Molecular and cellular alterations in tobacco smoke-associated larynx cancer

Mutation Research 445 Ž1999. 259–274 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres

Molecular and cellular alterations in tobacco smoke-associated larynx cancer K. Szyfter a

a,)

, Z. Szmeja b, W. Szyfter b, K. Hemminki c , J. Banaszewski b, R. Jaskuła-Sztul a , J. Louhelainen c

Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska ´ 32, Poznan´ 60-479, Poland b Clinic of Otolaryngology, K. Marcinkowski Medical UniÕersity, Poznan, ´ Poland c Center for Nutrition and Toxicology, Karolinska Institute, Huddinge, Sweden Received 15 November 1998; accepted 15 December 1998

Abstract Tumours of head and neck belong to the most frequent types of cancer world-wide. In Poland, mortality from larynx cancer among males has been continuously increasing during the last decades up to 8.4 deaths per 100,000 men in 1993, which exceeds epidemiological records from other countries. The aetiology of laryngeal cancer is strongly associated with exposure to carcinogens present in tobacco smoke. The review describes a sequence of molecular and cellular events from carcinogenic exposure, DNA adduct formation, detection of mutations in the p53 gene, loss of heterozygosity ŽLOH. in chromosomal loci encoding the p53 and p16 genes, and loss of control of the cell cycle. The section concerning DNA adducts includes a discussion of the role of such confounders as exogenous exposure, the age and sex of the subject, and disease progression. The significance of genetic factors as individual risk determinants is discussed in relation to bleomycin-induced chromosome instability and in connection with the occurrence of defects in genes encoding detoxifying enzymes. The question concerning the substantial difference between men and women in larynx cancer morbidity and mortality remains open, even when the significantly higher adduct formation in male DNA compared with female material was taken into account. Preliminary experiments suggest a role of the frequently observed loss of the Y-chromosome. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Laryngeal cancer; Tobacco smoking; DNA adduct; p53 mutation; Chromosome alteration; Genetic risk

AbbreÕiations: HNCs, head and neck cancers; scc, squamous cell carcinoma; PAH, polycyclic aromatic hydrocarbons; BPDE, benzoŽ a.pyrene-diolepoxide; SHBG, sex hormone-binding globulin; PCNA, proliferating cell nuclear antigen; GST, glutathione-Stransferase; NAT, N-acetyltransferase; EH, epoxide hydroxylase; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; FISH, fluorescence in situ hybridization; brc, breaks per cell ) Corresponding author. Tel.: q48-61-8233011; fax: q48-618233235; E-mail: [email protected]

1. Introduction Tumours of head and neck Žexcluding those of the oesophagus. belong to the most common types of cancer, taking the sixth position in the list of the most frequent cancers world-wide w1x. It is estimated that there are roughly 500,000 new cases annually of

1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 3 1 - X

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head and neck cancer ŽHNC. in the world. Besides medical aspects, there are many matters concerning molecular epidemiology and biology of HNC to be studied with adequate molecular methods. A model of multistep carcinogenesis preceding clinically recognised HNC, among which is cancer of the larynx, can be presented by the following scheme:

The above scheme explains the interest that researchers take in investigating particular steps of this process and in studying the relationships between them. In Poznan, ´ Poland, a project was established in 1992, which focused on DNA damage in tobacco smoke-associated larynx cancer. Shortly afterwards, this research effort turned into a multilaboratory cooperation aiming at the investigation of multistage carcinogenesis in the larynx and its interindividual variability and genetic background. The present article is an overview of our results and conclusions confronted with the findings of other investigators.

2. Larynx cancer epidemiology and aetiology HNCs are recognised as a common category primarily because of their anatomic contiguity w2x. Tumours can be located in many different sites including mouth, oral cavity, nasopharynx, larynx, oesophagus Žthere is, however, no common agreement in including oesophagus in this category. and other locations, with larynx appearing to be the most frequent target for tumourigenesis in the head and

neck region. Brain and eye tumours are classified outside the HNC category. In fact, even having excluded brain and eye, HNC represents aetiological and morphological diversity. On the other hand, one can find several common features of HNC w2x. First, the vast majority of HNCs develop in the same anatomical entity — the squamous epithelial lining of the upper aerodigestive tract. So it may be preferable to speak about head and neck squamous cell carcinoma ŽHNSCC.. Second, most HNCs appear to be resistant to cytostatic drugs, and chemotherapy is generally followed by severe, undesired side-effects. Hence, treatment of HNC usually comprises surgery or radiotherapy or a combination of both. Next, the prognosis of diagnosed HNC is poor, as is evident from a 5-year survival coefficient below 55%. It should be added that despite some progress in early diagnosis and subsequent treatment, the survival still remains low. Third, it was hypothesised that multiple primary head and neck tumours share a common clonal origin w3,4x; however, there is no general agreement on this point w5x. Finally, the aetiology of HNC is strongly associated with tobacco smoking and alcohol consumption w6–9x, an aspect that needs to be discussed in more detail. Tobacco and alcohol consumption are mentioned together, as smokers often tend to be also drinkers and vice versa. Moreover, the exposures to these factors seem to act synergistically. It should be noted, however, that this is not the case for lung cancer, where alcohol drinking does not contribute to an increased risk. The risk of cancer associated with combined exposure to tobacco and alcohol is more pronounced in the upper than in the lower part of the larynx, which can be explained by limited penetration of the respiratory tract with volatile components of beverages. A well-marked link between alcohol consumption and larynx cancer risk is only seen with strong alcoholic drinks such as vodka, brandy, whisky, etc. The cancer risk attributable to tobacco smoking w10x is related to the quality of the tobacco, cigarettes and cigars used. Tobacco products with high tar and nicotine levels have been proven to cause a higher increase of larynx cancer risk for the smokers than do the products with lower tar and nicotine levels. The protection of the respiratory tract through the use of filter cigarettes is beneficial for tobacco smokers. On the other hand, fibres can be

