Mutagenesis, Vol. 17, No. 1, 73-77,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Micronuclei and p53 accumulation in preneoplastic and malignant lesions of the head and neck
1 National Cancer Research Institute, Genova, Italy, 2 Institute of Mutagenesis and Differentiation of the CNR, Pisa, Italy and 3 Department of Oncology, Biology and Genetics, University of Genova, Genova, Italy
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Abstract |
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No single biomarker can predict the risk for malignant trasformation of precancerous lesions of the head and neck. Micronucleus frequency, nuclear p53 accumulation and mitotic index were determined in proliferating basal cells using paraffin-embedded specimens from normal, dysplastic and malignant tissues. p53 accumulation was detected by immunohistochemistry using pAb 1081 and pAb 240 antibodies. Micronuclei were scored in the same cell population and classified for the presence/ absence of p53 accumulation in the main nucleus. Fifty-three carcinomas and 15 precancerous lesions were studied. Both micronuclei and p53 accumulation were found in precancerous lesions, suggesting that they are early events in head and neck squamous cell carcinoma progression. The two biomarkers were not related to each other: indeed, micronucleus frequency was higher in p53-negative than in p53-positive cells. Three patients with precancerous lesions later developed carcinomas; all three cases showed high frequencies of both micronuclei and cells accumulating p53 protein.
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Introduction |
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It has been estimated that numeric chromosome imbalance is the most prevalent genetic change recorded among >20 000 solid tumors analyzed so far (Heim and Mitelman, 1995
). The significance of chromosome imbalance in cancer is not completely understood, but the consequent change in copy number of specific genes is likely to play a major role. In particular, tumor suppressor genes may be lost as a consequence of chromosome loss. Changes in the tumor suppressor gene p53 occur at a particularly high frequency in cancer. Many cancers express high levels of either mutant or stabilized normal p53. p53 is peculiar among tumor suppressor genes, because defective cells present a generalized genetic instability, such as gene amplification (Tilsty et al., 1992), chromosone breakage and chromosome imbalance (Bischoff et al., 1990). Loss of p53 tumor suppressor protein also results in centrosome amplification, which leads to aberrant mitosis and chromosome instability (Carrol et al., 1999). Chromosome imbalance may then both lead to and be augmented by p53 mutations. The recent observation that centrosome amplification occurs at a high frequency in p53-defective cells (Fukasawa et al., 1996) suggests a possible mechanism for chromosome imbalance.
Immunohistochemical studies have shown p53 overexpression to be an early event in head and neck squamous cell carcinoma (HNSCC), being found in dysplastic lesions and in carcinomas in situ, before the development of invasive carcinoma. p53 overexpression reflects not only gene mutation but also a normal response to DNA damage induced by genotoxic carcinogens (Boyle et al., 1993). Focal p53 overexpresssion has been found in human mucosa in relation to smoking.
We have previously reported that in the head and neck region micronuclei are found at increased frequencies from normal mucosa to potentially precancerous lesions to carcinoma, suggesting ever increasing chromosome instability (Casartelli et al., 2000). In this paper we wanted to answer the question of whether the increase in micronucleus frequency is matched by an increase in p53 overexpression. To this end, we analyzed the presence of nuclei accumulating p53 protein and the presence of micronuclei in epithelial cells of the basal layer of normal mucosa, precancerous lesions and carcinomas from 68 patients. Mitotic index (MI) was evaluated as indicative of the proliferating ability of the cells. Our results in basal cells confirmed a trend of micronuclei to increase from normal tissue to carcinoma. However, the increase in chromosome imbalance was not apparently correlated with p53 overexpression.
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Materials and methods |
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Patients
We studied a total of 68 patients, 53 cases of squamous cell carcinoma and 15 cases of precancerous lesions with various levels of dysplasia (mild to severe). Among cases of carcinoma, 18 presented dysplastic tissue in the vicinity of the lesions and 16 presented normal tissues in the same biopsy. Three cases of precancerous lesions evolved to carcinomas during the study. Biopsies of seven healthy people were analyzed as controls.
