) and many technical variables (76,9597) help determine the presence, pattern of distribution and intensity of p53 IHC activity. Wild-type p53 may be overexpressed as a consequence of acute exposure to a variety of DNA-damaging agents, including radiation (98103) and genotoxic chemicals (40,41,99,100). In the present study, induction of p53 in response to MBN-mediated DNA damage is the most likely contributor, in combination with mutational stabilization of p53 protein, to account for the high proportion of tumors expressing p53 IHC activity at day 3 post-MBN treatment. This hypothesis is supported by the following: (i) DNA damage is likely to approach maximum levels by 3 days following MBN administration, since (a) MBN is an indirect acting carcinogen which must undergo metabolic activation to reactive intermediates (104) following its gradual absorption from the mucosal surface and (b) previous studies have demonstrated that following systemic administration of MBN to rodents, bound metabolites of MBN reach maximum levels by approximately 3 days (42); (ii) the observed reduction in the proportion of carcinomas exhibiting p53 staining between 3 days and 7 weeks following the last MBN application is likely a consequence of tissue metabolism and clearing of MBN, with concomitant subsidence of DNA damage and repair and an accompanying reduction in the levels of induction of wild-type p53; (iii) the D07 antibodies used to demonstrate p53 overexpression in this study recognize wild-type as well as mutant forms of the p53 protein (43). In addition, there is no evidence to suggest that overexpression of the endogenous protein mdm-2 contributes to p53 stabilization (76,84) in the MBN–HBP model (33).

As expected from the results of a previous study (22), histological examination of the flat to irregular mucosa surrounding the MBN-induced carcinomas revealed a complete spectrum of hyperplastic, hyperkeratotic and dysplastic lesions, as well as carcinomas in situ and microinvasive SCC (see Figure 1Go). The precancerous nature of dysplastic lesions and carcinomas in situ is well established from clinico-pathological studies in man (105,106). In the present study, p53 IHC activity was observed in many of these structural precancerous lesions and in many non-dysplastic sites within the MBN-exposed mucosa. Thus the pattern of p53 staining in this animal model recapitulates those observed in structural precancerous lesions (64,87,97,108111), as well as in high cancer risk non-dysplastic squamous mucosa in man (64,108,111). It is reasonable to hypothesize (112) that in both man and this hamster pouch model (33): (i) persistent intra-epithelial p53 IHC lesions are material precursors for the subsequent development of at least some of the carcinomas also exhibiting p53 IHC activity; (ii) that loss of p53 function plays a mechanistic role in the development of carcinomas. In normal squamous mucosa, DNA damage resulting from carcinogen exposure leads to up-regulation of wild-type p53, which in turn contributes to inhibition of cell replication and apoptotic cell death (92,113). Presumably, mucosal sites lacking p53 function, such as those with persistent p53 IHC activity resulting from p53 mutation, have a selective growth advantage over the surrounding mucosa, resulting in their expansion during episodes of repeated carcinogen exposure and DNA damage. According to this working hypothesis, cellular replication at these sites, in the face of continual carcinogen-mediated DNA damage, in turn increases the risk of acquiring additional mutational events leading to a malignant phenotype (112).

Ha-ras mutations were observed in eight of 60 (i.e. 13.3%) MBN-induced carcinomas. The frequency of MBN-induced Ha-ras mutations observed here is in agreement with that of a previous study (33). Three of the 60 carcinomas in the present study exhibited double mutations involving both the Ha-ras and p53 genes (see Table IIIGo). Activating Ha-ras mutations occur most commonly at codons 12 and 61 and potentially at codons 13, 59, 63, 116 and 119 (48,49). Only four of the eight observed Ha-ras mutations occurred at codons which have been shown to activate this oncogene (specifically, two mutations at codon 12, one mutation at codon 13 and one mutation at codon 63) (see Table VGo). The functional significance, if any, of the other four mutations observed at codons 7 (three mutations) and 60 (one mutation) is unknown. The MBN-induced HBPC exhibited a lower frequency of Ha-ras than p53 mutations. This relationship is also characteristic of oral cancers observed in cigarette smoking populations (53).

Telomerase activity was observed in 92% (34/37) of the HBPC examined. In contrast to the temporal variation in levels of p53 IHC expression observed, the metabolic perturbations and toxicity related to recent MBN exposure appeared to have no effect on telomerase activation, since there was no appreciable difference in the level of telomerase activity or the frequency of carcinomas exhibiting telomerase activity between groups A and B (85 versus 96%). The high frequency of tumors exhibiting telomerase activation in this study parallels that seen in human tumors (46), including SCC of the oral cavity and head and neck (38,39,46,47,50). It has not been established in this model whether telomerase activation is related to acquisition of the neoplastic phenotype per se or is a function of enhanced cell replication (114) or other persistent alterations resulting from carcinogen exposure. Whereas ostensibly normal squamous mucosa usually lacks telomerase activity (38,115,116), it is frequently demonstrable in structural precancerous lesions of the head and neck (38,39,47,50,115). In contrast to the corresponding malignant lesions, benign neoplastic or hyperplastic lesions of uterus, prostate and breast characteristically lack telomerase activity (46,117). However, in oral and other head and neck mucosal sites, telomerase activity has been demonstrated in benign hyperplastic lesions (50,115), as well as in presumptive precancerous lesions such as dysplasia (i.e. premalignant hyperplasia). In hyperplastic head and neck lesions with a strong inflammatory component, infiltrating lymphocytes should also be considered as a potential source of telomerase activity (118). A recent study has suggested a relationship between telomerase levels and levels of cell proliferation during HBP carcinogenesis mediated by the polycyclic aromatic hydrocarbon 7,12-dimentlybenz[a]anthracene (34). Additional studies are required to critically examine the role of telomerase activation in neoplastic progression versus hyperplasia, both in experimental models (119) and in head and neck carcinogenesis.

This study demonstrates molecular similarities between MBN-induced HBP lesions and the analagous human lesions of the head and neck. The proximal aerodigestive tract has many characteristics which make this anatomical region amenable to development of novel strategies for cancer chemoprevention and therapy (1,120122). Perhaps chief among these is the accessibility of this anatomical region in general, and the oral mucosa in particular, for efficient delivery of chemopreventive and therapeutic agents and for effective clinical monitoring by visualization, palpation and periodic tissue (or cytological) sampling. The mounting evidence of molecular similarities between these two systems supports the MBN–HBP model as a promising in vivo system for testing potential therapeutic agents and for the development of individual and combinational therapies directed at specific molecular targets. p53 (120,123127) and telomerase (128,129) have frequently been proposed as potential molecular targets for cancer chemotherapy. The MBN–HBP model may be useful in the development of such novel chemopreventive and therapeutic modalities, by establishing their efficacy in vivo, prior to institution of clinical trials.


    Notes
 
2 To whom correspondence should be addressed Email: d-solt{at}nwu.edu Back


    Acknowledgments
 
We thank Janardan K.Reddy MD (Magerstadt Professor and Chairman of Pathology, Northwestern University) for Departmental support and D.S.R. Sarma PhD (University of Toronto) for valuable discussions during the preparation of the manuscript. We also wish to thank Vanessa Jones, Gail Alfred, Sandi Eggena, Carol Kiely and Janet Wilson for excellent technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 19, 2000; revised March 27, 2000; accepted March 29, 2000.





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