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released from the filters and penetrate into the respiratory tissue, thus becoming a further factor initiating tumorigenesis w11x. Tobacco smoking combined with alcohol consumption is undoubtedly the main causative agent in laryngeal cancer. Other human exposures to exogenous, chemical genotoxicants include environmental pollution and hazardous working conditions associated with such industries as metallurgy, petrochemistry, dye production, or occupations such as varnishing, asphalt laying and others w12x. Besides, there are indications of a viral origin for some types of HNC w13,14x. Some geographical differences were reported concerning the occurrence of particular types of HNC w1x. Nasopharyngeal cancer is very rare in the population world-wide except in China and to some extent, in southeast Asia. Oral cavity tumours are common among betel chewers, which is a habit in the Indian subcontinent. The occurrence of laryngeal cancer is not randomly distributed in Europe: it is very rare in Scandinavia and moderately frequent in Mediterranean countries; in Poland, Hungary and former Czechoslovakia, death rates from larynx cancer in males belong to the highest in the world w15x. In Poland, mortality from larynx cancer among males has been continuously increasing from 2.7 deaths per 100,000 in 1963, through 7.5r100,000 in 1989 and 8.4r100,000 in 1993 w16,17x, while the mortality among females is almost constant at a level below 0.5r100,000. A working hypothesis to explain the extraordinarily high morbidity and mortality from larynx cancer in Poland has put forward a combination of all the previously mentioned factors: Ži. a high consumption of poor quality, non-filter cigarettes Žthe number of cigarettes per capita sold in Poland is lower only than that in Mexico and China — the world leaders in tobacco smoking.; Žii. heavy environmental pollution resulting from forced industrialisation under communist reign; and Žiii. difficult working conditions in many branches of industry w6,15x. It must be concluded that larynx cancer as both a health and a social problem in Poland is a challenge itself to researchers. Another peculiarity of larynx cancer is the remarkably disproportionate distribution of its morbidity and mortality between men and women, as mentioned above for the Polish situation: in the world

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population, the malerfemale proportion of mortality due to larynx cancer is about 8–10:1 w1,16,18x.

3. Damage of genetic material induced by genotoxicants 3.1. DNA adducts in larynx cancer subjects The presence of carcinogen:DNA adducts in human cells is recognised as a proof of a previous exposure to a carcinogen w19,20x. A demonstration of aromatic DNA adducts in human laryngeal tissue was first described in 1993 w21,22x when DNA from this tissue was analysed by means of the 32 P-postlabelling technique w23,24x, which was commonly used for detection and quantification of different types of DNA adducts. ‘Bulky’ aromatic DNA adducts result from exposure to polycyclic aromatic hydrocarbons ŽPAHs., nitrohydrocarbons and aromatic amines. All of the above compounds require metabolic activation by cytochrome P450-dependent enzymes before they react with DNA w10,23x. The significance of the activation step of tobacco smoke genotoxicants in the case of larynx cancer was confirmed by Degawa et al. w25x, who found a correlation between the levels of CYP1A1, 2C and 3A4 and the amounts of aromatic DNA adducts in the human larynx. An association of the DNA adducts observed by 32 Ppostlabelling with PAH was deduced from co-migration of the main spot seen in the thin-layer chromatography ŽTLC. chromatogram with the standard adduct, BPDE-N 2-dGMP w21,22x. A more direct differentiation between DNA adducts derived from PAH and aromatic amines, based on variants of the 32 Ppostlabelling technique exploring nuclease-P1 digestion or butanol extraction for adduct enrichment, was not commonly used at that time. The recent follow-up study ŽJ. Banaszewski and L. Moller, manuscript in ¨ preparation. provides proof for the observed adducts to be mostly PAH:DNA adducts, as shown by means of a 32 P-postlabellingrhigh-performance liquid chromatography ŽHPLC. protocol with a number of PAH:DNA adduct standards. More recently, N 7-alkylguanine was found in DNA of larynx tissue, and the authors provided evidence that N-nitrosoamines present in tobacco smoke are capable, after metabolic activation, to