Nuclear p53 accumulation
Formalin-fixed and paraffin-embedded tissue sections were prepared at the Servizio di Anatomia e Citoistologia Patologica (SACIP) from surgical biopsies. Sections (4 µm thick) were cut from the paraffin blocks and deparaffinized as described previously (Harlow and Lane, 1988). After deparaffinization and dehydration, endogenous peroxidases were blocked with 0.6% hydrogen peroxide in methanol for 5 min at room temperature and washed three times with phosphate-buffered saline (PBS) for 10 min. Two antibodies were used, pAb 1801, recognizing both wild-type and mutant forms of p53, and pAb 240, recognizing only mutant forms of p53 (kindly provided by Prof. D.P.Lane). The slides were incubated overnight with the antibodies diluted 1:75 in PBS containing 0.1% bovine serum albumin (BSA). After incubation, the specimens were washed three times with PBS and non-specific binding sites were blocked with PBS, 3% BSA for 30 min at room temperature. The sections were then incubated with a biotinylated anti-mouse antibody diluted 1:100 in PBS, 1% BSA. After three washes in PBS, incubation with ABC complex (10 µg peroxidase/ml PBS) was performed for 30 min at room temperature. The specimens were then incubated with a 0.03% solution of diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide in PBS for 15 min at room temperature. After washing, sections were counterstained with Mayer's hematoxylin diluted 1:5 for 5–10 min and mounted with permanent medium. Accumulation of p53 was indicated by brown staining of the nucleus. Single cells were classified as p53+ and p53- depending on whether or not their nucleus stained brown in this assay. As a control, the slides were stained without the primary antibody. Positive control cases were retrieved from archival material, including cases previously know to be positive for p53 staining. The stained slides were examined under a microscope at 1000x magnification by two researchers.
Detection of micronuclei
Micronuclei were scored by standard criteria (Countryman and Heddle, 1976) in the same cell population and micronucleus counts were recorded separately in cells with and without nuclear p53 accumulation.
Determination of MI
Mitotic figures were counted in the same cell population and classified for the presence or absence of p53 accumulation, as done for micronuclei.
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Results |
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Micronucleus frequency in basal cells of oral mucosa: comparison between cells with and without nuclear p53 accumulation.
Micronuclei are formed during cell division and are a semi-permanent form of damage (Countryman and Heddle, 1976
). In Chinese hamster fibroblasts chemically induced micronuclei were found to be lost in a few days (Granetto et al., 1996). Micronuclei are therefore expected to be more frequent in the basal layer, where they are generated, than in more superficial layers of the mucosa and in exfoliated cells, an expectation which has received experimental confirmation (Casartelli et al., 2000). In exfoliated cells we found that micronucleus frequencies increase with the gravity of the lesion (Casartelli et al., 2000). We have now measured the micronucleus frequency in basal cells of normal and pathological oral mucosa and have found mean values (± SD) of 6.9 ± 3.2, 12.3 ± 6.8 and 10.1 ± 5.9 per 1000 cells for normal tissues surrounding pathological lesions, precancerous lesions and carcinomas, respectively. The micronucleus frequency was lower in normal tissues derived from control subjects than in normal tissues surrounding the lesions in patients, ranging from 0.15 to 0.9 per 1000 in a group of nine subjects. Micronucleus frequencies were measured in archived tissue sections. This might reduce the accuracy of the assay. However, there is no reason to expect systematic errors and random overestimations/underestimations should balance each other with the relatively high numbers of cells scored (see Table II).
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Table I
shows the fraction of specimens with a relatively low (<5%), medium (5–50%) or high (>50%) percentage of p53-positive (p53+) cells in normal and pathological tissues of patients. The `precancerous lesions' category includes lesions with various levels of dysplasia (mild to severe). In situ carcinoma and invasive cancer have been included under `carcinomas'. The data indicate that cells with nuclear accumulation of p53 are found even in normal tissue. However, specimens with a high percentage (>50%) of p53+ cells were more frequent in carcinomas than in normal tissue or precancerous lesions. Fisher's exact test indicated significant differences between carcinomas and normal tissue (P = 0.003) and between carcinomas and precancerous lesions (P = 0.032). Only one specimen with a percentage of p53-accumulating cells >50% was observed among 16 normal tissue samples from patients. No p53-accumulating cells were observed in tissues from seven control subjects.