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alkylate DNA, which includes the formation of N 7alkylguanine in the mucosa of the larynx w26x. With regards to carcinogenesis and DNA adducts, most research papers are concerned with the levels of DNA adducts in biopsies of cancer subjects compared to those of non-cancer donors as studied in breast w27x, bronchus w28x, and colon w29x. In larynx cancer patients, DNA adducts were detected in larynx tumour cells, in sections of larynx histopathologically recognised as normal and in peripheral blood lymphocytes w22,26,30x. As expected, the level of DNA adducts in blood cells was lower than that in larynx tissue directly exposed to tobacco smoke ŽTable 1.. The proportion of DNA adduct levels in tumour vs. non-tumour cells of the same organ is an interesting matter. Rapid proliferation of tumour cells facilitates the DNA repair process through a transcription-coupled repair mechanism on the one hand, but there are many reports of defective DNA repair in tumour tissue w31x. Comparing tumour biopsies and non-tumour larynx cells, we have found a significantly higher level of aromatic DNA adducts w30x and a non-significantly higher level of N 7-alkylguanine w26x in the former. When studying breast biopsies, Li et al. w27x found a fully comparable pattern of aromatic DNA adducts in tumour cells and in adjacent normal tissue, but the majority of tumours showed lower levels of DNA adducts than normal adjacent tissues. The only possible interpreta-

tion is that the whole organ is subject to DNA adduct formation, and the presence of DNA adducts is not the only reason for entering multistage carcinogenesis. This conclusion has been strengthened since the aromatic DNA adducts were analysed in the interarytenoid area Žnon-tumour.. This region of larynx was taken under investigation when in 3800 subjects treated for laryngeal cancer at Clinics of Otolaryngology of K. Marcinkowski Medical University in the years 1946–1996, only one case was found with primary tumour location. The level of aromatic DNA adducts in the interarytenoid area slightly exceeded that in tumour as well as nontumour tissue in the group of 33 subjects ŽBanaszewski et al., manuscript submitted.. The lack of pronounced differences in DNA adduct level in laryngeal tumour and non-tumour tissues may reflect the heterogeneous structure of the tumour. It reminds of a variable pattern of chromosomal aberrations in various sections of laryngeal tumours. The differences of chromosomal aberrations were demonstrated by microsatellite marker hybridization and interpreted in terms of field cancerization w32x. It could also be used to explain a variability of DNA adduct level in tumour material. The intertissue comparison of DNA adduct levels leads to one of the fundamental questions in molecular epidemiology related to the applicability of surrogate cells instead of the target tissue. An obvious

Table 1 Average DNA adduct levels in tissues of larynx cancer subjects shown as number of adducted nucleotides per 10 8 normal nucleotides The numbers of samples analysed are given in parentheses. Grouprtissue

Aromatic DNA adducts Ž x " SD.

N 7 -alkylguanines Ž x " SD.

Larynx cancer subjects Larynx tumour — all Smokers Non-smokers Larynx non-tumour — all Smokers Non-smokers Leukocytes — all Smokers Non-smokers

5.7 " 5.1 Ž41. 6.5 " 5.5 Ž30. 3.7 " 0.7 Ž4. 4.7 " 4.9 Ž36. 4.8 " 5.1 Ž30. 5.8 " 3.0 Ž4. 2.1 " 1.8 Ž12. 2.3 " 2.1 Ž11. nd

26.2 " 38.0 Ž44. 28.2 " 36.3 Ž39. 11.3 " 5.9 Ž5. 22.7 " 19.9 Ž33. 24.1 " 18.9 Ž31. 15.3 " 2.0 Ž3. 13.1 " 5.6 Ž9. 13.6 " 48 Ž8. nd

Non-cancer controls Leukocytes — all Smokers Non-smokers

1.3 " 2.1 Ž12. nd nd

9.2 " 5.9 Ž15. 9.7 " 5.9 Ž10. 5.1 " 2.8 Ž5.

K. Szyfter et al.r Mutation Research 445 (1999) 259–274

advantage of the use of surrogate cells is an easy non-invasive way of aspiration. Its validity, though, should first be established by determining a relationship between the levels of a given biomarker in both kinds of tissue. There is an almost universal agreement on the use of peripheral white blood cells as surrogate cell type, but it should be noted that some papers report a lack of correlation between the levels of aromatic DNA adducts in white blood cells and lung w33x or bronchial tissue w28x. In our study, we have found a strong correlation in larynx cancer subjects between DNA adduct levels in larynx tumour cells and in blood leukocytes, and a slightly weaker correlation between adduct levels in nontumour larynx cells and blood leukocytes w30x. 3.2. Confounders of the DNA adduct leÕel in larynx biopsies Tobacco smoking is recognised as a main causative factor in larynx cancer aetiology. Hence, attention was paid to the relationship between the extent of tobacco smoking and the DNA adduct level w26x. When subjects were divided into non-smokers Žincluding ex-smokers who already decided to quit

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smoking at least 5 years before surgery. and smokers categorised further according to number of cigarettes smoked per day, the average levels of aromatic adducts and N 7-alkylguanine increased, as shown in Fig. 1. This is in accordance with the data of other authors, who have found a positive correlation between tobacco smoking and DNA adduct levels in white blood cells w34x, bronchial tissue w28x, and lung w35x. Some other potential confounders modulating the level of DNA adducts in human laryngeal cells were studied. A weakly positive correlation was established between the subjects’ age Žranging from 38 to 78 years. and the level of N 7-alkylguanine w26x. In another study, we reported that an increase of the N 7-alkylguanine level was typically found in a group of male subjects of over 70 years of age Žunpublished.. Lower efficiency of N 7-alkylguanine removal from senescent tissue as compared to younger tissue was observed also by Gaubatz and Tan w36x in mouse kidney cells. Altogether, these observations fit the general phenomenon of a reduced capacity of the aged organism to remove DNA lesions w37x. Any link between DNA adduct level and cancer progression may have prognostic value. Average lev-

Fig. 1. The effect of tobacco smoking on DNA adduct formation in laryngeal tissues. DNA adduct levels Žper 10 8 nucleotides or 10 7 nucleotides for aromatic DNA adducts and N 7-alkyl-dGMP, respectively. are shown as a function of the number of cigarettes smoked per day.

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els of N 7-alkylguanine were calculated separately for groups of larynx cancer subjects categorised according to the TNM staging system w38x. Although the attempt was unsatisfactory because of the small number of patients in the early stage of tumourigenesis, there still were changes observed in the N 7-alkylguanine levels in relation to the tumour growth, reaching a maximum at the T3 stage. Metastasis to the adjacent lymph nodes is usually followed by a remarkable worsening of the patients’ general health, which is not reflected by changes in the N 7-alkylguanine levels. We take it as another proof for DNA adducts playing a significant role in the early stage of carcinogenesis w38,39x; nonetheless, their significance in late stages of cancer cannot be excluded. We have also compared DNA adduct levels in larynx tissues of subjects with primary and recurrent larynx tumours. The average levels of aromatic DNA adducts and N 7-alkylguanine both in tumour and in non-tumour larynx tissue were higher in recurrent biopsies ŽFig. 2.. One can speculate then on persistent impairment of DNA repair occurring during the course of tumorigenesis. 3.3. Sex-related damage of genetic material The question concerning the cause of the substantial difference between men and women in larynx cancer morbidity and mortality remains open. A few hypotheses were raised and experimentally tested. The first one is related to exogenous exposure, and refers to the more frequent tobacco smoking and alcohol consumption in the male than in the female population. However, in leading industrialised countries, it has been observed that adoption by men and women of similar lifestyle habits is not followed by drastic changes in larynx cancer epidemiology. An alternative explanation of this sex difference suggests involvement of hormonal regulation of larynx function, but sufficient experimental data have not been provided yet. We have compared the levels of aromatic DNA adducts and N 7-alkylguanine in tumour and nontumour larynx tissue in the groups of moderate tobacco smokers, both male and female ŽFig. 3.. For both types of tissue, the DNA adduct levels were higher in males. The differences were more pro-

Fig. 2. Average DNA adduct levels Žper 10 8 nucleotides or 10 7 nucleotides for aromatic DNA adducts and N 7-alkyl-dGMP, respectively. in laryngeal tissues in the groups of subjects with primary or recurrent tumours.

nounced in the case of N 7-alkylguanine, reaching a malerfemale ratio of 3.7 in larynx tumour tissue w40x. These results are in contrast with those of Ryberg et al. w41x, who reported higher levels of aromatic DNA adducts in lung biopsies from female lung cancer patients compared to male patients, after adjusting for tobacco smoke exposure. On the other hand, Phillips et al. w42x, studying bronchial biopsies from patients undergoing pulmonary surgery, have found that normal bronchial epithelial cells from males contained 1.9-fold more aromatic DNA adducts than cells from females. In the latter case, the results were not adjusted for tobacco smoke exposure. Also in nasal mucosa tissue from non-cancer smokers w43x, the level of aromatic DNA adducts was 3-fold higher in males than in females; in non-smokers, the difference was smaller Ž1.8-fold.. The analysis of DNA adducts in nasal mucosa was performed on a small group of subjects. We think that the discrepancy between men and women with regards to DNA adduct levels in lung, bronchus, nasal cavity and larynx reflects a difference between lower and upper respiratory tracts. This statement should be considered with caution, however, as exposure to tobacco smoke is not taken into account in the same way by the authors of the cited papers. In any case, tobacco smoke exposure in

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Fig. 3. Sex differences of average DNA adduct levels Žper 10 8 nucleotides or 10 7 nucleotides for aromatic DNA adducts and N 7-alkyl-dGMP, respectively. in laryngeal tissues.