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Accumulation of p53 in normal tissue could be the consequence not only of mutation but also of stress suffered by the cells, which remained blocked temporally in G1. The antibody pAb 240, which recognizes mutant forms but not wild-type p53 in its native form (Gannon et al., 1990
), was used to re-evaluate p53 accumulation previously detected with the antibody pAb 1801. Five of 25 tissue samples of precancerous lesions derived from 16 patients did not show nuclear immunostaining, suggesting that wild-type p53 accumulated in these nuclei; the remaining 20 samples showed nuclear accumulation, presumably of the mutant form of p53. The latter conclusion is weakened by the known ability of pAb 240 to react with wild-type p53 when its native form has been disrupted, e.g. during sample preparation. Nevertheless, the ability of pAb 240 to discriminate between different populations of p53 is not lost altogether (Gannon et al., 1990). Moreover, an ability of pAb 240 to discriminate among pAb 1801-positive cell populations in archived tissues of HNSCC has been reported (Piffko et al., 1995). As a second, indirect criterion, we also looked at p53 accumulation in the fibroblast nuclei of connective tissue. Accumulation of p53 in tissues proximal to the lesion is considered indicative of cellular stress rather than mutation (Ramael et al., 1992; Hall et al., 1993). The 20 precancerous lesions, suspected to contain mutant p53 on the basis of the pAb 240 immunostaining, had no p53 accumulation in the surrounding fibroblasts and inflammatory cells.
Micronuclei were scored on the same slides stained for p53 and their frequency was determined separately in p53+ and p53- cells. As shown in Table II
, there was no difference between the two cell populations in any of the three tissue categories (Mann–Whitney test). The frequencies of micronulei in cells accumulating and non-accumulating p53 were also compared using the 
2 test for each patient. In 12 of 15 specimens of precancerous lesions there was no difference in the frequency of micronuclei in the two cell populations. Two specimens showed a higher percentage of micronuclei in p53- cells and one a higher percentage in p53+ cells. Micronucleus frequencies were not significantly different in 26 of 28 cases of carcinoma. In two cases more micronuclei were found in p53+ cells. These data indicate that, in our group of patients, chromosome instability was already high in normal tissues and precancerous lesions irrespective of p53 accumulation.
MI was measured in p53+ and p53- basal cells (Table II). In control tissues only five mitoses were observed among a total of 13 275 nuclei (MI = 0.4). In tissues from patients we studied this parameter in relation to p53 accumulation. Significant differences between p53+ and p53- cells were found in all three types of tissue, with higher frequencies in p53- cells, suggesting a cellular response to some kind of stress. MI increased with the gravity of the lesions, but there was no difference in the proliferation rate of p53+ and p53- basal cells. Statistical analyses (Fisher's exact or 2 test) performed for each patient again showed no difference in MI between p53+ and p53- cells in 42 of 51 total cases (three classes pooled together). Among nine cases showing a significant difference in MI, six showed a higher frequency in p53+ cells. Aberrant mitoses were observed in both p53+ and p53- cells: pooling data from the different types of lesions, we observed 16 of 240 and 47 of 591 aberrant mitoses in p53+ and p53- cells, respectively. This difference was not significant (P = 0.66, Fischer's exact test).
Accumulation of p53, MI and micronuclei in patients with multiple lesions
We studied nine cases in which precancerous lesions and carcinomas were present in the same patient (Table III). These data show the high heterogeneity of different cases and also of samples obtained from different tissue areas of the same patient. In six cases (2 and 5–9 in Table III) a general trend of p53+ cells to increase with the gravity of the lesion is quite evident. However, that this is not an absolute rule is shown by cases 1 and 4. Intra-individual variation was particularly evident in cases 2 and 5, where biopsies from different areas of the same lesion showed large differences in the incidence of p53+ cells. In two cases (2 and 5 in Table III) micronucleus frequency and p53 accumulation varied concomitantly in precancerous lesions and carcinomas, but, in general, the correlation between p53+ cells, cell proliferation and micronuclei was poor.