the upper respiratory tract is direct and almost topical, in contrast to exposure conditions in the lower respiratory tract. Differences in activities of carcinogen-metabolising enzymes should be taken into account as well. Nevertheless, the morbidity differences found at the epidemiological level seem to be recognisable also by methods used in molecular epidemiology. Parallel to DNA adduct studies, we have also investigated a possible association between larynx cancer morbidity and the concentration of sex hormones in blood serum w44x. The serum concentrations of testosterone and sex hormone-binding globulin ŽSHBG. were compared with the average DNA adduct levels in the patient groups divided according to age. The only significant result was the association of a decreased level of SHBG with increased morbidity in males over 50 years old. Altogether, we failed to find, however, any significant correlation between the parameters studied. Hence, we have concluded that testosterone and SHBG concentration in serum are not suitable markers of male predisposition to laryngeal cancer w40x. In order to explain sex differences in larynx cancer morbidity, we have conducted an analysis of numerical aberrations of sex chromosomes. The frequent loss of the Y-chromosome reported in HNC was interpreted as a process accompanying aging

w45,46x. On the other hand, loss of the Y-chromosome was claimed to be directly associated with HNC w47x. We decided to study the latter possibility concerning a linkage between Y-chromosome loss and larynx cancer by comparing the presence of this chromosome in larynx tumour and non-tumour cells and in peripheral blood lymphocytes in a small group of larynx cancer subjects. For cytogenetic analysis of lymphocytes, a full classical karyotyping following 72-h culture of mitogen-stimulated cells was applied. Larynx cells were only incubated for 6 h and then analysed by use of the fluorescence in situ hybridization ŽFISH. technique, with a probe specific for the Y-chromosome centromere. As shown in Table 2, frequent numerical aberrations of the Y-chromosome were seen only in tumour tissue Žunpublished.. Although it is still not clear whether the observed aberration is a marker of cancer predisposition and has prognostic value, it seems worthwhile to explore Y-chromosome disintegration further. This work is in progress. 3.4. Mutations and loss of heterozygosity (LOH) in p53 and p16 tumour suppressor genes The DNA adduct analysis described above concerns the whole genome. It is well-known that many

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Table 2 Numerical aberration of the Y-chromosome in larynx cancer shown by full karyotyping in PBL and by the FISH technique in laryngeal cells Subject

Blood lymphocytes Žkaryotype.

Larynx non-tumour cells ŽFISH signal.

Larynx tumour cells ŽFISH signal.

1 2 3 4 5 6

46, XY 46, XY 46, XY 46, XY 46, XY 46, XY

single single single single single single

none single none double single none

lesions along the genome remain without effect, whereas some are crucial for carcinogenesis. The latter group includes lesions located in proto-oncogenes and tumour suppressor genes w48x. Mutations in the p53 tumour suppressor gene are frequent in HNC w49,50x but not in nasopharyngeal cancer w51x. Using single-strand conformation polymorphism ŽSSCP., we have analysed mutations in samples derived from 40 larynx cancer subjects w52x. An extra band was found in 15 samples, among which six transitions and seven transversions localised in exons 5–8 of the p53 gene were detected ŽTable 3.. Some of these mutations were found in codons known to be hotspots for mutagenesis in HNC. On the basis of lung cancer findings w53x, we assume that at least six of the detected mutations could be attributed to genotoxic activity of tobacco smoke. We have found four additional T ™ A transversions Žnot yet described in larynx cancer. that have not been connected with tobacco smoking. Although the group under study comprised many alcohol abusers, the occurrence of T ™ A transversions in association with alcohol exposure seems unlikely, as acetaldehyde Ža metabolite of ethanol. tends to interact with

Table 3 Mutations found in the p53 gene in larynx tumour tissue Type

Number Suggested origin

Transition A ™G Transversion A ™ T Transition C ™T Transition G ™ A Transversion G ™T Transversion T ™ A Transition T ™C

1 1 2 2 2 4 1

tobacco smoke ŽPAH. tobacco smoke Ž N-nitrosoamines. tobacco smoke ŽPAH. ?

exocyclic groups of A, G, and C but not T. Hence, alcohol consumption could contribute to the induction of the mutations observed, and in this way increase larynx cancer risk, but it does not explain the origin of the frequent T ™ A transversions. Overexpression of the p53 gene and accumulation of the p53 protein are regular responses of laryngeal cells to DNA damage w54,55x. The mutations in p53 gene do not necessarily lead to an inhibition of gene expression. There is always a chance that the remaining copy of the wild-type gene would compensate through synthesis of unchanged protein. However, many studies have documented the complete deletion of one copy and mutationŽs. in the other copy in such genes as p53 and p16. This LOH in regions encoding tumour suppressor genes is a severe defect blocking the function of relevant proteins. We have examined 48 samples from larynx cancer subjects for the occurrence of LOH and microsatellite instability in chromosome 9p near the p16 locus, in 17p at the p53 locus and in 17p at the locus D17S520, which is not involved in carcinogenesis w56x. By use of highly polymorphic microsatellite markers in a polymerase chain reaction ŽPCR.-based technique, it was found that the highest level of LOH was present at the p53 locus Žrange: 33–45%. followed by the p16 locus Ž28–38%. and the D17SS520 locus Ž22%.. The data suggest an important protective role of p53 in laryngeal cancer, exceeding that of p16. Similar results have been obtained by Gonzales et al. w57x. On the other hand, some papers claim a substantial role for the p16 protein in suppressing growth of head and neck carcinoma cells w58,59x. Recent findings associate LOH at the p16 protein with an early phase of carcinogenesis. Although most of the tumours we analysed could be classified as T3NM and T4NM,

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the finding concerning more frequent LOH at the p53 location than at p16 seems to be correct. At this point, it is worth adding that studies on tumour suppressor genes and their alterations are of clinical significance w60x. The discovery of LOH at p53 has become a starting point to design individual radiotherapy regimens. A gene-therapy protocol, based on the introduction of the wild-type p53 gene by recombinant adenovirus technology in order to suppress growth of head and neck tumour cells, has been tested in a mouse model w61,62x. 3.5. The loss of cell cycle control Tumour suppressor genes control cell proliferation via the mechanism of cell cycle regulation. Dysfunction of these genes allows a cell to enter uncontrolled proliferation that is a part of neoplastic transformation. The accumulation of p53 protein during progressive stages of carcinogenesis, i.e., from anaplasia and dysplasia to clinically developed cancer, has already been reported w54,55x. With the use of histochemical staining, we have determined levels of p53 protein in larynx tumour biopsies obtained from 120 donors in different stages of disease progression w63x. The level of p53 protein increased with tumour staging. In the same set of samples, we have determined two protein markers of cell proliferation, proliferating cell nuclear antigen ŽPCNA. and Ki67, the levels of which also showed a gradual increase. This means that the larynx cells, which are in the G0 phase under physiological conditions, have entered proliferation, giving rise to their uncontrolled multiplication and tumour growth. Our data are consistent with those of Lavieille et al. w64x who have studied a very similar model. The determination of protein markers for cell proliferation can also be applied to prognostics w65x or, more specifically, to the prediction of occult metastases in laryngeal cancer w66x. We further conclude that a considerable fraction of the p53 protein is the product of a mutated gene unable to exert its proper function in cell cycle control. This is partly confirmed by the results described above ŽSection 3.4., as all 40 samples analysed for p53 mutations w52x were obtained from subjects belonging to a larger group Ž120 persons. analysed for proliferation markers w63x.

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4. The significance of genetic factors in larynx cancer Larynx cancer is not recognised as a hereditary type of cancer mainly because of its clear aetiology associated with exogenous exposure to tobacco smoke as the main causative agent. On the other hand, not all tobacco smokers and not even all heavy smokers develop any type of cancer. Interindividual variability in susceptibility to tobacco smoke carcinogens can hide genetic predisposition. Carcinogenesis is a multistage process involving many non-genetic modulators that affect individual susceptibility. From the work of Copper et al. w67x, there is convincing evidence that unspecified genetic factors play a role in HNSCC. In this large retrospective study, the occurrence of cancer in over 600 first-degree relatives of HNC patients is compared with that in an unrelated control group of the same size. It was found that cancers of the respiratory tract and the upper part of digestive tract are 3.1-fold more frequent in first-degree relatives Ž8-fold in siblings. than in controls. These findings have been confirmed in reports on familial HNC in Brazilian w68x and Canadian w69x populations. Studies in this field are focused on the individual response to genotoxicants, which depends on the genetically determined activities of xenobiotic metabolising enzymes w70,71x. Hence, the heterogeneous response to genotoxic exposure as deduced from interindividual differences of a given marker level within the group of subjects exposed to similar doses of genotoxicant can be recognised as an indirect indication of genetic polymorphism. In the course of our studies, interindividual differences were already noticed, which would indicate potential genetic variability.

4.1. Variable pattern of DNA adduct leÕels The analysis of DNA adduct levels revealed large interindividual differences w22,26,30,38x, as shown in Table 4. Large differences have been noted for aromatic DNA adducts and N 7-alkylguanine in all tissues studied. The calculations concern only a group of moderate cigarette smokers, but exclusion of heavy smokers and non- and ex-smokers did not lead to a

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Table 4 Interindividual variation in the level of DNA adducts in biopsies of laryngeal subjects Tissue

Average level

Range of results

n-fold variation

Aromatic DNA adducts Larynx tumour 5.7"5.1 Larynx non-tumour 4.7"4.9 Leukocytes 2.1"1.8

0.3–22.1 0.1–23.8 0.7–8.4

73.7 238.0 12.0

N 7 -alkylguanines Larynx tumour Larynx non-tumour Leukocytes

0.3"224.5 0.4"64.9 1.5"26.5

748.3 162.3 17.7

26.2"38.0 22.7"19.9 13.1"5.6

Numbers indicate average levels of adducted nucleotides per 10 8 normal nucleotides.

drastic narrowing of the range of interindividual results. The differences in DNA adduct levels have been critically discussed by Hemminki w72x in the light of the assumption of Harris w48x, who explained the actual level of DNA adducts as a result of variable efficacy of counteracting processes of metabolic activation, detoxification and DNA repair. Having analysed two types of DNA adducts in biopsies of the same donors, we have attempted to correlate their levels. Fig. 4 shows the interindividual variation of DNA adduct levels found in tumour biopsies. Aromatic DNA adducts arise from exposure to PAH, whereas N 7-alkylguanines are formed by interaction of N-nitrosoamines with DNA. Correlation coefficients were found to be very low w26x, which means that formation and removal of both types of DNA adducts proceed independently under control of entirely different enzymes. The activation of PAH is mediated by CYP1A1, while N-nitrosoamines are activated by CYP2D6, CYP2E1 and other cytochrome P450-dependent enzymes, all of them encoded by genes in different loci w48x. Differences also emerge in the DNA repair process. Aromatic DNA adducts are removed by nucleotide excision repair, while ‘small’ DNA alkylations can be processed in various ways. Therefore, when aromatic adducts and DNA alkylations are formed under the same conditions of complex exposure, these lesions are formed and removed independently according to the genetically determined activity of enzymes involved w48,70x.