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p53 accumulation, MI and micronuclei in lesions that later on evolved to carcinoma
Among biopsies present in the SACIP tissue repository, three cases with an evolution from dysplasia to carcinoma were singled out (Table IV
). The data indicate moderate to high frequencies of cells accumulating p53 and of micronuclei in dysplastic lesions of the three cases. Carcinomas did not show higher frequencies of p53+ cells or micronuclei compared with the lesions from which they evolved.
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Discussion |
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The distribution of cases with p53 accumulation among normal tissue, precancerous lesions and carcinomas is in line with data already published by various authors (Ahomadegbe et al., 1995
; Reis et al., 1998). Our data, however, are in general greater than the data obtained by pooling different reports with high numbers of patients. Accumulation of p53 is an early biomarker, already being present in cells defined as normal by histopathological parameters. About 80% of precancerous lesions were classified as p53+, a result similar to that described by Rich et al. (1999). The fraction of p53+ carcinomas was not higher than the fraction of p53+ precancerous lesions (78%), but the percentage of cases with >50% p53+ cells was increased in carcinomas.
An advantage of our experimental approach is that it is possible to correlate different biomarkers, in our case micronuclei and nuclear p53 accumulation, at the level of single cells of the proliferating layer. Defining the cause of p53 accumulation was beyond the aim of our study, but a next step will be to sequence p53 in our samples. Micronuclei, a marker of chromosome instability, should in theory be related to p53 protein: they are expected to increase in cells with functional alterations of p53. In esophageal carcinoma, another epithelial tumor, p53 mutation is considered a very early event in the progression (Montesano and Hainaut, 1998). On the other hand, chromosome aberrations are also described as precocious in these types of tumors (Califano et al., 1996). In our study, micronucleus frequency was not higher in cells with nuclear p53 accumulation than in cells not accumulating p53. Micronucleus counts were already high in morphologically defined normal tissue of patients and in tissues with low levels of dysplasia, in comparison with normal tissue of healthy subjects. Our data suggest that chromosome loss is an early event not necessarily subsequent to stabilization of p53 protein.
Accumulation of p53 under stress is a transient phenomenon causing arrest of the cell cycle in G1/S. Unlike p53 accumulation due to DNA damage, stress-induced accumulation is not expected to involve micronucleus formation. It has been reported that p53 mutations are not found in all HNSCCs with nuclear accumulation (Ahomadegbe et al., 1995; Reis et al., 1998). Using two different antibodies and scoring fibroblasts surrounding the lesions we could exclude only a few cases, in both precancerous lesions and in carcinomas, from those classified as containing mutant forms of p53. The MI data indicated, as expected, higher proliferation in carcinoma cells, compared with dysplastic and normal tissues surrounding the lesion and in comparison with normal tissues from controls, influenced by p53 status (Table II).
The availability of multiple biopsies from the same patient (different areas with different levels of dyplasia or carcinoma) are a precious material to understand the significance of biomarkers as predictors of progression. However, the study of the nine cases in our hands was disappointing. A very heterogeneous pattern emerged, from which no clear trend could be evinced. In three more cases we were able to follow up the evolution of dysplastic lesions to carcinomas. High frequencies of micronuclei together with high frequencies of p53-accumulating cells were found in dysplastic tissues of these patients, but these frequencies did not further increase in the resulting carcinomas. These very limited data do not suggest that cancer cells originated from cell subpopulations in the precancerous lesions with higher genomic instability.
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Acknowledgments |
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This work was partially supported by the Ministero della Sanità and MURST.
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Notes |
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4 To whom correspondence should be addressed at: National Cancer Research Institute, Largo R. Benzi 10, I-16132 Genova, Italy. Tel: +39 10 5600251; Fax: +39 10 5600992; Email: 'stefania bonatti'abbondan{at}hp380.ist.unige.it
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Received on July 4, 2001; accepted on September 19, 2001.
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