4.2. SensitiÕity to bleomycin-induced chromosome breaks Genomic instability in cancer comprises both cleavage at hidden chromosome breakpoints emerging under the action of a mutagen w73x and apparent chromosome rearrangements which arise during the course of carcinogenesis and subsequently give rise to aberrant cell function w74x. We have undertaken a study on chromosome sensitivity to bleomycin exposure in vitro in line with the suggestion to apply the test for the identification of subjects characterized by an increased risk to develop HNC w71,72x. Established in the laboratory of Spitz et al. w76x and since then known as the ‘bleomycin test’, the method has been used throughout our study w77x. The concept of the method is to estimate the number of chromatid breaks per cell Žbrc. induced by bleomycin in peripheral blood lymphocytes proliferating in vitro. In agreement with the results of other authors w75,78x, we found that bleomycin-induced damage in 35 larynx cancer subjects Žaverage brc s 0.72. significantly exceeded that in the matched controls Žbrc s 0.42.. The number of spontaneous chromatid breaks was also higher in larynx cancer subjects Žbrc s 0.20. than in healthy

Fig. 4. Individual levels of aromatic DNA adducts and N 7-alkyldGMP in laryngeal tumour biopsies of larynx cancer subjects.

K. Szyfter et al.r Mutation Research 445 (1999) 259–274

controls Žbrc s 0.17., although the difference did not reach the level of significance. Extending the study group to 72 subjects and 60 controls has generally confirmed these findings, and has also demonstrated the genetic heterogeneity that is encountered during analysis of a range of individual results. As can be seen from the distribution patterns of individual bleomycin-induced breaks ŽFig. 5., laryngeal cancer subjects and controls suggestively differ with respect to chromosome instability. Another relevant point is that brc indices ) 0.8, or ) 1, classified according to Hsu w75x to be indicative of genuine instability Žbrc. or overinstability, respectively, were found in the group of larynx cancer subjects but never in controls ŽDabrowski et al., manuscript submitted.. 4.3. Genotypes of detoxifying enzymes A considerable part of metabolically activated xenobiotic material is detoxified and removed from the cell and finally removed through urine or bile. We have assumed that differences in the activity of detoxifying enzymes could modulate a biologically active dose of carcinogen more effectively than differences in activating enzymes. The significance of some detoxifying enzymes as modifiers of genetic risk of cancer has been already described w71,79x.

269

We have decided to study the genotype of the polymorphic detoxifying enzyme, glutathione-Stransferase ŽGST., instead of estimating the enzymatic activity itself w80x. GST occurs in one microsomal and four cytosolic isoforms encoded by different genes at different loci w81x. To determine the distribution of defective genes potentially responsible for poor detoxification, we have employed a PCR-based method w82,83x. A group of 179 larynx cancer subjects was compared with 180 controls with respect to the distribution of GST M1 and GST T1 genotypes. The results show that: Ži. the percentage of ‘null’ genotypes in the control group Ž57.7% for GST M1, 21.7% for GST T1. is comparable to other data concerning Caucasians w79x; and Žii. none of the studied GST ‘null’ genotypes is significantly overrepresented in larynx cancer subjects. At this point, the conclusion is that none of the genes studied separately plays a role in individual susceptibility to develop larynx cancer. When tobacco smoking was taken into account, however, the percentage of GST M1 ‘null’ and GST T1 ‘null’ genotypes showed a tendency to increase in the following order: heavy smokers Ž46.3.1 and 19.4% for GST M1 and GST T1, respectively., moderate smokers Ž51.1 and 16.0%. and non-rex-smokers Ž55.6 and 22.2%.. This result at least seems to indicate that defective GST M1 activity cannot be excluded as a determinant of

Fig. 5. Distribution of spontaneous and bleomycin-induced chromosome breaks in peripheral blood lymphocytes proliferating in vitro.

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270

Table 5 Occurrence of ‘risk’ genotypes of detoxifying enzymes in larynx cancer subjects Genotype

GST M1 Žy. GST M3 BrB GST T1 Žy. GST P1 ‘slow’ NAT2 ‘slow’ EPHX1 ‘low’ a EPHX1 ‘low’ b

Larynx cancer subjects Ž n s 214.

Bladder cancer subjects Ž n s 45.

Controls Ž n s 240.

n

n

111 2 39 31 112 30 102

%

OR Ž95% CI.

51.5 0.9 18.2 14.5 52.3 14.0 47.8

0.9 Ž0.8–1.6. 0.3 Ž0.1–1.5. 0.7 Ž0.8–2.1. 1.3 Ž0.7–1.4. 1.0 Ž0.7–1.9. 1.1 Ž0.6–2.0. 1.3 Ž0.8–1.9.

25 4 7 11 28 10 21

risk of smoking-related larynx cancer. On the other hand, in the case of larynx cancer, high exposure to tobacco smoke carcinogens may mask the significance of genetic factors in larynx cancer risk. The data and conclusions described above were confirmed in a study conducted among a group extended to 217 cases and 240 controls ŽR. JaskulaSztul, 1998, PhD thesis.. Because of the recognised association of GST ‘null’ genotypes with a bladder cancer risk w84x, blood samples collected from bladder cancer patients were included as a positive control. Apart from inclusion of larger groups into the study, the analyses were extended to GST M3, GST P1, N-acetyltransferase 2 ŽNAT2. and epoxide hydroxylase ŽEH.. The cases and control groups were large enough to demonstrate a significant overrepresentation of the following ‘risk’ genotypes: GST P1 and EPHX1 in larynx cancer subjects and GST M3, GST P1, NAT2 and EPHX1 in bladder cancer patients ŽTable 5.. The involvement of different genotypes as determinants of the risk of the cancer types under study may reflect differences between the

%

OR Ž95% CI.

n

%

OR Ž95% CI.

55.6 8.9 15.5 24.5 62.2 22.2 46.7

1.0 Ž0.5–1.9. 3.9 Ž1.0–11.3. 1.6 Ž0.7–3.8. 2.2 Ž1.1–5.5. 1.4 Ž0.–2.8. 2.1 Ž0.8–5.6. 1.4 Ž0.6–3.3.

132 7 55 29 128 34 102

55.0 2.9 22.9 12.1 53.3 14.2 43.8

1.0 1.0 1.0 1.0 1.0 1.0 1.0

direct Žalmost topical. exposure to tobacco smoke in the larynx and the indirect exposure in the urinary bladder. The study concerning detoxifying enzymes confirmed: Ži. a tendency of the gene defects to occur in combination Žwhich was the case for GST M1 ‘null’ and NAT2 ‘slow’ in larynx cancer.; and Žii. a masking of the genetic factor by heavy tobacco smoking. A further analysis of GST genotypes explored the significance of the demonstrated combined occurrence of ‘risk’ genotypes w85x. The aim of the work was to correlate a biomarker of exposure to tobacco smoke with the multiplicity of gene deficiences, by calculating the levels of aromatic DNA adducts in the various groups divided according to the number of ‘risk’ genotypes. It was shown that the mean DNA adduct level increased with the number of defective genes ŽTable 6.. Possibly, a deficiency in two or more pathways of detoxification could be recognised as a real risk factor in laryngeal cancer. The final conclusion from this part of the investigations is that the role of a genetic factor as a

Table 6 Mean levels of aromatic DNA adducts Žper 10 8 normal nucleotides. in laryngeal mucosa calculated in groups of subjects according to multiplicity of defects of detoxifying enzymes genes Multiplicity of gene defect

Number of subjects

Tumour

Non-tumour

None Single Double Triple

9 18 10 4

25.8 " 12.0 30.8 " 28.6 48.8 " 44.4 58.4 " 59.1

36.6 " 34.2 33.4 " 40.1 38.8 " 49.3 68.9 " 59.7

K. Szyfter et al.r Mutation Research 445 (1999) 259–274

271

Table 7 Summary of molecular and cellular alterations studied in laryngeal cancer Stage

Phenomena studied

Tobacco smoke carcinogens DNA adducts

PAH, N-nitrosoamines, dose effect aromatic DNA adducts ŽBPDE-N 2 -dGMP., N 7 -alkyl-dGMP; dose effect, targetrsurrogate tissue correlation, confounders p53 ŽG ™ T, G ™ A, C ™ T, T ™ A. LOH at p53 and p16; loss of Y p53 abortive expression PCNA, Ki67 laryngeal scc

Gene mutations Chromosome aberrations Altered gene function Uncontrolled cell proliferation Pathology

determinant of larynx cancer risk is difficult to demonstrate on the basis of genotypes of detoxifying enzymes.

Acknowledgements The authors would like to express their gratitude to the colleagues from Poznan, ´ Huddinge, Helsinki, Bergen and Linkoping who contributed through ex¨ perimental work and discussion of the knowledge of biology of larynx cancer.

5. Conclusions An extensive review on the molecular biology of HNC was published in 1995 by Koch w86x. The author emphasized the frustration of physicians who are getting an increasingly better understanding of the sequence of events in HNC on the one hand, while at the same time, the basic knowledge contributes relatively little to clinical practice. We are afraid that this is still the case. Nevertheless, we want to present a further approximation to understanding the various steps from carcinogenic exposure to a clinically recognised laryngeal tumour. Altogether, the schematic sequence of events presented in Fig. 1 on the basis of the studies performed can now be illustrated with some clear-cut findings compiled in Table 7. Moreover, the magnitude of changes induced by exogenous factors is modulated by host factors. The influence of a factor, such as a subject’s sex, is still difficult to explain. Concerning the significance of genetic factors, no conclusive data have been provided yet to demonstrate the role of a specific gene or group of genes. We strongly believe that a better understanding of the molecular biology and epidemiology of laryngeal cancer will contribute to improvement of the patients’ treatment, which is still far from satisfactory.

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